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Sangeetha S. et al. / International Journal of Biopharmaceutics. 2011; 2(1): 1-7.

ISSN 0976 - 1047

2229 - 7499

IJB
International Journal of Biopharmaceutics

Journal homepage: www.ijbonline.com

PHARMACOKINETIC EVALUATION OF QUININE IN TABLET

FORMULATION FROM HERBAL EXTRACT
S. Sangeetha, N. Manjunatha, Malay K Samanta and Mukesh Malik.
Department of Pharmaceutics, JSS College of Pharmacy, Ooty, Tamil Nadu, India.

ABSTRACT

The rationale of this project work is to formulate oral herbal tablets of Cinchona officinalis extract containing
quinine and also with pure synthetic quinine, further to determine the pharmacokinetic profile for both and compare. The
concentration of quinine in the extract was estimated by HPTLC with comparison to synthetic form. Tablets were prepared
from both extract and synthetic form through direct compression technique by varying the process and formulation
parameters. The pre and post compression parameters were evaluated for the formulated batches. The pharmacokinetic
studies were conducted in rabbit models of either sex. The pharmacokinetic profile of the extract formulation was compared
with the pharmacokinetic profile of the pure quinine formulation. The tablets formulated possessed good pre and post
compressional properties. The invitro release studies showed that it followed first order release kinetics. Even though there
was no significant differences with p value between the pharmacokinetic data obtained for pure and extract quinine tablets,
the absorption was slightly increased in case of extract. Therefore for the predicted dose is a very high amount of the extract
containing Quinine needed to be given to attain the required therapeutic range.

Keywords: Quinine, Cinchona officinalis, Pharmacokinetics, Tablet.

INTRODUCTION consist of natural plant material and not a synthesized

Herbal drugs have been in use by different
civilizations in different parts of the world for centuries
to fight a large number of diseases. Many of these are in
common use even today. With the emerging interest in
the world to adopt and study the traditional system and to
exploit their potentials based on different healthcare
systems, the evaluation of the rich heritage of the
traditional medicine is essential. In conventional
medicine, most remedies consist of synthetic drug
preparation (Joy et al., 2001). Since herbal formulations
*Corresponding Author
S. Sangeetha
E mail id: sangeethamadhesh@gmail.com
chemical, herbal remedies are less likely to cause
unpleasant side effects than conventional pharmaceutical
drugs. Thus, herbal remedies are prcised safer, gentler
and lower costing than conventional drugs (Blumenthal
et al.,1998) .Cinchona - a genus of family Rubiaceae,
native to tropical South America have been important
antimalarial drug for more than 350 years (Calixto et
al., 2000) (Gupta, 2000). The principal alkaloid, quinine
is chiefly used in the treatment of falciparum malaria
resistant to other antimalarials (quinacrine, chloroquine,
and primaquine). In addition to its antimalarial activity,
quinine shows antibacterial, antipyretic, mild oxytocic,
local anesthetic, cardiovascular stimulant and analgesic
properties, and it decreases the excitability of motor
endplate. Quinine is used to prevent cardiac arrhythmias

Sangeetha S. et al. / International Journal of Biopharmaceutics. 2011; 2(1): 1-7.
and is used in tonic beverages, which are mixed with
alcohols for bitter taste. Quinine is one of the most useful
alkaloids for pharmaceutical purposes. The
pharmacokinetics of quinine have been studied for
extensively in patients with acute falciparum malaria and
also in young volunteers but pharmacokinetics of quinine
in the form of Cinchona extract has not been studied. For
a given dose of any herbal medicine, its physiological
effect (or that of its constituents) will be governed by
pharmacokinetic parameters the absorption,
distribution, metabolism and excretion of its various
components (Samanta et al., 2000). Knowledge of herbal
pharmacokinetics can provide valuable information to aid
practitioners in prescribing herbs safely and effectively. It
may also enable useful predictions to be made, for
example regarding possible interactions between herbal
remedies and conventional pharmaceuticals and also for
prediction of dosage regimen. (Shobha Rani ., 2000),
(Atul Bhattaram et al., 1996), (Jonathan et al., 2000).
Also combination of quinine with other Cinchona
alkaloids in the extract may show to have a synergistic
effect. So it may be also useful in cases where
plasmodium falciparum is becoming less susceptible to
quinine. In the present study, the pharmacokinetics of
quinine in extract form was compared with the synthetic
quinine in tablet formulation
MATERIAL AND METHODS
Materials
Cinchona officinalis Bark: Local market (Ooty),
Quinine sulphate: Preet Remedies (Baddi). All other
chemicals were of analytical grade.
Preparation of Cinchona bark Extract
The powdered Cinchona bark (50g) was mixed
with calcium oxide (30gms), water (40 ml) and 5%
sodium hydroxide to form a paste and kept overnight.
Then they obtained paste was packed in Soxhlet
apparatus and extracted with methanol for 8 hrs at 600C.
Methanolic extract was shaken with three successive
portions of 25ml of 5% sulphuric acid. The acidified
extract was basified with ammonia solution (pH 10) and
liberated alkaloids were extracted with three successive
20ml portion of chloroform. The chloroform extracts
were combined. The chloroform was distilled off to yield
total alkaloids. It was dried to constant weight at 800 C
and kept in dessicator (Kokate., 2009), (Goonetillake et
al., 1984).
Quantification of the extract by HPTLC
The quantification was carried out at max of
236nm (CAMAG Linomat IV, Switzerland) after
development of pre-coated plates of 4x 10 cm size (Silica
gel 60 F 254, E. Merck) as described by Ehab A and co-
workers (HimanshuMisra et al., 2008).
2
Preparation of samples
100mg of extract was weighed accurately
and dissolved in 1ml of methanol in a volumetric flask.
The flask was shaken for 30 mins, the solution filtered
through whatman filter paper (Ravishankara et al., 2001).
Preparation of standard solutions
The standard quinine solution was
prepared at a concentration of 1mg/ml with methanol in
the similar manner and used for the analysis.
Application of standard and sample solutions
For the application of the solutions, pre-
coated plates of 5 x 10 cm size (Silica gel 60 F 254,
E.MERCK) were used. The standards and the sample
solutions were applied on different tracks of the plate. A
thin band of 6mm width was applied using Linomat IV
(automatic TLC applicator, CAMAG, Switzerland).
Chromatogram development
The suitable solvent system selected for
the quantification of quinine was ethyl acetate: di ethyl
amine in the ratio 88: 12 (v/v) and it was used for the
development of chromatogram. The plates were
developed in the twin-trough chamber. After the
development of the chromatogram, plates were taken out,
dried using hair drier and observed under UV light.
Densitometric scanning
The developed plates were scanned using
densitometer at 236 nm wavelength. The quantification
of the quinine was calculated with reference to standard
solution (Vinod et al., 2003)
Formulation of Tablets
Tablets of both Synthetic quinine (300mg) and the
extract (equivalent to 16.71mg of quinine) were
compressed (Rotary tablet compressor, 10 station,
Rimek, Ahmedhabad, India) using 12mm standard
concave punches by direct compression technique with
varying the process and formulation parameters. All the
powder mass was passed through BSS-80 mesh and
mixed geographically for 20 min. Prior the compression,
the powder mixture was evaluated for angle of repose,
bulk density and compressibility index and drug content
(Kohli DPS et al., 1998) (Lachman Leon, 1987). The
tablets were then punched. The table 1 and table 2 gives
the formulae of different batches.
Pre-compression parameters
The powder mixtures were studied for angle of
repose, bulk and tapped density, compressibility index
and drug content. The angle of repose was determined by
funnel method using the below formulae where h and r
was the height and radius of the powder cone

Sangeetha S. et al. / International Journal of Biopharmaceutics. 2011; 2(1): 1-7.
(USP, 2006). The bulk and tapped density was
determined by taking the known quantity of slightly
shaken powder mixture. These powder samples were,
introduced to 50ml measuring cylinder to record the bulk
density (Aulton, 1988). This was then allowed to fall
under its own weight onto a hard surface from the height
of 2.5 cm at 2 sec interval. The tapping was, continued
until no further change in volume was, observed. The
density was, calculated based on the weight of powder to
volume occupied by it. The compressibility index was
calculated using the below formula. Drug content was
determined by analyzing the quantity of quinine extracted
into phosphate buffer pH 3 from 200 mg of powder. The
samples were analyzed spectroscopically at max of
254nm and expressed in terms of percentage.
Angle of repose = tan-1(h/r).
Compressibility index (%) = 100(TD-BD)/TD. Where
TD and BD represent tapped and bulk density
respectively.
Post-compression parameters
The post-compression parameters like weight
variation (n=20), drug content (n=10), friability
(n=tablets to whole weight of 6.5 g), disintegration time
(n=6) and hardness was determined. The test for post
compression parameters except hardness was, performed
as per IP 2007. The drug content was, estimated for
individual tablets. The hardness and friability was
calculated using Monsanto hardness tester (Cadmach,
Ahmedabad, India) and friability testing apparatus
(Indian equipments, Mumbai, India). The n represents
the number of tablets used for the test.
In vitro drug release
The In Vitro dissolution studies of the developed
formulation were carried out using USP apparatus type II
(Electro lab, Mumbai, India) at 100 rpm. The dissolution
medium consisted of 900 ml of 0.1N HCl acidic buffer
pH 1.2 from 0 to 1 hour for the developed formulation
maintained at 37C 0.5C. The drug release at different
time intervals was measured by UV-visible
spectrophotometer (Shimadzu) at 254nm. It was made
clear that none of the ingredients used in the matrix
formulations interfered with the assay. The release
studies were conducted in triplicate and the mean values
were plotted versus time.
Pharmacokinetic Studies
Animals
Experiments were carried out on New-Zealand
white rabbits of either sex 1.5 0.2 kg (mean SD). The
animals were overnight fasted prior to treatment but had
free access to tap water. None subject was receiving any
other drug at least two weeks before commencement of
the study and no other drug was permitted throughout the
3
duration of the study. They were randomly assigned into
5 groups (3 animals per group). Two groups received
quinine sulphate tablets, two groups received cinchona
extract tablets and one group as control. All experiments
adhered to the institutional animals ethics committee
(JSSCP/IAEC/Projects/01/2008-09).
Administration and Collection of blood samples:
The dose for the study taken was 21mg and
42mg of quinine from quinine sulphate tablets and extract
tablets respectively calculated according to body weight
and body surface area of rabbit with the help of
conversion factor with respect to the absolute human
dose. The rabbits received the dose orally through an
intra-gastric tube as 0.3% w/v CMC suspension. Blood
samples (1 ml) were collected in heparinized tubes from
the marginal ear vein at 0, 0.5, 1, 2, 4, 6, 8, 12, 24 h after
the drug administration. The samples were centrifuged at
3500 rpm for 10 min to separate the plasma and they
were transferred into airtight containers and stored at -20
0.2 C until analyzed (Ghosh MN, 2005).
Preparation of plasma samples
The quinine was extracted from plasma samples
(1ml) obtained from study subjects by addition of 200 l
of perchloric acid to precipitate plasma proteins. The
resulting solution was vortexed for 5 minutes and
centrifuged at 4000 rpm for 10 minutes. The supernatant
layer was separated and analyzed by using Shimadzu
gradient HPLC system (Sethi P D, 2000).
Determination of quinine in plasma
The quinine was extracted from plasma samples
(1ml) obtained from study subjects by addition of 200 l
of perchloric acid to precipitate plasma proteins. The
resulting solution was vortexed for 5 minutes and
centrifuged at 4000 rpm for 10 minutes. The supernatant
layer was separated and analyzed by using Shimadzu
gradient HPLC system with LC-20 AD 230V Solvent
delivery system (Pump), Manual Injector 25l
(Rheodyne), SPD-M20A 230V Photo diode array
detector and LC solutions data station. Separation was
achieved at room temperature on a Phenomenex Gemini
C18 (250x4.6mm i.d., 5) column. The mobile phase was
a mixture of phosphate buffer 50mM (pH3.0) and
acetonitrile (80:20). The flow rate was 1.0 ml/min. The
detection was done at 254 nm using SPD 20AD Diode
Array Detector (Gerhard Dongowski et al., 2005), (Udo
Huber Pharmaceuticals, 1998).
Statistics
All data were expressed as mean value
standard deviation (S.D.). Statistical analysis was

Sangeetha S. et al. / International Journal of Biopharmaceutics. 2011; 2(1): 1-7.
performed using ANOVA test (Tukeys test). Mean
differences were considered as statistically significant at
a level of p < 0.05.
RESULTS AND DISCUSSION
The extraction of quinine from Cinchona
officinalis bark was done with methanol at 60C for 36
hrs, and followed by treatment with sulphuric acid and
ammonia. Then finally added with chloroform and was
evaporated to dryness at 40C using rotary evaporator
concentrated under vacuum and crude extract was
obtained. The extract was, quantified for the quinine by
HPTLC with comparison with the peak area obtained for
0.5 mg/ml methanolic solution of pure quinine. The
application volume of extract and synthetic solution was
5 l respectively.
The pre-coated plates developed using mobile phase
consisting of ethyl acetate: di ethyl amine in the ratio 88:
12 (v/v) was, scanned by densitometer at wavelength of
236nm. The typical densitogram is shown in figure
1.With the obtained peak area at Rf value of 0.820.02 it
was estimated that 5.16% of quinine is present in the
extract. The results are tabulated in Table 3.
Various batches of tablets were, compressed by
direct compression technique and the Table 4 and 5
displaces their pre and post-compression parameters. The
hardness of all the formulations was, greater than 3.05kg
(USP 2008). The angle of repose gives a qualitative
assessment of internal and cohesive frictional forces. All
the batches had an angle of repose less than 30 showing
good flow potential. The size and shape of the particles
reflects the density of the material. The density is directly
proportional to the number of spherical particles present
whereas inversely to the size of the particles. The
granules produced the adequate flow and stable packing.
In our formulation MCC was used in both
quinine pure and in Cinchona officinalis extract
Table 1: Formulation table For Extract (500mg) Tablet
Extract + Excipients QE1
324
Extract (Eq. to 16.71mg of Quinine)
Micro crystalline cellulose 166mg
Cross Caramellose 5mg
Aerosil 5mg
Disintegration
Remarks Time: 40min15sec
Hardness: >8kg/cm2
4
formulations QP1, QP2, QP3 and QP4, this batches
showed an optimum disintegration for the tablets
prepared with pure quinine but were as in the extract
form (QE1) the tablets showed disintegration of more
than 30mins with hardness of 4kg/cm2. But on increasing
the concentration of Cross Caramellose the batches QE2,
QE3 and QE4 showed a drop in disintegration and the
results were found to be optimum. The ideal formulations
selected for further study was QP4 in quinine pure form
and QE4 in extract form.
The invitro dissolution study was carried out for
1hour and drug release at different time intervals was
measured by HPLC (Shimadzu) at 250 max. The study
was, carried for QP4 and QE4 as the DT was suitable for
the study. The QP4 released almost 98% of the drug at
the end of 60 min and the QE5 released about 90% of
drug at the end of 60 min. Both the batches showed
satisfactory invitro release.
A single dose study, for two concentrations of
formulated extract and pure quinine containing three
rabbits in each group, for each concentration of the
formulations were carried out. The blood levels of drug
were estimated by validated HPLC method and
pharmacokinetic parameters such as peak plasma
concentration (cmax), time of peak concentration (tmax),
area under the plasma concentration - time curve (AUC0-
24)and AUC0-), elimination rate constant (keli),
elimination half-life (t1/2), etc. were calculated. The
pharmacokinetic profile of the quinine in extract tablet,
obtained after the oral administration to the rabbit was
found to be comparable with that of synthetic quinine.
The pharmacokinetic parameters obtained from the study
are given in Table 6. All statistics were made by means
of one way ANOVA followed by Tukeys test by using
Graphpad Prism version 5 software. Non-significant
variation (P<0.05) was found when area under curve
(AUC) of extract formulation was compared with pure
and marketed formulation.
QE2 QE3 QE4
324 324
324
(Eq. to 16.71mg of (Eq. to 16.71mg of
(Eq. to 16.71mg of Quinine)
Quinine) Quinine)
161mg 156mg 151mg
10mg 15mg 20mg
5mg 5mg 5mg
Disintegration Disintegration Time: Disintegration Time:
Time: 32min 25sec 14min38sec 7min35sec
Hardness: >7kg/cm2 Hardness: 5kg/cm2 Hardness:5kg/cm2
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Sangeetha S. et al. / International Journal of Biopharmaceutics. 2011; 2(1): 1-7.

Table 2: Formulation table For Pure Drug (500mg) Tablet

Drug +
QP1 QP2 QP3 QP4
Excipients
324 324 324 324
Pure Drug (Eq. to 300mg of (Eq. to 300mg of (Eq. to 300mg of (Eq. to 300mg of
Quinine) Quinine) Quinine) Quinine)
Micro crystalline
166mg 161mg 156mg 151mg
cellulose
Cross caramellose 5mg 10mg 15mg 20mg
Aerosil 5mg 5mg 5mg 5mg
Disintegration Disintegration Disintegration Disintegration
Remarks Time:12min50sec Time:8min45sec Time:3min15sec Time: 1min35sec
Hardness:7kg/cm2 Hardness: 6kg/cm2 Hardness:5kg/cm2 Hardness: 5kg/cm2

Table 3: Quantification of quinine in the herbal extract by HPTLC

S.No Sample Rf Peak area Quinine(%w/w)

1 Cinchona officinalis Extract 0.82 20181.4
2 5.16
Quinine sulphate 0.82 15632.8

Table 4: Evaluations of powder

S.No Parameters Unit Pure Quinine sulphate Cinchona officinalis Extract

0
1 Angle of repose (degree) 28.20.753 33.470.619
2 Bulk density g/cm3 0.4610.006 0.5120.013
3 Tapped density g/cm3 0.5300.008 0.6320.022
4 Compressibility % 13.051.268 18.851.604
5 Drug content % 97.750.531 93.821.062

Table 5: Evaluations for Formulated tablets

Standard Quinine
S.No Parameters Unit Cinchona officinalis extract formulations
sulphate formulations
1 Average weight mg 499.530.115 499.50.1
2 Hardness Kg/cm2 5.160.288 5.660.288
3 Friability % 0.2860.041 0.180.02
4 Disintegration min 3.580.34 13.38 0.16
5 Drug Content % 96.270.509 92.150.740

Table 6: Pharmacokinetic studies

Standard Quinine Sulphate Cinchona officinalis Extract

S.No Parameters Formulation Formulation
Dose: 21mg Dose: 42mg Dose: 21mg Dose: 42mg
1 Cmax (g/ml) 2.71 2.77 2.37 2.59
2 Tmax(hr) 3 3 3 3
3 Keli(h-1) 0.133 0.120 0.083 0.141
4 t1/2(h) 5.221 5.784 6.345 6.345
5 AUCo-t(g.h/ml) 14.348 18.722 16.887 12.606
6 AUC0-inf(g.h/ml) 15.249 20.046 20.632 13.359
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Sangeetha S. et al. / International Journal of Biopharmaceutics. 2011; 2(1): 1-7.

Figure-1: Typical densitogram of Quinine (A) and Cinchona officinalis extract (B)
A B

Figure 1: Invitro Drug Release

CONCLUSION quinine dosage forms. So we can formulate as herbal

It was predicted that the dosage of herbal
tablets was very high, as the calculated quantity of
quinine contained in each tablet was very minimum to
attain the reported quinine dosage in adult humans (300
to 600mg). Therefore it has been concluded from the
present study, that it was practically impossible to
formulate an herbal tablet of quinine containing its
extract form only, as it was approximately 17 tablets in
the present form needed to be consumed per day to get its
minimum therapeutic efficacy. Thereby it may not be a
suitable alternate when compared to that of the synthetic
dosage forms such as matrix tablets and pellets and
thereby it may be a suitable alternate when compared to
that of the synthetic Quinine dosage form.
Acknowledgement
The authors acknowledge the All India Council
for Technical Education, India for the financial assistance
to carry the above work under the scheme of National
Facilities in Engineering and Technology with Industrial
Collaboration with Apex Laboratories Limited, Chennai,
India.
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