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Thus a simple, sensitive and reproducible derivative
UV spectrophotometric method for quantitative
determination of ciprofibrate was developed and
this method is suitable for the determination of
ciprofibrate in tablets and could be used in a quality
control laboratory for routine sample analysis.
It can be concluded that the proposed first‑order
derivative UV spectrophotometric and HPLC
method is suitable for the analysis of ciprofibrate in
commercial tablets.
ACKNOWLEDGEMENTS
We thank the Principal, R. C. Patel Institute of
Pharmaceutical Education and Research, Shirpur for
providing the facilities to carry out the research work.
REFERENCES
Fig. 4: Typical HPLC chromatogram of ciprofibrate The chromatogram
was developed in mobile phase consisting of 90:10 methanol:water,
pH 3.7 at 232 nm and a flow rate of 1.0 ml/min.
TABLE 5: COMPARISON OF THE THREE METHODS FOR
THE DETERMINATION OF CIPROFIBRATE IN TABLETS,
ANOVA TEST
Amount taken (9 µg/ml)
Amount found*
CV
Zero order
9.13
1.54
First order
8.98
1.60
RP‑HPLC
8.91
0.70
*Average of six experiments, ANOVA, P = 0.0226; F = 4.935
relatively high recovery value, 99.58‑99.68% (Table 3),
was obtained. Furthermore, the results obtained with
HPLC were in good agreement with those obtained
using the first order derivative UV spectrophotometric
method. Finally, results obtained using all three
methods for the determination of ciprofibrate in
tablets were subjected to ANOVA test (Table 5) which
indicated that there were no significant differences
between results.
1. Sweetman SC. Martindale The Complete Drug Reference. 35th ed.
London: The Pharmaceutical Press; 2000. p. 1118‑9.
2. Budavari S, editors. The Merck Index. 14th ed. Whitehouse Station, NJ,
USA: Merck and Co., Inc.; 1996. p. 386.
3. Robert WM, Bersot TP. Drug Therapy for Hypercholesterolemia
and Dyslipidemia. In: Hardman JG, Limbird LE, Gilman AG,
editors. Goodman and Gilman’s The Pharmacological Basis of
Therapeutics. 10th ed. New York: McGraw‑Hill Professional; 2001.
p. 971‑1002.
4. British Pharmacopoeia. (A‑I), London: The Stationary Office Medicinal
and Pharmaceutical Substances; 2005. p. 483‑4.
5. Luhua F, Xingming Y, Juan Y. Determination of contents of ciprofibrate
and its related substances by HPLC. China Pharm 2006;5:1‑4.
6. Masnatta LD, Cuniberti LA, Rey RH, Werba JP. Determination of
bezafibrate, ciprofibrate and fenofibric acid in human plasma by
high‑performance liquid chromatography. J Chromatogr B 1996;687:437‑42.
7. Anderson NH, Johnston D, Vojvodic PR. The enantiomeric resolution
of ciprofibrate and related compounds by HPLC using chiral stationary
phases. J Pharm Biomed Anal 1992;10:501‑5.
8. Huttemann H, Blaschke G. Achiral and chiral determination
of ciprofibrate and its glucuronide in human urine by capillary
electrophoresis. J Chromatogr B 1999;729:33‑41.
9. Misztal G, Komsta L. Determination of bezafibrate and ciprofibrate in
pharmaceutical formulations by densitometric and videodensitometric
TLC. J Planar Chromatogr 2005;18:188‑93.
Accepted March 20, 2012
Revised March 14, 2012
Received June 14, 2011
Indian J. Pharm. Sci., 2012, 74 (2): 168-171
Potential Antileishmanial Effect of Three Medicinal Plants
A. ELTAYEB* AND K. IBRAHIM
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, P.O. Box 1996, Sudan
*Address for correspondence
E‑mail: [email protected]
March - April 2012
Indian Journal of Pharmaceutical Sciences
171
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Eltayeb and Ibrahim: Antileishmanial Effect of Three Medicinal Plants
The antileishmanial activity of three organic solvent extracts and water residue of the plants: Acacia nilotica
(Mimosaceae) (husk), Ambrosia miratima (Astraceae) (aerial shoot) and Azadarichta indica (Meliaceae) (leaves) were
tested in vitro against Leishmania donovani promastigotes. The study revealed that the extracts of A. nilotica and
A. miratima have effectious antileishmanial activity at concentrations (IC50) less than 8 µg/ml, while the extracts
of A. indica lack antileishmanial activity. The chromatographic analysis of the ethyl acetate extract of A. nilotica,
the most potent extract, resulted in four TLC fractions. Three of these fractions possessed antileishmanial activity.
Phytochemical study of the potent fractions revealed the presence of poly hydroxyl compounds.
Key words: Acacia nilotica, Ambrosia miratima, antileishmanial activity, Azadarichta indica
Leishmaniasis is an infection due to protozoa
belonging to the genus Leishmania. Leishmaniasis
is considered as a major public health problem
in developing countries such as Sudan [1,2] . In
Sudan where the primary health care is not always
affordable, the use of herbal medicine is a common
practice. However, very few studies were carried out
to investigate the potential use of these medicinal
plants in treatment of parasitic diseases[3,4].
(reversed phase C18) 5 µm, 15×4.6 mm. Mobile phase
consisted of water and acetonitrile (30:70); flow rate was
1 ml/min and detection was on UV detector at 271 nm.
The plant materials were collected according to
their traditional use in folk medicine and a
voucher specimen was kept in the Department of
Pharmacognosy, Faculty of Pharmacy, University
of Khartoum. The aqueous solution of the dried
methanol extracts of the test plants were partitioned
by liquid‑liquid partitioning using petroleum ether,
dichloromethane and ethyl acetate. The three organic
solvent fractions and water residue were dried and
dissolved in RPMI‑1640 medium to prepare test
concentrations of 0.5, 5, 25, 50 and 100 µg/ml.
Human peripheral blood mononuclear cells from
heparinized blood were isolated using lymphoprep
solution (Pharmacia Biotech). Each cell of 96 well
round bottomed microtitre plate received 0.3×106/ml
peripheral blood mononuclear cell (PBMC) in 200
µl of RPMI 1640 containing the plants extracts,
concentration of 0.5, 5, 25, 50 and 100 µg/ml; 10 µl
of phyto‑haemaggulitinin (PHA, 47 µg/ml, Murex
diagnostic, England) was added into all microplate
wells except blank control ones, the positive control
ones receive PHA but no plant extract was added to
those wells.
Promastigotes of the identified strain of Leishmania
donovani archidadi Mon 82 were grown in RPMI‑1640
medium enriched with 20% foetal calf serum. Two
hundred microlitre of each test extract (concentration
0.5‑100 µg/ml) was added to 96 well plate (Nunc
microtitre plate). To one raw, 200 µl of plane
RPMI‑1640 medium was added and this was kept
as control. Then 50 µl of promastigotes suspension
(3×10 6/ml) was added to all wells. Parasites were
allowed to multiply at 24º for 24 h. Then promastigotes
were harvested in a counting slide and counted under
light microscope in comparison with control cells.
The ethyl acetate extract of A. nilotica (husk) was
fractionated by TLC on silica gel (GF) plates using a
solvent system of acetone and petroleum ether (1:1).
The isolated TLC fractions were detected for their purity
using Knauer HPLC machine in a Hypersil column
172
The UV spectrum of the aqueous solution (20%) of the
TLC fractions having potent antileishmanial activity was
measured at 120 nm/min scan speed on Perkin Elmer
Lamda 2 UV/Vis spectrophotometer. FTIR machine
(Shimadzu‑800) was used to study the FTIR spectrum of
KBr discs of dried crystals of the three above fractions.
The results of the present study reveal that, the
extracts of A. nilotica husk and A. miratima aerial
shoot have a potent antileishmanial activity in vitro
against L. donovani promastigotes at concentrations
(IC50) less than 8 µg/ml (Table 1). On the other hand
the extracts of A. indica leaves did not give significant
antileishmanial activity. As shown in Table 2 the thin
layer chromatography of the ethyl acetate extract
of A. nilotica husk indicated the presence of three
fractions 1, 2 and 3 of R f values: 0.70, 0.41 and
0.24, these fractions possessed potent antileishmanial
activity with IC50 concentration less than 5 µg/ml.
Where as the fourth fraction Rf value 0.00 (at base
line) exhibited poor antileishmanial activity IC 50
concentration more than 20 µg/ml. The performance of
the different sensitivity and toxicity tests in this study
were analyzed using SPSS computer software using
Indian Journal of Pharmaceutical Sciences
March - April 2012
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TABLE 1: THE ANTILEISHMANIAL EFFECT OF THE MEDICINAL PLANTS EXTRACTS
Plant extract solvent
Acacia nilotica/Ethyl acetate
Acacia nilotica/Dichloromethane
Ambrosia maritima/Ethyl acetate
Ambrosia maritime/Dichloromethane
Ambrosia maritime/Petroleum ether
Ambrosia maritima/Water residual
Azadarichta indica/Ethyl acetate
Azadarichta indica/Dichloromethane
Azadarichta indica/Petroleum ether
Azadarichta indica /Water residual
Y=a+bx
Y=‑2.13+2.413x
Y=‑0.69+1.03x
Y=‑0.45+0.761x
Y=‑1.1+1.65x
Y=‑1.43+2.63x
Y=‑0.45+0.98x
Y=‑4.05+0.90x
Y=‑2.84+0.68x
Y=‑3.36+0.81x
Y=‑3.34+0.93x
IC50 (µg/ml)
7.65
4.6
4.01
4.65
3.49
2.9
31900.0
14865.0
14095.0
3818.9
IC90 (µg/ml)
62.28
624.0
325.0
100.0
23.9
520.0
8857169
>25432544
>7323899
>871619
R2
0.924
0.625
0.590
0.999
0.687
0.188
0.835
0.410
1.000
0.939
TABLE 2: THE ANTILEISHMANIAL ACTIVITY OF TLC FRACTIONS (1‑4) OF ACACIA NILOTICA DRIED ETHYL
ACETATE EXTRACT
TLC Fractions of A. nilotica ethyl acetate extract
Fraction (1) Rf=0.70
Fraction (2) Rf=0.41
Fraction (3) Rf=0.24
Fraction (4) Rf=0.00
Y=a+bx
Y=‑1.39+2.04x
Y=‑0.819+1.83x
Y=‑0.3+1.46x
Y=‑1.497+1.46x
TABLE 3: IR ANALYSIS RESULTS OF TLC FRACTIONS
OF THE ETHYL ACETATE EXTRACT OF ACACIA
NILOTICA HUSK
Fraction 1
Fraction 2
Fraction 3
+
+
+
+
+
+
+
+
+
‑
+
+
+
+
+
+
+
+
+
‑
‑
Putative
chemical
group
O‑H
C‑H(aliphatic
origin)
–C=O
–C=C–
–CH3 and
–CH2‑
C‑O
(different
environments)
C‑Cl
Absorption
band
(cm‑1)
3500‑3200
2950
1750‑1650
1650‑1600
1450‑1400
Multiple
bands
1300‑1000
600
the standard agent (pentostam) which exhibit relatively
high IC50 of 156 µg/ml.
The HPLC chromatogram of fraction 1 showed a single
principle peak at Rt of 3.28 min with minor impurities,
while chromatograms of fraction 2 and 3 show multiple
peaks. The spectroscopic analysis of the TLC fractions
of the ethyl acetate extract of A. nilotica husk provided
the following results: The UV spectrum of aqueous
solutions of fraction 1 showed absorption bands at
278.6, 262.3 and strong end absorption at 228.3 nm.
With fraction 2 the UV spectrum revealed strong end
absorption at 210 nm and shoulders at 423.2, 378 and
276 nm. The UV spectrum of fraction 3 gave strong
end absorption with a single shoulder at 277.7 nm in
aqueous solution. The IR spectrum absorption bands
of TLC fractions of the ethyl acetate extract of Acacia
nilotica husk are listed in Table 3.
March - April 2012
IC50 (µg/ml)
4.80
2.80
1.60
22.39
IC90 (µg/ml)
57.80
44.80
51.66
712.20
R2
0.496
0.832
0.966
0.525
The present work aimed at screening the
antileishmanial activity of some Sudanese medicinal
plants by in vitro studies to identify their active
extracts or principles. The ethyl acetate extract of
A. nilotica husk showed a promising antileishmanial
activity. The findings of the present study agree with
the earlier reports where, the ethyl acetate extract
of A. nilotica husk had showed potent antiparasitic
activity against Plasmodium falciprum. A. nilotica
is widely used in folk medicine to treat a variety
of diseases, where the gum stem bark, leaves and
fruits are used for treatment of colds, bronchitis,
pneumonia, ophthalmia, diarrhea and hemorrhage[5].
In a preliminary phytochemical screening of the ethyl
acetate extract of A. nilotica husk, the presence of
tannins and phenolic compounds was confirmed when
a blue‑black color was observed when ferric chloride
reagent was added to a solution of the extract.
Several studies stated that many plant species have
immunomodulatory action[6]. The ethyl acetate extract
of A. nilotica husk, at low concentrations revealed
an increase of human lymphocytes cells count. This
may be an indication of the ability of this extract to
enhance human immunity. TLC fractioning of the
ethyl acetate extract of A. nilotica husk using silica
gel GF as stationary phase and a solvent system of
acetone: petroleum ether (1:1) was carried out. Four
fractions were isolated of Rf values of 0.70, 0.41, 0.24
and baseline spot. The first three fractions were found
to posses antileishmanial activity with IC50 value of
4.8, 2.8, 1.6 µg/ml and IC90 value of 57.8, 44.8 and
Indian Journal of Pharmaceutical Sciences
173
www.ijpsonline.com
51.66 µg/ml, respectively. The isolated TLC fractions
were detected for their purity using Knauer HPLC
machine. Fraction 1 was found to be the most pure,
while fraction 2 and 3 showed multiple peaks as an
indication of the presence of other compounds in each
fraction. The high antileishmanial activity of fraction
2 and 3 may be attributed to synergistic action of
these additional compounds.
ACKNOWLEDGEMENTS
A potent antileishmanial activity was observed when
L. donovani was treated with A. maritima extracts
(IC50 <5 µg/ml). Further in vivo studies are needed to
confirm the bioactivity of this plant.
1. Desjeux P. Leishmaniasis: current situation and new perspectives. Comp
Immunol Microbiol Infect Dis 2004;27:305–18.
2. Anonymous. Control of the Lesishmanaisis. Report of a WHO Expert
Committee. World Health Organ Tech Rep Ser 1990;793:1‑158.
3. Khalid SA, Farouk A, Geary TG, Jensen JB. Potential antimalarial
candidates from African plants: An in vitro approach using Plasmodium
falciparum. J Ethanopharmacol 1986;15:201‑9.
4. Ibrahim AM. Anthelmintic activity of some Sudanese medicinal plants.
Phytother Res 1992;6:155‑7.
5. Watt JM. Leguminosae. In: Watt JM, Brandwijk MG, editors. The
Medicinal and Poisonous Plants of Southern Africa. Edinburgh: E and
S Livingstone Ltd.; 1962. p. 546.
6. Labadie RP, van der Nat JM, Simons JM, Kroes BH, Kosasi S,
van den Berg AJ, et al. An ethnopharmacognostic approach to the
search for immunomodulators of plant origin. Planta Med 1989;
55:339‑48.
7. Fatima F, Khalid A, Nazar N, Abdalla M, Mohomed H, Toum AM,
et al. In vitro Assesment of anti‑cutaneous leishmaniasis activity of
some Sudanese plants. Turk Parazitol Derg 2005;29:3‑6.
8. Fernando S. Herbal food and Medicine in Siralanka. New Delhi:
Navrang Booksellers and Publishers; 1982. p. 114.
The present study concludes that A. indica leaves
lack anti leishmanial activity (IC50 value >1 mg/ml).
In a previous study, it was reported that the methanol
extract of A. indica leaves exhibited a moderate
antileishmanial activity[7]. In fact, A. indica preparations
are used locally in treatment of many parasitic diseases.
Oil extracted from the seeds is used in both man and
animals as an anthelmintic, and in scabies, abdominal
ulcers, rheumatism and muscular pain[8].
The extracts of the tested plants illustrates the diversity
of antiprotozoal compounds found in those plants. Some
of them may act on systems present in the parasite
which are absent in the host cells. These are worthy of
further study and perhaps semi synthetic modification in
order to improve their therapeutic/toxic ratios.
Authors acknowledge the help of Dr. Ibrahim M. A.
Sudanese, Botanist, Department of Pharmacognosy,
Faculty of Pharmacy, University of Khatoum for plant
authentication.
REFERENCES
Accepted 23 March 2012
Revised 16 March 2012
Received 18 May 2011
Indian J. Pharm. Sci., 2012, 74 (2): 171-174
Development and Evaluation of Patient Information
Leaflets (PIL) Usefulness
R. ADEPU* AND M. K. SWAMY1
Department of Pharmacy Practice, JSS College of Pharmacy, Mysore‑570 015, 1Department of Pharmacy Practice, SAC
College of Pharmacy, B.G. Nagara‑571 448, India
Adepu and Swamy: Usefulness of Patient Information Leaflets
A prospective study was conducted to develop, validate and assess the usefulness of the patient information leaflets
for selected diseases among the patient population. Flesch readability ease score, Baker Able leaflet design criteria
were applied to develop the patient information leaflets. The leaflets were validated for both content and translation.
Eligible patients meeting the study criteria were enrolled in the study. Suitably designed knowledge, attitude and
practice questionnaire was administered at base line followed by patient education complimented with an information
leaflet. After a month, knowledge, attitude and practice questionnaire was administered once again to assess the
influence of education and usefulness of patient information leaflets on knowledge, attitude and practice scores.
The mean readability score of the information leaflets is 80 and Baker Able leaflet design criteria score is 22. Post
*Address for correspondence
E‑mail: [email protected]
174
Indian Journal of Pharmaceutical Sciences
March - April 2012