Sub-acute toxicological studies of pongamol isolated from Pongamia pinnata

Abstract

The sub-acute toxicity of pongamol isolated fromPongamia pinnata (Fam. Papilionaceae) wasstudied on long Evan’s rats. The studies included the gross observation such as changes in body weight,hematological profiles (total count of red blood cell, white blood cell and platelet, differential count ofwhite blood cell, erythrocyte sedimentation rate and haemoglobin percentage), biochemical parameters ofblood (serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, serum alkaline phosphatase, serum bilirubin, creatinine and urea) and histopathology of the liver, kidney, heart and lungof both control and experimental groups of rats. The changes in body weight, hematological andbiochemical parameters were statistically not significant after administration of pongamol in a dose of300 μg/rat/day for consecutive 14 days when compared to that of control group rats. Histopathologicallyno abnormality was found on liver, kidney, heart and lung of experimental group rats after treatment whencompared to that of control group rats. This preliminary study suggests that the isolated compoundpongamol may be used safely for chronic toxicological studies and clinical trial.

Full text

Research Journal of Medicine and Medical Sciences, 2(2): 53-57, 2007
© 2007, INSInet Publication
Corresponding author: Alam Khan, Department of PharmacyUniversity of Rajshahi, Rajshahi- 6205, Bangladesh
Fax: +880-721-750064. Tel: +880-721-750041/4110.
E-mail: alamkhan792002@yahoo.co.in
53
Sub-Acute Toxicological Studies of Pongamol Isolated from Pongamia pinnata
Md. Abdullahil Baki, Alam Khan , M Abdul Alim Al-Bari,
12*2
Ashik Mosaddik, G. Sadik and K.A.M.S.H. Mondal
221
Department of Pharmacy, University of Rajshahi, Rajshahi 6205, Bangladesh
2
Department of Zoology, University of Rajshahi, Rajshahi 6205, Bangladesh
1
Abstract: The sub-acute toxicity of pongamol isolated from Pongamia pinnata (Fam. Papilionaceae) was
studied on long Evan’s rats. The studies included the gross observation such as changes in body weight,
hematological profiles (total count of red blood cell, white blood cell and platelet, differential count of
white blood cell, erythrocyte sedimentation rate and haemoglobin percentage), biochemical parameters of
blood (serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, serum alkaline
phosphatase, serum bilirubin, creatinine and urea) and histopathology of the liver, kidney, heart and lung
of both control and experimental groups of rats. The changes in body weight, hematological and
biochemical parameters were statistically not significant after administration of pongamol in a dose of
300 µg/rat/day for consecutive 14 days when compared to that of control group rats. Histopathologically
no abnormality was found on liver, kidney, heart and lung of experimental group rats after treatment when
compared to that of control group rats. This preliminary study suggests that the isolated compound
pongamol may be used safely for chronic toxicological studies and clinical trial.
Key words: Pongamol, Hematological parameters, Biochemical parameters, Histopathological study,
Pongamia pinnata.
INTRODUCTION
Toxicologically is the aspect of pharmacology
that deals with the adverse effect of bioactive
substance on living organisms . In order to establish
[1]
the safely and efficiency of a new drug, toxicological
studies are very essential experiment in animals like
mice, rat, guinea pigs, dog, rabbit, monkey etc under
various condition of drug . No drug is used
[1]
clinically without its clinical trial as well as toxicity
studies . Toxicological studies help to make decision
[1]
whether a new drug should be adopted for clinical
use or not . Depending on the duration of drug
[2]
exposure to animals toxicological studies may be
three types (acute, sub-acute and chronic
toxicological studies) . In acute toxicity studies,
[3-5]
single dose of drug is given in large quantity to
determine immediate toxic effect. Acute toxicity
50
studies are commonly used to determine LD of
drug or chemicals. In sub-acute toxicity studies,
repeated doses of drug are given in sub-lethal
quantity for a period of 14 to 21 days. Sub-acute
toxicity studies are used to determine effect of drug
on biochemical and hematological parameters of
blood as well as to determine histopathological
changes. In chronic toxicity studies, drug is given
in different doses for a period of 90 days to over a
year to determine carcinogenic and mutagenic
potential of drug.
Pongamia pinnata (Linn.) Merr. (Syn. Pongamia
glabra, Fam. Papilionaceae), locally known as
Karaja, is a small evergreen tree, that is
widely distributed in Bangladesh, India, China
Philippine and Australia . Seed extract of this plant
[6-7]
has hypotensive effects and produce uterine
contractions . Powdered seed is used in bronchitis,
[6]
chronic fever, whooping cough and chronic skin
diseases and painful rheumatic joints . Seed oil is
[6-7]
used in scabies, leprosy, piles, ulcers, chronic fever,
lever pain and lumbago . Pongamol was isolated
[7]
from seed of this plant. Induction of quinone
reductase by pongamol , Anticonvulsant effect of
[8]
pongamol , CNS-depressant activity of pongamol
[9] [10]
and pongamol promoted increased sensitivity to
sound and touch were reported in literature.
[10]
Because of UV radiaton absorbing properties of
pongamol, it is used in Indian Ayurvedic herbal
medicine and cosmetic preparation for skin protection
and in treatment of rheumatic pain . Although a
[11]
number of biological effect and uses of pongamol

Res. J. Medicine and Med. Sci., 2(2): 53-57, 2007
54
were reported in literature, its toxicological
evaluation has not been explored yet. Therefore, in
the present study an attempt was made to evaluate
sub-acute toxicities of pongamol.
MATERIAL AND METHODS
Isolation of Pongamol: The seed of the plant was
collected from of Rajshahi University of Bangladesh
and identified by Professor A.T.M. Naderuzzaman,
Department of Botany, University of Rajshahi,
Bangladesh where its voucher specimen (No.
ET3467) was deposited. The seed (200 g) were air-
dried, ground into powder, extracted with petroleum
ether (2 L) in a Soxhlet apparatus (Quickfit,
England) , filtered and concentrated. Pongamol was
[12]
isolated from this extract using chromatographic
techniques (TLC, PTLC, Column). The structure was
confirmed on the basis various spectral data and their
comparison with published spectral data .
[13]
Experimental Animals and Their Collection: The
experiment was carried out on Evan’s male rats.
They were 6 months old, weighing between 115-137
g. They were collected from the Animal Branch of
International center for Diarrhoeal Disease Research,
Bangladesh (ICDDR,B).
Maintenance of Rats: The rats were housed in iron
cages (considering group) under temperature and
light controlled condition . They were fed a
[14]
balanced diet and tap water. The animals were
15
maintained in this condition for 15 days before
experiment to adjust with food and environment.
Grouping of Rats: Individual weight of the rats was
taken and they were grouped in two, randomly. The
rats of group B (4 rats, average weight 121.5 g)
were used for experiment while those of group A
(4 rats, average weight 122.7 g) were used as
control.
Administration of Sample: The compound,
pongamol was dissolved in distilled water with the
help of tween-80 as co-solvent, so that each 0.3 ml
solution contains 300 µg pongamol . Each rat of
[1]
experimental group (group B) was administered with
0.3 ml sample solution (contain 300 µg compound)
daily, for 14 consecutive days and each rat of control
group (group A) was administered with 0.3 ml
isotonic vehicle daily, for 14 consecutive days.
Intraperitoneal route was used for these
administrations .
[1]
Blood Collection: For hematological study (total and
differential blood cell count, ESR and percent
hemoglobin determination), blood was drawn from
the tail vein of both groups before drug
administration, at 7 day and after completion of
th
treatment. For biochemical study, blood was collected
at 15 day after completion of treatment from the
th
jugular veins of each mouse . Then all rats were
[1]
sacrificed and liver, kidney, heart and lung were
removed for histological study .
[1]
Gross General Observation: During the whole
experimental period their behavior, central nervous
system (CNS) excitation, CNS depression, muscular
weakness, salivation, diarrhoea and food intake were
observed. The body weight of each rat of Group A
and B were measured before drug administration and
after treatment prior to sacrificing the animals.
Investigation of Hematological Profiles: The
hematological parameters determination like total
count (TC) of RBC, WBC and platelet, differential
count (DC) of WBC, ESR and haemoglobin
percentage were performed just before drug intake
and at 7 day of treatment and after completion of
th
treatment. Haemoglobin percentage was estimated by
Sahli’s acid hematin method using Sahli’s
hemometer . Total count of RBC, WBC and
[16]
platelet was carried out by automated cell counter
(Beckman Coulter, China). Differential count (DC) of
WBC was carried out by microscopy using
Leishman’ stain . ESR was Determined by
[16]
Westergren’s method using Westergren tube and
stand .
[16]
Investigation of Biochemical Parameters:
Biochemical parameters (serum glutamate oxaloacete
transaminase, serum glutamate pyruvate transaminase,
serum alkaline phosphatase, serum bilirubin,
creatinine and urea) were determined at 15 day after
th
completion treatment. Determination of Biochemical
parameters was carried out using automated
biochemical analyzer (Vitros-250, Johnson and
Johnson Co. USA).
Histopathological Investigation: Histopathological
investigation was carried out at 15 day after
th
completing treatment and collection of blood. Both
experimental and control groups rats were sacrificed
and liver, kidney, heart and lung were isolated for
investigation. The tissues were separately sliced in
piece, fixed in 10% formaline for 3 days, processed,
stained, mounted on glass slides and observed under
high power microscope at the Department of
Pathology, Rajshahi Medical College, Bangladesh.

Res. J. Medicine and Med. Sci., 2(2): 53-57, 2007
55
RESULTS AND DISCUSSIONS
Average body weights of all rats before and
after treatment were presented in Table 1. After
14 days control group was gained weight 1.96% and
experimental group gained weight 5.46%. The change
in body weight for both control and experimental
group were insignificant. The hematological profiles
of the experimental and control group rats were
determined before treatment, at 7 day of treatment
th
and after treatment and compared to check the
hematological disorders after intraperitoneal
administration of pongamol. No mentionable change
in the values of RBC count, WBC count, platelet
count, differential WBC count, ESR and haemoglobin
percentage of experimental rats were observed
when compared to that of control group rats
(Table 2 and 3). Biochemical parameters of blood
(SGOT, SGPT, alkaline phosphatase, serum bilirubin,
creatinine and urea) were determined after treatment
by pongamol and compared to that of control group
rats to check any change of these parameters. It was
found that most of the parameters were slightly
changed with respect to control group rats but remain
within the normal range (Table 4). These indicate
insignificant adverse effect of pongamol on liver and
kidney functions. After 14 day of drug
administration, the animals of both control and
experimental groups were sacrificed and the liver,
kidney, lung, spleen and heart were isolated and
examined under a microscope. No abnormality was
detected in the organs of both control and
experimental animals, indicating that compound has
no significant adverse effect on cellular structures.
Table 1: Effect of pongamol on body weight of rats after intraperitoneal administration.
Body weight (g) Body weight (g)
before treatment after treatment t value at 5%
Group of mice n = 4, M ± SD Calculated‘t’ value significance Remark
n = 4, M ± SD % Change
Control 51 ± 1.22 52 ± 1.87
(51, 53, 50, 50) (54, 55, 51, 52) +1.96 0.42 2.447 NS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Experimental 46.7 ± 1.73 49.25 ± 1.79
(48, 45.2, 44.8, 8.8) (51, 48, 47, 51) +5.46 0.36 2.447 NS
M = mean weight of mice; SD = standard deviations; n = number of mice; ‘+’= increase, ‘-’ = decrease; t = significance of difference
between experimental and control group and NS = not significant. Individual values presented within bracket.
Table 2: Haematological profile of control group rats. Normal rats Treated with vehicle
---------------------- ----------------------------------------------------------------
1st day 7th day 14th day
Haematological parameters n = 4, M ± SD n = 4, M ± SD n = 4, M ± SD
Total RBC count (million/cc) 3.8 ± 0.07 4.0 ± 0.07 3.975 ± 0.08
(3.8, 3.9, 3.8, 3.7) (4.0, 4.1, 4.0, 3.9) (4.0, 4.1, 3.9, 3.9)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Total WBC count (thousand/cc) 5.85 ± 0.17 5.95 ± 0.23 5.975 ± 0.29
(5.9, 6.1, 5.7, 5.7) (6.1, 6.2, 5.6, 5.9) (6.2, 6.3, 5.8, 5.6)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Differential count of WBC in % Neutrophil 48 ± 1.58 48 ± 1.41 46 ± 0.71
(47, 46, 49, 50) (46, 48, 50, 48) (46, 45, 47, 46)
----------------------------------------------------------------------------------------------------------------------------------
Lymphocyte 38.5 ± 1.80 38.5 ± 1.12 41.75 ± 1.48
(39, 36, 41, 38) (38, 37, 40, 39) (42, 40, 44, 41)
----------------------------------------------------------------------------------------------------------------------------------
Monocyte 1 ± 0.71 1.25 ± 0.43 2.5 ± 0.5
(2, 0, 1, 1) (1, 1, 2, 1) (2, 2, 3, 3)
----------------------------------------------------------------------------------------------------------------------------------
Eosinophil 3 ± 0.71 3.25 ± 0.83 3.5 ± 1.12
(4, 3, 2, 3) (4, 2, 3, 4) (5, 2, 4, 3)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Platelet count (no./cc) 272250 ± 7595.2 276500 ± 5722.8 284500 ± 5220.2
(270000, 285000, (281000, 283000, (288000, 291000,
265000, 269000) 269000, 273000) 278000, 281000)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Haemoglobin (%) 58.75 ± 1.48 60 ± 1.22 61.5 ± 2.06
(58, 57, 61, 59) (60, 58, 61, 61) (63, 58, 62, 63)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
ESR (mm/1st hour) 20.5 ± 1.12 21 ± 0.71 21.5 ± 0.5
(21, 22, 19, 20) (21, 21, 20, 22) (22, 21, 21, 22)
M = mean; SD = standard deviations; n = number of mice; data represented in the form of M ± SD and individual values presented
within bracket.

Res. J. Medicine and Med. Sci., 2(2): 53-57, 2007
56
Table 3: Haematological profile of rats treated with pongamol
Normal rats Treated with pongamol
----------------------- ----------------------------------------------------------------
1st day 7th day 14th day
Haematological parameters n = 4, M ± SD n = 4, M ± SD n = 4, M ± SD
Total RBC count (million/cc) 3.325 ± 0.170 3.525 ± 0.170 3.725 ± 0.170
(3.5, 3.3, 3.4, 3.1) (3.7, 3.5, 3.6, 3.3) (3.9, 3.7, 3.8, 3.5)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Total WBC count (thousand/cc) 5.45 ± 0.208 5.45 ± 0.191 5.9 ± 0.374
(5.5, 5.2, 5.7, 5.4) (5.6, 5.4, 5.2, 5.6) (5.8, 5.5, 6.4, 5.9)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Differential count of WBC in % Neutrophil 44.75 ± 1.707 45.75 ± 1.707 47.25 ± 2.061
(43, 45, 44, 47) (44, 46, 45, 48) (45, 49, 46, 49)
----------------------------------------------------------------------------------------------------------------------------------
Lymphocyte 32.5 ± 2.645 32.25 ± 1.5 32.5 ± 1.290
(35, 34, 29, 32) (33, 31, 34, 31) (31, 34, 32, 33)
----------------------------------------------------------------------------------------------------------------------------------
Eosinophil 32.5 ± 1.290 3.25 ± 0.957 3.25 ± 0.957
(2, 3, 4, 5) (4, 3, 2, 3) (2, 3, 4, 4)
----------------------------------------------------------------------------------------------------------------------------------
Monocyte 1 ± 0.7071 1.75 ± 0.829 2.5 ± 0.5
(2, 1, 0, 1) (3, 2, 1, 1) (3, 2, 3, 2)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Platelet count (no./cc) 245750 ± 2861.38 250000 ± 3391.16 251500 ± 2291.28
(245000, 250000, (252000, 254000, (250000, 255000,
246000, 242000) 249000, 245000) 252000, 249000)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Haemoglobin (%) 52 ± 1.870 53 ± 1.870 55 ± 2.549
(51, 50, 55, 52) (52, 51, 56, 53) (53, 52, 57, 58)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
ESR (mm/1st hour) 24 ± 1.224 25 ± 1.224 25.25 ± 0.829
(25, 24, 25, 22) (26, 25, 26, 23) (26, 25, 26, 24)
M = mean; SD = standard deviations; n = number of mice; data represented in the form of M ± SD and individual values presented
within bracket.
Table 4: Effect of pongamol on biochemical parameters of rats blood after intraperitoneal (i.p.) administration of 300 mg/rat/day for 14
consecutive days.
Control group Experimental group
Biochemical parameters n = 4, M ± SD n=4, M ± SD % of change tc ts Remark
SGPT (IU/L) 8.75 ± 0.957 9.75 ± 0.957
(9, 10, 8, 8) (9, 11, 10, 9) -11.428 0.975 2.447 NS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
SGOT (IU/L) 10.525 ± 0.411 10.97 ± 0.818
(10, 10.5, 10.6, 11) (10, 11, 12, 10.9) -4.228 2.186 2.447 NS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
SALP (IU/L) 4.175 ± 0.287 4.225 ± 0.298
(4.5, 4.2, 3.8, 4.2) (4.3, 4.5, 3.8, 4.3) +1.754 0.241 2.447 NS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Serum bilirubin (mmol/L) 0.272 ± 0.035 0.286 ± 0.031
(0.24, 0.28, 0.25, 0.32) (0.24, 0.30, 0.28, 0.32) +6.206 0.621 2.447 NS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Creatinine (mg/dl) 0.587 ± 0.009 1.5 ± 0.355
(0.58, 0.60, 0.58, 0.59) (1.3, 2, 1.5, 1.2) +60.86 5.119 2.447 NS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Urea (mg/dl) 17.75 ± 0.957 53 ± 1.825
(17, 18, 17, 19) (51, 52, 54, 55) +66.50 25.34 2.447 NS
M = mean; SD = standard deviations; n = number of mice; t = significance of difference between experimental and control group;
tc = Calculated value; ts = t value at 5% level of significance and NS = not significant. Individual values presented within bracket.
Different biological effects such as induction of
quinone reductase , Anticonvulsant effect , CNS-
89
depressant activity were reported for pongamol. The
10
results of sub-acute toxicity studies have shown no
abnormalities on body weight, hematological and
biochemical parameters of blood and on
histopathological slides. The biological effect of
pongamol and its present toxicological studies
suggest that pongamol can be safely subjected to
chronic toxicological studies and clinical trial.

Res. J. Medicine and Med. Sci., 2(2): 53-57, 2007
57
REFERENCES
1. Alam, A.H.M.K., R. Islam, K.A. Salam,
M.M. Manir, M.A. Baki, M.A. Hossain and
G. Sadik, 2006. Toxicological studies of N-trans-
ferloyl-4methyldopamine isolated from
Achranthes ferruginea. Pakistan Journal of
Biological Sciences, 9: 1052-1055.
2. Anisuzzaman, A.S.M., N. Sugimoto, G. Sadik
and M.A. Gufor, 2001. Sub-acute toxicity study
of 5-hydroxy-2(hydroxy-methyl) 4H-pyran-4-
One, isolated from Aspergillus fumigatus.
Pakistan Journal of Biological Sciences,
4: 1012-1015.
3. Klaassen, C.D., 1996. Principle of Toxicology
and Treatment of Poisoning. In: Goodman and
Gilman’s The Pharmacological Basis of
Therapeutics, eds., Hardman, J.G., L.E. Limbird,
P.B. Molinoff, R.W. Ruddon and A.G. Gilman.
9 ed., McGraw-Hill Companies, United States,
th
America, pp: 63-74.
4. Katzung, B.G., 1994. Basic and Clinical
Pharmacology. 6 ed., Prentice-Hall International
th
In., California, pp: 879-881.
5. Khan, A., 2004. Phytochemical and
Pharmacological investigations on roots
of Laportea crenulata Gaud and Rhizome
of Drynaria quercifolia J. Smith. M. Pharm.
Thesis Paper, Rajshahi University, Bangladesh,
pp: 72.
6. Ghani, A., 1998. Medicinal plants of
Bangladesh. Asiatic society of Bangladesh,
Dhaka, Bangladesh, pp: 270.
7. Kirtikar, K.R. and B.D. Basu, 1994. Indian
medicinal plants. Vol. 1, 2 ed., Dehra Dun
nd
publisher Ltd, India, pp: 830-832.
8. Chang, L.G., C. Gerhauser, L. Song,
N.R. Farnsworth, J.M. Pezzuto and
A.D. Kinghorn, 1997. Activity-guided isolation
of constituents of Tephrosia purpurea with
the potential to induce the phase II enzyme,
quinine reductase. Journal of Natural Product,
60: 869-873.
9. Basu, S.P., J.K. Mandal and N.S. Mehdi, 1994.
Anticonvulsant effect of pongamol. Indian
Journal of Pharmaceutical Science, 56: 163-167.
10. Mahli, S.S., S.P. Basu, K.P. Sinha and
N.C. Banerjee, 1989. Pharmacological effects of
karanjin and pongamol [from seed oil of
Pongamia pinnata]. Indian Journal of Animal
Science, 59: 657-660.
11. Ingredient guide, Function and active
cosmetic ingredients for skin and hair,
2006. Ashford, Kent, England, pp: 9.
www.questcosmeticactives.com.
12. Bhal, B.S. and A. Bhal, 1992. A text book of
organic chemistry. 13 ed., Schand and
th
Company Ltd, India, pp: 5-14.
13. Sinha, B., A.A. Natu and D.D. Naravati, 1982.
Prenylated flavonoides from Tephrosia purpurea
seeds. Phytochemistry, 21: 1468-1470.
14. Khanna, N., A. Ray, M. Alkondon and P. Sen,
1989. Effect of β-adrenoceptor antagonists and
some related drugs on maximal electroshock
seizures in mice. Indian Journal of Experimental
Biology, 27: 128-130.
15. Hawks, P.B., L. Oser and W.H. Summerson,
1954. Practical Physiological Chemistry. 13 ed.,
th
McGraw Hill Book Company, US, pp: 394-395.
16. Ghai, C.L., 1999. A textbook of Practical
Physiology. 5 ed, Jaypee Brothers Medical
th
Publishers (p) Ltd, New Delhi, pp: 24-27, 77-79.