effect of centella asiatica on arsenic induced oxidative stress and metal distribution in rats

CENTELLA ASIATICA IN ARSENIC TOXICITY 213

Copyright © 2006 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 213–222

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2006; 26: 213–222Published online 3 January 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1131

Effect of Centella asiatica on arsenic induced oxidativestress and metal distribution in rats

Richa Gupta and S. J. S. Flora*

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior-474002,India

Received 29 July 2005; Revised 19 October 2005; Accepted 31 October 2005

ABSTRACT: Concomitant oral supplementation of Centella asiatica (100, 200 or 300 mg kg−−−−−1, orally once daily) during

arsenic exposure (20 ppm in drinking water for 4 weeks) was investigated in rats for its protective value. The animals

exposed to arsenic (III) showed a significant inhibition of δδδδδ-aminolevulinic acid dehydratase (ALAD) activity, a marginal

decrease in glutathione (GSH) and an increase in zinc protoporphyrin (ZPP) level in blood. Hepatic and renal glutathione

(GSH) decreased, while oxidized glutathione (GSSG) and thiobarbituric acid reactive substance (TBARS) levels increased

significantly in the liver, kidney and brain. The activities of brain superoxide dismutase (SOD) and catalase decreased

marginally on arsenic exposure. Concomitant administration of Centella asiatica showed a significant protective action on

inhibited blood ALAD activity and restored the blood GSH level, whereas most of the other blood biochemical para-

meters remained unchanged on Centella asiatica supplementation. Interestingly, most of the hepatic biochemical variables

indicative of oxidative stress showed protection. There was, however, a significant protection observed in the altered kidney

GSSG level and hepatic and brain TBARS. Only a marginal beneficial effect of Centella asiatica on blood and liver

arsenic concentration was noted, particularly at the highest dose studies (300 mg kg−1). No effect of Centella asiatica

on most of the altered renal biochemical parameters was noted. The results thus lead to the conclusion that simultane-

ous supplementation of Centella asiatica significantly protects against arsenic-induced oxidative stress but does not influ-

ence the arsenic concentration in these organs. It can thus be suggested that co-administration of Centella asiatica protects

animals from arsenic-induced oxidative stress but exhibits no chelating property. Further studies are recommended for

determining the effect of co-administration of Centella asiatica during chelation therapy with a thiol chelator. Copyright

© 2006 John Wiley & Sons, Ltd.

KEY WORDS: arsenic toxicity; oxidative stress; protection; Centella asiatica

desorption from iron oxide or up flow of arsenic contain-

ing geothermal water (Thomas et al., 2001; Welch et al.,

1999).

The accepted treatment for poisoning with arsenicals is

the administration of a chelating agent such as 2, 3-

dimercapto-1-propanol (BAL), a dithiol compound. How-

ever, recent advances in the treatment of arsenic toxi-

city have shown that meso 2,3-dimercaptosuccinic acid

(DMSA) and sodium 2,3-dimercaptopropane 1-sulphonate

(DMPS) are orally effective, dithiol chelating agents

useful for treating arsenic poisoning (Flora et al., 1995;

Aposhian and Aposhian, 1990). These chelators are com-

paratively less toxic than BAL and consequently both

DMSA and DMPS can be administered in much higher

doses than BAL (Kreppel et al., 1990; Aposhian and

Aposhian, 1989). Chelation therapy, however, is compro-

mised with many side effects and is generally ineffective

for treating established arsenical peripheral neuropathies

and many other clinical implications and it may not pre-

vent their development in all cases. The potential role

for oxidative stress in the injury associated with arsenic

poisoning suggests that antioxidants may enhance the

efficacy of a treatment protocol designed to mitigate

arsenic-induced toxicity. It was recently reported that the

Introduction

Arsenic is distributed widely in nature and mainly trans-

ported in the environment by water. Humans are exposed

to inorganic arsenic through environmental, medicinal

and occupational sources. The main source of arsenic ex-

posure for the general population is ingestion of drinking

water with a high level of arsenic (ATSDR, 1990; WHO,

1992). Chronic exposure to arsenic (III) causes a wide

range of toxic effects and this metalloid is classified as a

carcinogen in humans (IARC, 1987). Long-term occupa-

tional exposure to arsenic has been associated with an

increased prevalence of cancer of the buccal cavity,

pharynx, lung, kidney, bone, large intestine and rectum

(Enterline et al., 1995; Thomas et al., 2001). Environ-

mental exposures to inorganic arsenic commonly occur

through the consumption of drinking water drawn from

groundwater sources that contain inorganic arsenic dis-

solved from arsenic containing sulphide mineral,

* Correspondence to: S. J. S. Flora, Division of Pharmacology and Toxi-

cology, Defence Research and Development Establishment, Jhansi Road,

Gwalior-474002, India.

E-mail: [email protected]; [email protected]

214 R. GUPTA AND S. J. S. FLORA


combined administration of n-acetylcysteine and succimer

led to a rapid mobilization of arsenic (Flora, 1999).

Several naturally occurring dietary or non-dietary

constituents, and parts of several species of edible plants

having pharmacological activity, influence antioxidant

enzymes and provide protection against free radicals

induced damage (Gupta and Flora 2005a,b; Scartezzini

and Speroni, 2000).

In the Indian system of medicine Ayurveda, Centella

asiatica (Umbelliferae) syn Hydrocotyl asiatica has been

used in various parts of India for different ailments such

as headache, body ache, insanity, asthma, leprosy, ulcers,

eczemas and wound healing (Shukla et al., 1999; Suguna

et al., 1996). Centella asiatica occurs in marshy places

throughout the country up to 200 m. Centella asiatica is

commonly known as Indian Pennywort in English, Gotu

kola in Chinese, Brahmi in Hindi and Manduukaparni,

Maanduuki in Ayurveda. In the course of pharmacologi-

cal studies, the plant showed CNS depressant activity,

antitumour and an inhibitory effect on the biosynthetic

activity of fibroblast cells (Veechai et al., 1984; Quan

et al., 1982; Babu et al., 1995). The whole plant of C.

asiatica has been shown to be beneficial in improving

memory (Vaidyaratnam, 1994) and it is reported to im-

prove the general mental ability of mentally retarded chil-

dren (Kakkar et al., 1984). Nalini et al. (1992) reported

that fresh leaf juice improves the passive avoidance task

in rats. However, studies on the different extracts of the

whole plant on paradigms of learning and memory are

lacking. Kumar and Gupta (2002) reported that the aque-

ous extract of the whole plant of C. asiatica had two pro-

nounced effects, i.e. improving the learning and memory,

and an antioxidant property by decreasing lipid peroxida-

tion and augmenting the endogenous antioxidant enzymes

in the brain. Gupta et al. (2003) recently also recom-

mended the potential role of an aqueous extract of

Centella asiatica as an adjuvant to antiepileptic drugs with

an added advantage of preventing cognitive impairment.

The present study was undertaken to see if the oral

intake of a crude aqueous plant extract of Centella

asiatica could modify (i) the oxidative stress induced by

arsenic, (ii) provide protection to biochemical changes in

the blood, liver, kidneys and brain and, (iii) prevent up-

take of arsenic in blood and soft tissues.

Materials and Methods

Chemical and Plant Material

Sodium arsenite (NaAsO2, molecular weight 129.9,

Sigma Chemical, USA), δ-aminolevulinic acid (Sigma

Chemical, USA) and all other chemicals were of analyti-

cal grade and were purchased from Merck (Germany),

BDH Chemical (Mumbai, India) or Sigma (USA). Pro-

fessor Y. K. Gupta, Department of Pharmacology, All

India Institute of Medical Sciences, New Delhi, India,

supplied the aqueous plant extract of Centella asiatica.

Details on the preparation of plant extract, authentication

and the correct botanical identity was previously reported

(Kumar and Gupta, 2002; Gupta et al., 2003).

Animals and Treatment

The study was carried out using male Wistar rats weigh-

ing approximately 120 g (6–7 weeks old). All animals

were obtained from animal house facility of Defence

Research and Development Establishment (DRDE),

Gwalior. The Animal Ethical Committee of DRDE,

Gwalior, India approved the experimental protocol. All

experiments were performed on male Wistar rats weigh-

ing 120 ± 10 g group-housed in stainless steel cages (5

rats per cage) in an air-conditioned room with tempera-

ture maintained at 25 ± 2 °C and 12 h alternating day and

night cycles and relative humidity of 45%–55%. The rats

were allowed standard rat chow diet (Amrut Feeds,

Pranav Agro, New Delhi, India; metal contents in ppm

dry weight: Cu 10.0, Zn 45.0, Mn 55.0, Co 5.0, Fe 75.0)

and drinking water ad libitum throughout the study.

Experimental Design

Forty animals were randomized into eight groups of

five rats each and were treated as below for a period

of 4 weeks: Group I, normal animals; Group II,

Centella asiatica, 100 mg kg−1 day−1, orally; Group III,

Centella asiatica, 200 mg kg−1 day−1, orally; Group IV,

Centella asiatica, 300 mg kg−1 day−1, orally; Group V,

Arsenic, 20 ppm in drinking water; Group VI, Arsenic (as

in group V) plus Centella asiatica 100 mg kg−1 day−1,

orally; Group VII, Arsenic (as in group V) plus Centella

asiatica, 200 mg kg−1 day−1, orally; Group VIII, Arsenic

(as in group V) plus Centella asiatica, 300 mg kg−1

day−1, orally.

A statistician was consulted before the start of the

experiment for the minimum number of rats required

to give viable statistical and reproducible data and for

statistical analysis. The doses for arsenic and Centella

asiatica were selected on the basis of previously pub-

lished studies (Gupta et al., 2003; Kumar and Gupta,

2002; Sharma and Sharma, 2002).

The food and water intake was recorded and the rats

were weighed every week. After the administration of the

last dose, the animals were given a 1 day rest and were

killed under light ether anaesthesia. Blood was collected

by cardiac puncture in heparinized tubes. Liver, kidneys

and brain were removed, rinsed in cold saline, blotted,

weighed and used for various biochemical variables

and metal analysis. Half portions of the liver and brain

and one of the kidneys from each rat were processed



immediately for biochemical estimation and the remain-

der was stored at −20 °C before wet acid digestion with

HNO3 for estimation of arsenic contents.

Biochemical Assay

Blood δ-aminolevulinic Acid Dehydratase (ALAD)

The activity of blood ALAD was assayed according to

the procedure of Berlin and Schaller (1974). 0.2 ml of

heparinized blood was mixed with 1.3 ml of distilled

water and incubated for 10 min at 37 °C for complete

hemolysis. After adding 1 ml of standard δ-ALA, the

tubes were incubated for 60 min at 37 °C. The reaction

was stopped after 1 h by adding 1 ml of trichloroacetic

acid (TCA). To the supernatant, an equal volume of

Ehrlich reagent was added and the absorbance was re-

corded at 555 nm after 5 min.

Blood Zinc Protoporphyrin

Zinc protoporphyrin (ZPP) was determined in a drop

of blood with the help of a haematofluorimeter (Model

2060, Aviv, Lakewood, USA) and a calibrated glass slide

(Grandjean, 1979).

Blood Glutathione (GSH)

Analysis of blood GSH concentration was performed by

a modified method of Jollow et al. (1974). 0.2 ml of

whole blood was added to 1.8 ml of distilled water and

incubated for 10 min at 37 °C for complete haemoly-

sis. After adding 3 ml of 4% sulphosalicylic acid, the

tubes were centrifuged at 2500 rpm for 15 min. To the

supernatant 0.2 ml of 10 mM solution of 5,5′ dithiobis-

(2 nitro benzoic acid) (DTNB) was added in the presence

of phosphate buffer (0.1 M pH 7.4). The absorbance

recorded at 412 nm was used for calculation of GSH

concentration (Ellman, 1959).

Tissue Reduced Glutathione (GSH) and Oxidized(GSSG) Glutathione

Kidney, liver and brain GSH and GSSG estimations were

performed as described by the method of Hissin and Hilf

(1973). Briefly, 0.25 g of tissue sample was homogenized

on ice with 3.75 ml of 0.1 M phosphate–0.005 M EDTA

buffer (pH 8.0) and 1 ml of 25% HPO3 which was used

as a protein precipitant. The homogenate (4.7 ml) was

centrifuged at 100 000 g for 30 min at 4 °C. For the GSH

assay, 0.5 ml supernatant and 4.5 ml phosphate buffer

(pH 8.0) were mixed. The final assay mixture (2.0 ml)

contained 100 µl supernatant, 1.8 ml phosphate–EDTA

buffer and 100 µl O-phthaldehyde (OPT; 1000 µl ml−1 in

absolute methanol, prepared fresh). After mixing, the

fluorescence was determined at 420 nm with an excitation

wavelength of 350 nm using a spectrofluorimeter (Model

RF 5000 Shimadzu, Japan).

For the GSSG assay, 0.5 ml of supernatant was in-

cubated at room temperature with 200 µl of 0.04 mol l−1

N-ethylmaleimide solution for 30 min. To this mixture,

4.3 ml of 0.1 mol l−1 NaOH was added. A 100 µl sample

of this mixture was taken for the measurement of GSSG

using the procedure described above for GSH assay,

except that 0.1 mol l−1 NaOH was used as the diluent

instead of phosphate buffer.

Superoxide Dismutase

Superoxide dismutase (SOD) activity in the brain was

assayed spectrophotometrically as described by Durak

et al. (1996). Briefly, 2.8 ml of reactive mixture (xanthine

0.3 mM, EDTA 0.67 mM, 150 µM nitrotetrazolium blue

chloride (NBT), sodium carbonate 0.4 M, bovine albumin

30 mg 30 ml−1) was added to 0.1 ml sample and 50 µl

xanthine oxidase (10 µl in 2 M ammonium sulphate), in-

cubated at 25 °C for 20 min and mixed with 0.1 ml 8 M

copper chloride. The colour reaction was measured at

560 nm.

Catalase

Catalase activity in the brain was assayed following the

procedure of Aebi (1984) at room temperature. 100 µl

of tissue extract was placed on an ice bath for 30 min

and then for another 30 min at room temperature. 10 µl

Triton-X 100 was added to the each tube. To a cuvette

containing 200 µl phosphate buffer and 50 µl of tissue

extract, was added 250 µl of 0.066 M H2O2 (in phosphate

buffer) and the decrease in optical density was measured

at 240 nm for 60 s. The molar extinction coefficient of

43.6 M cm−1 was used to determine the CAT activity. One

unit of activity is equal to the mol of H2O2 degraded

min−1 mg−1 protein.

Thiobarbituric Acid Reactive Substances (TBARS)

Tissue lipid peroxidation was measured by method of

Onkawa et al. (1979). Tissue homogenate was incubated

with 8.1% SDS (w/v) for 10 min followed by addition of

20% acetic acid (pH 3.5). The reaction mixture was in-

cubated with 0.6% TBA (w/v) for 1 h in a boiling water

bath. The pink chromogen so formed was extracted in

butanol pyridine solution and read at 532 nm. The

amount of TBARS was calculated using a molar extinc-

tion coefficient of 1.56 × 105M cm−1.

Metal Estimation

The arsenic concentrations in the blood, liver, kidneys

and brain were measured after wet acid digestion using a



Table 1. Body weight and average food and water intake in different groups

Body weight Mean food Mean watergain (g) intake (g) intake (ml)

Normal animals 79.9 ± 4.53 23.5 ± 2.9 27.2 ± 0.97

C. asiatica, 100 mg 82.1 ± 5.98 24.6 ± 2.2 25.3 ± 1.4

C. asiatica, 200 mg 76.9 ± 4.63 22.1 ± 3.6 23.2 ± 2.2

C. asiatica, 300 mg 77.1 ± 5.64 21.8 ± 4.3 21.6 ± 1.8

Arsenic 72.3 ± 3.99 24.6 ± 4.9 25.2 ± 2.0

Arsenic + C. asiatica, 100 mg 66.4 ± 2.93 25.4 ± 3.8 24.2 ± 3.3



Values are mean ± SE; n = 5.

Table 2. Protective value of simultaneous supplementation of Centella asiatica against ar-senic induced changes in some haematological variables in rat

WBC RBC HGB HCT

Normal animals 12.4 ± 0.84 7.0 ± 0.13 14.1 ± 0.59 45.3 ± 1.32

C. asiatica, 100 mg 13.8 ± 0.93 6.9 ± 0.10 14.6 ± 0.67 43.9 ± 0.97

C. asiatica, 200 mg 14.1 ± 0.23 7.8 ± 0.36 13.7 ± 0.12 43.9 ± 0.97

C. asiatica, 300 mg 14.6 ± 0.87 7.5 ± 0.83 13.9 ± 0.19 47.3 ± 1.93

Arsenic 15.4 ± 2.16 7.3 ± 0.19 12.4 ± 0.44 42.4 ± 1.38

Arsenic + C. asiatica, 100 mg 15.3 ± 2.39 7.1 ± 0.13 12.5 ± 0.21 42.1 ± 0.56



MCV MCH MCHC PLT

Normal animals 64.4 ± 1.53 20.1 ± 0.47 31.2 ± 0.22 662 ± 32.6

C. asiatica, 100 mg 60.9 ± 1.97 21.6 ± 1.23 29.6 ± 0.97 598 ± 36.3

C. asiatica, 200 mg 60.9 ± 1.97 21.6 ± 1.23 29.6 ± 0.97 598 ± 36.3

C. asiatica, 300 mg 67.2 ± 1.35 22.3 ± 0.83 32.3 ± 0.78 636 ± 45.2

Arsenic 63.9 ± 1.58 19.8 ± 0.53 31.0 ± 0.62 740 ± 44.3

Arsenic + C. asiatica, 100 mg 65.7 ± 1.04 20.3 ± 0.38 30.8 ± 0.34 727 ± 20.2



Values are mean ± SE; n = 5.

WBC, white blood cells as × 103 µl−1; RBC, red blood cells as × 106 µl−1; haemoglobin as g dl−1; haematocrit as %; MCV, mean

cell volume as fl; MCH, mean cell haemoglobin as pg; MCHC, mean cell haemoglobin concentration as g dl−1; PLT, platelet

count as 103 µl−1.

microwave digestion system (CEM, USA, model MDS-

2100). Arsenic was estimated using a hydride vapour

generation system (Perkin Elmer model MHS-10) fitted

with an atomic absorption spectrophotometer (AAS,

Perkin Elmer model AAnalyst 100). Zinc, copper and

iron contents were also measured in the digested tissue

samples using AAS (Parker et al., 1967).

Statistical Analysis

Data are expressed as mean ± SEM. Data comparisons

were carried out using one-way analysis of variance fol-

lowed by Tukey’s post test to compare means between

the different treatment groups. A difference between

unexposed (with or without treatment) with a value of

P < 0.05 was considered significant.

Results

Effects of Arsenic, Centella asiatica eitherIndividually or in Combination on Body Weight,Food, Water Intake and Blood HaematologicalVariables

No significant changes in body weight, food and

water intake of animals in different groups were noted

(Table 1). The full haematological status of the animals

in different groups is given in Table 2. No changes

in any of the blood haematological variables were noted

following exposure to arsenic, except for a small increase

(statistically non significant) in WBC and PLT counts.

No effect of the combined administration of Centella

asiatica with arsenic on these variables was also

observed.



Table 3. Protective value of simultaneous supplementation of Centella asiaticaagainst arsenic induced changes in some haematological variables in rat

ALAD (nmol ZPP (µmol GSH (mg ml−1)min−1 ml−1 mol−1 heme)

erythrocyte)

Normal animals 8.09 ± 0.31 67.0 ± 3.45 3.29 ± 0.07

C. asiatica, 100 mg 7.95 ± 0.20 63.6 ± 1.34 3.14 ± 0.25

C. asiatica, 200 mg 6.45 ± 0.86 67.5 ± 3.42 3.23 ± 0.16

C. asiatica, 300 mg 6.68 ± 0.40 61.5 ± 4.11 3.61 ± 0.39

Arsenic 1.95 ± 0.30a 87.2 ± 1.34a 2.82 ± 0.18a

Arsenic + C. asiatica, 100 mg 2.95 ± 0.16b 87.4 ± 3.46 3.24 ± 0.12



Values are mean ± SE; n = 5.a P < 0.05 compared with normal animals; b P < 0.05 compared with arsenic exposed animals.

Effects of Centella asiatica on HaematopoieticVariables

The effects of arsenic exposure and co-administration of

Centella asiatica on some selected biochemical variables

of the haematopoietic system are presented in Table 3.

No effect of Centella asiatica administration in normal

animals at any dose level, on blood ALAD, ZPP or GSH

levels was noted. On the other hand, arsenic exposure led

to a significant fall in blood ALAD activity and GSH

concentration, while it produced a significant increase in

ZPP level. Co-administration of Centella asiatica signifi-

cantly increased the inhibited blood ALAD but had no, or

marginal, effect on GSH and ZPP levels. The elevated

blood arsenic concentration showed a marginal decrease

in the animals co-administered the highest dose of

Centella asiatica (300 mg kg−1) (Fig. 1).

Effects of Centella asiatica on Hepatic OxidativeStress

Table 4 shows the effects of Centella asiatica at variable

doses on arsenic induced changes in some biochemical

variables suggestive of hepatic oxidative stress. Arsenic

exposure produced a significant depletion of hepatic

Figure 1. Simultaneous supplementation of Centella asiatica with arsenic on arsenic concentration in blood, liver,kidneys and brain of male rats. Values are mean ± SE; n ===== 5. *P <<<<< 0.05 compared with normal animals; † P < 0.05compared with arsenic exposed animals



Table 4. Protective value of simultaneous supplementation of Centella asiatica againstarsenic induced hepatic oxidative stress in rat

GSH (µg g−1) GSSG (µg g−1) TBARS (nmolmg−1 protein)

Normal animals 1.91 ± 0.08 0.94 ± 0.04 2.7 ± 0.63

C. asiatica, 100 mg 1.95 ± 0.10 0.88 ± 0.08 3.1 ± 0.15

C. asiatica, 200 mg 1.85 ± 0.06 0.99 ± 0.04 3.3 ± 0.26

C. asiatica, 300 mg 1.86 ± 0.09 0.94 ± 0.11 3.6 ± 0.19

Arsenic 1.17 ± 0.04a 1.88 ± 0.02a 5.3 ± 0.18a



Arsenic + C. asiatica, 300 mg 0.75 ± 0.06b 1.26 ± 0.03b 3.5 ± 0.16b


GSH, while GSSG and TBARS levels showed an

increase accompanied by an increase in liver arsenic

concentration. No effect of Centella asiatica on these

variables was noted when administered to normal ani-

mals. On the other hand, co-administration of this herbal

product with arsenic had no beneficial effect on liver

GSH but marginally reduced GSSG and TBARS (at the

highest dose). These changes were also accompanied by

a significant depletion of hepatic arsenic concentration in

animals concomitantly administered arsenic and Centella

asiatica (300 mg kg−1) (Table 4, Fig. 1).

Effects of Centella asiatica on Renal OxidativeStress

Exposure to arsenic produced a significant depletion of

kidney GSH levels while, it lead to a significant increase

in GSSG level (Table 5). The TBARS level also in-

creased, but only marginally, on arsenic exposure.

Centella asiatica had no effect on these variables when

administered in normal animals, however, when co-

administered to animals exposed to arsenic, it signific-

antly reduced the GSSG level, but no effect on GSH and

TBARS was noted. Interestingly, no effect of Centella

asiatica on increased kidney arsenic concentration was

noted (Table 5, Fig. 1).

Effects of Centella asiatica on Brain OxidativeStress

Brain GSH and GSSG levels remained unchanged, while

the TBARS level increased significantly on arsenic expo-

sure (Table 6). Co-administration of Centella asiatica had

no effect on GSH and GSSG either individually or when

co-administered with arsenic. Interestingly, the elevated

brain TBARS level responded favourably to Centella

asiatica. The brain arsenic level also showed a marginal

depletion on Centella asiatica administration particularly

at the highest dose level (Fig. 1).

The brain SOD activity decreased marginally, while

the catalase activity decreased significantly on arsenic

exposure (Table 6). No adverse or beneficial effect of

Centella asiatica on SOD or catalase activities was noted,

except that the animals co-administered arsenic and

Centella asiatica (100 and 300 mg kg−1) showed signifi-

cant protection (Table 7).

Table 5. Protective value of simultaneous supplementation of Centella asiatica againstarsenic induced renal oxidative stress in rat


Normal animals 1.52 ± 0.20 0.80 ± 0.03 3.79 ± 0.36

C. asiatica, 100 mg 1.64 ± 0.24 0.78 ± 0.08 3.16 ± 0.15

C. asiatica, 200 mg 1.34 ± 0.24 0.79 ± 0.04 3.76 ± 0.16

C. asiatica, 300 mg 1.60 ± 0.19 0.74 ± 0.11 3.63 ± 0.22

Arsenic 0.84 ± 0.05a 1.27 ± 0.02a 5.27 ± 0.35a

Arsenic + C. asiatica, 100 mg 0.84 ± 0.06 0.81 ± 0.01b 4.33 ± 0.19b

Arsenic + C. asiatica, 200 mg 0.96 ± 0.03 0.79 ± 0.03b 5.10 ± 0.15

Arsenic + C. asiatica, 300 mg 0.90 ± 0.05 0.85 ± 0.02b 6.84 ± 0.68




Table 6. Protective value of simultaneous supplementation of Centella asiaticaagainst arsenic induced brain oxidative stress in rat


Normal animals 1.06 ± 0.01 0.72 ± 0.01 4.20 ± 0.16

C. asiatica, 100 mg 1.04 ± 0.04 0.71 ± 0.08 4.06 ± 0.12

C. asiatica, 200 mg 1.04 ± 0.02 0.77 ± 0.04 3.76 ± 0.16

C. asiatica, 300 mg 1.06 ± 0.09 0.74 ± 0.01 3.93 ± 0.12

Arsenic 0.90 ± 0.05 0.73 ± 0.02 7.93 ± 2.03a


Arsenic + C. asiatica, 200 mg 1.05 ± 0.03 0.66 ± 0.03 2.55 ± 0.18b

Arsenic + C. asiatica, 300 mg 0.94 ± 0.12 0.86 ± 0.04 2.65 ± 0.16b


Effects of Centella asiatica on Essential MetalStatus

The effect of arsenic and Centella asiatica either alone or

when co-administered with arsenic on blood and tissue

essential status is given in Fig. 2. Arsenic exposure pro-

duced no significant effect on the levels of these essen-

tial metals except for a significant increase in the kidney

copper concentration (Fig. 2a). Co-administration of

Centella asiatica (200 and 300 mg kg−1) with arsenic

led to a pronounced increase in blood and kidney copper

concentration, while the blood zinc concentration also

showed a marginal increase (Fig. 2b). No effect of

Centella asiatica on the blood and tissue iron concentra-

tion was noted (Fig. 2c).

Discussion

Centella asiatica mentioned as ‘Madhya Rasayana’ in

Ayurvedic texts of the Indian system of medicine has

been described for its intelligence promoting property

(Chopra et al., 1956). Animal studies and clinical trials

related to its effects on the central nervous system (CNS)

have shown promising results in improving memory and

in improving the negative effects of fatigue and stress

(Mukerji, 1953). Centella asiatica is reported to possess

anti-ulcer (Tan et al., 1997), cytotoxic (Sarma et al.,

1995), antileprotic, antifeedant and wound healing prop-

erties (Srivastava et al., 1997). It has also been reported

to protect against membrane peroxidation (Padma et al.,

1998) and lipid peroxidation (Chandraprabha et al.,

1996). Despite the fact that Centella asiatica is an impor-

tant medicinal plant, its efficacy in relation to protecting

animals from the toxic effects of metals or metalloid has

not been studied before. The present study revealed its

antioxidant protection against arsenic although only mar-

ginal evidence of its ability to chelate arsenic was noted.

Further, the effects on biochemical variables suggestive

of oxidative stress were also not consistent. The blood

ALAD activity which decreased significantly on exposure

to arsenic responded favourably to co-administration of

Centella asiatica, while the blood GSH and ZPP level

remained statistically unchanged after treatment with

Centella asiatica. ALAD is a sulphydryl containing

enzyme involved in haem synthesis and an important

Table 7. Protective value of simultaneous supplementation ofCentella asiatica against arsenic induced brain oxidative stress in rat

SOD (unit min−1 Catalase (unitmg−1 protein) min−1 mg−1

protein)

Normal animals 2.27 ± 0.17 2.59 ± 0.26

C. asiatica, 100 mg 2.29 ± 0.11 2.71 ± 0.18

C. asiatica, 200 mg 2.18 ± 0.11 2.67 ± 0.14

C. asiatica, 300 mg 2.03 ± 0.05 2.44 ± 0.21

Arsenic 1.70 ± 0.05a 1.93 ± 0.04a

Arsenic + C. asiatica, 100 mg 2.38 ± 0.11b 2.29 ± 0.14b

Arsenic + C. asiatica, 200 mg 1.73 ± 0.05 1.32 ± 0.19

Arsenic + C. asiatica, 300 mg 2.45 ± 0.20b 1.66 ± 0.25




Figure 2. Simultaneous supplementation of Centella asiatica with arsenic on essential metals (zinc, copper andiron) concentration in blood, liver and kidneys of male rats. Values are mean ± S.E.; n = 5. *P < 0.05 comparedwith normal animals; † P < 0.05 compared with arsenic exposed animals



bio-marker for arsenic toxicity. This is a very sensitive

enzyme and a small change in arsenic leads to a signifi-

cant alteration in the activity of this enzyme. On the other

hand, reduced glutathione (GSH) which plays a pivotal

role in protecting cells against reactive oxygen species

and the ZPP level suggestive of iron depletion/anaemia

might require more time to return to a normal level fol-

lowing administration of Centella asiatica. GSH plays an

important role in the detoxification of arsenic through

different mechanisms, facilitating arsenic uptake by the

cells, modulation of arsenic methylation reaction and

stimulation of extraction of methylated arsenic com-

pounds. A small increase in WBC and PLT counts ac-

companied by a significant increase in ZPP was observed

after arsenic exposure. As the synthesis of haem was

interrupted due to inhibition of ALAD activity, the level

of ZPP and to some extent haemoglobin also showed

depletion on arsenic exposure.

One of the most notable observations of the present

study was the hepato-protective role of Centella asiatica,

which is in accordance with an earlier report by Appa

Rao et al. (1969). They suggested that the plant extract

of Centella asiatica prevented liver damage. They also

attributed this effect to the depletion of serum acid

phosphatase activity. Free radicals have been suggested to

be the most likely factor responsible for producing vari-

ous toxic effects in chronic arsenic exposure (Flora,

1999). The antioxidant potential of Centella asiatica has

recently been reported by Kumar and Gupta (2002). They

reported that the aqueous extract of Centella asiatica at

a dose of 200 and 300 mg kg−1 showed a significant de-

crease in the brain levels of MDA, which is the end

product of lipid peroxidation and a measure of free radi-

cal generation. They also reported a simultaneous and

significant increase in the levels of glutathione, a

tripeptide found in all cells, that reacts with free radicals

to protect cells from superoxide radicals, hydroxyl radi-

cals and singlet oxygen (Schultz et al., 2000). In this

study, there was a significant decrease in the activity

of SOD on arsenic exposure, which was significantly

increased by Centella asiatica particularly at the lower

dose level. SOD is the only enzyme that uses superoxide

anions as a substrate and produces hydrogen peroxide as

a metabolite, which is more toxic than the superoxide

radical and has to be removed by catalase (Kumar and

Gupta, 2002; Harman, 1991; Carrillo et al., 1992).

Centella asiatica was effective in increasing the inhibited

brain catalase activity particularly at the lower dose.

These changes, besides the effective role in decreasing

arsenic induced TBARS levels in the liver and brain, may

be due to its antioxidant property. Centella asiatica

contains asiaticoside, madecassic acid, thankunoside,

thankunic acid, asiatic acid, brahmoside, brahminoside,

brahmic acid, isobrahmic acid, centoic acid, centillic acid

(Srivastava et al., 1997) and some flavonoid glycosides,

namely, 3-glucosylquercetin and 3-glucosylkaempferol

and an alkaloid hydrocotyline (Khare, 2004). It is also

reported that Centella asiatica and its three compounds

madecassic acid, asiatic acid and asiaticoside have wound

healing properties in normal as well as in diabetic ani-

mals because of their antioxidant nature (Shukla et al.,

1999; Khare, 2004). Zainol et al. (2003) reported that C.

asiatica had a high antioxidative activity, as good as that

of α-tocopherol. They also reported that the total phe-

nolic content according to the Folin-Ciolteau method

varied from 3.23 to 11.7 g 100 g−1 dry sample. These re-

sults suggest that phenolic compounds are the major con-

tributors to the antioxidant activities of C. asiatica.

It could be concluded from the present results that sup-

plementation of Centella asiatica possessed a significant

protective value as a potent antioxidant activity against a

few arsenic sensitive biochemical variables in blood such

as ALAD activity and hepatic oxidative stress. However,

it had only limited or moderate ability to chelate arsenic

and kidney and brain oxidative injury. Minor side effects

such as an imbalance in essential metals (like copper,

zinc and iron) might be of some concern. Based on these

results, it is suggested that supplementation of Centella

asiatica might prove beneficial in achieving optimum

effects of chelation therapy. Further studies are in

progress in our laboratory in this direction.

Acknowledgement—The authors thank Mr K. Sekhar, Director of theestablishment for his support and encouragement.

References

Aebi H. 1984. Catalase. In Methods in Enzymology, Packer L (ed.).Academic Press: Orlando, FL, 125–126.

Aposhian HV, Apsohian MM. 1989. New developments in arsenic tox-icity. J. Am. Coll. Toxicol. 8: 1297–1305.

Aposhian HV, Aposhian MM. 1990. Meso 2, 3 dimercaptosuccinicacid: chemical, pharmacological and toxicological properties of anorally effective metal chelating agent. Ann. Rev. Pharmacol. Toxicol.

30: 279–306.Appa Rao, MVR, Usha SP, Rajagopalan SS, Sarangan R. 1969. Six

months results of a double blind trial to study the effect ofMandookparni and Punarnava on normal adults. J. Res. Indian Med.

2: 79–85.

Figure 3. Structure of some active constituent ofCentella asiatica (T. Kartnig, 2002. Clinical applicationof Centella asiatica (L.) Urb. In Herbs, Spices andMedicinal Plants, Vol. 3, Craker LS, Simon JE (eds). 148)



ATSDR. 1990. ATSDR Case Studies in Environmental Medicine.Agency for Toxic Substance and Disease Registry: Atlanta, GA.

Babu TD, Kuttam G, Padikkala J. 1995. Cytotoxic and antitumourproperties of certain taxa of Umbelliferae with special referenceto Centella asiatica (L.) Urban. J. Ethnopharmacol. 48: 53–57.

Berlin A, Schaller KH. 1974. European standardized method for thedetermination of delta aminolevulinic acid dehydratase activity inblood. Z. Klin. Chem. Klin. Biochem. 12: 389–390.

Carrillo, Kanai S, Nokubo M, Ivj GO, Sato Y, Kitani K. 1992. (−)Diprenyl increases activities of superoxide dismutase and catalase instriatum but not in hippocampus: the sex and age related differencesin the optimal dose in the rats. Exp. Neurol. 166: 206–215.

Chandraprabha D, Annapurani S, Murthy N. 1996. Inhibition of lipidperoxidation by selected medicinal plants. Indian J. Nutr. Dietet. 33:128–132.

Chopra RN, Nayer SL, Chopra IC. 1956. Glossary of Indian Medicinal

Plants. CSIR: New Delhi, 58.Durak I, Canbolat O, Kavutcu M, Ozturk HS, Yurtarslani Z. 1996.

Activities of total, cytoplasmic and mitochondrial superoxidedismutase enzymes in sera and pleural fluids from patients with lungcancer. J. Clin. Lab. Anal. 10: 17–20.

Ellman GL. 1959. Tissue sulphydryl groups. Arch. Biochem. 82: 70–77.

Enterline PE, Dat R, Marsh GM. 1995. Cancers related to exposure toarsenic in copper smelter. Occup. Environ. Med. 52: 28–32.

Flora SJS. 1999. Arsenic induced oxidative stress and its reversibilityfollowing combined administration of n-acetylcysteine and meso 2,3 dimercaptosuccinic acid in rats. Clin. Exp. Pharmacol. Physiol. 26:865–869.

Flora SJS, Dube SN, Kannan GM, Arora U, Malhotra PR. 1995. Thera-peutic potential of meso 2,3-dimercaptosuccinic acid and 2,3-dimercaptopropane 1-sulfonate against chronic arsenic poisoning inrats. Biometals 8: 111–117.

Grandjean P. 1979. Occupational lead exposure in Denmark: screeningwith a haematofluorimeter. Br. J. Indust. Med. 36: 52–58.

Gupta R, Flora SJS. 2005a. Protective value of Aloe vera against sometoxic effects of arsenic in rats. Phytother. Res. 19: 23–28.

Gupta R, Flora SJS. 2005b. Therapeutic value of Hippophae

rhamnoides L. against sub-chronic arsenic toxicity in mice. J. Med.

Food. 8: 353–361.Gupta YK, Kumar V, Srivastava AK. 2003. Effect of Centella asiatica

on pentylenetetrazole-induced kindling, cognition and oxidative stressin rats. Pharmacol. Biochem. Behav. 74: 579–585.

Harman D. 1991. The ageing process: major risk factor for disease anddeath. Proc. Natl Acad. Sci. 88: 5360.

Hissin PJ, Hilf R. 1973. A fluorometric method for the determinationof oxidized and reduced glutathione in tissues. Anal. Biochem. 74:214–216.

IARC. 1987. Monograph on the Evaluation of Carcinogenic Risks to

Humans: Overall Evaluations of Carcinogenicity: An updating of

IARC Monographs, Vol 1 to 42. IARC: Lyon.Jollow DJ, Mitchell JR, Zamppaglione Z, Gillette JR. 1974.

Bromobenzene induced liver necrosis. Protective role of glutathioneand evidence for 3, 4-bromobenzene oxide as the hepatotoxicmetabolites. Pharmacology 11: 151–155.

Kakkar P, Das B, Vishwanathan PN. 1984. A modifiedspectrophotometric assay of superoxide of superoxide dismutase.

Ind. J. Biochem. Biophys. 21: 130–132.Khare CP. 2004. Encyclopedia of Indian Medicinal Plants. Splinger

Verlag, Heidelberg, Germany 138–139.

Kreppel H, Reich FX, Szinicz, Fichti B, Forth W. 1990. Efficacy ofvarious dithiol compounds in acute arsenic poisoning in mice. Arch.

Toxicol. 64: 387–392.Kumar V, Gupta YK. 2002. Effect of different extracts of Centella

asiatica on cognition and markers of oxidative stress in rats. J.

Ethnopharmacol. 79: 253–260.Mukerji B. 1953. Indian Pharmaceutical Codex. Council of Scientific

and Industrial Research, New Delhi, India, 60–61.Nalini K, Arroor AR, Karanth KS, Rao A. 1992. Effect of Centella

asiatica fresh leaf aqueous extract on learning and memory andbiogenic amine turnover in albino rats. Fitoterapie LXIII: 232–237.

Onkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides inanimal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351–358.

Padma PR, Bhuvaneswari V, Silambuchelvik. 1998. The activities ofenzymatic antioxidants in selected green leaves. Indian J. Nutr.

Dietet. 35: 1–3.Parker MM, Humoller FL. Mahler DJ. 1967. Determination of copper

and zinc in biological material. Clin. Chem. 13: 40–48.Quan S, Chen K, Toshito R, Yoshikim. 1982. Antitumor agents 135.

Structure and stereochemistry of polocandrin, a new cytotoxictriterpene from Polansia dodicandra. J. Nat. Prod. 55: 1488–1497.

Sarma DNK, Khosa RL, Chansauria JPN, Sahai M. 1995. Antiulceractivity of Tinospora cordifilia Miers and Centella asiatica Linn.extracts. Phytother. Res. 9: 589–590.

Scartezzini P, Speroni E. 2000. Review on some plants of Indiantraditional medicine with antioxidant activity. J. Ethnopharmacol. 71:23–43.

Schultz JB, Linderau J, Dichgens J. 2000. Glutathione, oxidative stress,and neurodegeneration. Eur. J. Biochem. 276: 4904–4911.

Sharma J, Sharma R. 2002. Radioprotection of Swiss albino mouse byCentella asiatica extract. Phytother. Res. 16: 785–786.

Shukla A, Racik AM, Jain GK, Shankat R, Kulshreshta DK, DhawanBN. 1999. In vitro and in vivo wound healing activity of asiaticosideisolated from Centella asiatica. J. Ethnopharmacol. 65: 1–11.

Srivastava R, Shukla YN, Kumar S. 1997. Chemistry and pharmacol-ogy of Centella asiatica. A review. J. Med. Arom. Plant Sci. 19:1049–1056.

Suguna L, Sivakumar P, Chandrakasan G. 1996. Effects of Centella

asiatica extract on dermal wound healing in rats. Indian J. Exp. Biol.

34: 1208–1211.Tan PV, Njimi CK, Ayufor JF. 1997. Screening of some African

medicinal plant antiulceroginic activity — Part I. Phytother. Res. 11:45–47.

Thomas DJ, Styblo M, Lin S. 2001. The cellular metabolism and sys-temic toxicity of arsenic. Toxicol. Appl. Pharmacol. 176: 127–144.

Vaidyaratnam PSV. 1994. Indian Medicinal Plants: A Compendium of

500 Species, Vol. 2, Orient Longman: Anna Salai, Madras, India, 52.Veechai AD, Senmi J, Gassan G, Mohinoaro M. 1984. Effect of

Centella asiatica on the biosynthetic activity of fibroblast in culture.Farmacie Edi. 39: 355–364.

Welch AH, Helsel DR, Focazio MJ, Watkins SA. 1999. Arsenic in theground water supplies of the United States. In Arsenic Exposure and

Health Effects, Chappel WR, Abernathy CO, Calderon RL (eds).Elsevier: Amsterdam, 9–17.

WHO. 1992. Guidelines for Drinking Water Quality. Recommendation.

Vol. 1. 2nd edn. WHO: Geneva, 41.Zainol MK, Abd-Hamid A, Yosuf S, Muse R. 2003. Antioxidative

activity and total phenolic compounds of leaf, root and petiole of fouraccessions of Centella asiatica (L.) Urban. Food. Chem. 81: 575–581.

effect of centella asiatica on arsenic induced oxidative stress and metal distribution in rats

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