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Hepatoprotective effect of camel urine against Carbontetrachloride (DOC)

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					       Hepatoprotective effect of camel urine against Carbontetrachloride induced
                                   hepatotoxicity on rats



           Salawa M. E. Khougli1, El-Hassan2 A. M, Mohamed3, O. Y and Majid4, A. A.


      1.    Central Veterinary Research Laboratories, Khartoum.
      2.    Collage of Pharmacy, National Ribat University.
      3.    Faculty of Pharmacy, University of Khartoum.
      4.    National Centre for Research, Khartoum.



Abstract

        Oral administration to rats of 4ml/100g BW of 24 hour adult she-camel urine (24
HCU) and 2ml/100g BW of young early morning urine (EMU); one hour before Carbon
tetrachloride (CCL4) injection result in a remarkable reduction in the death toll to 0 and
40% respectively. 8 ml of the (24HCU) was found lethal to rats in less than 55 minutes
following oral administration. Different doses of the above camel urine and the latter
chloroformic extract were assessed for their effect on rat liver transaminases activity.
Reduction of the high activity of alanine amino-transferase (ALT) and aspartate amino-
transferase (AST) induced by CCL4 and the ameliorate of the liver hepatotoxicity were
noticed after the administration of 4ml, 2ml and 0.2ml respectively. A linear decrease of
ALT activity with double dosing Female Camel urine Chloroformic Extract (CE) was
observed and addition of these components one hour after CCL4 administration gave the
same results. These results indicate that, camel urine, as crude or chloroformic extract
play an important role as an antioxidant and efficiently act as a protective agent against
liver damage.

Introduction:
        Urine is not a waste product, but a purified sterile by-product of blood filtration,
medically referred to as plasma ultra filtrate made by kidneys. It is rather an
extraordinary valuable physiological substance, (Martha, 2000, Armstrong, 1971). It has
been shown throughout the history of medical science till today that urine has a profound
medical uses, (Martha, 2000), such as effectiveness against allergies, psoriasis and all
skin problems. Also Natalie (2002) reported the effect of urine on fertility, fever, burns
and tuberculosis.
        The liver, the key organ of metabolism and execration, is continually exposed to a
variety of xinobiotic and therapeutic agents, hence the disorder associated with it are
numerous and variable. Liver and kidney are target organs for (CCL4)* toxicity. CCL4 is
a colorless volatile liquid with characteristic sweet odor, it is miscible with most aliphatic
and stable in the presence of air and light. Decomposition may produce phosgene, CO
*
    Carbontetrachloride.


                                                                                            1
and hydrochloric acid ( (Krone et al, 1991). The final step in the biotransformation of
CCL4 is catalyzation by cytcrome P-450 enzyme leading to formation of reactive
trichloromethyl radical. In the oxidative biotransformation the most important pathway is
the formation of the most reactive trichloromethyl peroxide, which radical covalently
links to macromolecules and lipid peroxidation occurs via metabolic intermediate of
CCL4, (Nagi et al, 1999). For the management of liver ailment, {Handa et al, 1986) and
Raja (2002) reported that, plants and other natural products are proved to be
hepatoprotective agents.
        The aims of the present study is to make more considerable research work on
camel urine, and to assess camel urine remedy that is claimed in traditional medicine, and
its pharmacological role of liver protection against chemically induced hepatotoxicity.

Materials and methods
1-Materials:
   a/ Source of urine
   Urine was collected from camel premises at the Central Veterinary Research
   Laboratories, Khartoum. And from camels kept on free range at Butana and Gezira
   regions.
    b/ Sample collection
   Urine samples were collected by Tashweel technique described by O’hag (1998) or
   during normal urination. 24 hours urine and early morning urine were collected from
   adult and young camels, respectively.
    C/ Chloroformic extract of camel urine
       This was prepared by mixing equal volumes of urine and chloroform in a
   volumetric flask, the mixture was allowed to shake for three hours at room
   temperature (8-shape horizontal shaker). The mixture was poured in a separating
   funnel, till two layers were clearly separated. The lower chloroformic layer was
   displaced in a weighed beaker and left to complete dryness at room temperature (120
   ml of urine gives 0.5g of chloroformic extract). For intrapretonial injection the extract
   was dissolved in corn oil (15% v/v at dose of 0.1/100g BW) to the rats to induce liver
   hepatotoxicity.
   d/ Experimental animals
       75 wistar albino rats, weighing 90-250 grams of either sex were used in this study,
   they were provided with balanced diet and water ad libitum. The rats were divided
   into 15 groups of 5 rats each and every 5 groups were allowed to a separate treatment.
   Group1 of treatment1 was orally administered 1ml of 2% sodium carboxymethyle
   cellulose (CMC)* and group1 of treatments 2 and 3 received 1ml of corn oil, group2
   in all treatments were injected with 0.1ml CCl4, they act as control. One hour before
   CCL4 injection group3 of three treatments were drenched orally with 2ml of adult
   (24HCU), 2ml (EMU)† was injected with 0.1ml (EMU),chloroformic extract {CE} of
   camel urine respectively. Group4 and 5 of treatments 1 and 2, one hour before CCL4
   injection, received 4 and 8ml of (24HCU) and (EMU) respectively, while those of
   treatment 3 received 0.2 and 0.4 (CE), as shown in Table1.


*
    SodiumCarboxyMethiylCelulose.
†
    Early Morning Urine.


                                                                                          2
   Table (1) Different treatments of camel urine one hour before CCL4 injection

     Group N0.          T1                  T2                 T3
     Control 1          CMC 1ml             Corn oil 0.1ml     Corn oil 0.1ml
     Control 2          CCL4 0.1ml          CCL4 0.1ml         CCL4 0.1ml
                        Adult (24HCU)       Young (EMU)        (CE)
     3                  2 ml                2 ml               0.1 ml
     4                  4 ml                4 ml               0.2 ml
     5                  8 ml                8 ml               0.4 ml

   e/ CCL4 elimination
       Five Wistor albino rats were injected with a single dose 0.1ml/100g BW of 15%
   v/v CCL4 in corn oil to study its elimination by collecting blood samples for four
   days from orbital plexus under light anesthesia. The enzymes ALT and AST were
   measured for four times in 24 hours interval.
   f / Blood sera
       Puncturing orbital plexus 24 hour after administration of CCL4 drew Blood
   samples. Serum was separated by centrifugation at 4000 rpm for 5 minutes and stored
   at -20C for further analysis. Sera samples were biochemically examined for the
   activities of ALT and AST, as described by Reitman and Frankel (1957).
   g / Statistical analysis
   Results of biochemical estimations have been presented as mean ± SD and percentage
   variations against CCL4 controls were calculated. Percentage was calculated by
   considering enzyme level difference between CCL4 treated and control rats as 100%
   level of reduction, (Chakrabborti and Handa, 1986). The variation present in a set of
   data was analysed through one way analysis of variance (ANOVA).
   Results
       100% mortalities were encountered in rats injected with CCL4 only, while those
   groups of rats receiving 2 and 4ml of (24HCU) result in 60 and 100% recovery
   respectively. The 8ml of adult (24HCU) was found lethal to rats in less than 55
   minutes after administration as shown in Table 2.

   Table (2) CCL4 toxicity and % recovery achieved by adult (24HCU)

  Treatment          Log-dose     %recovery      Probit            Calculated
                                                                   probit
  CCL4 +0mL          -            0.00           -                 -
  CCL4 + 2ml         0.3010       60             5.25              4.2
  CCL4 + 4mL         0.6021       100            7.33              7.9
  CCL4 +8ml

                  CCL4 elimination from the body measured by assessing ALT and AST
for four days (mean ±SD) as shown in Table (3) and Fig (1), elevated levels reduced to
normal after 96 hours.




                                                                                      3
Table (3) CCL4 elimination
 Time/           24hours          48hours         72hours            96hours
 enzyme
 ALT IU/L        88.0±18.3        65.33±5.6       48.27±2.41         28.67±6.5
                 6                8
 AST IU/L        208.1±8.9        181.27±8.       161.93±10.55       146.67±6.11
                 5                73

        The serum enzyme levels were highly elevated in group of rat administered
with CCL4. The administration of camel urine one hour before CCL4 brought about
significant lowering of enzyme level (Table 4and5). The 4ml of (24HCU) reduced
AST by 72.5% Alt by 90.3% compared to CCL4 group and were close to the values
of these enzymes in normal rats. The relevant percentage lowering of the enzymes by
2ml (EMU) and 0.4ml or less (CE) for AST and ALT were 143.4%, 92.2% and
58.6%, 83.3% respectively.




Fig.2: Carbon tetrachloride elemination




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Table (4) Effect of (24HCU), (EMU) and (CE) on AST iu/L activity one          hour
after CCL4 induced hepatotoxicity
 Treats/Drugs       (24HCU)      (EMU)     (CE)        Overall Total          Sig
 0.1ml corn oil     19.0±2.47    39.0±3.72 116.8±14.27 58.27±44.39d           *
 15%CCL4            32.80±4.09 51.1±3.85 157.6±2.07 80.5±57.05a               *
 2ml(24hcu),        33.6±3.91    42.6±9.94 150.0±7.18 75.4±55.16              *
 1ml(emu), 0.1(ce)
 4ml(24hcu),        22.8±4.15    33.75±3.0 120.0±7.71 58.85±45.72d            *
 2ml(emu), 0.2(ce) (72.46%)      6         (92.15%)
                                 (143.38%)
 8ml(24hcu),        33.5±11.9    43.25±5.5 135.0±5.57 70.58±47.94b            *
 4ml(emu), 0.4(ce)               6
 Overall mean       28.34±8.53 41.94±7.8 135.88±18.0 68.72±49.66
                                 5         7

(24hcu) 24hour adult camel urine, (emu) young early morning urine, (ce) young early
morning urine chloroformic extract.
No. of rats in each group = 5. Values in parenthesis indicate percentage recovery.
Values are means ± SD
Means with same superscript in column not significat

Table (5) Effect of (24HCU), (EMU) and (CE) on ALT iu/L activity one          hour
after CCL4 induced hepatotoxicity
 Treats/Drugs       (24HCU) (EMU)         (CE)        Overall Total           Sig
 0.1ml corn oil     17.2±3.03 18.22±5.02 28.4±6.19    21.1±6.94d              **
 15%CCL4            43.0±2.72 37.0±4.53   69.0±12.27 46.33±18.14a             **
 2ml(24hcu),        28.2±3.42 22.2±2.39   43.8±2.59   31.4±9.78b              **
 1ml(emu),
 0.1ml(ce)
 4ml(24hcu),        19.7±4.92 20.13±2.87 39.4±3.51    20.79d
 2ml(emu),          (90.3%)    (58.6%)    (70.4%)
 0.2ml(ce)
 8ml(24hcu),        24.4±7.96 23.0±3.54   35.2±5.25   27.53±7.83
 4ml(emu),                                (83.25%)
 0.4ml(ce)
 Overall mean       24.4±2.29 20.74±11.5* 43.16±15.49
                    *          *          *

(24hcu) 24hour adult camel urine, (emu) young early morning urine, (ce) young early
morning urine chloroformic extract.
No. of rats in each group = 5. Values in parenthesis indicate percentage recovery.
Values are means ± SD
Means with same superscript in column not significant




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Discussion
            The results of the antihepatotoxic effect of camel urine against CCL4
   intoxicated rats, revealed a highly significant hepatoprotective effect P<0.05. This
   was evidenced by 100% recovery of the CCL4 intoxicated rats by 4ml/Kg body
   weight of twenty four hour collected adult camel urine, 2ml/Kg BW of early morning
   urine and 0.2ml of chloroformic extract. Higher doses were found lethal to rats
   specially that of 24 hours collection, this might be due to absorption of large quantity
   of chemically active substances which lead to accumulative effect. Also long standing
   of urine might cause some chemical changes, which affect the physiological capacity
   and the level of absorption by endothelial cells. The dose of choice was the 4ml/Kg
   BW of the (24HCU) since 2ml is too small and 8 ml is too large and cause rupturing
   of the rat gut wall.
   Although serum enzyme levels are not a direct measure of hepatic injury, they give a
   picture of the status of the liver. The lowering of enzyme levels is a definite
   indication of hepatoprotective action of camel urine. There are no published data
   concerning camel urine hepatoprotective activity, whereas similar findings were
   reported by many authors, dealing with some plants such as (Ballanitis agypticaca,
   Mellen et al, 1987; Robbin, 1967; Ali et al, 2001). Solanum nigieum plant has a
   protective effect as reported by Rana and Avadhood,mnvc Calotropis procera roots
   chloroformic extract do posses a hepatoprotective effect (Basu et al, 1992). Rhazya
   stricta, Bolanitis aegyptiaca, Halophylum tuberculatum, hepatoprotective effect were
   reported by Ali et al, (2001), Raja, (2002). The data of the lowering percentage
   indicate that 4ml (24HCU) is the major contributor to antihepatotoxic activity of the
   camel urine. On the bases of these results we could clearly suggest that camel urine
   has an active component(s) which may have an important role as an endogenous
   antioxidant and/or could act as cytoprotective agent against tissue damage mediated
   by the toxic substances.
.
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