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Pakistan Journal of Nutrition 9 (1): 52-56, 2010
ISSN 1680-5194
© Asian Network for Scientific Information, 2010
Corresponding Author: S. Janakat, Department of Nutrition and Food Technology, Jordan University of Science and Technology, P.O.
Box 3030, Irbid, Jordan
52
Hepatoprotective Activity of Desert Truffle (Terfezia claveryi) in
Comparison with the Effect of Nigella sativa in the Rat
S. Janakat and M. Nassar
Department of Nutrition and Food Technology,
Jordan University of Science and Technology, P.O. Box 3030, Irbid, Jordan
Abstract: Hepatoprotective activity of Terfezia claveryi aqueous, methanolic and petroleum ether extracts
was evaluated in the rat using a potent hepatotoxin carbon tetrachloride (CCl ) in comparison with the
4
hepatoprotective activity of a reference plant Nigella sativa. The extracts were administrated via gavage three
days prior to CCl intoxication followed by two additional doses one hour and four hours after CCl injection.
4 4
Twenty four hours after intoxication, blood samples were collected and serum bilirubin concentration,
Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST)
activities were measured. Body weight was measured then livers were excises and livers were weighed. The
aqueous, methanolic and petroleum ether extracts of T. claveryi and N. sativa lowered all liver function tests
significantly. However, the aqueous extract of T. claveryi almost normalized the effect of CCl and was as
4
effective as the petroleum ether extract of the reference plant N. sativa. Moreover, the aqueous extract of T.
claveryi normalized CCl induced hepatomegaly, which was comparable to the effect of petroleum ether
4
extract of N. sativa. These results demonstrate that aqueous extract of T. claveryi possesses a very powerful
hepatoprotective activity against CCl and it is as effective as petroleum ether extract of the reference plant
4
N. sativa.
Key words: Truffles, Terfezia claveryi, Nigella sativa, hepatoprotective, bilirubin, ALP, AST, ALT
INTRODUCTION
Truffles grow naturally in many parts of the world
including particular localities of the Arabian Desert (Al-
Delaimy, 1977). Truffles are considered one of the
oldest foods used by the Arabs. They are well known for
their nutritional importance especially when compared
with meat and fish (Bokhary and Parvez, 1993). The
Bedouins use truffles as a substitute for meat in their
diet. Its preparation and cooking methods are similar to
those of meat (Al-Delaimy and Abu-Ghraib, 1970).
Truffles are healthy foods that are low in calories and fat
and rich in fiber, proteins, vitamins and minerals. Their
protein content is higher than that of most vegetables Sample preparation: Terfezia claveryi which is dark
and their amino acid composition is comparable to that brown red in color, small in size and round in shape was
of animal proteins (Gazzani et al., 1998a; Gazzani et al., purchased from local markets of Baghdad. The sample
1998b; Murcia et al., 2002). was washed carefully, peeled and preserved at -20 C
Truffles are traditionally used in folk medicine for the until use. Nigella sativa seeds were purchased from the
treatment of eye ailments in Iraq, Saudi Arabia and the local market of Irbid. The sample was sorted from
Eastern Badia of Jordan (Janakat et al., 2004). impurities, washed and air-dried then was kept at room
Furthermore, truffles have been used as convalescent temperature until use.
for several centuries due to their high content of
antioxidants such as vitamin A, C, ß-carotene and many Chemicals: Bilirubin, ALP, ALT and AST kits were
phenolic compounds, which are very specialized purchased from Cromatest, Spain. CCl was purchased
scavengers of peroxy radicals and are able to reduce from Pharmacos LTD, England.
and chelate ferric ions, which induce lipid peroxidation
(Gazzani et al., 1998a; Gazzani et al., 1998b; Murcia et Test animals: Male Wister albino rats weighing 170-200
al., 2002). The effect of truffles in general and of T. g were obtained from the Animal House Unit at Jordan
claveryi in particular on liver functions was not University of Science and Technology. The animals were
documented earlier. Since the overall incidence of liver
diseases in the general population is about 1%
(Rochling, 2001) and since truffles are very rich source
of antioxidant then most probably truffles will act as a
hepatoprotective agent. Therefore the present study was
undertaken to evaluate the hepatoprotective activity of
aqueous, methanolic and petroleum ether extracts of T.
claveryi in comparison with a reference plant N. sativa
extracts against experimental liver damage inflicted by
CCl .
4
MATERIALS AND METHODS
o
4
Pak. J. Nutr., 9 (1): 52-56, 2010
53
housed in suspended screen wire cages in an air- Analysis System (SAS, 2004). Least significant
conditioned room at 20±3 C and maintained on tap difference was calculated by Students t-test. Different
o
water and standard diet ad libitum. All animal superscriptsdiffer significantly p<0.05.
experiments conformed to local animal care regulations.
Preparation of extracts: Frozen Iraqi truffles were
homogenized using 1:3 (w/v) of each solvent (distilled
water, methanol or petroleum ether), using a household
blender on full speed for one minute. Whereas, N. sativa
seeds were first milled using a household electric mill
then the sample was mixed with each solvent using a
household blender on full speed. The homogenates
were refrigerated overnight, filtered through cheesecloth
and then were centrifuged at 4000 rpm for 15 min. The
supernatants were then dried using rotary evaporator.
The dried matter of the aqueous and methanolic extracts
were re-suspended using distilled water while the dried
matter of the petroleum ether extracts were re-
suspended using paraffin oil and kept at -20 C until use
o
(Nielsen et al., 1997; Janakat et al., 2004).
Experimental design: Hepatotoxicity was induced in rats
using a (1:1) mixture of CCl :olive oil, administrated
4
intraperitoneally at a single dose of 2 ml CCl /kg body
4
weight (Janakat and Al-Merie, 2002a,b). Rats were
divided into groups of five. The control group consisted
of normal untreated rats (negative control). The other
four groups were intoxicated with CCl as described
4
above. Intoxicated groups were treated either with T.
claveryi or with N. sativa extracts (aqueous, methanolic,
or petroleum ether). One intoxicated group did not
receive any extracts (positive control). The test groups
were treated twice daily with the extracts using
intragastric tube for three days. On the fourth day, the
rats were intoxicated with CCl :Olive oil mixture
4
intraperitoneally, followed by two additional doses of
truffle extracts after 1 and 4 h of CCl injection. The
4
negative and positive control groups received distilled
water instead of the extracts. Blood samples were
collected 24 h after CCl administration (Janakat and Al-
4
Merie, 2002a,b).
Assessment of liver function: Rats were anaesthetized
with ether and then decapitated for blood collection.
Serum was separated by centrifugation at 3000 rpm for
10 min. The level of total serum bilirubin and the activity
of ALP, ALT and AST were assayed according to the
methods of Jendrassik and Groff (1938), Bergmeyer and
Brent (1974), Reitman and Frankel (1957) and Berger
and Rudolf (1963), respectively (Jendrassik and Groff,
1938; Bergmeyer and Brent, 1974; Reitman and Frankel,
1957; Berger and Rudolf, 1963).
Statistical analysis: Data were analyzed using analysis
of variance of the complete randomized design (ANOVA)
using the General Linear Model (GLM) of the Statistical
RESULTS AND DISCUSSION
Effect of T. claveryi extracts on liver function tests:
Table 1 depicts the effect of T. claveryi extracts on liver
function tests. As expected the positive control group
which was intoxicated with the potent hepatotoxin CCl
4
had significantly higher bilirubin concentration (0.50
mg/dl) in comparison to the negative control group (0.14
mg/dl). This comes in accordance with the all
researchers findings since the classical article of
Recknagel, 1967 to the present day (Recknagel, 1967;
Muchizuki et al., 2009). The elevation of these
parameters is attributed to significant free radical
mediated hepatotoxicity leading to cell necrosis, fibrosis
and cirrhosis. The mechanism by which CCl causes
4
damage involves the biotransformation of CCl by
4
cytochrome P450 system into a trichloromethyl free
radical (CCl ), which in turn is transformed into a more
3
.
reactive trichloromethyl peroxyl radical (CCl O ) leading
3 2
.
to lipid peroxidation and hepatocellular injury [18].
Moreover, ingestion of T. claveryi extracts caused a
strong significant reduction in all liver function tests
performed. Serum bilirubin level decreased from 0.5 to
0.16, 0.31 and 0.4 mg/dl in aqueous, methanolic and
petroleum ether extracts respectively. Whereas, the
activity of ALP decreased from 144-70, 105 and 126 U/L
respectively, ALT decreased from 791-111, 356 and 511
U/L respectively and AST decreased from 795-188, 420,
and 612 U/L respectively. This can be attributed to the
high antioxidants contents in T. claveryi, such as vitamin
C and ß-carotene (Gazzani et al., 1998a; Gazzani et al.,
1998b; Murcia et al., 2002) which stop the mounting of
peroxyl radical formation and preventing plasma
membrane bleb formation, which conserve the integrity
of the plasma membrane from rupturing and cytosolic
enzymes such as ALP, ALT and ASP from being
released into the blood stream (Mehendale et al., 1994).
Effect of N. sativa extracts on liver function tests:
Table 2 depicts the effect of N. sativa aqueous,
methanolic and petroleum ether extracts on liver function
tests. Elevated bilirubin level induced by CCl decreased
4
significantly when aqueous, methanolic and petroleum
ether extracts of N. sativa were used (from 0.49-0.34,
0.42 and 0.21 mg/dl respectively). The activity of ALP
decreased from 142-105, 133 and 81 U/L respectively,
ALT decreased from 781-385, 553 and 196 U/L
respectively and the activity of AST decreased from 790-
404, 601 and 210 U/L respectively. As evident form the
above mentioned results all extracts were
hepatoprotective, yet the hydrophobic extract was the
most potent, this can be attributed to the volatile oil
which is abundant in N. sativa seeds that has been
Pak. J. Nutr., 9 (1): 52-56, 2010
54
Table 1: Effect of T. claveryi extracts on liver function tests
Aqueous Methanolic Petroleum
Group -ve control +ve control extract extract ether extract
BRN (mg/dl) 0.14±0.003 0.50±0.009 0.16±0.005 0.31±0.011 0.40±0.007
e a d c b
ALP (U/L) 46±1.034 144±1.035 70±1.409 105±1.611 126±1.034
e a d c b
ALT (U/L) 108±1.234 791±2.566 111±1.235 356±2.45 511±1.232
d a d c b
AST (U/L) 170±0.889 795±2.18 188±3.905 420±1.235 612±1.235
e a d c b
ALP; Alkaline Phosphatase, ALT; Alanine Aminotransferase, AST; Aspartate Aminotransferase, BRN; Bilirubin,-ve control; Normal rats,
+ve control; CCl intoxicated rats. Values are expressed as mean±SEM (n = 5). P-values were calculated by Students t-test.
4
Means with superscripts (b,c,d,e) differ significantly from the positive control group, p<0.05
Table 2: Effect of N. sativa extracts on liver function tests
Aqueous Methanolic Petroleum
Group -ve control +ve control extract extract ether extract
BRN (mg/dl) 0.12±0.005 0.49±0.009 0.34±0.041 0.42±0.008 0.21±0.009
e a c b d
ALP (U/L) 46±1.409 142±1.784 105±2.523 133±1.596 81±1.684
e a c b d
ALT (U/L) 111±1.502 781±1.491 385±1.491 553±2.016 196±1.491
e a c b d
AST (U/L) 171±1.235 790±1.491 404±1.127 601±1.008 210±1.127
e a c b d
ALP; Alkaline Phosphatase, ALT; Alanine Aminotransferase, AST; Aspartate Aminotransferase, BRN; Bilirubin,-ve control; Normal rats,
+ve control; CCl intoxicated rats. Values are expressed as mean±SEM (n = 5). P-values were calculated by Students t-test.
4
Means with superscripts (b,c,d,e) differ significantly from the positive control group, p<0.05
shown to contain many antioxidants such as
thymoquinone, monoterpenes (El-Tahir et al., 1993). N.
sativa seeds extracts were also found to cause
immunomodulatoion (El-Kadi and Kandil, 1987), act as
anti-inflammatory agent (Houghton et al., 1995) anti-
tumor agent (El-Daly, 1998) and prevents liver fibrosis,
cirrhosis and decreases liver enzymes elevation
induced by the potent hepatotoxin CCl in the rat (Kanter
4
et al., 2005; Turkdogan et al., 2001; Turkdogan et al.,
2003), the hepatoprotective effect of N. sativa was
attributed to the presence of highly potent antioxidants
such as thymoquinone, carvacrol, t-anethol and 4-
terpineol, phytosterols, phenols and tocopherols, which
prevent the transformation of CCl to trichloromethyl free
4
radical and trichloromethyl peroxyl radical (Houghton et Fig. 1: Effect of T. claveryi extracts on Liver weight/body
al., 1995; Ramadan et al., 2003; Daba and Abdel- weight ratio. -ve control; Normal rats, +ve control;
Rahman, 1998; Burits and Bucar, 2000; Dakhakhny et CCl intoxicated rats. Aq; Aqueous, Meth;
al., 2000). Methanolic, Ether; Petroleum ether. Values are
Effect of T. claveryi extracts on liver weight/body calculated by Students t-test. Means with different
weight ratio: Figure 1 depicts the effect of T. claveryi superscripts (a,b) differ significantly (p<0.05)
extracts on Liver Weight/Body Weight Ratio (LW/BW).
CCl intoxicated rats developed pronounced in the methanolic and petroleum ether extracts, this
4
hepatomegaly in comparison with the normal control, inhibited the biotransformation and mounting of CCl to
LW/BW almost doubled in the positive control. This CCl and CCl O thus decreasing the need for
hepatomegaly can be attributed to the action of detoxification enzymes and transporters (Recknagel et
Constitutive Androstane Receptor (CAR), which is a al., 1989; Huang et al., 2005).
central regulator of xenobiotic metabolism. CAR
activation induces hepatic expression of detoxification Effect of N. sativa extracts on liver weight/body weight
enzymes and transporters which increases liver size ratio: Figure 2 depicts the effect of N. sativa extracts on
(Huang et al., 2005). The ingestion of T. claveryi liver LW/BW ratio. Once again CCl intoxicated rats
aqueous extract normalized the effect of CCl on LW/BW developed pronounced hepatomegaly in comparison
4
ratio, whereas methanolic extract decreased LW/BW with the normal control which is attributed to the action
ratio significantly while petroleum ether extract was of CAR which increases the expression of detoxification
ineffective. This indicates that the quality and quantity of enzymes and transporters that leads to increased liver
antioxidants in the aqueous extract was superior to that size (Huang et al., 2005). Aqueous and methanolic
4
expressed as mean±SEM (n = 5). P-values were
4
3 3 2
. .
4
Pak. J. Nutr., 9 (1): 52-56, 2010
55
Fig. 2: Effect of N. sativa extracts on Liver weight/body
weight ratio. -ve control; Normal rats, +ve control;
CCl intoxicated rats. Aq; Aqueous, Meth;
4
Methanolic, Ether; Petroleum ether. Values are
expressed as mean±SEM (n = 5). P-values were
calculated by Students t-test. Means with different
superscripts (a,b) differ significantly (p<0.05)
extracts of N. sativa did not affect the significant increase
induced by CCl . Whereas, the ingestion of N. sativa
4
petroleum ether extract normalized the effect of CCl on
4
liver weight/body weight ratio which indicates the
abundance of fat soluble antioxidants such as
tocopherols, phytosterols, and phenols in N. sativa
crude oil plays a major role in the prevention of
hepatomegaly (Ramadan et al., 2003).
Conclusion: The aqueous extract of T. claveryi is as
potent as the effect of the reference plant N. sativa seeds
petroleum ether extract and can be used to prevent liver
damage induced by oxidative stress.
ACKNOWLEDGMENTS
We would like to express our gratitude to the Deanship
of Research at Jordan University of Science and
Technology for the financial support of this work, grant
number (156/2004).
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