Int. J. Pharmacognosy, 29 (1991), No. 4, pp. 296-301
© Swets & Zeitlinger
Hepatoprotective and Safety Evaluation Studies
S. RAFATULLAH, J.S. MOSSA, A.M. AGEEL, M.A. AL-YAHYA
and M. TARIQ*
Medicinal, Aromatic and Poisonous Plants Research Center and Departments of
Pharmacognosy and Pharmacology, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh-11451, Saudi Arabia
The effect of the ethanol extract of sarsaparilla (Smilax regelii) has been studied on carbon tetra-
chloride (CCi4)-induced hepatocellular damage in rats. Pretreatment with an ethanol extract of sar-
saparilla significantly inhibited CCI4-induced biochemical changes. Acute and chronic toxicity studies
were also undertaken to determine the safety of prolonged use of sarsaparilla. Acute administration
of sarsaparilla extract in the dose range of 0.5 to 3.0 g/kg did not produce any adverse effects or
mortality in mice over a period of 24 hours. Animals treated with sarsaparilla extract (100 mg/kg/
day) for a period of 90 days in drinking water showed no symptoms of toxicity. There was no
significant change in body weight and hematological parameters in the chronically treated animals as
compared to the control group. These findings suggest that sarsaparilla, besides having hepatoprotective
potential, has no untoward effects in rodents.
The uses of sarsaparilla (Smilax regelii Killip & Morton, Fam. Liliaceae) as tonic,
antirheumatic, diuretic, and for the treatment of skin and liver diseases have long
been known (Al-Baitar 1871; Ghani 1921; Kritikar and Basu 1918; Nasir 1882).
This plant was introduced in Europe by Spaniards in the early sixteenth century
(Trease and Evans, 1972). Sarsaparilla was included in the British Pharmacopoeia
in 1864. In 1942, the drug was added to the United States Pharmacopoeia (Wallis,
1955). In Greco-Arab and Indian systems of medicine, the drug is still used for
the treatment of liver diseases, inflammation and renal deficiency (Ghani, 1921;
Raza, 1933; Albert, 1952). Besides its medicinal use, sarsaparilla is often used as
a flavouring agent in non-alcoholic drinks (Stuart, 1979). A decoction made
from the roots is used as a vehicle in the preparation of syrups which have been
reported to have cooling properties (Clause, 1971). Recently, Ageel et al. (1989)
have observed significant anti-inflammatory and anti-rheumatic activity of an
ethanol extract of sarsaparilla in rats. However, the much claimed activity against
liver disorders has not been scientifically documented. The present investigation
has been undertaken to study the hepatoprotective and toxic potential of an
ethanol extract of sarsaparilla in rodents.
MATERIALS AND METHODS
The roots of sarsaparilla were procured from a local market and identified at the Medicinal, Aromatic
and Poisonous Plant Research Centre of King Saud University.
The powdered drug (500 g) was extracted using percolation with 96% ethanol. The solvent was
removed at low temperature under reduced pressure and the extract stored in a refrigerator prior to
pharmacological and toxicity studies. The extract yield was 11.6% (w/w) in terms of starting mate-
rial. All doses are expressed in terms of the extract.
Animals and exposure conditions
Male Wistar albino rats, weighing approximately 185-200 g and Swiss albino mice, weighing 25-30
g (home bred), were fed Purina diet and tap water ad libitum. The animals were housed at a tem-
perature of 25 "C + l'C, with a 12-h light/dark cycle (light from 0600 to 1800 h).
Carbon tetrachloride (CCl4)-induced hepatocellular damage
The rats were divided into three groups of six rats each. The first group served as control; the second
and third groups were injected intraperitoneally with CC14 in a dose of 3 ml/kg body weight (10%
solution of CC14 in light liquid paraffin). The third group was pretreated orally for 3 consecutive days
with 500 mg/kg of the ethanol extract of sarsaparilla (5 ml/kg) before the injection of CC14 on the fourth
day. The animals were anaesthetized with ether 18 h after the CC14 injection, and blood was collected
by heart puncture for biochemical evaluations. Livers were excised and fixed in buffered formalin for
The biochemical parameters included liver glycogen (Montgomery, 1957); serum transaminases
(Rietman and Frankel, 1957), serum bilirubin (King, 1951), serum cholesterol (Zlatkis et al. 1953) and
blood urea (King, 1951).
Safety evaluation studies
Acute toxicity testing was performed on 3 groups of mice consisting of 6 animals per group. The
ethanol extract was administered orally in doses of 500 mg, 1 g and 3 g/kg body weight. The
behavioural changes, symptoms of toxicity and mortality were observed for 24 h (Shah et al., 1988).
A total of 40 mice (20 male and 20 female) were randomly allotted to different treated and control
groups. The extract in each case was administered in drinking water. The dose selected was 100 mg/
kg body weight per day, which is 1/5 of the pharmacologically active dose (Parmar et al., 1987), for
a period of 3 months (WHO, 1967). The animals were observed for symptoms of toxicity and
mortality. The average pre- and post-treatment body weights of the animals were recorded and
compared with the control group. At the end of the treatment, 5 animals from each group were
sacrificed 24 h after the last exposure. The blood was analysed for RBC and WBC counts, and
haemoglobin level, with a Contraves degicell 3100h.
The preliminary phytochemical screening of the roots of sarsaparilla was conducted to determine the
presence or absence of alkaloids, cardiac glycosides, flavonoids, tannins, coumarins, anthraquinones,
saponins, volatile oil, volatile bases, cyanogenic glycosides, glucosinolates, sterols and/or triterpenes
according to the methods described by Famsworth (1966).
RESULTS AND DISCUSSION
The treatment of animals with CC14 produced a significant increase in serum
transaminases (Table 1). The hepatocellular enzymes, especially GPT, are tightly
bound to particular organelles, and an hepatotoxin like CC14 is known to disturb
S. RAFATULLAH ET AL.
Table 1. Effect of pretreatment with ethanol extract of sarsaparilla (500 mg/kg) on CCl4-induced
hepatocellular damage in rats.
Groups Liver GOT GPT Bilirubin Cholesterol Blood urea
Glycogen (I.U./Litre) (I.U./Litre) (mg/100 ml) (mg/100 ml) (mg/100 ml)
Control 18.20 ± 1.06 30.53 ± 3.23 26.50 ± 2.39 1.05 ± 0.18 264.0 ±16.31 59.0 ± 14.26
CC14 6.40 + 0.43<= 55.47 + 4.73b 58.21 ± 5.72b 2.75 ± 0.29b 334.0 ± 26.94a 160.0 ± 13.87b
CC14+ 17.60 ±3.21b 47.97 ±4.39 30.85 + 4.62° 1.20 ± 0.18b 267.2 ± 19.30° 131.0 ±24.20
a = P < 0.05; b = P < 0.01 and c = P < 0.001. Student's t-test.
Six animals were used in each group.
the membrane integrity and produce leakage of this enzyme, suggesting tissue
destruction (Choudhury and Poddar, 1983; Bang, 1958). Treatment of animals
with extract significantly inhibited the CCl4-induced rise in GPT. This enzyme is
one of the best indices of liver injury. Liver GPT activity represents 90% of the
whole body content (Cornelius, 1963). Unlike GPT, the extract failed to inhibit
the CCl4-induced increase in GOT activity. However, this enzyme is not totally
specific to the liver. Its activity may be increased in pathogenic conditions of
skeletal muscle, diaphragm, and heart muscle, as well as liver injury. It is also
increased in stress situations (Cornelius, et al. 1959).
There were also increases in serum bilirubin, cholesterol and urea levels.
Elevation of these parameters following CCl4-induced hepatotoxicity is well
documented (Osar et al, 1965; Nisa, 1985). Pretreatment of the rats with sarsa-
parilla extract significantly protected the liver against CCl4-induced heptocellular
metabolic changes. These findings were further confirmed by our histopathological
studies in which the animals treated with the extract before the CC14 challenge had
well-defined cellular borders with minor mononuclear infiltration; only a few
cells showed architectural abnormalities. In contrast, the liver of the rats treated
with CC14 showed centrilobular necrosis with mononuclear infiltration in the
portal area with fatty deposition, and a loss of cell boundaries.
The chemical constituents of Sarsaprilla responsible for hepatoprotection and
their mechanism of action are not clear. Our phytochemical studies have revealed
the presence of a high concentration of saponins along with flavonoids, tannins,
sterols and triterpenes. Treaesche et al. (1969) have isolated saponins from radix
sarsaparilla, a species closely related to Smilax regelii. Saponins have recently
been shown to possess highly significant immunomodulating activity, promoting
the ability of the animals to combat pathogens and xenobiotics (Bomford, 1988;
Chavali and Campbell, 1987; Tanaka and Kasai, 1984; Price and Johnson; 1987).
The immunostimulating action of sarsaparilla was also suggested in an earlier
report from our laboratory in which Ageel et al. (1989) showed a significant in-
hibition of carrageenan-induced edema and cotton pellet granuloma. On the other
hand, some flavonoids have been reported to have anti-oxidant activity (Harborne,
1967; Das and Ratty, 1986). Kiso et al. (1983) attributed hepatoprotective activity
of several plants to their antioxidant properties. However, further studies are
required to determine the active constituents and their mechanism(s) of action.
Our results on acute toxicity of the ethanol extract of sarsaparilla, following a
single oral dose (0.5, 1.0 or 3 g/kg) produced no significant adverse effects nor
mortality in the animals during 24 hours. Sarsaparilla is widely used as a vehicle
and large quantities are employed in the manufacture of nonalcoholic drinks
(Trease and Evans, 1972), suggesting frequent exposure of people to the plant for
a longer period. Chronic treatment of mice in the dose of 100 mg/kg/day for 90
days did not show any significant change in the behaviour and growth rate of the
animals. The gain of the body weights of the mice in the treated and control
Table 2. Effect of chronic treatment (90 days) of an ethanol extract of sarsaparilla on the body
weight of mice.
Treatment Pretreatment body Post-treatment body
and dose weight Mean ± S.E. weight Mean + S£.
(100 mg/kg/day) Male Female Male Female
Control 28.3 ± 1.0 27.8+ 1.2 31.1 ± 1.2 30.0 ±1.5
Sarsaparilla 32.7 ±0.7 21.1 ± 0.9 33.4 ± 1.6 27.1 ± 1.2*
* P< 0.01, Student's t-test.
Twenty mice were used in each group.
Table 3. Effect of chronic treatment (90 days) of an ethanol extract of sarsaparilla on haemoglobin
level, RBC and WBC counts in mice.
Treatment Oral dose Haemoglobin
mg/kg/day (mg/lOOml) RBC (XI03) WBC (X 106)
Control 13.10 ±0.53 6.83 ± 0.30 4.28 ±0.60
extract 100 11.4 ± 0.23 6.92 ±0.33 4.92 ±0.60
Five animals were used in each group.
S. RAFATULLAH ET AL.
groups were similar. However, the gain in body weight of extract-treated female
mice was higher as compared to control animals (Table 2).
Sarsaparilla has been used as a tonic and an appetite stimulant in Unani
system of medicine (Raza, 1933). Our studies on hematology and survival fol-
lowing the chronic treatment of mice with sarsaparilla extract revealed no sig-
nificant change in RBC or WBC counts and haemoglobin levels of the animals as
compared to control group (Table 3). The rate of mortality in the test groups over
a period of 90 days was same as in the control group (20%, each group). These
findings suggest that the sarsaparilla, besides having hepatoprotective potential,
does not cause any deliterious effect on animals.
This study was supported, in part, by the King Abdulaziz City for Science and Technology (KACST).
Thanks are due to Mr. Altaf Hussain Naqvi, for his excellent typing of the manuscript.
AGEEL, A.M., MOSSA, J.S., AL-YAHYA, M.A., AL-SAID, M.S. and TARIQ, M. (1989). Drugs
Exptl. Clin. Res., 15 (8), 369-72.
AL-BAITAR, I. (1871). Jame-al-Mufradat-al-Advia-wal-Aghzia, Amra Press, Egypt, 111, 124.
ALBERT, F.H. (1952). Economic Botany, McGraw-Hill Book Company, U.S.A., p. 441.
BANG, N.U. (1958). J. Am. Med. Assoc., 168, 156-60.
BOMFORD, R. (1988). Phytotherap. Res., 2 (4), 159-64.
CHAVALI, S.R. and CAMPBELL, J.B. (1987). Int. Arch. Allergy Appl. Immunol. 84, 129.
CHOUDHURY, B.R. and PODDAR, M.K. (1983). Meth. and Find Expd. Clin. Pharmacol., 5 (10), 727-
CLAUSE, E.P., VARO, E.T. and LYNN, R.B. (1971). Pharmacognosy, Henry Kimpton Publishers,
CORNELIUS, C.E. (1963). Nature, 200, 581.
CORNELIUS, C.E., BISHOP, J., SWITZERLAND, J. and RHODE, E.A. (1959). Cornell Vet., 49,
DAS, N.P. and RATTY, A.K. (1986). In: Plants Favonoids in Biology and Medicine; Biochemical,
Pharmacological and Structure-activity Relationship. (V. Cody, E. Middleton, J. Harborne,
Eds.) A.R. Liss, New York, 243-247.
FARNSWORTH, N.R. (1966). J. Pharm. Sci. 55, 225-72.
GHANI, M.A. (1921). Khazeenat-al-Advia, Matbaa Naval Kishore, Lucknow, India, 111, 116-7.
HARBORNE, J.B. (1967). Comparative Biochemistry of the Flavonoids. Academic Press, London,
KING, E.J. (1951). Microanalysis in Medical Biochemistry, (2nd Ed.), Churchill Livingstone, Lon-
KISO, Y., SUZUKI, Y., WATANABE, N., OSHIMA, Y. and HIKINO, H. (1983). Planta Medica, 49,
KRITIKAR, K.R. and BASU, D.B. (1918). Indian Medicinal Plants, Sudhindra Nath Basu, Allahabad,
India, 11, 1286.
MONTGOMERY, R. (1957). Arch. Biochem. Biophys. 67, 376-86.
NASIR, M. (1882). Mufradat-e-Nasir Mai Takmila, Mataba Alvi Mohammed Ali, Lucknow, India,
NISA, M., TARIQ, M. and AKBAR, S. (1985). IRCS Med. Sci., 13, 150.
OSAR, B.L. (1965). Hawk's Physiological Chemistry (14th ed). Tata McGraw Hill, New Delhi, 504.
PARMAR, N.S., TARIQ, M., AL-YAHYA, M.A., AGEEL, A.M. and AL-SAID, M.S. (1985).
Fitoterapia, 52 (5), 670.
PRICE, K.R., JOHNSON, I.T. and FENWICK, G.R. (1987). CRC Crit. Rev. Food Sci. Nutr. 26, 27.
RAZA, A.K. (1933). Tazkiratul-Hind, Yadgaar Razaee, Shamsul-Islam Press, Hyderabad, India, 80-
REITMAN, S. and FRANKEL, S. (1957). Am. J. Clin. Pathol., 28, 56-63.
SHAH, A.H., QURESHI, S., TARIQ, M. and AGEEL, A.M. (1988). Phytother. Res., 3 (1), 25-29.
STUART, M. (1979). The Encyclopedia of Herbs and Herbalism, Orbis Publishing Ltd., London,
TANAKA, O. and KASAI, R. (1984). Prog. Chem. Nat. Prod., 46, 1.
THE BRITISH PHARMACEUTICAL CODEX (1934). The Pharmaceutical Press, London, 933-934.
THE PHARMACOPOEIA OF THE UNITED STATES OF AMERICA (1942). (12th Rev.), 400-1.
TREAESCHE, R., GUNDOLF, L. and GUNTER, W. (1969). Chem. Ber., 702 (4), 1253-69.
TREASE, G.E. and EVANS, W.C. (1972). Textbook of Pharmacognosy, The University Press,
WALLIS, T.E. (1955). Textbook of Pharmacognosy, J. & A. Churchill Ltd., London, 423-25.
WHO REPORT OF A SCIENTIFIC GROUP (1967). Principles for Pre-clinical Testing of Drug
Safety. World Health Technical Report Series, 341, WHO, Geneva, Switzerland.
ZLATKIS, A., ZAK, B. and BOYLE, J.A. (1953). J. Lab. Clin. Med., 4,486.
Received February 9, 1990
Accepted July 3, 1990