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DRUGS EXPTL. CLIN. RES. XXX(5/6) 213-220 (2004)
HEPATOPROTECTIVE PROPERTIES OF COMMIPHORA OPOBALSAMUM
("BALESSAN"), A TRADITIONAL MEDICINAL PLANT OF SAUDI ARABIA
AL-HOWIRINY T.A.,
1
AL-SOHAIBANI M.O.,
2
AL-SAID M.S.,
1
AL-YAHYA M.A.,
1
EL-TAHIR K.H.,
1
RAFATULLAH S.
1
1) Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud
University, Saudi Arabia.
2) Department of Pathology, College of Medicine, King Khalid University Hospital, King Saud University, Saudi
Arabia.
Summary: The hepatoprotective activity of an ethanolic extract of Commiphora opobalsamum ("Balessan")
was investigated in rats by inducing hepatotoxicity with carbon tetrachloride:liquid paraffin (1:1). This extract has
been shown to possess significant protective effect by lowering serum transaminase levels (serum glutamate
oxaloacetate transaminase and serum glutamate pyruvate transaminase), alkaline phosphatase and bilirubin.
Pretreatment with an extract of Balessan prevented the prolongation of the barbiturate sleeping time associat-
ed with carbon tetrachloride-induced liver damage in mice. On the other hand, CO
1
/induced low-level nonpro-
tein sulfhydryl concentration in the liver was replenished by the Balessan extract. These data suggest that the
plant C. opobalsamum may act as an antioxidant agent and may have a hepatoprotective effect.
Introduction
Balessan is the Arabic or local name of Commi-
phora opobalsamum (L.) Engl., family Burseraceae.
This plant is one the most ancient plants, with a mag-
Address for correspondence: S. Rafatullah, Medicinal, Aro-
matic and Poisonous Plants Research Center (MAPPRC),
College of Pharmacy, King Saud University, RO. Box 2457,
Riyadh 11451, Saudi Arabia.
Fax: 00966-1-467-6383 E-mail: srafat@ksu.edu.sa
nificent history of healing, and was a valuable medic-
inal agent in ancient Arabia. It grows wild in countries
on both sides of the Red Sea (1). It has been used in
diseases of liver, stomach and urinary tract. A decoc-
tion or tincture is used by local traditional healers for
the treatment of chest, stomach and kidney com-
plaints; to promote digestion; and to relieve rheuma-
tism, scurvy and jaundice (2). Abdul-Ghani and Amin
(3) have reported an antihypertensive activity of an
aqueous extract of this plant in rats. However, there is
0378-6501/2004/5/6 00213+7 $02.50/0
© 2004 Bioscience Ediprint Inc.
Al-Howiriny T.A. et al.
a dearth of scientific data on this plant and therefore
the present investigation was undertaken to evaluate
the antihepatotoxic potential of an ethanolic extract in
laboratory animals.
Materials and methods
Plant collection and extraction. The aerial parts of
the plant were collected from the Farasan Island of
the Red Sea (Saudi Arabia) in March 2002 and were
identified by our taxonomist Dr. Atiqur Rahman (Col-
lege of Pharmacy, King Saud University). A voucher
specimen (#14312) was deposited at the herbarium
of the College of Pharmacy for future reference. Pow-
dered shade-dried aerial parts of the plants were
macerated in 96% ethanol for 36 h. Solvent elimina-
tion was carried out under reduced pressure which
yielded a brownish semisolid compound. A solution
of the extract was made in distilled water for admin-
istration to animals.
Animals. Wistar albino rats, of either sex and ap-
proximately the same age (8-10 weeks), weighing
180-200 g, obtained from the Experimental Animal
Care Center, College of Pharmacy, King Saud Uni-
versity, Riyadh, were used. Swiss albino mice were used
for studies of sleeping time. The animals were kept in
constant temperature (22 ± 2 °C), humidity (55%)
and light-dark conditions (12/12 h light/dark ratio). The
animals were provided with Purina chow and free ac-
cess to drinking water ad libitum.
Phytochemical screening. A phytochemical anal-
ysis of the aerial parts of Balessan was conducted for
the detection of alkaloids, cardiac glycosides, flavo-
noids, tannins, anthraquinones, saponins, volatile oil
and cyanogenic glycosides, glucosinolates, coumarins,
sterol and/or triterpenes (4).
Induction of acute hepatotaxicity by carbon tetra-
chloride (COJ. Male Wistar rats were divided into four
groups containing six animals in each group. Group I
was kept as a control group. Groups II, III and IV re-
ceived 0.25 ml of CCI
4
in liquid paraffin (1:1) per 100 g
body weight intraperitoneally (5). Group II received
only CCI
4
treatment. Groups III and IV were treated
with 250 and 500 mg/kg of ethanolic extract of
Balessan, respectively. Drug treatment was started 5
days prior to CCI
4
administration and continued until
the end of the experiment. After 48 h, following CCI
4
administration, animals were sacrificed using ether
anesthesia. Blood was collected by heart puncture
and the serum was separated. The liver was immedi-
ately removed and a small piece was fixed in 10%
formalin for histopathological assessment.
Assay of serum glutamic-oxaloacetic transaminase
(GOT), glutamic-pyruvate transaminase (GPT), alka-
line phosphatase (ALP) and total bilirubin activities.
The collected blood was centrifuged at 3,000 rpm for
10 min to separate the plasma. The plasma was ana-
lyzed for the biochemical parameters including GOT,
GPT, alkaline phosphatase and total bilirubin (6, 7).
Estimation of nonprotein sulfhydryl groups (NP-SH).
The activity of NP-SH was measured according to the
method of Sedlak and Lindsay (8). The liver tissue was
homogenized in ice-cold 0.02 M ethylenediaminete-
traacetic acid (EDTA). Aliquots of 5 ml of the homo-
genates were mixed in 15 ml test tubes with 4 ml of
distilled water and 1 ml of 50% trichloroacetic acid.
The tubes were shaken intermittently for 10-15 min and
centrifuged at 3,000 g. Two milliliters of supernatant
were mixed with 4 ml of 0.4 M Tris buffer, pH 8.9, and
0.1 ml of 0.4% (5,5-dithio-bis- [2-nitrobenzoic acid])
(DTNB) was added and the sample was shaken. The
absorbance was read within 5 min of addition of DTNB
at 412 nm against a reagent blank with no homo-
genate.
Hepatoprotective properties of Balessan
Measurement of phenobarbital-induced sleeping
time. Mice were divided into four groups of 10 ani-
mals each. Group I received the vehicle (0.3 ml of
saline); group II received CCI
4
only. Groups III and IV
received Balessan extract (250 and 500 mg/kg orally).
Thirty minutes after the administration of the extract,
animals of groups II, III and IV were treated with sodi-
um phenobarbital (50 mg/kg, intraperitoneally). The
time interval between the onset and the regaining of
the righting reflex was measured as sleeping time (9).
Histopathalogical studies. The liver tissue was fixed
in 10% ethanol buffered formalin and processed through
graded ethanol, xylene and impregnated with paraf-
fin wax; sections were made by microtome. After stain-
ing with hematoxylin-eosin stain, the sections were
examined under a research microscope by a person
who was not aware of the experimental protocols. The
different histopathological indices were screened (10).
Statistical analysis. The data were statistically ana-
lyzed using Student's f-test.
Results
The preliminary qualitative phytochemical screen-
ing of aerial parts of Balessan revealed the presence
of flavonoids, tannins, sterols and/or triterpenes.
The effects of ethanolic extract on CCI
4
-induced
hepatotoxicity in rats are shown in Table I. Rats sub-
jected to the CCI
4
regimen alone developed signifi-
cant hepatocellular damage as evidenced by a sig-
nificant elevation in serum activities of GOT, GPT, ALP
and bilirubin concentrations compared with normal
values, which have been used as reliable markers of
hepatotoxicity. Oral administration of an ethanol extract
of Balessan (250 and 500 mg/kg) exhibited a signifi-
cant reduction in CCI
4
-induced increased levels of
SGOT, SGPT, ALP and serum bilirubin concentrations.
NP-SH contents in liver were significantly decreased
following the administration of CCI
4
. Treatment with
Balessan extract (either dose) significantly reversed
the NP-SH level (Table II).
There was a significant lowering of phenobarbital-
induced sleeping time following the administration of
the Balessan extract (500 mg/kg) in the CCI
4
-induced
acute liver injury model (Table III). In contrast, the lower
dose (250 mg/kg) showed an insignificant reduction
in sleeping time.
Histological observations supported the results
obtained from liver enzyme assays. Confluent hepat-
ic cell necrosis and karyorrhexis and karyolysis of
hepatocytes were also noted in the control CCI
4
-
treated rat livers. Extensive hepatic cell steatosis was
seen. No confluent necrosis was observed in either of
the groups treated with Balessan extract (Figs. 1-4).
Table I Effect of an ethanol extract of Balessan on some enzymes and bilirubin in rats with CCI
t
-induced liver damage
Treatment (n = 6) Dose mg/kg GOT mg/kg
GPT mg/dl ALP mg/dl Bilirubin mg%
Control
N. saline 151.00 ± 9.76
91.4 ± 3.94 399.16 ± 46.60 0.72 ± 0.025
CCI
4
1.25 ml/kg
592.25""
a
± 49.35
474.41 **"
a
± 35.67
729.65*"
a
± 33.65
1.69*'*
a
±0.12
Balessan + CCI
4
250
256.41'"" ± 13.74
123.66"'" ± 6.60
509.33*"" ± 23.06
1.45 ± 0.003
Balessan + CCI
4
500
200.08"*" ± 30.80 115.58"*" ± 9.99
489.00 """ ± 24.55
1.06"*" ± 0.03
"*p < 0.001 Student's f-test;
a
as compared with the control (normal saline) group; "as compared with the CCI
4
group. GOT = glutamic-
oxaloacetic transaminase; GPT = glutamic-pyruvate transaminase; ALP = alkaline phosphatase.
215
Al-Howi.riny T.A. et al.
Table II Effect of an ethanolic extract of Balessan on the level of nonprotein sulfhydryl (NP-SH) groups in the liver of rat treated with CCI
4
Treatment {n = 6) Dose mg/kg orally NP-SH (mean ± SE) mol/g of tissue
Control normal saline - 1.62 + 0.13
Control CCI
4
- 0.95 ± 0.04"'
a
Balessan extract + CCI
4
250 1.26 ± 0.008"
b
Balessan extract + CCI
4
500 1.50 ± 0.07""
b
a
As compared with the control (normal saline) group;
b
as compared with the control (CO.). "p < 0.01; '"p < 0.001 Student's f-test.
Discussion
The efficacy of any hepatoprotective drug is
essentially dependent on its capability to either
reduce harmful effects or to maintain the normal
hepatic physiological mechanisms that have been
unbalanced by the hepatotoxin (5). The results of the
present study reveal that the ethanolic extract of
Balessan possesses significant hepatoprotective
and antioxidant activities against CCI
4
-induced liver
damage in rats. It has been observed that CCI
4
is bio-
transformed by the cytochrome P-450 system to the
trichloromethyl free radical. This free radical may
react again with oxygen to form a trichloromethyl per-
oxyl radical, which may attack lipids on the mem-
brane of endoplasmic reticulum. The trichloromethyl
peroxyl free radical leads to lipid peroxidation, the
disruption of Ca
++
homeostasis and, finally, results in
cell death (11,12). Therefore, leakage of large quan-
tities of enzymes into the blood stream are often
associated with massive necrosis of the liver (13).
Administration of CCI
4
results in a rapid increase of
serum GOT GPT and ALP levels (14). Serum GOT can
be found in the liver, cardiac muscle, kidney, brain,
pancreas, lungs, skeletal muscle, leukocytes and ery-
throcytes (in decreasing concentrations) (15), whereas
the highest concentration of Serum GPT is found in the
liver. In tissues, Serum GPT occurs in two locations,
the cytosol and mitochondria (16). Serum GPT ap-
pears to be a more sensitive and specific test of acute
hepatocellular damage than Serum GOT (14). There-
fore, the possible hepatoprotective mechanism of
Balessan extract on CCI
4
-induced liver injuries may be
due to the following factors: (i) inhibition of cytochrome
P-450 activity; (ii) prevention of lipid peroxidation; (iii)
stabilization of the hepatocellular membrane; and (iv)
enhancement of protein synthesis (17).
Furthermore, alkaline phosphatase (ALP) is the
prototype of these enzymes that reflects the patho-
logical alteration in biliary flow (18). CCI
4
-induced ele-
vation of this enzymatic activity in serum is in line with
Table III Effect of the ethanolic extract of Balessan on duration of phenobarbital sleeping time of mice treated with CCI
4
Treatment (n = 6) Dose mg/kg Sleeping time (mice) Reduction in sleeping time
Only phenobarbital
50
33.2 ± 2.33
-
CCI
4
+ phenobarbital
-
132.2 ± 6.15"*
a
-
CCI
4
+ Balessan extract + phenobarbital 250 + 50
127.8 ± 4.77
b
3%
CCI
4
+ Balessan extract + phenobarbital 500 + 50
109.00 ± 5.12'
b
18%
216 'p < 0.05, '"p < 0.001 Student's f-test.
a
As compared to the phenobarbital group;
b
as compared to the CCI„ + phenobarbital group.
Hepatoprotective properties of Balessan
Fig. 1 Control animal (no treatment) portal space (in the center)
surrounded by normal liver parenchymal cells. Hematoxylin-eosin
x200.
the high level of serum bilirubin content (17). The
extract-mediated suppression of the increased ALP
activity with the concurrent depletion of raised biliru-
bin level suggests the possibilities of the extract
being able to stabilize biliary dysfunction in the rat
liver, thereby indicating its effectiveness in maintain-
ing the normal functional status of the liver (20). Our
observations in the present study also indicate that
treatment with CCI
4
caused a significant reduction in
Fig. 2 Liver parenchyma after CCI,, treatment (exposure); central
vein of the lobule surrounded by confluent hepatic cell necrosis. Note
karyorrhexis and karyolysis of hepatocytes. Hematoxylin-eosin x 200.
NP-SH concentration in the rat liver. An ethanolic
extract of Balessan, however, offered a significant
replenishing of the NP-SH level. Thus, sulfhydryl
seems to have a role hepatoprotection through its
antioxidant potential (21, 22). Additionally, phenobar-
bitone-induced sleeping time is significantly pro-
longed in liver damage and this parameter may be
employed as a measure of functional status of the
hepatic drug-metabolizing system (23).
Al-Howiriny T.A. et al.
Fig. 3A Liver parenchyma after CCI
4
exposure and Balessan 250 mg/
kg treatment. No confluent necrosis is evident. Instead, the pericen-
tral area displays extensive hepatic cell steatosis as well as inflam-
matory infiltrate. Hematoxylin-eosin x 200.
Regarding the effect of CCI
4
on liver cells and the
protective effect of Balessan, no confluent necrosis
was observed in either Balessan extract-treated
groups, which supports our biochemical findings.
The chemical constituents of Balessan, respon-
sible for its hepatoprotective activity against chemi-
cal injury, is not known. However, Balessan contains
a number of phytochemical constituents, including
flavonoids, saponin, volatile oils, sterol and/or tri-
terpenes. All of these constituents are known to
Fig. 3B Liver parenchyma after CCI
4
exposure and Balessan 250
mg/kg treatment. No confluent necrosis is evident. Instead, the
pericentral area is localized around central vein. Hematoxylin-
eosin x 200.
exhibit antioxidant activity, offer protection against
cell damage and possess free radical scavenging
effects (24, 25). Interestingly, some Commiphora
species have been shown to possess diversified
activities through various mechanisms. These in-
clude Commiphora molmol and Commiphora
mukul, which showed pharmacological effects that
included anti-inflammatory, antihepatotoxic, antic-
holesterolemic, antiulcer and cytotoxic actions (26-
29).
Hepatoprotective properties of Balessan
Fig. 4 Liver parenchyma after CCI
4
exposure and treatment with
500 mg/kg of Balessan extract. There is no evidence of necrosis or
bile retention. The rim of steatotic hepatocytes appeared and sur-
rounds the periportal space. Hematoxylin-eosin x 200.
In conclusion, this study demonstrates that Ba-
lessan possesses significant hepatoprotective and
antioxidant effects in rats. Further studies are neces-
sary to isolate the active chemical component(s) and
to elucidate its exact mechanism(s) of action.
Acknowledgment
The authors are grateful to the Research Center,
College of Pharmacy, KSU, grant CPRC#123, and to
Mr. Malik Sawood for his assistance.
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