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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 9, Num. 3, 2005, pp. 77-80

Journal of Applied Sciences & Environmental Management, Vol. 9, No. 3, 2005, pp. 77-80

Anti-Oxidant Protective Effects of Cassia Alata in Rats Exposed to Carbon Tetrachloride

1,*WEGWU, M O; 1AYALOGU, E O; 2SULE, O J

1 Department   of Biochemistry, University of Port Harcourt, P.M.B 5323, Port Harcourt E-mail: wevic2000@yahoo.com
2 Departments of Medical Biochemistry, NigerDeltaUniversityWilberforceIsland, BayelsaState

Code Number: ja05062

ABSTRACT:

The protective activity of Cassia  alata against carbon tetrachloride  (CCl4) – induced   hepatotoxicity  in  rats  was  assessed.  Crude  extracts of petals of the plant in 0.5% ethanol were administered by intubation for 14 days prior to injection of 0.5ml Kg-1 CCl4 into the rats.  Serum aspartate   aminotransferase  and  alanine  aminotransferase  levels, 18 hours after CCl4 administration, decreased significantly (P < 0.05) in rats treated with the extracts than in CCl4 – treated rats only.  These findings suggest that Cassiaalata may be protective against hepatotoxin-induced liver damage in rats.@JASEM

A number of chemicals, including CCl4 are  known  to cause necrosis  of cell or tissue.  The damage or death  of  tissue usually  results  in the leakage of  the  enzymes  in the  affected  tissue(s)  into the blood  stream (Sieger et. al., 1985; Obi  et. al., 2001).  Serum  or  plasma  enzyme  levels have  been used  as  markers  for  monitoring  chemically  induced  tissue damages  (Lin  and Wang, 1986; Ngaha  et. al., 1989). The  enzymes  L-Alanine aminotransferase  (L-ALT) [E.C. 2.6.1.2] and  L-Aspartate  aminotransferase  (L-AST)  [E.C. 2.6.1.1] are  important  enzymes  that  are often employed in assessing  liver  injury (Jaeger et  al., 1975;  Ngaha et. al., 1989;  Obi  et. al., 1998). The biotransformation of CCl4 to metabolites is a  cytochrome  P450-mediated  reaction  that  initiate  lipid  peroxidation and  attendant  tissue  damage.   Shenoy et al (2001) reported  that  the oxidative  damage  through free  radical  generation is  among  the  various  mechanisms   involved  in the hepatotoxic  effects  of  CCl4.

Some plant extracts have been shown to have protective antioxidant effects and are therefore hepatoprotective.  Such   plants  would  include H. rosasinensis  (Obi et. al., 1998), Celosia  argentea  (Koji et. al., 1996),  Ginkgo  biboba  (Shenoy  et  al., 2001), among  others.  Cassia  alata,  a soft  wooden  plant,  common  in  villages  and  clearings  within  the Delta zone  of Nigeria  has  been reported by Elenwo (1997)  to have some antimicrobial properties.  The leaves and stem bark are taken orally after maceration and filteration for the treatment of heart burn, in some localities (Elenwo, 1997).  The flowers and leaves are used for the treatment of ringworms and eczema.

This study  was  therefore  designed  to  evaluate  the anti-oxidant protective effects  of  Cassia alata  in  rats exposed to carbon tetrachloride (CCl4 ).   

MATERIALS AND METHODS

Animals:  Twenty  adult  male  Wistar  albino  rats  (200-250g)  used  in this study  were obtained  from the Animal House,   Department  of Biochemistry, University  of  Port  Harcourt, Port Harcourt,  Nigeria. They were housed  in standard  cages  (Griffin and George Modular Cage  System) and left  to acclimatize  for  7-days  to  laboratory  conditions  before the commencement   of the  experiment.  During  the acclimatization,  the animals  were  fed  with  pelleted  rat chow  and  water  ad libitum.

Chemicals: Absolute ethanol and formic  acid  were  the products  of  BDH Chemical Company  Ltd  (Poole,  England) while  CCl4 and chloroform  were from May and  Baker (Dagenham, England).  Other  materials  include  vegetable  oil  (Mazola produced  for CPC, UK) and rat  chow  (Pfizer  Nigeria  Plc).

Preparation of extracts.     The petals of Cassia alata were collected from Omuhuechi,  Aluu  in Rivers –State, Nigeria. Preparation of  the extracts  followed  the method of Obi et. al. (1998). 50g of petals of  the plant  were  macerated  in a mortar  and introduced into a 250ml beaker.  Initial extraction was achieved using 150ml of  formic  acid, ethanol,  water  (1:10:9 v/v/v) mixture. The extract was filtered after three  hours  through  cheese  cloth.  This was repeated thrice  and the filtrates  were  pooled. An equal volume  of petroleum  ether  (bp 40-60°C) was  employed  in the  ‘washing’ of the pooled  filtrate  in a  separatory  funnel.  The  separatory  funnel  and  its  content  was  shaken  vigorously  (manually at 5min. intervals) for  2 hours and allowed to stand  for  12 hours  in  order  to  eliminate  carotenoids  and   chlorophyll  from  the  extract.  The washed  extract  was left  at  4°C while the  petroleum  ether  layer  was  discarded .  The  organic   solvents  were evaporated  at  70°C  using  low  temperature  hot   plate  (B&T, Searle Company) and the   residue  filtered  through  a 0.45 micron  filter  paper  using  a  vacuum  pump  (Compach).  The   concentrate was  mixed  with 5% (v/v)  ethanol and  left  at  4°C in a  refrigerator  until used.

Experimental Procedure: The   rats  were  divided  into  five experimental  groups  with each consisting  of four rats.  Rats  in  groups  1  and 2  received  2.5ml/kg body weight  of 5% (v/v) aqueous  ethanol  by  gavage.   The   rats  in groups  3,4 and 5  were given 2.5ml/kg body  weight of 1.0, 5.0 and 10.0% (v/v) solution  of the crude extract concentrate in aqueous ethanol,  respectively, by  gavage.  These treatments were for 14 days. CCl4 dissolved in vegetable oil was administered intraperitoneally   to rats  in groups  2, 3, 4 and 5 at  a dose  of  0.5ml/kg body  weight  in 0.5ml  of  vegetable  oil  on  the  14th day, after  the  administration   of the extracts  of  Cassia   alata.  The rats in group 1 received 0.5ml of vegetable oil only intraperitoneally. Eighteen hours after the administration of  CCl4, the  rats  were anaesthetized  in  a  chloroform  saturated  chamber.   Blood samples  were  obtained  by  cardiac  puncture    from each  rat by means of  a 5ml  hypodermic syringe  and  needle  and placed  in an anticoagulant free  bottle.

The  serum was  used  for  the assay  of the hepatic  enzymes  activities  (L-AST  and L-ALT) using  the Humazym  M UV-test  kits.  The  mean values  of the control  and  test serum activities  of  a given enzyme were  compared  using  the student’s  t-test (Zar, 1984).  The significance level was set at  P≤ 0.05.

RESULTS AND DISCUSSION

Serum L-AST and  L-ALT  induced  by CCl4 treatment  and the effects  of  treatment   with  various  concentrations  of  extracts  of  Cassia  alata  prior  to treatment of rats  with CCl4  are shown  in  Tables 1 and 2, respectively. In  the  rats  treated  with CCl4  only, there  was  a significant (P≤0.05)  increase  in the activities  of the enzymes.  A  concentration-dependent  decrease  (P≤0.05) in  the  activities  of both L-ALT and L-AST was observed  in rats  pretreated  with  extracts  of Cassia alata  prior  to  administration  of  CCl4  compared  with CCl4 – treated  rats alone.  There was  no  significant difference (P≤0.05) in  the  activities  of  the enzymes  in the  control  rats (Group 1) with  those  that  received 10% extract with CCl4 treatment  (Group 5) for  L-ALT.  A similar  trend  was  observed  for  L-AST  in  rats  in  Group  4  (5.0% extract)  and Group 5 (10% extract).

Table 1. Effects of crude extracts of Cassia alata on serum L-ALT activity (U/l/mean mg protein) in CCl– induced liver damage.

GROUP

CCl4ml/kg

EXTRACT (%)

MEAN

S.E.M

1.

-

-

102.06a

1.01

2.

0.5

-

213.75b

1.32

3.

0.5

1.0

143.39c

1.53

4.

0.5

5.0

119.29d

1.34

5.

0.5

10.0

109.72a

0.52

  • Means with different superscripts are significantly different at the 0.05 level
  • Values are means for 4 replicates (n=4)
  • S.E.M = standard error of the mean.

One  commonly  used  hepatotoxin  in the  experimental  study  of  liver  diseases  is  CCL4 (Koji  et. al., 1996;  Obi et. al., 1998; Shenoy et al., 2001).  The  toxicity of CCl4  to the liver  of  mammals  is  largely  as  a  result  of   the  active  metabolite,  trichloromethyl  radical  (Johnston  and  Kruening, 1998). The trichloromethyl radical binds to tissue  macromolecules  and thus  induce  peroxidative  degradation  of  membrane  lipids  of the endoplasmic reticulum  which  are rich in polyunsaturated fatty acids.  Shenoy  et. al. (2001) postulated   that  such  development  would   ultimately  lead  to  the formation of lipid  peroxides  that  in turn yield  other  products, among  which is  malondialdehyde  (MDA).  MDA has  been  demonstrated  to cause  damage  to the membrane.  Hence, one  of  the principal  causes  of  hepatotoxicity  of  CCl4 is the  lipid  peroxidative  degradation  of  biological membrane  (Kaplowitz et al., 1986; Cotram  et. al., 1994).

Our preliminary investigations on the toxicity of CCL4 in male wistar albino rats indicate that administration of CCL4  concentrations above O.6 mg/kg was lethal within 24-hours. Also CCL4 levels below O.4mg/kg was not lethal and liver damage was not obvious. These findings informed the application of O.5mg/kg in this study to induce liver damage in rats. The CCL4 dose adopted in this work is in agreement with that of Obi et.al.(1998) who induced liver damage in rats by administering CCL4 levels of  O.5mg/kg         

Table 2 Effect of crude extracts of Cassia alata on serum L-AST activity (U/l/mean mg protein) in CCl4 – induced liver damage

GROUP

CCl4 ml/kg

EXTRACT (%)

MEAN

S.E.M

1.

-

-

107.26a

1.04

2.

0.5

-

   227.32b 

1.53

3.

0.5

1.0

140.38c  

1.30

4.

0.5

5.0

116.44a

1.03

5.

0.5

10.0

110.75a

2.08

  • Means with different superscripts are significantly different at the 0.05 level
  • Values are means for 4 replicates (n=4)
  • S.E.M = standard error of  the mean.

The  results  in this  study  indicate  that CCl4  caused  an increase  in the serum levels  of  marker  enzymes: ALT  and AST.  Such  elevation  is  indicative  of liver  injury,  especially,  the rise  in L-ALT  activity (Lin and  Wang, 1986; Reinke et. al., 1988). The  injection  of  0.25ml of  1.0,  5.0 and 10.0% crude  extracts / kg body weight  of  rats  for  14 days  prior  to administration  of  0.5ml/kg  CCl4 progressively  caused  a  decline  in hepatotoxicity  than with CCl4 alone.  This is evidenced in the marked decrease in serum L-AST and L-ALT  activity  relative  to the group treated  with CCl4 only. Shenoy et. al. (2001) reported  an  elevation  in the  levels  of MDA in  liver  of  rats  treated with CCl4 only.  They  attributed the  increase  in MDA  levels  to enhanced  lipid  peroxidation,  leading    to tissue  damage and failure  of antioxidant  defence  mechanisms  to prevent    the formation  of  excessive  free  radicals. The  results  in this  study  suggest  that  pretreatment   of rats  for 14 days  with  Cassia  alata  prior  to  CCl4 administration  significantly (P ≤ 0.05) reversed  these  changes.  It  would  be  deduced,  therefore,  that  the antioxidant  effects of crude extracts  of  Cassia  alata  could  possibly  be  its  mechanism of  hepatoprotection.

In order to eliminate contamination with lipids, including lipid soluble materials, 50% aqueous ethanol slightly acidified with formic acid was employed in anthocyanin extraction present in the Cassia alata (Obi et al., 1998).  The extraction condition reported in this work ensured that the carotenes, tocopherols, chlorophyll, carotenoids, sterols, aglycones and catecols (Ibrahim and Baron, 1989; Van Sumere, 1989) were not present in the extract. Therefore, the anti-oxidant that is present in the extract is anthocyanin. This is in agreement with Obi et al. (1998) who reported the protection of H. rosasinensis anthocyanin against CCl4 –induced liver damage in rats. The anthocyanin in the extract may have prevented liver damage by breaking the sequence of events between the reductive dechlorination of CCl4; abstraction of hydrogen ion from unsaturated fatty acids in the membrane; and peroxide formation.  The anthocyanin may also have inhibited the activities of cytochrome P450 Isoenzymes required for trichloromethyl radical production

Appreciation: The  authors wish  to express  their   profound  gratitude  to  the University  of Port  Harcourt  for the Senate  Research Grant  No UPH/DVC.AC/11/SD.022/COM.4 in    partial support  for  this  work

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