Indian Journal of Pharmaceutical Education The Official Journal of Association of

Indian Journal of Pharmaceutical Education
The Official Journal of Association of
Pharmaceutical Teachers of India
(Registered under Registration of Societies Act XXI of 1860
No. 122 of 1966- 1967, Lucknow)
Web Site -
ISSN - 0019 - 5464
e-mail : [email protected]
Abstracted in International Pharmaceutical Abstract
Vol. 39(5)*
B.G. Nagavi, Ph.D.
1997 - till date
Number 02
Invited Editorial
P. Gundurao, Ph.D.
A.V. Kasture, Ph.D.
Jan 1981 - Jun 1984
Restructuring Pharmacognosy in Emerging Global Scenario - P.Velayudha Panicker
Letter to the Editor
Past Editors
M.N.A. Rao, Ph.D.
April - June 2005
Pharmacy Education in India - U.B. Hadkar
Tutorial Review
Insights into Artificial Neural Networks and its implications for Pharmacy – a
Tutorial Review: Part - 3
- Sathyanarayana Dondeti, K. Kannan and R. Manavalan
Review Article
Emerging Challenge of Herb-Drug Interaction - U. N. Harle, N. J. Gaikwad
Need of Upgradation of Diploma in Pharmacy Curriculum - Mehta Nikhil K, Asnani
Alpana, Pathak Ajay V and Rabra Vandana
A.N. Saoji, Ph.D.
March 1980 - Dec 1980
Study of the Dimer of Imipramine Radical as Laboratory Marker for Antioxidant
Evaluation - M.K. Tripathy, D.K. Tripathi and U.N. Dash
Leveraging through Marketing in Modern Times - Manthan J., Udupa N
C.L. Lakhotiya, Ph.D.
Jan 1979 - Jan 1980
Ribozymes: The Trans Acting Tools for RNA Manipulation
- A. Nayak, and D. V. Kohli
Microwave Assisted Rapid Quantitative Analysis of Aspirin, Calcium Carbonate
& Riboflavin - Shrishailappa Badami, Natasha Joseph Chelapureth, Deepa S,
Sandhya Deepa E, Elna Merilla Bose, Vijeesh Govindan and Suresh B
Institutional Excellence – An Approach to Contend in Global World
- A. M. Godbole and S. A. Sreenivas
CoMFA-A 3D QSAR Approach and its Applications - Sanmati K. Jain
Spectrometric Method for Determination of Carbocysteine - P. N. Sanjay Pai,
Gopalkrishna Rao, B. Balakrishna, Hamid Khan and K. Pasha
C.T. Chopde, Ph.D
Sept 1978 - Dec 1978
P. Gundurao, Ph.D.
1975 - 1978
B.M. Mithal, Ph.D.
1967 - 1974
Ph.D Thesis
Study of anti-ulcer activity of some herbal drugs - Siddhraj Singh Sisodia
Book Review
Indian J.Pharm. Educ. 39(2) April - June 2005
* Number in bracket indicates the new number after registration of IJPE with registrar of news papers for India , New Delhi.
Invited Editorial
Restructuring Pharmacognosy in Emerging Global Scenario
P.Velayudha Panicker
Professor of Pharmacognosy (Retd), Chalayil, Kavalam 688 506
Received on 17.01.2005
From the present status, Pharmacognosy has a greater
task to perform in the emerging scenario of fulfilling the
social need of health care system of India 2020 A.D. Being
a part of the clan since 1958, except for phytochemistry
and synthetic pharmaceutical chemistry contributing a
great deal to industry, I am not yet convinced how far
has a Clinical pharmacist achieved any significant triumph
from social point of view as an inevitable unit in hospital
The opinion that is being conveyed here is an
extension of my article ‘Herbal pharmacist-a link between
tradition and modernity’ published in IJPE in 2002, to
explore the role of a herbal pharmacist in hospital
environment as distinctive and individualistic from the
design and working of a phytochemist and propose a
restructuring process of pharmacognosy curriculum at
least in post-graduate level. An herbal pharmacist is to
be trained in traditional formulations [Ayurveda, Unani
or Sidha depending on regional needs] in the P.G. level
overcome these hurdles to practice their traditional ‘YingYang’ concept under the same roof of modern therapy.
I believe that as a branch of science of longevity,
Ayurveda formulate a recipe only after diagnosis from
patient counseling and this is synonymous to Dispensing
practice of yesteryears. A market product of Ayurveda
was hardly available about a century back. Ayurveda
training even today advocates flexible combination or
formulation decided on the conclusion of physician after
diagnosis of patient. The reason for this, on one side is
its basic principle of patient oriented holistic outlook and
on the other side, the difficulty in procuring genuine raw
materials leading to finished products of uniform
standards. Looking from another angle, many modern
practitioners look down the traditional systems with
aversion and brush aside as unscientific. A molecular
level of activity need not always be insisted; instead the
efficacy of multiple components if proved; shall be
to devise quality control of raw materials, finished
products and improvise preparative technique in
collaboration with traditional physicians in hospitals. The
accepted and documented even forgetting the chemistry
syllabus has to be accordingly designed so that the herbal
pharmacist has to act as a link between traditional and
modern physicians.
systems [at least in private hospitals] co-exist and codify
I have had occasion to witness in many places in
South India-especially in Kerala where many Ayurvedic
Vaidyasala simultaneously maintains practice of modern
through modern physicians. Herbal pharmacist has a
medicine. It is also true that a number of modern private
hospitals do practice a holistic traditional system side by
side. But there again, they do not have a common meeting
molecular level taking priority area to continue prevailing
place to exchange and interact the end result in a scientific
way of documentation. This is where a herbal pharmacist
can play a role in a creative way to develop formulation
technique in tune with bedside patient counseling.
There are some unsympathetic hurdles that may
come on the way to make this rendezvous of two systems
difficult. The Chinese however to a certain extent have
of molecules.
These hurdles can be surpassed only if both the
the documentation of clinical data of such modified
traditional formulations to bring out clinical results
crucial role in linking these systems. Further, it
encourages a phytochemist to work on herbal drugs in
phytochemical research program of Pharmacognosy.
The opinion expressed is the continuation of my
endeavour to bring the attention of academicians and
pharmacognosists to justify the history of Indian
pharmacy and the urgency in preserving the vast wealth
in treatise in proper perceptive of global context without
which there is no rationale in writing history of Indian
pharmacy and therapy.
Indian J.Pharm. Educ. 39(2) April - June 2005
Letter to the Editor
Pharmacy Education in India
U.B. Hadkar
Mumbai Education Trust’s Institute of pharmacy, Bandra(W), Mumbai 400 050
Received on 01.03.2005
I wish to congratulate Mr. Shantanu Kale for his
excellent article on pharmacy education. He was bold
enough to state problems arising out of the two statutory
bodies: Pharmacy Council of India and All India Council
for Technical Education. He has correctly pointed out
the consequences that would result if the D. Pharm course
is closed down.
The AICTE, the PCI, University and the state
governments hold inspection of teaching institutions for
the approval, the extension of approval and affiliation
and extension of affiliation and this takes a lot of time in
completing the formalities. I have to make a suggestion
in this regard. The above stated authorities should evolve
a common format for the inspection and should send the
inspection committee comprising of representative of each
one of the authorities so that there is no loss of time in
conducting separate inspections. This will enormously
cut down the unnecessary paper work and the college
administrators will be greatly relieved.
As per the existing norms & standards laid down by
the AICTE, and PCI, the teaching institutions have to
invest lacs of rupees on buying the journals as well as
costly instruments. Effort should be made to have a
centralized building in each of the cities like Mumbai,
Kolkata, Chennai etc., having all the costly instruments,
Indian J.Pharm. Educ. 39(2) April - June 2005
such as NMR, FTIR, HPLC etc., and also a centralized
library should be housed in the same building having
the collection of various journals-national and
international. The funds necessary for raising such a
centralized building and for the maintenance of the same
may be collected from the various pharmacy colleges in
the city or in that state. This will considerably reduce the
financial burden on the pharmacy colleges and will save
foreign exchange. The colleges can have the e-library
facility so that the teachers & students can have access
to different journals in the college library. This will also
minimize the requirements of space to be allotted towards
such facilities in individual institutions. The centralized
library can also have the internet facilities and the elibrary. The students and teachers who use the facilities
may also be charged a nominal fee per year OR the
modalities may be worked out.
I hope that office bearers of AICTE, PCI, University
and Government offices start thinking on the above lines
and each city will have a centralized building for
instrumentation and library.
I appeal to different associations like Association of
Pharmacy Teachers of India, office bearers of AICTE &
PCI to give a serious thought to my above suggestions.
Pharm. Education
Tutorial Review
Insights into Artificial Neural Networks and its implications for
Pharmacy – a Tutorial Review: Part-3
Sathyanarayana Dondeti* , K. Kannan and R. Manavalan
Received on 19.6.2004
Department of Pharmacy, Annamalai University, Annamalainagar 608 002
Accepted on 25.11.2004
In this third part of the tutorial review series some typical problems relevant to pharmacy have been chosen for
discussion to provide some insights and at the same time to serve as a pointer to the implications of neural networks as
relevant to pharmaceutical scientists. A brief survey of relevant applications from scientific literature is presented at the
appropriate juncture to enable interested readers to dig deeper in their respective field of application. The next and final
part of the series would encompass data processing, performance evaluation, some essential mathematics and other
paraphernalia in handling neural networks.
Drug Analysis
ANNs can be used to build empirical multivariate
calibration models for the simultaneous estimation of
multi-component formulation by spectrophotometry.
Absorbance values, at a series of selected wavelengths,
of a solution containing the analytes as multivariate input
and the corresponding concentrations of the analyte as
the target forms a training pair. The training pairs of a
large number of solutions containing varied proportions
of the analytes form a training set (or calibration set).
Training set for calibration can be generated
experimentally with reference methods such as the wet
chemistry or obtained from synthetic mixtures prepared
from standard solutions with known concentrations. The
input layer and output layer contains neurons
corresponding to the number of wavelength points
selected and the number of analytes in the sample
respectively. The ANN may contain one or more hidden
layer with or without bias depending upon the problem
at hand. The flexibility of ANNs and their ability to
maintain their performance even in the presence of
significant amounts of noise in the input data are highly
desirable 1, 2, since perfectly linear and noise free data
sets are seldom available in practice, thus making it
suitable for multivariate calibration modeling. The authors
have published their studies on the application of ANN
for multi-component sample analysis 3, 4, 4a, 4b.
Simultaneous determination of multi-components of
six amino acids using a linear neural network (LNN)
algorithm is reported by Yin 5 et al. They selected 17
wavelength points from the original UV spectral data with
343 wave length points (193.85 to 296.45nm with 0.3nm
interval) as input for the LNN to build a neural network
model with accurate results as evidenced by split-sample
validation and leave-one-out cross validation.
Simultaneous quantitative estimation of
acetaminophen and phenobarbital in pharmaceutical
preparations was done with kinetic spectrophotometric
data using three models namely, ANN, ANN coupled
with principal component analysis (a method for
dimension reduction) (PC-ANN) and partial least squares
(PLS) models was reported by Ni6 et al. The method relies
on the different kinetic rates of the analytes in their
oxidative coupling reaction with 3-methylbenzothiazolin2-one hydrazone (MBTH) in the presence of hydrochloric
acid and the Fe(III) oxidant. The absorbance was
measured and recorded from 440 to 740 nm every 60
seconds from 15 to 435 seconds. The performance of the
various models was compared with a validation set of
overlapping spectra containing kinetic data of binary
mixtures with different concentration ratios of
acetaminophen and phenobarbital. Investigation of these
models with simulated data and validation measurements
from synthetic binary mixtures of the analytes showed
that PC-ANN is a more effective calibration method with
better prediction (lower relative prediction error [RPE]).
PC-ANN was found to be the most efficient of the three
models since it not only simplifies the training procedure
of the ANN but decreases the contribution of experimental
noise and other minor extraneous factors due to the
inclusion of only the significant principal components in
the model.
Prediction of drug content and hardness of intact
tablets using ANNs and Near Infrared spectroscopy has
been reported by Chen7 and his co-workers. Duponchel8
et al, have developed a neural network approach to correct
for spectral differences between two spectrometers.
Spectral response of a given instrument is modeled from
another before the use of the calibration equations. In
this way, the time-consuming step of recalibration for
the second spectrometer is avoided and the initial error
prediction level is retrieved.
Population Pharmacokinetic Data: Applicability of using
a neural network approach to analyze population
pharmacokinetic data was reported by Chow9 et al. Data
* For Correspondence.
Indian J.Pharm. Educ. 39(2) April - June 2005
were collected retrospectively from pediatric patients who
had received tobramycin for the treatment of bacterial
infection. The information collected included patientrelated demographic variables (age, weight, gender, and
other underlying illness), the individual’s dosing regimens
(dose and dosing interval), time of blood drawn, and the
resulting tobramycin concentration. This network had
seven input variables (i.e., age, gender, illness, weight,
dose, dosing interval, and time of blood drawn). The
values of age, weight, dose, dosing interval, or time of
blood drawn were normalized linearly to range between 0
and 1. Gender and illness variables had values of 0 or 1,
with male 0, female 1 and oncology 0, cystic fibrosis 1,
respectively. The logarithm of the tobramycin
concentration was used as output values, which were
normalized to result in output values ranging from 0 to 1.
After experimentation, eight and seven hidden nodes
were selected to provide the interconnections between
input and output for neural networks test I and test II,
respectively. Seven hidden nodes were found to be
optimum. Each node in a layer is connected to every
node in the next layer. Each of the 622 patients’ clinical
information, tobramycin dosing regimens, and time of
blood drawn along with corresponding tobramycin
concentration measurements was treated as input and
output data pair. The network training started with a
random set of connection weights. The delta rule 10 was
used as the error correction formula to adjust the
connection weights each time the network saw a new
input-output pair. The method suggested by Rumelhart 11
was adopted to incorporate a tuning (or validation) data
set to prevent generalization problems. Convergence of
training (termination of training) was determined when
the network showed a minimum sum of square error in
predicting the tobramycin concentrations in the tuning
data set. This work showed that neural networks were
capable of capturing the relationships between patientrelated factors and plasma drug levels from routinely
collected sparse within-patient pharmacokinetic data.
This further documents the applicability of neural
networks to population pharmacokinetic data analysis.
Prediction of Plasma Levels of Amino glycoside
Antibiotic: Prediction of plasma peak and trough levels
of an amino glycoside antibiotic in patients with severe
illness in an intensive care unit by a novel approach was
reported by Nishizawa 12 et al. Knowledge of the
pharmacokinetic (PK) and pharmacodynamic PD
parameters is helpful for selecting the initial dosage
regimen and subsequent adjustment of drug dosage.
However, because the PK and PD characteristics of these
patients may be far from the normal physiological
condition, it is difficult to predict or to estimate the PK
and PD parameters. If plasma levels of drugs in these
patients could be predicted from selected physiological
Indian J.Pharm. Educ. 39(2) April - June 2005
measures, it would be of great assistance in determining
the appropriate dosage regimen without the need for the
determination of blood concentration experimentally.
Plasma levels were predicted based on the values of 15
physiological measurements using an artificial neural
network (ANN) simulator. Physiological measurements
were collected for 30 patients who were given Arbekacin
sulfate (ABK) 100 mg of ABK was administered by
intravenous drip infusion for one hour in each 12 hour
period. Blood samples were taken from the patients just
after administration (peak level) and just before the next
administration (trough level). The concentration of ABK
in plasma was determined by a fluorescence polarization
immunoassay. The following 15 physiological
measurements were collected for each patient: age, body
weight, rectal temperature, mean arterial pressure, heart
rate, respiratory rate, arterial pH, serum sodium
concentration, serum potassium concentration, serum
creatinine concentration, hematocrit, white blood cell
count, volume of parenteral fluid, volume of urine and C
Reactive Protein (CRP). A data set of 15 physiological
measurements for 30 patients was used to develop the
model. The ANN structure consisted of three layers: an
input layer comprised of 15 processing elements
(necessarily same as the number of variables), a hidden
layer comprised of 10 processing elements with a sigmoid
function as an activation function, and an output layer
of two processing elements (peak and trough levels). One
additional “bias” neuron was incorporated to act on the
hidden and output layers. The ANN was trained according
to the delta rule back-propagation using “error backpropagation learning”13. The training of the network was
stopped when the maximum error between observed and
predicted values had decreased to a value less than 0.01.
The network had been trained 10 times, each time using
new random sets of initial weights. The goodness fit was
evaluated by root of mean squared error (RMSE). The
correlation coefficients between observed and predicted
values obtained by ANN prediction using standardized
data sets were r = 0.825 and r = 0.854 for peak and trough
levels, respectively. These results indicate that ANN
shows better performance in prediction of amino glycoside
antibiotic plasma levels from patients’ physiological
measurements than MLRA. Prediction of plasma levels
of antibiotic in patients with severe illness by ANN was
superior to the standard statistical method.
Standardization of input data was found to be important
for better prediction. ANN has some advantages over
standard statistical methods, as it can recognize complex
relationships in the data.
Similarly Yamamura 14 has demonstrated the
superiority of ANN over multiple regression analysis in
predicting the plasma concentration of aminoglycosides
(arbekacin sulfate and amikacin sulfate) in severely ill
patients by. In this study plasma level was predicted by
ANN model using parameters related to the severity of
patient’s condition.
Gentamicin peak predictions: Gentamicin peak predictions
were made using neural network model by Smith and
Brier15. Peak observations from 392 patients were used to
train, test, and validate the feedforward neural network.
Inputs were demographic and drug dosing information.
Data were collected from dosing records on 392 patients
that received Gentamicin and had the pharmacy follow
drug dosing. Information on patient age, height, weight,
serum creatinine and gender were recoded from which
creatinine clearance, body surface area and ideal body
weight were calculated. The dose of Gentamicin received,
dose interval, and the subsequent steady-state peak and
trough were also recorded. The total data set was
subdivided randomly into three sets. The first set
consisted of 200 records and was used as training set.
The second data set was termed the testing data set and
was used to periodically test the predictive performance
of the model and consisted of 92 records; the final data
set consisted of 100 records and was used to validate
the predictive model. They concluded that neural
networks can be built using large number of parameters
that have good predictive performance.
Pharmacokinetic-Pharmacodynamic model: A novel
model-independent approach to analyze pharmacokinetic
(PK) and pharmacodynamic (PD) data using ANNs has
been reported by Gobburu 16 and his co-workers using
simulated data with standard mathematical relations
governing the PK and PD of hypothetical drugs. The
dose, time and logarithm of drug concentration served
as input variables and PD effect as the output variable
with two hidden layer neurons. The prediction quality of
a network was assessed by employing the correlation
coefficient between the output and desired (target) as
the numerical performance index. The pharmacological
effect was assumed to be free of any contributions from
an active metabolite. The trained network was used to
predict the PD effect at a higher dose. Similar tests were
conducted when the drug was administered extravascularly and in another instance when the drug was
given an intravascular injection. ANNs were found to be
successful in predicting the PD profiles accurately in both
Quantitative Structure-Pharmacokinetic Relationships
(QSPR): The application of neural networks to predict
the pharmacokinetic properties of beta blockers in human
has been reported by Gobburu and Shelver17. The beta
blockers included in their study were acebutolol,
alprenolol, atenolol, bufuralol, metoprolol, nadolol,
pindolol, propranolol, timolol and tolamolol. The
pharmacokinetic parameters (targets) studied include
fraction drug bound plasma (fb), steady-state volume of
distribution based on the total drug in plasma (Vss),
steady-state volume of distribution based on the unbound
drug in plasma (Vuss), renal clearance (CLr), non renal
clearances (CLnr), and mean residence time (MRT).
Table 1: Input and Output Parameter for the Neural
Output Parameters
Input Parameters
Logarithm of Fraction
pK a and logarithm of Ksf
Volume of Distribution
pK a and logarithm of Ksf
Logarithm of Renal
Logarithm of Ksf and
logarithm of fb
Logarithm of Non-renal
Mean Residence Time
pK a and Ksf
pK a and Ksf
As predictor (input) variables, they used pK a ,
octanol/buffer (pH 7.4) partition coefficient determined
by the shake flask method (Ksf), and the fraction of the
drug bound in plasma (fb). A separate neural network
was developed for prediction of each of the
pharmacokinetic parameter. Table 1 illustrates the data
sets used for prediction of various pharmacokinetic
parameters. In all cases five hidden neurons were used.
The neural network-predicted values showed better
agreement with the experimental values (8% mean
difference) than those predicted by multiple regression
techniques (47% mean difference).
Intestinal absorption prediction: A three layer, feed
forward neural network has been developed for the
prediction of human intestinal absorption (HIA%) of drug
compounds from their molecular structure 18. The data set
contained 86 drug and drug-like compounds whose
molecular structure was described with six descriptors.
This model does not produce an exact rank ordering, but
is clearly differentiates the well absorbed compounds from
the poorly absorbed ones and thus illustrates the
potential of using QSPR methods to aid drug
development process.
QSAR Applications
QSAR of Carboquinones: Aoyatoma 19, 20 et al. chose a
data set that had previously been studied by Yoshimoto21
et al. using traditional modeling techniques such as
MLRA in order to compare those results with the results
of neural networks The QSAR relationship study was
designed to predict the minimum dose of drug
(carboquinone and its analogues) required to produce a
40% extension of the lives of test animals (mice) that had
been inoculated with lymphoid leukemia L-1210 cells. This
minimum effect dose depends on the concentration, C,
Indian J.Pharm. Educ. 39(2) April - June 2005
of the substance necessary to give the desired effect,
and is given as log(1/C). The more effective the drug is,
the smaller will be the concentration necessary. Since
the required concentration of different drugs vary over
several orders of magnitude, log (1/C) (the logarithmic
transformation) has been chosen as a measure of effective
dose. Altogether 35 different carboquinones were
selected; for all of them, a total six variables were used
as inputs. The variables (four) describing the combined
influence of substituents are molar refractivity,
contribution to hydrophobicity, sum of the constants for
field effect and sum of the constants for resonance effect.
The variables describing the influence of one substituent
include the molar refractivity and the contribution to
hydrophobicity. The output consisted of the minimum
effective dose which is given by log (1/C). Thus the
neural network consisted of 6 input nodes with one output
node. Optimum hidden nodes were found to be 12 and
the network was trained with back-propagation algorithm.
The log (1/C) values were compared with those obtained
by MLRA 21 on the same set of 35 compounds. The results
of the ANN were significantly better than those obtained
by MLRA. A book edited by J. Devillers 22 is a good
source of information for those interested in QSAR and
drug design applications.
Classification of the Activity of Capsaicin and Its
Analogues: An ANN to model the structure-activity
relationship of a large series of capsaicin analogues was
reported by Hosseini23 et al. Back-propagation artificial
neural networks (ANNs) were trained with parameters
derived from different molecular structure representation
methods, including topological indices, molecular
connectivity, and novel physicochemical descriptors to
model the structure-activity relationship of a large series
of capsaicin analogues. In order to present the ANN with
as much useful information as possible from structure,
the authors investigated a variety of structural
parameterization methods. These were molecular
connectivity, topological indices and charged indices,
connection table theory, and a simple atomic
decomposition of molecules into atom type. These were
used in conjunction with novel physicochemical
descriptors and some simple atomic descriptors
developed in this study. The design of the dataset for
training and testing the ANN is a bit specialized and
complex to be discussed here. Interested readers can refer
to the original paper. The ANN QSAR model produced a
high level of correlation between the experimental and
predicted data. After optimization, the developed model
correctly classified 34 of 41 inactive compounds and 58
of 60 compounds of 101 capsaicin analogues. The results
demonstrated the capability of ANNs for predicting the
biological activity of drugs, when trained on an optimal
set of input parameters derived from a combination of
Indian J.Pharm. Educ. 39(2) April - June 2005
different molecular structure representations.
Antimicrobial Activity: Topological connectivity indexes
were used to detect the microbiological activity in a group
of heterogeneous compounds 24 . Step wise linear
discriminant analysis models and ANN models were
developed. Though both methods are appropriate to
differentiate between active and inactive compounds, the
artificial neural network was better and showed in a test
set a prediction success of 98% while the linear
discriminant analysis showed only 92%.
QSAR of Carbonic anhydrase: Development of
quantitative structure-activity relationship and
classification models for a set of carbonic anhydrase
inhibitors have been reported by Marttioni25 et al.
Formulation Development
Optimization of Formulation Parameters for Liposomes:
Optimization of the formulation parameters of Cytarabine
liposomes prepared by the lipid film hydration method
using artificial neural networks (ANN) and multiple
regression analysis has been very recently reported in
an online journal by Indian investigators, Subramanian26
and coworkers. The process variables such as drying
time, rotation speed, temperature, vacuum applied, and
hydration time are kept constant, while the formulation
variables, drug/lipid (phosphatidyl choline [PC] and
cholesterol [Chol]) molar ratio (X1), PC/Chol in percentage
ratio of total lipids (X2), and the volume of hydration
medium (X 3), which have been predicted to play a
significant role in enhancing the percentage drug
entrapment (PDE) are taken as variable parameters and
the PDE was selected as the dependent variable. The
third independent factor, volume of hydration medium, is
also a very important factor as the drug is introduced
into the liposomes after dissolving in the hydration
medium. As model formulations, 27 formulations were
prepared. A multilayer feed-forward back-propagation
network using Levenberg-Marquardt’s learning rule was
used to predict PDE of the liposomal formulations. Three
causal factors, drug:lipids (X1), PC:Chol (X2), and volume
of hydration medium (X3) were used as each unit of input
layer. The output layer was composed of one response
variable PDE. The optimization methods developed by
both ANN and multiple regression analysis were validated
by preparing another 5 liposomal formulations. The
predetermined PDE and the experimental data were
compared with predicted data by paired t test, no
statistically significant difference was observed. ANN
showed less error compared with multiple regression
Theophylline controlled release tablet: Formula
optimization of theophylline controlled-release tablet
based on artificial neural networks has been reported by
Takayama 27 et al. 16 kinds of theophylline tablets were
prepared. The amounts of Controse, the mixture of
hydroxypropylmethyl cellulose with lactose, cornstarch
and compression pressure were selected as causal factors.
The release profiles of theophylline were characterized
as the sum of the fast and slow release fractions. A set
of release parameters and causal factors were used as
tutorial data for ANN. The optimization technique
incorporating ANN showed a fairly good agreement
between the observed values of release parameters and
the predicted results.
Optimization of Aspirin extended release tablets: Aspirin
extended release tablet formulations have been optimized
using ANN by Ibric 28 et al. The effects of concentration
of Eudragit L 100 and compression pressure as formulation
and process variables was studied on the in vitro release
profile of aspirin from matrix tablets. The release profiles
predicted by the generalized regression neural networks
coincided well with the experimental values.
Ketoprofen Hydrogel optimization: A novel simultaneous
optimization technique incorporating an artificial neural
network (ANN) was applied to a design of a ketoprofen
hydrogel containing O-ethylmenthol by Takayama 29 e t
al. For the optimization study, the amount of ethanol and
O-ethylmenthol were selected as causal factors. A rate
of penetration lag time and total irritation score were
selected as response variables. A set of causal factors
and response variables was used as tutorial data for ANN
and the nonlinear relationships between the causal factors
and the response variables were represented well with
the response surface predicted by ANN.
Salbutamol sulfate osmotic pump tablet optimization: In
a study by Wu 30 et al. a formulation optimization
technique using ANN has been reported. Thirty different
formulations of salbutamol sulfate osmotic pump tablets
were prepared, and their dissolution tests were performed.
The amounts of hydroxypropyl methylcellulose (HPMC),
polyethylene glycol 1500 (PEG1500) in the coating
solution, and the coat weight were selected as the causal
factors. Both the average drug release rate for the first 8
hr and the correlation coefficient of the accumulative
amount of drug released and time were obtained as
release parameters to characterize the release profiles. A
part of the data of release parameters and causal factors
was used as training data for the ANN, and another part
of data was used as test data. The optimal formulation
produced by the technique gave the satisfactory release
profile since the observed results coincided well with the
predicted results thus demonstrating ANNs to be quite
useful in the optimization of pharmaceutical formulations.
Controlled release formulation: An artificial neural
network (ANN) and pharmacokinetic simulations in the
design of controlled-release formulations with predictable
in vitro and in vivo behavior has been reported by Chen31
et al. Seven formulation variables and three other tablet
variables (moisture, particle size and hardness) for 22
tablet formulations of a model sympathomimetic drug were
used as the ANN model input, and in vitro dissolutiontime profiles at ten different sampling times were used as
output. The optimized ANN model was used for prediction
of formulations based on two desired target in vitro
dissolution-time profiles and two desired bioavailability
profiles. For three of the four predicted formulations there
was very good agreement between the ANN predicted
and the observed in vitro and simulated in vivo
properties. This work illustrates the potential for an
artificial neural network, along with pharmacokinetic
simulations, to assist in the development of complex
dosage forms.
Polymorphic Ranitidine determination: Ranitidine
hydrochloride is antihistaminic drug that exist in two
polymorphic forms known as Form 1 and Form 2. Diffuse
reflectance IR spectral analysis 32 and X-Ray diffraction33
were combined with ANNs as a data modeling tool for
the qualitative and quantitative control of ranitidine-HCl.
The ANN was trained to recognize specific patterns of
constituents of the formulations from the overall spectral
pattern. When the classification ANN was exposed to
complex tablet formulation samples containing only
ranitidine-HCl Form 1 crystal modification, it successfully
identified and quantified all components in tablet
formulation down to a concentration of 0.7± 1.88%. There
was no need to extract the active ingredient and Form 1
was successfully quantified in the presence of tablet
excipients and additives.
Tablet granulation: Prediction of a model granulation and
tablet system characteristics from the knowledge of
material and process variables utilizing ANNs has been
reported by Kesavan34 and Peck. The formulation design
contained the following variables: granulation equipment,
diluent, method of binder addition, and the binder
concentration. The material, process, granulation
evaluation, and tablet evaluation data of the formulations
were used as the data set for training and testing of the
neural network models. A comparison of the neural
network prediction performance with that of regression
models was also done. Both the granulation model and
the tablet model converged fairly rapidly in the training
step. In the testing step, the predictions for all granulation
model variables (geometric mean particle size, flow value,
bulk density, and tap density) were satisfactory. In the
tablet model, the predictions for disintegration and
thickness were also satisfactory. The predictions for
hardness and friability were less than satisfactory. Two
situations where the neural network may not perform
adequately are discussed. The neural network prediction
is better or comparable to regression methods for all the
predicted variables in this study.
Indian J.Pharm. Educ. 39(2) April - June 2005
ANN applications for pharmaceutical formulation
development have also been reported by several
researchers 31, 35-38. ANN models showed better fitting and
predicting abilities in the development of solid dosage
forms 39, 40 in investigations of the effects of several factors
(such as formulation, compression parameters) on tablet
properties (such as dissolution) compared to the
response surface methodology. Fluidized bed granulation
processing has been modeled by Murtoniemi 41, 42 et al.
The use of ANNs to examine the relative importance of
formulation and processing variables in determining the
dissolution profile of sustained release (SR) dosage form
has been reported by Leane 43 et al. A review by
Takayama 44, 45 et al. on the use of ANN as a novel method
to optimize pharmaceutical formulations could be of use
to formulation developers. Sun46 et al. have reviewed the
basic ANN structure, the development of the ANN model
and an explanation of how to use ANN to design and
develop controlled release drug delivery systems are
Prediction of Aerosol Deposition in Human
Formulation scientists have been concerned with the
problem of how best to administer medical aerosols to
maximize delivery of therapeutic agents to infected regions
of lung diseased patients and aerosol deposition in
humans has been subjected to many years of experimental
and theoretical investigation. A reliable method for
predicting the pattern of aerosol particle deposition within
the human lungs, using artificial neural networks was
reported by Nazir47 et al. They used experimental data
(regional and total deposition fractions of inhaled
particles of defined sizes under well-defined breathing
conditions) from the most extensive in vivo studies in
human subject studies involving human volunteers
published by Heyder48 et al. to train neural networks for
prediction of regional and total aerosol deposition
patterns in human lungs. The deposition data presented
in their paper represent mean values for lung deposition
fractions of mono-disperse unit density aerosols for three
healthy subjects during nose and mouth breathing. In
each laboratory test, the subjects were asked to inspire
at a prescribed constant flow rate up to a given tidal
volume, then exhaled at a similar flow rate, with no
irregular pauses between inspiratory and the expiratory
periods of a breath. Particle diameter, breathing cycle
period, and mean inspiratory flow rate were provided to
the network as input and the target output consisted of
the aerosol particle regional deposition fractions. The
network inputs were selected through a simple
consideration of the known factors influencing the site
and extent of aerosol deposition, viz., the sizes of the
particles, their residence time within the airways, and the
Indian J.Pharm. Educ. 39(2) April - June 2005
velocity of the air in which they are borne along within
the airways49. An MLP network consisting of three input
nodes, four hidden nodes, and one output node was
found adequate for predicting bronchial deposition,
whereas both laryngeal and alveolar deposition data
necessitated the use of a 3–5–1 configuration. The three
networks were trained to provide predictions for laryngeal,
bronchial, and alveolar deposition fractions, respectively.
The total deposition fraction for a given set of breathing
conditions was computed simply as the sum of the
outputs produced from the corresponding regional
deposition ANNs.
Secondary Structure of Protein
The secondary structure of protein is of utmost
importance to its biological activity and there is much
interest in predicting the secondary structure of proteins
from their primary structure. There are three types of
secondary structures: alpha-helix, beta-sheet and random
coil. In a -helix structure, the protein chain turns
continuously in the same direction to form a spiral; in a
b-sheet, two or more parts of the same chain are aligned
parallel in space; the term coil collects all the other more
or less irregular 3-dimensional arrangements of amino
acids. Qian and Sejnowski are the pioneers in the use of
back-propagation neural networks to predict the
secondary structure of polypeptides from amino acid
sequence 50. The basic assumption is that the identities
of an amino acid and its neighbours determine the
secondary structure of that neighbourhood. A sort of
‘window scan’ over a polypeptide segment might in
principle give the secondary structure of the chain. Each
amino acid is coded as a 21-bit string with one specific
bit turned on and the other all zero. The 21st position of
the bit vector is reserved for a special code called a spacer
to handle situations when the moving window is at the
beginning or end of protein chain. The input was a
sequence of 13 amino acids and the output is 3 units
representing the secondary structures in which the central
amino acid is embedded. The training set consisted of
106 proteins, having altogether 18,105 amino acid residues
and the corresponding specifications of the kind of
secondary structure it is embedded in. Another 15
proteins with a total of 3,520 amino acids and their known
participation in a secondary structure are taken as test
set. The ANN gave 62.5% right answers on the test set,
a remarkable improvement over the method of Chou5 1
and Fasman with a predictive ability of 50 %. An overview
by Rost52on this is excellent.
HPLC optimization: The usefulness of ANNs for
response surface modeling in HPLC optimization53-55 was
compared with linear regression methods. Retention
mapping describes the chromatographic behavior of
solutes by response surface, which shows the relationship
between the chromatographic behavior of solutes and
components of the mobile phase. The capacity factor of
every solute in the sample can then be predicted, rather
than performing many separations and simply choosing
the best one obtained. Experiments confirmed that
predicted capacity factors of solutes obtained by ANNs
were better than those obtained with multilinear stepwise
regression model.
Electrophoretic mobility: It is the most important
parameter governing the separation of solutes in capillary
zone electrophoresis. Qianfeng56 et al. built a new model
by means of a multilayer neural network using extended
delta-bar-delta algorithm to estimate complex property of
electrophoretic mobilities of aliphatic carboxylates and
amines from simpler experimental properties. The molecular
weight (W), molecular volume (V), the code (+1 or -1) of
acid and base and pK value were used as input parameters
to predict electrophoretic mobility. The optimum artificial
neural networks model was found to be internally valid
and could give excellent prediction results yielding
accurate predictions for a large external set.
Bacterial fingerprinting: Three rapid spectroscopic
approaches for whole-organism fingerprinting – pyrolysis
mass spectrometry (PyMS), Fourier transform infra-red
spectroscopy (FT-IR) and dispersive Raman microscopy
– were used to analyse a group of 59 clinical bacterial
isolates associated with urinary tract infection by
Goodacre 57 et al. The artificial neural network (ANN)
approaches exploiting multi-layer perceptrons or radial
basis functions could be trained with representative
spectra of the five bacterial groups so that isolates from
clinical bacteriuria in an independent unseen test set
could be correctly identified. Comparable ANNs trained
with Raman spectra correctly identified some 80% of the
same test set.
Physicochemical properties of organic compounds: The
literature describing neural network modeling to predict
physicochemical properties of organic compounds from
the molecular structure is reviewed from the perspective
of pharmaceutical research by Taskinen58 et al. The
standard three-layer, feed-forward neural network is the
technique most frequently used, although the use of other
techniques is increasing. Various approaches to describe
the molecular structure have been successfully used,
including molecular fragments, topological indices, and
descriptors calculated by semi-empirical quantum chemical
methods. Some physicochemical properties, such as
octanol-water partition coefficient, water solubility,
boiling point and vapour pressure, have been modeled
by several research groups over the years using different
approaches and structurally diverse large training sets.
The prediction accuracy of most models seems to be
rather close to the performance of the experimental
measurements, when the accuracy is assessed with a test
set from the working database. Results with independent
test sets have been less satisfactory. Implications of this
problem are discussed.
Clinical Medicine: Recently prediction of HIV drug
resistance with neural networks has been reported by
Drachici 59 et al. They demonstrated that the drug
resistance can be predicted from either the structural
features or protein sequence of the HIV protease. Novel
artificial neural network for early detection of prostate
cancer has been reported by Djavan 60 et al. An
evolutionary artificial neural networks approach for breast
cancer diagnosis has been reported by Abbass61 and his
team. A neural computational aid to the diagnosis of acute
myocardial infarction has been studied by Baxt 62 et al. A
review by Khan63 et al. on ANN and medicine may be of
interest to those involved in clinical pharmacy.
The above survey is by no means exhaustive but
should be sufficient to indicate the implications of ANNs
in pharmacy. It is hoped that it would serve as
motivational factor to lead the interested reader into
contemplating some applications in his area of interest in
pharmaceutical sciences. The next and final part (part-4)
of the tutorial review would cover the grey areas
untouched till now such as data processing, performance
evaluation, some relevant mathematics, ANN software
and resources on neural networks in print and Internet.
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Indian J.Pharm. Educ. 39(2) April - June 2005
Review Article
Emerging Challenge of Herb-Drug Interaction
U. N. Harle and N. J. Gaikwad
Department of Pharmaceutical Sciences, University Campus, Nagpur 440 033
Received on 2.11.2004
Accepted on 13.1.2005
The use of herbal products is rising leading to co administration of herbals with medicinal drugs and the likelihood
of a clinically relevant interaction is raising high. The objective of this review is to explore such interaction and tabulate
new methodology useful to predict new interactions. A number of potent pharmacological interactions have been
recognized between phytochemicals and medicinal drugs and some are the case reports in clinical practise. Herbal
medicines and vegetables, which modulate physiological parameters like cytochrome P450s enzyme system, efflux
transporters PgP or modulation of Ah receptors are selected to categorise them in various chemical class. Among the
wide range of phytochemicals, piperine has been identified in clinical trials that increases plasma concentration of
phenytoin, propranolol, and theophylline by inhibiting CYP3A4 activity and modulating gastric emptying time. Furthermore,
bergamottin, hypericin and hyperforine were investigated for having the ability to increase bioavailability of many
medicinal drugs by modulating CYP3A4 activity. Some herbal alkaloids (vincristine, rutaecarpine, evodamine and
dehydroevodiamine), class of AQ, triterpenoids, and flavonoids serve as substrates, inducers or inhibitors of CYP’s.
Herbs are classified according to the chemical class and their physiological targets results in interaction emergencies.
The current misconception about safety of herbs is becoming invalid as the phytochemicals are able to modulate various
physiological factors and interact with medicinal drugs and therefore these interactions should be rigorously tested.
Exposure of the human body to exogenous chemicals
is a continuous, inevitable and unavoidable process.
Among the vast majority of chemicals to which man is
exposed are nature based chemicals of plant origin. It
has been estimated that more than 99% of the chemicals
ingested by humans are naturally occurring 1. Herbal
remedies are administered on chronic basis, which
dramatically increase the intake of phytochemicals that
would otherwise be consumed at low levels. The first
report of herb-drug interaction published in 1970s, stated
that consumption of cruciferous vegetables resulted in
lower plasma levels of analgesic drug phenacetin 2. It was
suggested that indoles, which are present in such
vegetables enormously, could be responsible for
interaction2. In this report, many herb-drug interactions
have been reported and reviewed. But it has been
discerned that many medicinal herbs and pharmaceutical
drugs are therapeutic at one dose and toxic at another.
Therefore, interactions between herbs and drugs may
increase or decrease the pharmacological or toxicological
effects of either component. Reviews published
previously in the same context suggested, herb-drug
interaction as a theoretical possibility and of lesser
importance has given room to the clinical relevance and
significance of phytochemicals responsible for herb-drug
interaction. Many of the reviewers have stated that ‘herbal
medicines are ubiquitous: the dearth of reports of adverse
events and interactions probably reflect a combination
of under-reporting and the benign nature of most herbs
used’3 . The true prevalence of drug interactions is
substantial but unknown 4. But till date there are various
clinical relevant herb-drug interactions reported that call
Indian J.Pharm. Educ. 39(2) April - June 2005
our attention towards the possibility of clinical
compliance from herb-drug interaction.
The case report has stated that herbs traditionally
used to decrease glucose concentrations in diabetes have
precipitated hypoglycaemia when taken in combination
with conventional medicinal hypoglycaemic agents 5.
The clinical relevance of phytochemicals is not
focused by many review published on herb drug
interactions. An attempt has been made through this
review to admit the relevance of phytochemicals,
responsible for interaction. Phytochemicals apart from
medicinal herbs, cruciferous vegetables containing
phenethyl isothiocyanate and sulforephane are also
capable of modulating cytochrome P 450 and interfere
with metabolism of medicinal drugs5. However, the picture
is more complex as cruciferous vegetables also influence
enzyme systems other than CYP family, so the effects of
these vegetables on metabolism of a certain drug will be
very much dependent on the nature of the active
phytochemicals present in such vegetables 3. For example,
cruceferious vegetables containing indoles also modulate
the activity of enzymes other than CYP family, upregulating conjugation reactions such as glutathione, Stransferases and glucuronyl transferases 2, and downregulating the flavin monooxygenase system6. In clinical
practice, polypharmacy is common, and thus, all ingested
phytochemicals in the form of herbs as food, are potent
to interact with each other.
Most of the possible herb-drug interactions may be
classified in two major categories viz pharmacokinetic and
pharmacodynamic interactions.
Pharmacokinetic Interactions
Despite of the limitations of the in vitro/in vivo
studies, as well as animal vs. human studies, there are
many studies reported for the pharmacokinetic herb-drug
interactions. Factors, which can modulate the
pharmacokinetic of the herb or drugs, are discussed as
The physical passage of herbs or drugs from the
outside to the inside of the body occurs by means of
intestine. The majority of all absorptions occur in the
intestine, where herbs or drugs must pass through the
intestinal wall to enter the blood. Several mechanisms
may interfere with the absorption of drugs through the
intestine. The influence of herbs on the absorption of
drugs is well narrated by many of the reviewers but there
are also reports of change in absorption of herbs by co
administration of the drugs. For example, drugs such as
cholestyramine, colestipol and sucralfate bind to certain
herbs, forming an insoluble complex, and decrease
absorption of both substances because the size of the
insoluble complex is too big to pass through the intestinal
There are reports of herb-herb interaction precipitated
by those drugs, which change the pH of the stomach.
With the subsequent decrease of stomach acid, herbs
may not be broken down properly, leading to poor
absorption in the intestine. Drugs, which change the pH
of stomach by affecting the secretion of stomach acid,
are antacids, cimetidine, famotidine, nizatidine, ranitidine
and omeprazole 7 . Furthermore, herbs, which affect
gastrointestinal motility, may even alter the absorption
of drug. Slower gastrointestinal motility means the drug
stay in the intestine for a longer period of time and there
will be an increase in absorption. Conversely, faster
gastrointestinal motility means the drugs stay in the
intestine for a shorter period of time and there may be a
decrease in absorption. Agar is well known laxative drug
that causes dehydration in moderate doses. Drugs such
as metoclopramide and cisapride increase gastrointestinal
motility which may possibly decrease absorption of herbs;
and drugs such as haloperidol decrease gastrointestinal
motility and may increase absorption of herbs. Therefore,
it may be necessary to alter the dosage of herbs when
the patient takes a drug that affect the gastrointestinal
motility and change overall absorption.
Any herbal laxative or bulk-forming agents will speed
intestinal transit, and thus may interfere with the
absorption of almost any intestinally absorbed drug. The
most popular laxative herbs are anthranoid-containing
herbs such as senna (Cassia senna and C angustifolia)
and cascara sagrada (Rhamnus purshiana). Dried exudate
from the aloe vera (Aloe barbadensis) leaf (not gel) also
contains anthranoids and is used as a laxative. Aloe vera
gel, found within the leaves, is used topically for burns
and cuts, and is sometimes recommended to treat ulcers
and other disorders. The gel (or juice made from the gel)
does not contain anthranoids, but the laxative leaf may
contaminate some oral preparations. Less commonly used
anthranoid-containing plants are frangula (R hamnus
frangula), yellow dock (Rumex crispus), and Chinese
rhubarb (Rheum officinale). Various herb-drug interaction
and major phytochemicals are capsulised in table-1.
Table-1: Herb-drug interaction and the resultant complications
(Aconitum napellus)
(Agrimonia eupatoria)
(Aloe vera)
(Angelica dahurica)
Agarose; Agaropectine
Interaction and comments
Antihypertensives and cardiac glycosides: Increased
toxicity and death may occur68.
Electrolyte solution and tannic acid: Dehydration69.
Anticoagulants: Decrease in clotting time70 .
Berbaloin; Aloeemodine; aloin
Xanthotoxin; Osthol;
Cardiac glycosides and antiarrhythmic agents: Potentiating
effect on heart by reducing potassium via laxative effect71.
Tolbutamide: P
rolongedhalf-life and reduced the
(Ocimum sanctum)
Astragalan; Bassorin;
Cyclosporine, azathioprine, methotrexate: Impair
immunosuppressive activity71, 73.
Linolol; Estragole
Insulin: Potentiation of hypoglycaemic effect21 .
Indian J.Pharm. Educ. 39(2) April - June 2005
Betel nut
(Areca catechu)
(Vacinium myrtillus)
Black haw
(Viburnum prunifolium)
(Rhamnus cathartica)
(Arctium lappa)
Capsicum pepper
(Capsicum frutescens)
Cascara Sagrada
(Rhamnus purshiana)
(Nepeta cataria)
Devil's claw
Dong quai (Angelica
(Ephedra sinica)
Cinnamic acid; Benzoic
Scoplin; Scopoletin
(Allium sativum)
compounds- Thioester
allyl sulphide, allicin,
diallyl suphide, diallyl
(Ginkgo biloba)
(Panax ginseng)
Flupenthixol and procyclidine: Rigidity, bradykinesia, jaw
tremor 74.
Fluphenazine: Tremor, stiffness, akithesia74.
Prednisone and salbutamol: Inadequate control of asthma75.
Anticoagulants, antiplatelete agents: Potentiation of the
Anticoagulats: Increase in the anticoagulant effect77.
Spartteine; Scoparoside
Increased the effect of antiarrhythmics, antihypertensives
and cardiac glycosides78.
Antiarrhythmics, cardia glycosides, corticosteriods and
Thiazide diuretics: Hypokalemia 79 .
Antidiabetics: Hypoglycemia80 .
Antiarrhythmic and cardiac glycosides: Hypokalemia81.
Cascarosides; Barbaloin
Antiarrhythmic and cardiac glycosides: Hypokalemia82.
Nepetalactone; Thymol
Sedatives: Sedation potentiation83.
Sedatives: Sedation Potentiation84 .
Kempferol; Luteolin;
Warfarin: Increased and prolonged action85.
Osthole ; Psoralen ;
Warfarin: Increased and prolonged action86, 87.
MAO inhibitors: Concurrent use causes hypertension.
Cardiac glycosides or halothane: Cardiac arrhythmia
Caffeine intensifies cardiovascular side effects 89.
Warfarin: Postoperative bleeding90, 91, spontaneous spinal
epidural haematoma92.
Diallyl suphide, diallyl disulphide suppresses hepatic
Ginsenosides and two
Digoxin: Raised digoxin concentrations88 .
Aspirin: Spontaneous hyphema94-96.
Paracetamol and ergotamine/caffeine: Bilateral subdural
haematoma97, Subarachnoid haemorrhage98. Warfarin:
Intracerebral haemorrhage99.
Thiazide diuretic: Hypertension 100.
Warfarin: Decreased INR101 .
Phenelzine: Headache and tremor102, mania 103.
Alcohol: In mice, ginseng increases the activity of alcohol
dehydrogenase and aldehyde dehydrogenase104.
Ginseng is reported to inhibit cDNA expression of CYP450
enzymes119 .
Indian J.Pharm. Educ. 39(2) April - June 2005
Grapefruit juice
Karela or bitter melon
(Momordica charantia)
Liquorice (Glycyrrhiza
Papaya (Carica
Psyllium (Plantago
(Rheum palmatum)
(Ayurvedic mixed-herb
St John's wort
Glycyrrhetinic acid;
Glycyrrhizin acid.
Rhein; Senosides; Gallic
Aqueous extract of
Evolvulus alsinoides
Linn, Convolvulus
Pluricaulis Choisy
Hypericin; hyperforin
Diazepam: Increased plasma level and activity51.
Vinblastine: Decrease plasma level15 .
Chlorpropamide: Glycosuria106.
Prednisolone: Increases concentration in the plasma107.
Hydrocortisone: Glycyrrhetinic acid potentiates of
cutaneous vasoconstrictor response121.
Oral contraceptives: Hypertension, oedema and
Glycyrrhizin increase the activity of CYP2B, CYP1A 122.
Warfarin: Increased INR 85.
Lithium: Decreased lithium activity102 .
Cardiac glycosides and antiarrhythmic agents:
Phenytoin: loss of seizure control117.
Paroxetine: Lethargy/incoherence118.
Sertraline: Mild serotonin syndrome105.
Nefazodone: Mild serotonin syndrome105.
Theophylline: Decreased activity120.
Phenprocoumon: Modulate activity108.
Oral contraceptive (ethinyloestradiol): Breakthrough
bleeding109. Cyclosporin: Decrease plasma levels by
stimulating CYP3A4 activity and increased expression of
Intestinal Pgp 14, 110.
Protease inhibitors: Decrease plasma concentration of
Oral contraceptives: Increased deactivation of steroid
through CYP3A-mediated metabolism66.
Alprazolam: Plasma level lowered112.
Anticoagulants: Decreased plasma level and loss activity of
warfarin113 .
Amitriptyline: Decrease the AUC114.
Aspirin: Increased bioavailability 115.
Tamarind (Tamarindus Tartaric acid; Potassium
Valerian (Valeriana
Valeric acid; Terpinols
Alcohol: Reduces the adverse effect of alcohol116.
officinalis )
Tricyclic antidepressants: Hypertension117.
Note: INR=International Normalized Ratio; AUC=Area Under the Concentration/time curve.
Effect of phytochemicals on P-glycoprotein
Efflux transporters proteins present in the intestine
also affect absorption of drug. P-glycoprotein (Pgp) is a
plasma membrane-bound drug efflux protein found
primarily in drug-eliminating organs and presumably
functions as a detoxifying transporter because it actively
extrudes xenobiotics from the body8. In the small intestine,
P-gp has been localized to the apical membrane of the
intestinal epithelial cells, having a role of effluxing the
compounds back into the intestinal lumen.
Pharmacokinetic studies of paclitaxel, digoxin, and CsA
in mdr1a knockout mice have revealed the importance of
intestinal P-gp in limiting the oral bioavailability of these
drugs 9. Phytochemicals are also known to interact with
the ATP-dependent transporter proteins such as the
intestinal P-glycoprotein and other multidrug resistance
Indian J.Pharm. Educ. 39(2) April - June 2005
proteins that facilitate the efflux of the drugs11-13. St.
John’s wort administration was reported to decrease the
blood/plasma concentrations of cyclosporine, indinavir
and digoxin. The mechanism of interaction is the direct
effect of St. John’s on intestinal P-glycoprotein/MDR1
(in human and rats), hepatic CYP3A2 (in rats), and
intestinal and hepatic CYP2A4 (in humans) 14. Inhibition
of vinblastine efflux is also reported to be mediated by Pglycoprotein by grapefruit juice components in coca-2
cells 15. Therefore, to minimize the risk of interaction, it is
best if the drugs and the herbs are taken separately by
approximately two hours.
At present, herb-drugs interaction is not reported
by affecting distribution. Interactions occur during the
distribution phase if the drugs are highly protein-bound.
For example, warfarin is an anticoagulant highly bound
to protein and has a very narrow range of safety index.
Warfarin interacts with various drugs, vitamins and food
materials. Some known examples that interact with warfarin
include aspirin, ibuprofen, vitamin K, some types of tea,
green leaf vegetables and the like. These items interact
with warfarin by either enhancing its effectiveness and
thus leading to prolonged bleeding, or by decreasing its
effectiveness and thus increasing the risk of blood clots
in the vessels, both of which may be very dangerous to
the patients. Agrimonia eupatoria has been reported to
interfere with the efficacy of anticoagulants 5. Therefore
patients who are taking warfarin need to be exceedingly
cautious when taking herbs concurrently. Unfortunately,
it is extremely difficult to predict whether an individual
herb will interact with warfarin, because there are very
few tests or experiments documented for prediction of
such interactions. The best precautionary measure is
close observation of the patient’s condition. If the patient
shows abnormal signs of bleeding and bruises, then the
dosage of herbs may need to be adjusted and the
patient’s medical doctor should be contacted immediately.
The body cannot discriminate a natural chemical
emanating from the garden and a synthetic chemical
generated in a laboratory, and consequently it reacts to
both in the same way. As the chemicals cannot exploit
beneficially by the body, they are recognized as being
foreign (xenobiotics), and potentially deleterious, so the
immediate response of the body is to eliminate them. A
large number of phytochemicals that gain access to the
systemic circulation tend to be lipophilic, and
consequently difficult to excrete; the body renders these
hydrophilic through metabolism to facilitate their
excretion16. The most versatile enzyme system involved
in metabolism of phytochemicals and medicinal drugs is
Indian J.Pharm. Educ. 39(2) April - June 2005
the cytochrome P45017. Since the same enzyme system
is responsible for the metabolism of naturally occurring
chemicals and medicinal drugs, the herb-drug interactions
need not surprise us. Phytochemicals at the dietary levels
can modulate the hepatic and extra hepatic expression of
cytochrome P450 levels resulting in marked changes in
the metabolism of drugs that lead to adverse drug
Effect of phytochemicals on cytochrome P450
Cytochrome P450 (CYP) is the most important Phase
I drug-metabolizing enzyme system, responsible for the
metabolism of various xenobiotics including therapeutic
drug and phytochemicals 19,20. The relative abundance of
the hepatic CYPs in humans has been determined as
CYP1A2 (13%), 2A6 (4%), 2B6(<1%), 2C (20%), 2D6 (2%),
2E1 (7%), and 3A4(30%)21, 22. The significance of the
individual CYP enzyme in human drug metabolism varies,
with CYP3A, CYP2D, and CYP2C being responsible for
the metabolism of 50, 25, and 20%, respectively, of the
currently known drugs 21, 23. Drug interactions can
frequently arise when drugs are co administered and one
drug modulates the metabolic clearance of the second
drug by inhibition or induction of a specific CYP enzyme,
possibly leading to some fatal interactions24, 25. Many
phytochemicals have been found to modulate the
expression and catalytic activity of various CYP isoforms.
For example, broccoli up-regulated a number of
cytochrome P450 enzymes in the liver and colon of the
rats 26. It was eventually established that indol-3-carbinol,
which is present in the form of glucosinolate and
glucobrassicin, are released by the action of the enzyme
myrosinase in the stomach affects the cytochrome P450
system 27-29 . Cruciferous vegetables containing
isothiocynate compounds, such as phenethyl
isothiocynate and sulforaphane also modulate cytochrome
P450 activity1, 5, 30, 31. Phytochemicals in tea, at dietary
levels of intake, are capable of altering the hepatic
cytochrome P450 profile 32. Grapefruit constituents,
furanocoumarins, in general, are known to inactivate
cytochrome P450 through mechanism-based process33-35.
Other constituent, bergamottin has also proved to reduce
CYP4A activity more effectively than its dihydroxylated
A. Piperine
Piperine is a pungent alkaloid present in Piper nigrum
Linn, was reported to have antidiarrhoeal 36 , antiinflammatory 37, immune-enhancing, anticonvulsant and
antioxidant activity38. Piperine inhibited gastric emptying
of solids/liquids in rats and gastrointestinal transits in
mice in a dose- and time-dependent manner36. Piperine
inhibited CYP3A4 activity in humans39. Piperine may work
as a drug bioavailability enhancer, which is evident by
the increase in the Cm a x and AUC of phenytoin,
propranolol, and theophylline by co administration of
piperine40, 41.
B. Flavonoids
Flavonoids are a diverse group of phytochemicals
that are produced by various plants including medicinal
herbs (e.g., Silybum marianum, Alpinia officinarum,
Hypericum perforatum) 42 . Flavonoids are structurally
classified into eight groups: flavins, flavvanones,
isoflavanones, flavones, isoflavones, anthocyanides,
chalcones, and flavonolignans. Flavonoids exhibit
antibacterial, anti-viral, anti-inflammatory, anti-angiogenic,
analgesic, hepatoprotective, cytostatic, apoptotic,
estrogenic activity and possess ability to modulate
several enzymes and cell receptors 38. Many flavonoids
have been reported as potent inducers of various
CYPs 43-45. The mechanism for the induction of CYPs by
flavonoids may involve direct stimulation of gene
expression via a specific receptor and/or CYP protein or
mRNA stabilization 25, 46 . Flavonoids like 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), induce CYPs via
binding to aryl hydrocarbon (Ah) receptor, a ligand
activated transcription factor47. For example quercetin,
one of the most abundant naturally occurring flavonoid,
bind as an antagonist to Ach receptor48. Flavonoids
present in grapefruit juice significantly increased the oral
bioavailability of most dihydropyridines (e.g., felodipine),
terfenadine, saquinavir, cyclosporine, midazolam,
triazolam, and verapamil 49, 50 .
Bergamottin, a
furanocoumarin isolated from grapefruit juice, has been
reported to increase diazepam bioavailability51.
C. Triterpenoids
Triterpenoids exist in many herbal medicines, exhibits
anticancer, anti-allergic, immunomodulatory, antihypertensive, hepatoprotective, antiviral, hypoglycemic,
antifungal and molluscicidal activities 52, 53. a-Hederin (a
triterpenoid saponin) significantly decreased the total
hepatic CYP content, and the activity of microsomal
ethoxyresorufin O-deethylase (CYP2B), and alanin
hydroxylase (CYP2E1) in the mouse54. Oleanolic acid, a
triterpenoid, dose dependently reduce total liver
microsomal CYP and cytochrome b content55. Further,
there is insufficient data of triterpenoid-drug interaction
therefore further studies are required to assess the
influence of triterpenoids on drug metabolism.
D. Anthraquinones
Anthraquinones (AQ) exists in variety of herbs,
known to interact with CYPs in three ways: 1) as a
substrate of CYPs; 2) as an inducer of CYPs; and 3) as
inhibitor of CYP. For example, emodine, present in laxative
herbs, is metabolized to 2-hydroxyemodin mainly by
CYP1A256. Emodine treatment induces CYP1A1 and
CYP1B1 mRNA in human lung carcinoma and breast
cancer MVF-7 cells 38. But AQ administration has shown
no significant effect on intestinal drug metabolizing
enzymes 38.
E. Polyphenols
Polyphenols are family of compounds occurring in
black and green teas, variety of medicinal herbs, fruits
and vegetables. In vitro animal studies have indicated
that polyphenols have chemoprotective, antifatigue,
neuroprotective, antioxidant, anticancer, and
hypolipidemic activities 57-59. Polyphenols may modulate
CYPs in two ways: 1) modulating the expression; and 2)
modulating the activity. For example, catechin are the
major polyphenol, increased the expression of CYP1A2
in rats 60. Gallic acid, a polyphenol found in wine and
herbal tea, inhibits CYP3A activity in human liver
microsomes 61. Ellagic acid is a naturally occurring plant
polyphenol found to decrease the hepatic CYP content,
cytochrome reductase activity and CYP2E1-catalyzed pnitrophenol hydroxylation62.
F. Alkaloids
Alkaloids exist in number of herbal medicines and
serves as a substrate, inducer or inhibitor of various
CYPs. For example, some coumarine-type of alkaloids are
substrates of CYP2A663. However, cephaeline and emetine
are potent inhibitors of CYP2D6 and CYP3A464. Some
alkaloids (e.g., rutarcarpine, evodiamine, and
dehydroevodiamine) isolated from evodia rutaecarpa
inhibited 7-methoxyresorufin O-demethylase and 7ethoxyresorufin O-deethylase (both are CYP1A) activities
in mouse liver microsomes and interfere with the
metabolism of tolbutamide, chlorzoxazone and nifedipine65.
In addition to the liver, the kidney is also responsible
for eliminating herbs and drugs from the body. Herbs
and drugs acting on the Pgp present in the tubular
membrane can affect the elimination of either or both.
Further, some herbs are known diuretic agents can affect
the excretion of medicinal drugs. The nephrotoxic drug
induces kidney damage results in slow rate of elimination
leading to an accumulation of herbs and drugs in the
body. Important examples of drugs that damage the
kidneys include amphotericin B, methotrexate, tobramicin
and gentimicin. Therefore, as a prophylactic measure, it
may be necessary to lower the dose of herbs to avoid
unnecessary and unwanted side effects.
Pharmacodynamic Interactions
Pharmacodynamic interactions are generally more
difficult to predict and prevent than pharmacokinetic
interactions. Most of the pharmacodynamic interactions
Indian J.Pharm. Educ. 39(2) April - June 2005
known are documented through actual cases as opposed
to laboratory experiment results. The best way to prevent
pharmacodynamic interactions is to watch the patient
closely and monitor all clinical responses including signs,
symptoms and any abnormal reactions. Examples of
pharmacodynamic interaction include additive and
antagonistic interactions. Betel nut (Areca catechu)
interact with Flupenthixol and procyclidine, Fluphenazine
and results in Rigidity, bradykinesia, jaw tremor74. Nepeta
cataria potentiates sedation by oral sedative agents 83.
Co administration of herbals with medicinal drugs is
frequent and the likelihood of a clinical relevant interaction
is high. Despite of the data shown by most of the literature
it is likely that such interactions are common than generally
thought, but are under-reported and appearance of
adverse effects may be attributed to the disease for which
the treatment is taken. Therefore, interaction between
herbal remedies and medicinal drugs are not any more
just a theoretical possibility. The interaction between
cyclosporine and St John’s wort is now well-documented66
and the underlying mechanisms are reasonably well
understood, but further interactions can only be avoided
if this under-researched crucial area of pharmacology
receives proper attention. A number of other potent
pharmacological interactions have been recognized in this
decade and cytochrome P450s enzyme system, efflux
transporters Pgp and Ah receptors are found to be major
targets for herb-drug interaction. There are many sources
of phytochemicals, including herbal medicines, vegetables
and food materials and it is not feasible to check the
intake. Therefore knowingly or unknowingly these agents
act on the physiological targets resulting in herb-drug
interaction and any untoward effect is considered as
adverse effect of drug. These phytochemicals interacting
with medicinal drugs are identified. Piperine has been
shown in clinical trials to increase plasma concentration
of phenytoin, propranolol, and theophylline40, 41. The
mechanism of interaction may be by inhibiting CYP2A4
activity and decreasing gastric emptying time of drugs36, 39.
Flavonoid present in grapefruit juice inhibits the activity
of CYP and PgP49. Bergamottin, a furanocoumarine from
grapefruit juice, was investigated for its ability to increase
bioavailability by modulating CYP activity 51 . Two
components of St John’s wort, hypericin and hyperforine
are believed to increase CYP3A4mRNA levels 67. Clinical
studies have documented that St John’s wort reduces
the plasma concentration of cyclosporine, amitriptyline,
digoxine, indivarin, nevirapine, oral contraceptives,
warfarin, phenprocoumarin, theophylline, simivastin, and
nortriptylline. Some herbal alkaloids (vincristine,
rutaecarpine, evodamine and dehydroevodiamine) are
serves as substrates, inducers or inhibitors of CYPs.
Indian J.Pharm. Educ. 39(2) April - June 2005
However, phytochemicals belonging to the class of AQ,
triterpenoids, and flavonoids may interact with CYPs in
three ways: as substrates, as an inducers or inhibitors
and modulating the expression. Previously herbals were
considered as a food ingredient and not an agent of
medical standards. The second misconception that the
herbals are safe and cannot interact with medicinal drugs
should totally be ruled out. Herb-drug interaction may
lead to several emergencies in clinical practise and some
may be more fatal that the complications resulted from
drug-drug interaction.
Herbal products are not subjected to proper scientific
testing and no proper awareness is available among
practitioners and patients for herbal safety and herb-drug
interaction. Clearly, the notion that phytochemicals are
problem-free is incorrect, and therefore some degree of
regulation is desirable.
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Letters, 145, 1999, 35042.
Pharm. Education
Need of Upgradation of Diploma in Pharmacy Curriculum
Mehta Nikhil K, Asnani Alpana, Pathak Ajay V and Rabra Vandana
J.L. Chaturvedi College of Pharmacy, Nagpur
Received on 15.5.2004
Modified on 16.9.2004
Accepted on 25.11.2004
In order to meet international standards, our country has to take steps for the improvement of Pharmacy education
system. The proposed paper “Need of Upgradation of Diploma in Pharmacy curriculum” looks into the matter that
diploma course should not be abolished rather upgraded. Revision of Pharmacy curriculum, in terms of upgradation is
essential. The present study considers the lacunae in the existing Diploma in Pharmacy courses and suggests various
measures to upgrade it. In addition, the study advocates that diploma in pharmacy should not be abolished.
Under the provision of Pharmacy Act-1948, Pharmacy
Council of India after approval from Central Government
made the regulations that prescribes minimum standard
of education required for Pharmacist. These regulations
were called as Education Regulation (ER)1. On the basis
of recommendations of WHO Consultative Committee,
Pharmacy Council of India and Central Government have
proposed to abolish Diploma in Pharmacy Course in the
country and advocate that, Degree in Pharmacy should
be considered as the minimum qualification for registration
as Pharmacist (ER-2001) 2. Although, the complete
Education Regulation is of significance, the present study
considers only few significant norms and thereby this
could be considered as the limitations of the study. The
norms considered are:
This has been a matter of opinion that diploma course
in Pharmacy should be abolished and degree in Pharmacy
should be considered the eligibility for dispensing
chemist. The experienced survey conducted among 100
professionals in the field of Pharmacy reveals that the up
gradation of curriculum of Diploma in Pharmacy should
be given consideration rather than abolishing the
curriculum. Therefore, the considered hypothesis has
been rejected. The emphasis has been given on the upgradation generally because the individuals opined that
diploma in Pharmacy provides faster employment and
entrepreneurship. Unless, prescription authorities are
given to retail pharmacist, there is no meaning in
upgrading the requirements of eligibility of practicing
pharmacy from diploma to degree. However, during the
survey the following shortcomings have been observed
in the existing Diploma in Pharmacy curriculum.
Nature and period of course
Nature and period of practical training to be
undertaken after the completion of regular course
(not less than 750 hours) covered in a minimum of
three months in an institution, hospital, pharmacy
or dispensary recognized by Pharmacy Council of
Analysis taught has no practical values.
Outdated compounding.
Allied subjects like Drug Store & Business
Management and Pharmaceutical Jurisprudence
have been limited only up to theoretical
Lack of Industry - Institute interactions.
The area for the present study has been considered
as Nagpur. Experienced survey was done of concerned
Pharmacists (inclusive of retail chemist, teachers, diploma
and degree students of pharmacy). The total sample size
was taken as 100 and the time of the study was academic
year 2002-2003. The limitations of the study have already
been mentioned. The research design has a specific nature
of process of Demand characteristics. Mean value of
opinion has been considered and statistical test has been
applied to check the hypothesis “Abolition of Diploma
Course in Pharmacy is the effective method to raise the
standards of Pharmacy education”. The hypothesis has
been interpreted and conclusions have been made. The
present study is suggestive in nature.
Lack of Hospital - Institute interactions.
Lack of Market - Institute interactions.
Practical training of 750 hours not up to the mark.
Lack of modernity and technical up-gradations in
many institutes.
Lack of professionalism in the professional course
(B.Pharm) and Diploma holders.
The subjects of the examination and the standards
attained therein.
10. Lack of Spirit of Entrepreneurial Development in the
During the survey, when asked to the respondents
the following suggestions have been observed and they
are opined below.
Indian J.Pharm. Educ. 39(2) April - June 2005
Lack of professionalism has been a curse to retail
chemist and most of the times they are compared
with Grocery walas. If it has been made mandatory
on the part of retail/wholesale chemist to have their
own quality control in their retail / wholesale
outlets, the significance of analysis taught to them
would be increased and the responsibility of
controlling and assuring the quality of product sold
by them would be extended to retailers / wholesalers
from manufacturers. Also, it would offer an
employment to others, who cannot afford to start
their own outlets and the quality system in the
country pertaining to pharmaceutical products and
services will be upgraded.
Compounding of old traditional medications4 should
be extended to compounding of medications
containing new molecules, dosage forms like
aerosols, and Ayurvedic preparations (considering
product patent regime from year 2005 onwards) so
that the chemist will be benefited with the advanced
knowledge. Likewise, Synthesis of newer molecules
should be added to the curriculum. The syllabus in
this context should be revised with quicker
Allied Subjects like Drug Store and Business
Management and Pharmaceutical Jurisprudence are
restricted to theoretical considerations because of
various reasons:
· Teachers during their graduation or post
graduation do not have adequate orientation
regarding these subjects. Mere passing paper
of 100 marks is not sufficient for teaching these
· No appropriate allocations or eligibility criteria
made for teacher specialization in M.Pharm
(Pharmaceutical management) or MBA or M.
Pharm (Quality Assurance) have been made,
leading to lack of orientation of such subjects
among the student.
market- institute interactions. ER-91 which states about
“nature and period of practical training to be undertaken
after the completion of regular course” needs
modification. This training should be made mandatory in
between first and second year of the curriculum (as in
the case of B. Pharmacy where industrial training can be
done after third year of the course).6 There should be
compulsory preparation and submission of report of
training by the student and viva voce be considered the
norms for examining the candidate. The Committee be
constituted for the purpose of examining these candidates
which should be comprising of:
Teacher of Drug Store and Business Management,
Pharmaceutical Jurisprudence or Hospital and Clinical
Pharmacy of the institute of candidate as the case
may be depending upon the type of training in
market, industry or hospital be the internal examiner.
Teacher of Drug Store and Business Management,
Pharmaceutical Jurisprudence or Hospital and Clinical
Pharmacy of the other institute as the case may be
depending upon the type of training in market,
industry or hospital be the external examiner.
Any Retail or Hospital Pharmacist or Industrial
pharmacist as the case may be as an expert.
Institute should have to supply the list of their
candidates undergoing training to Pharmacy Council
of India and MSBTE depending upon which surprise
visits can be held by Inspector of State Pharmacy
Council or Executive Committees.
The trainer (from retail, industry or hospital as the
case may be) are made to submit their reports in the
prescribed format7 (suggested along with the present
paper) to the institutes and which could be taken as one
of the tool for evaluating candidates performance.
· Communication Skills and Professional English
and Entrepreneurial Development should be
added as the subjects in the curriculum to raise
the standards of individuals.
The question cropped up during the interview was
why retailers, industry or hospital be interested in being
a part of training? The application of Victor Vroom’s
Theory of Motivation8,9 solves the problem i.e. “person
does the things in which he foresee his advantage”.
Government of India should take the step for increasing
interest of the retailers, industry or hospital in training
by availing concessions in income tax as a rebate per
candidate trained or certain kind of monetary benefits
provided by the sharing of institute, PCI, MSBTE and
Government of India. The remuneration as best training
centre can also be one of the motivating factors.
The extension of these subjects can be done by
increasing industry-institute, hospital-institute and
Thus, the upgraded diploma in pharmacy would yield
faster employment and promote entrepreneurship.
· Lack of Spirit of Entrepreneurial Development in
the curriculum of Drug Store and Business
Indian J.Pharm. Educ. 39(2) April - June 2005
Suggestive Format for Trainers
(to submit their reports to Principal of the Institute)
Name and Address (Place of training)
Name of the Candidate
Name of the Institute
Name of the Trainer
Registration no.of Trainer
Place of Training
Telephone No.
I) Please evaluate the performance of candidate (out of 3) in each case:
1. Learning Capability:
2. Alertness:
3. Initiativeness:
4. Technical Knowledge:
5. Behaviour of Customers:
6. Accounting Skills
7. Legal Knowledge:
8. Inventory Management Skills:
9. Routine Working Skills:
10. Behaviour with inspectors:
Total out of 30 :
II) Please mention the various contents covered during the training programme.
III) Recommendations: Promoted / Not to be Promoted
Signature of Trainer
Universal Law Publishing Co., Pharmacy Act 1948
with Short notes, Sec 10, 1999, 7.
Nagpur University, Nagpur, Faculty of Medicine,
Ordinance No. 153, point 22, 4.
Bag, M M, Abolition of Diploma Courses, Pharma
Academia, Express Pharma Pulse Special Feature,
December 18, 2003, 68
Zikmund William, Business Research Method,
Singapore:Thomson Asia, 512.
Mehta, R.M, Drug Store and Business Management,
New Delhi: Vallabh Prakashan, 3rd Ed., 118.
Maharashtra State Board of Technical Education,
Mumbai Syllabus of Diploma in Pharmacy, 9
Maharashtra State Board of Technical Education,
Syllabus of Diploma in Pharmacy, 16
Mehta, N. K, Industrial Psychology and Sociology
(for First year B. Pharm Students), Nagpur: VBD
Publications, 85
Pattanayak Biswajeet, Human Resource
Management, Motivational Theories, New Delhi:
Prentice Hall of India, 2001, 188.
Indian J.Pharm. Educ. 39(2) April - June 2005
Pharm. Chemistry
Study of the Dimer of Imipramine Radical as Laboratory Marker for
Antioxidant Evaluation
M.K. Tripathy*, D.K. Tripathi* and U.N. Dash**
*P.G. Department of Pharmaceutics, Sri Jayadev College of Pharmaceutical Sciences, Naharkanta
** Former Director, University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar
Received on 08.06.2004
Modified on 30.11.2004
Accepted on 05.02.2005
A laboratory marker has been considered as a tool to quantify the in vitro antioxidants property. In this study,
considering the non-enzymatic mechanism of antioxidants defense the oxidative reaction of Imipramine (IMP) hydrochloride
with Fe (III) ions as the oxidative substance and the response of the reaction to different antioxidants was considered.
Vitamin C was used as the reference antioxidant and four different extracts of Terminallia chebulla were used to test
their antioxidant potential. The study was based on the hypothesis that, dibenzoazepine undergoes oxidative reaction
with formation of coloured products, which can be quantified spectrophotometrically. The study revealed the development
of a blue colour, which is because of the dimer resulted due to the dimerization of two-cation radicals, of the oxidized
product imipramine, gave chromophores at 670 nm. Vitamin C as an antioxidant being a powerful electron donor reacted
with the cation radical and helped to get stabilized and prevent the formation of a coloured dimer as evident by the
decrease in absorbance. The different plant extracts also behaved in the same fashion in the reaction mixture and
stabilized the radical. As the antioxidants prevent the radicals produced in the system from formation of dimer and the
antioxidant activity following the reaction pathway are also accurately measurable on repeat tests, the system is proposed
as a novel method and the dimer as a laboratory marker for antioxidant evaluation.
The role of antioxidants in health and disease is now
well recognized. Antioxidants reduce or eliminate the
cytotoxic reactive oxygen species (ROS) that are
generated in the tissues in several patho-physiological
conditions. Biological tissues maintain critical balance
between antioxidant reserve and the ROS, which are
continuously produced in the cells. In patho-physiological
conditions, this balance is upset because of reduction in
antioxidants reserve and excess production of ROS, which
play a crucial role in many degenerative diseases. The
potential for antioxidants therapy therefore continues to
grow at a staggering pace1.
In this context, much thrust has been given on the
phyto constituents having antioxidant activity, hence their
screening becomes very important. A laboratory marker
has been considered as a tool to quantify the in vitro
antioxidant property. In this study, considering the nonenzymatic mechanism2 of antioxidant defence, we
envisaged, to study the oxidative reaction of imipramine
hydrochloride with Fe (III) ions as the oxidative substance
and the response of the reaction to different antioxidants.
In this study, vitamin C was used as the reference
antioxidant and four different extracts of Terminallia
chebulla were used to test their antioxidant potential.
The study is based on the hypothesis that the
dibenzoazepine derivatives undergo oxidative reaction
with the formation of coloured products. The extent of
Indian J.Pharm. Educ. 39(2) April - June 2005
formation of coloured product can be quantified
spectrophotometrically. In case of imipramine
hydrochloride which reacts with Fe(III) ions at higher
temperature with formation of a blue coloured product.
The progress of reaction depends on the concentration
and type of the acid used, concentration of antioxidant,
temperature and type of heating. Below is the mechanism
of reaction.3
Pure imipramine hydrochloride was procured as a gift
sample from Torrent Research Centre, Ahmedabad. All
other reagents were of Analytical Grade and the solutions
were prepared with double distilled water. Forty-five test
tubes graduated at 10 ml were divided into 5 groups (A,
B, C, D and E). Each group containing 9 test tubes and
were marked as T1, T2, …..., T9. Group A represented the
reaction module with vitamin C as reference antioxidant4, 5,
Group B to E represented the reaction module with the
four different extracts of Terminallia chebulla in
petroleum ether (40-60), chloroform, alcohol (95 % v/v)
and water respectively. In each group one test tube
marked as T1 was used as the control where no
antioxidant was added and the rest eight test tubes were
used for the tests where eight different volumes such as
0.1, 0.2, ……, 0.8 ml with a strength of 500 mg/ml of the
different samples were added. In each test tube, one ml
of 4 ´ 10-2 mole/litre FeCl3 solution was placed and to
that 1.5 ml of 10 mole/litre CH3COOH volume of 1 ´ 10-4
mole/litre imipramine hydrochloride solution was added.
In case of control, the total volume was diluted to the
mark (10 ml) with distilled water and in case of test sample
tubes (T2-T9) the respective volume of test samples were
added and the final volume was made up to the mark
with double distilled water. The reaction components in
each tube were mixed thoroughly with vortex mixer and
then heated at 75 ± 0.5o C for 5 minutes in a
thermostatically controlled waterbath. After cooling to
room temperature, the absorbance of each solution was
measured at 670 nm against reagents blank without using
imipramine in a Genesys 2 TM Thermo spectronic, double
beam U.V visible Spectrophotometer, U.S.A.
Preparation of Plant Extracts
Dried fruits of Terminallia chebulla were collected
locally. They were identified with the help of the
Department of Drabya Guna Vigyan, Government
Ayurvedic Medical College, Puri, Orissa. The specimens
were cut into small pieces, dried at 40-50oC for 48 hours
and powdered. The powder weighing 120 g was subjected
to successive extraction with petroleum ether (40-60),
chloroform, alcohol (95 % v/v) and water using soxhlet
apparatus for 8-10 hours. The extracts with different
solvents were completely evaporated at 40oC over
waterbath and finally under vacuum. The residues 0.380 g
for petroleum ether, 1.080 g for chloroform, 8.050 g for
ethanol and 1.780 g for water were subjected to
antioxidant activity.
As evidenced from (Table–1 & Fig-1), Group A shows
that the absorbance has decreased gradually from 0.433
to 0.048 due to the presence of the antioxidant, vitamin C
in an increasing order. This establishes that vitamin C
being a powerful electron donor stabilizes the cationic
radical of imipramine hence prevents the formation of the
dimer, the colored complex. Similarly the gradual decrease
in absorbance was found in each of group B, C, D and E
which represent the different extracts of Terminallia
chebulla in petroleum ether, chloroform, alcohol, water
respectively. This indicates that each of these extracts
contain a principle having antioxidant property and it
can be assumed that the mechanism of action is similar
to that of vitamin C, which is a known antioxidant.4 , 5
Since in each of the cases maximum absorbance of 0.433
has been observed when the dimerization of imipramine
radical took place under the experimental conditions
without any hindrance and if this absorbance is
considered to represent 100% dimerization, then reduction
of absorbance, i.e. blockade of dimer formation, to 0.048
can be interpreted as 88.92 % antioxidant activity by the
standard antioxidant, vitamin C. Table-1 shows that all
the four different extracts of Terminallia chebulla
represented as group B,C,D and E also reduced the
absorbance from 0.433 to 0.142, 0.064, 0.008 and 0.008
respectively. Out of all the five groups, group D and E,
i.e.alcoholic and water extracts of Terminallia chebulla
showed the maximum antioxidant activity by reducing
the absorbance to the minimum of 0.008. When the
activity of each group is correlated with the
concentration, except the alcoholic extract, all other three
extracts and vitamin C showed their maximum activity at
a concentration of 40 mg/ml (400 mg in 10 ml), while the
alcoholic extract shows its maximum activity at a
concentration of 25 mg/ml (250 mg in 10 ml) and it blocks
the dimer formation to the extent of 98.15%. Table-2
shows the interpreted results in terms of antioxidant
activity. Table-2 also indicates that petroleum ether extract
exhibited its antioxidant activity up to 67.20% at a
concentration of 40 mg/ml and chloroform extract at the
same concentration exhibited its activity up to 85.22%.
Both aqueous and alcoholic extracts exhibited better
results, that is each of the two could block the dimer
formation up to 98.15% and between these two, alcoholic
extract showed to be better because at a concentration
of 25 mg/ml it was active. The results are shown as an
average of 4 repeat experiments where in a good extent
of reproducibility was observed as indicated by low
standard deviation.
Indian J.Pharm. Educ. 39(2) April - June 2005
Table-1: Showing the response of reaction mixture to the addition of antioxidants
Test Modules
Group A
0.433 ± 0.014
0.401 ± 0.006
0.388 ± 0.002
0.314 ± 0.017
0.277 ± 0.012
0.186 ± 0.014
0.124 ± 0.008
0.084 ± 0.006
0.048 ± 0.004
Absorbance (Average ± S.D.)
Group B
Group C
Group D
0.433 ± 0.014
0.433 ± 0.014
0.433 ± 0.014
0.403 ± 0.022
0.363 ± 0.014
0.222 ± 0.014
0.389 ± 0.026
0.351 ± 0.012
0.162 ± 0.008
0.376 ± 0.026
0.309 ± 0.008
0.092 ± 0.012
0.306 0.019
0.295 0.006
0.044 ± 0.004
0.296 ± 0.017
0.229 ± 0.004
0.008 ± 0.002
0.242 ± 0.030
0.159 ± 0.008
0.196 ± 0.006
0.096 ± 0.006
0.142 ± 0.012
0.064 ± 0.002
Group E
0.433 ± 0.014
0.301 ± 0.010
0.262 ± 0.008
0.214 ± 0.004
0.156 ± 0.006
0.123 ± 0.003
0.094 ± 0.004
0.036 ± 0.002
0.008 ± 0.001
Table-2: Antioxidant activity in terms of % blockade of dimer formation
Fig. 1 : Showing the response of reaction mixture in different test modules to the addition of antioxidants
Indian J.Pharm. Educ. 39(2) April - June 2005
Different researchers have used different in vitro
models to evaluate the antioxidant activity of samples. In
this study dimerization of imipramine radical has been
taken as the in vitro model and vitamin C as the reference
standard. It has been concluded that vitamin C responded
to the model following nonenzymatic mechanism and the
different extracts of Terminallia chebulla contain
antioxidant principle which could block the activity of
imipramine radical in a reproducible manner. This model,
because of its simplicity and reproducibility is proposed
to be a novel method for antioxidant evaluation using
the imipramine dimer as the laboratory marker.
One of the authors (M.K.Tripathy) is thankful to the
management of Sri Jayadev College of Pharmaceutical
Sciences, Naharkanta for encouraging the research
activities and providing the necessary facilities for
carrying out the research work.
Das, D, “Antioxidant and Redox Signaling ” Lecture
Note, in International Conference on Role of Free
Radicals in Health and Disease, Cardiovascular
Research Centre, University of Connecticut, School
of Medicine, Farmington, USA, Feb. 10-12, 2003.
Chatterjee, P, Free radicals in Health and Disease, J.
International Medical Sciences Academy, 10, 1999,
Karpinska, J and Starczewska B, Reactions in
Imipramine-Pyrocatechol – Violet and Imipramine
Iron (III) ion Systems, Pharmazie, 54(1), 1999, 41-43.
Ray, K, Rudra, S, De, AU, Sengupta, C, Evaluation
of Ascorbic acid as an inhibitor of lipid peroxidation
induced by Cefotaxime Sodium and Metoprolol,
Indian J. Pharm.Sci, 61,1999, 44-47.
Patil, S, Jolly C and Narayanan, S, Free radical
scavenging activity of Acacia Catechu and Rotula
Aquatica: implications in cancer therapy, Indian
Drugs, 40(6), 2003.
Indian J.Pharm. Educ. 39(2) April - June 2005
Pharm. Marketing
Leveraging Through Marketing in Modern Times
Manthan J., Udupa N.*
Manipal College of Pharmaceutical Sciences, MAHE, Manipal 576 104
Received on 05.08.2004
Modified on 19.10.2004
World is undergoing radical changes. Last five
decades have seen enormous development. Technological
advancement, deregulation, globalization, elimination of
trade barriers provides endless opportunities. World is
shrinking and so do markets. Change is occurring at a
rocketing speed. What is new today may be obsolete
tomorrow. Government regulations are liberalized that
pave the way for marketing professionals. World has
become a global village and products developed in one
country are available throughout the world. Marketing is
a dynamic field and thus, marketing professionals have
to be dynamic too. They constantly need to amend new
products, new market segments etc. Demographic shifts
are taking place. There is increase in number of old people
all around the world and thus, their health concerns need
to be addressed. Average life expectancy of people in
India was 59 years in 1990s, which has increased to 64
years at present but this is less as compared to US where
average life expectancy is 77 years. This is due to
awareness regarding health and better medication.
Pharmaceutical Marketing
One may ponder how Pharmaceutical marketing is
different from general marketing. As we know that we do
not need a prescription to purchase items of daily livingbe it food, clothes or house. Here the buyer is the sole
decider of which product to purchase, when and in what
number. But you need a prescription to purchase
medicines, which are used to treat certain symptoms or
diseases. Thus, the significant difference is that in
Pharmaceutical marketing the marketer reaches target
consumer through intermediate customer who, in this
case, is a doctor. Pharmaceutical marketing deals with a
person who is more conversant and knowledgeable in
his field. A doctor decides which drug(s) to prescribe for
what indication and in what quantity. Buyer has no choice
in deciding which drug to buy and which not for a
particular illness. Therefore, it is no exaggeration to say
that Pharmaceutical marketing is unique.
Marketing – an essential part of industry gamut
Marketing professionals are accused of creating
needs. People always say, “Marketers get people to buy
things they don’t want.” As Philip Kotler, a prominent
Indian J.Pharm. Educ. 39(2) April - June 2005
Accepted on 29.12.2004
marketing guru, aptly says, “Marketers do not create
needs. Needs preexist marketers.” Thus, marketing
professionals try to unearth the latent needs of people
and cater to those needs, as people themselves are not
aware of their needs. An example of Viagra would be the
best fit for justification. Pfizer, world’s top Pharmaceutical
Company identified the need of people to improve their
sexual potential. Though people were interested in buying
such product, they were not able to speak candidly. Pfizer
found this latent need and leveraged on it. Thus, Pfizer’s
product Viagra became a blockbuster and generated more
than a billion dollars sale for the company. Another
example is that of ranitidine from Glaxo. Through
marketing skills, Glaxo was able to generate more than a
billion dollars as revenues from the sale of this single
product. This product became a blockbuster product.
Marketing also depends on the brand image of the
product. Companies have strived through the years to
create distinct Brand image in the minds of their
customers. Once the brand image is created half the battle
is won. But it is very difficult to create brand image in
this competitive era. Before a company builds a brand
image, it has to identify its market, inform decision makers
about its profile and give them compelling reasons to
care. A successful brand is one, which clearly satisfies
physician’s needs.
None can deny the role and importance of marketing
in Pharmaceuticals. Merely a good product developed
by R&D would not suffice to make product a success.
Best products developed by R&D have not been able to
see the daylight in the marketplace. For example Johnson
& Johnson introduced a new sanitary napkin “Modest”
in the 70’s to take on the pioneer “Comfit”, which was
marketed by competitor. The product of Johnson &
Johnson never took off and finally was withdrawn.
Another example is from cephalosporins market. So many
cephalosporins have been developed but only some
molecules have met with the success in the marketplace
due to sustained marketing efforts made by companies.
Cefprozil is one among such cephalosporins that never
really was accepted in the market. No matter how good
your product is, it would not be a success if marketing
department of a company were fragile. You need a sharp
mind to leverage on a good product developed by R&D.
As Charles Revson of Revlon observed: “ In the factory,
we make cosmetics; in the store we sell hope.” This is
true in our context also. A pharmaceutical company makes
medicines in their factory but sell hope; hope of healthy
living, hope of life in case of life threatening diseases
like AIDS and Cancer. The responsibility lies on the
professionals not to shatter this hope. In addition, the
responsibility of Pharma marketing professional is that
he should not play with the life of people by simply
marketing a good for nothing product.
Marketing Interface
Marketing interface builds an image for the company
– a corporate image as we call it. The task of marketer
simply does not end here. Marketing works in tandem
with other departments of a company; in fact, activities
of other departments depend on the marketing department.
Marketing department has to deal with internal as well as
external environment. (See Fig.1). Marketing department
gives the data on future requirements of market based
on which the production department sets out its
schedule. It also collects feedback from market and gives
information regarding changes in dosage form, packaging,
strength of a product, texture, color and flavor in case of
liquid dosage forms, method of administration of drugs,
change in salt of molecule. Based on this information
R&D department initiates its activities to modify existing
product and meet the market requirements. Marketing also
works in tandem with distribution and logistics department
of the company, as availability of drugs in market place
is also important. Time is one of the important factors in
marketing activity. Earlier you are in the market, more are
the benefits. People remember names of those who are
the first in their field. Ask what is the name of the first
person to fly solo across the North Atlantic? The answer
is Charles Lindbergh. Ask the name of the second person.
Few can remember. Therefore, time factor plays a pivotal
role in marketing. It is generally said that if you want win
the marketing battle, you need to be first in the market.
But this may not always be applicable to pharmaceutical
marketing. With more than 80,000 odd pharmaceutical
brands and new products added at breakneck speed, it is
necessary for Indian companies to clearly differentiate
their products with that of competitors. The key here is
product differentiation, which helps company to create
competitive advantage over competitors and establish
the product even if the company is late entrant in the
market for a particular product or therapeutic segment.
Marketing in general and pharmaceutical marketing
in particular has a social responsibility. As pharmaceutical
products deals with healthcare of society and provides
better hope of healthy living, it is marketers’ prime
responsibility to fulfill their obligations. While detailing
to doctors regarding the products, representative of the
company should bear in mind that they have to be
judicious in detailing of the product since, wrong product
or product information to the doctors can be fatal for the
patient. Responsibility to inculcate these virtues lies with
the marketing department of the companies and
companies should devise a strategy to implement this to
its bottom level i.e. to the representative level. Once
company’s image is established as a provider of authentic
information, half the battle is won to capture market share.
It is generally observed that currently almost all the
companies are following niche marketing to grab the
maximum market share and therefore are creating divisions
catering to the needs of doctors for various therapeutic
segments. This helps them to be focused and allocation
of the resources can be justified.
Marketing is also undergoing radical changes and
newer techniques are developed by the companies to
increase their reach to the doctors. E-detailing is one
such concept developed by the companies. Here rather
than face to face conversation with doctors, doctors are
contacted through web based virtual detailing where
doctors can interact on the web. This method saves the
money and busy doctors can access the information
regarding drugs at any time, depending on their
convenience. But this concept is still not developed in
India unlike United States. Further, Customer Relation
Management has become buzzword in the industry.
Companies are striving to retain customers rather than
increasing their customer base. This also provides
companies endless opportunities to grow in their
In a nutshell, it can be said that the role of marketing
cannot be ignored in this fiercely competitive world where
companies are vying with one another to gain better
market share. Lose your focus and you are out of the
arena and there is someone to replace you. So, in these
turbulent times where companies are finding it difficult
to survive, let alone grow, companies should leverage
on marketing as an essential tool.
“Companies should think of millennium as an
opportunity to increase mindshare and heartshare.”
Philip Kotler
Indian J.Pharm. Educ. 39(2) April - June 2005
Research and
Public Relations
Fig 1 : Internal and External Marketing Interface
Subba Rao, S.V.R., The Product, In: Handbook of
Pharmaceutical Marketing in India, Panther
Publishers, 1998, 3.1 to 3.3.
Ries Al, Trout Jack, Getting into the mind, In:
Positioning - The battle for your mind, McGraw Hill,
2001, 19.
Lidstone John, and Collier Terry, Product Review,
In: Marketing Planning for the Pharmaceutical
Industry, Gower, 51.
Indian J.Pharm. Educ. 39(2) April - June 2005
Kotler Philip, Understanding Marketing
Management-Marketing in the twenty first century,
In: Marketing Management, Prentice Hall India, 2000,
Smarta Raja, New Marketing Approaches, In:
Revitalizing the pharmaceutical market, Response
books, 1999, 43.
Davidson Terry and Sivadasa Eugene, Details Drive
Success, Marketing Health Services, spring 2004,
Pharm. Biotechnology
Ribozymes: The Trans Acting Tools for RNA Manipulation
A. Nayak, and D. V. Kohli*
Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar-470 003
Received on 14.08.2004
Accepted on 22.01.2005
Ribozymes are small and versatile nucleic acids that can cleave RNAs at specific sites. Ribozymes have enormous
potential in basic research and biotechnology with special promise for applications in human medicine. In this context,
a catalytic RNA is designed to specifically recognize, interact with, and alter a target molecule. These molecules have
great potential to be used as effective gene therapeutic agents. The best-characterized ribozymes are hydrolytic nucleases
that perform site-specific cleavage of a target RNA. The ability of ribozymes to recognize and cut specific RNA molecules
makes them exciting candidates for human therapy. Ribozymes can be used both to down regulate and repair pathogenic
genes that may lead to disease conditions. In some instances, short-term exogenous delivery of stabilized RNA is
desirable, but many treatments will require viral-mediated delivery to provide long-term expression of the therapeutic
catalyst. Current gene therapy applications employ variations on naturally occurring ribozymes.
Until about 20 years ago, all known enzymes were
proteins. But then it was discovered that some RNA
molecules can act as enzymes; that is, catalyze covalent
changes in the structure of substrates (most of which
are also RNA molecules). Catalytic RNA molecules are
called ribozymes. A ribozyme can be thought of as a
chimeric RNA molecule consisting of two stretches of
antisense RNA (AR) flanking a nucleolytic motif. The
AR component, referred below as the flanking
complementary regions (FCRs), provide the target
selectivity. Two generic types have been exploited, the
“hammerhead” ribozyme 8 and the “hairpin” ribozyme 9 .
Both can cleave GUC sites, but the hammerhead can also
cleave at GUA, GUU, CUC and UUC with comparable
efficiency10 and sometimes at AUC11, 12.
Types of Ribozymes
Five classes of ribozymes have been described based on
their unique characters in the sequences as well as threedimensional structures. They are denoted as (i) the
Tetrahymena group I intron, (ii) RNase
P, (iii) the hammerhead ribozyme
(Fig.1), (iv) the hairpin ribozyme,
and (v) the hepatitis delta virus
ribozyme. They may catalyze selfcleavage (intramolecular or “in-cis”
catalysis) as well as the cleavage of
external substrates (intermolecular or “in-trans” catalysis).
Fig 1: Hammerhead Ribozyme
Self-splicing group I introns catalyze their excision
and the ligation of flanking exon sequences via two
sequential transesterification reactions13. Group I introns
from T4 phage14 and Tetrahymena thermophila15,16 have
been bisected, and the separated components have been
coexpressed in various cell types. Formation of natural
base pairings and presumably, tertiary interactions
between the two components in vivo resulted in transsplicing reactions analogous to the naturally occurring
cis-splicing reactions. Demonstration that the
Tetrahymena ribozyme catalyzes the ligation of exons in
trans has led to speculation that these molecules could
be used for therapeutic repair of defective transcripts 17.
Alteration of endogenous myotonic dystrophy protein
kinase transcripts and repair of sickle b-globin transcripts
by trans-splicing ribozymes in mammalian cells have
recently been demonstrated. However, this type of
ribozyme interacts with the intended target mRNA via a
base-paired helix (P1) of only 6 bp and shows relatively
poor specificity, with products of illegitimate splicing seen
in vivo18.
Mode of Action of Ribozymes
The discovery that RNA can act as a biological
catalyst, as well as a genetic molecule, indicated that
Indian J.Pharm. Educ. 39(2) April - June 2005
as gene inhibitors 20. Ribozyme gene therapy may be used
as an adjunct to chemotherapeutic drugs affecting viral
suppression, and facilitating immune restoration without
problems of patient compliance. RNA enzymes; ribozymes
are being developed as treatments for a variety of
diseases ranging from inborn metabolic disorders to viral
infections and acquired diseases such as cancer. The
hammerhead ribozyme, one of the smallest ribozymes
identified, is able to induce site-specific cleavage of RNA,
with ribozyme and substrate being two different
oligoribonucleotides with regions of complementarity. Its
ability to down-regulate gene expression through RNA
cleavage makes the hammerhead ribozyme a candidate
for genetic therapy. This could be particularly useful for
dominant genetic diseases by down-regulating the
expression of mutant alleles. The group I intron ribozyme,
on the other hand, is capable of site-specific RNA trans
there was a time when biological reactions were catalysed
in the absence of protein-based enzymes. It also provided
the platform to develop those catalytic RNA molecules,
called ribozymes, as trans acting tools for RNA
manipulation. Viral diseases or diseases due to genetic
lesions could be targeted therapeutically through
ribozymes, provided that the sequence of the genetic
information involved in the disease is known 19.
In mammalian cells, genetic instructions are usually
revised by RNA splicing before they are translated to
proteins. A trans-splicing group I ribozyme can be
employed to intentionally modify the sequence of
targeted transcripts in tissue culture cells. Such analysis
should facilitate the logical development of safe,
therapeutic ribozymes that can repair mutant RNAs
associated with a variety of inherited diseases.
Ribozymes can be targeted to cleave specific RNAs
(Fig. 2), which has led to much interest in their potential
Ribozyme joins with RNA message
RNA message
Ribozyme cuts the RNA message
Cut introduced into RNA message
A large proportion of the RNA message is cut. Thus a corresponding protein is not produced.
Cut cleaved RNA messages
Fig 2 : Mode of Action of Ribozyme
Indian J.Pharm. Educ. 39(2) April - June 2005
-splicing. It can be engineered to replace part of an RNA
with sequence attached to its 3' end. Such application
may have importance in the repair of mutant mRNA
molecules giving rise to genetic diseases. However, to
achieve successful ribozyme-mediated RNA-directed
therapy, several parameters including ribozyme stability,
activity and efficient delivery must be considered.
Ribozymes are promising genetic therapy agents and
should, in the future, play an important role in designing
strategies for the therapy of genetic diseases 21.
The possibility of designing ribozymes to cleave any
specific target RNA (Fig.3) has rendered them valuable
tools in both basic research and therapeutic applications.
They have been exploited to target viral RNAs in
infectious diseases, dominant oncogenes in cancers and
specific somatic mutations in genetic disorders. Most
notably, several ribozyme gene therapy protocols for HIV
patients are already in Phase 1 trials. More recently,
ribozymes have been used for transgenic animal research,
gene target validation and pathway elucidation22.
Dominant Retinal Degenerative Disease: In dominant
forms of retinal degeneration, patients have a healthy
functioning gene and a gene with a disease-causing
mutation. The mutant gene produces a dysfunctional
toxic protein that damages the photoreceptor cell.
Ribozymes are molecules containing genetically encoded
information that disrupt the mutant gene’s ability to
produce the harmful protein. With the diseased gene
inactivated, the healthy gene can supply the
photoreceptor cell with the needed protein.
Cancer: The ability of ribozymes to recognize and cut
specific RNA molecules makes them exciting candidates
for cancer therapy. Already, a synthetic ribozyme that
destroys the mRNA encoding a receptor of Vascular
Endothelial Growth Factor (VEGF) is under clinical trials.
VEGF is a major stimulant of angiogenesis, and blocking
its action may help starve cancers of their blood supply.
Hammerhead ribozymes can be of great importance
to overcome drug resistance in chemotherapy. These
ribozymes targeted the RNA product of the MDR1 gene,
the multi-drug resistance gene, and the cells then became
sensitive to the antitumor drug, vincristine. The same
abstract also described a ribozyme targeting an
overexpressed growth factor gene that self- stimulated
tumor growth; the ribozyme blocked formation of the
growth factor and inhibited cell division. Further studies
Next Cycle
Fig 3 : Steps of Ribozyme Action
Indian J.Pharm. Educ. 39(2) April - June 2005
with these substances should be interesting and of great
Ribozymes expressing prostate cancer cells were
characterized by a significant reduction of survivin
expression compared to parental cells transduced with a
control ribozyme; the cells became polyploid, underwent
caspase-9-dependent apoptosis and showed an altered
pattern of gene expression, as detected by oligonucleotide
array analysis. Survivin inhibition also increased the
susceptibility of prostate cancer cells to cisplatin-induced
apoptosis and prevented tumor formation when cells were
xenografted in athymic nude mice. These findings suggest
that manipulation of the antiapoptotic survivin pathway
may provide a novel approach for the treatment of
androgen-independent prostate cancer23, 30.
Retinitis Pigmentosa: A mutant gene calls for the
creation of a protein that damages the eye’s light-sensitive
rod cells as in transgenic rat model of retinitis pigmentosa
containing a dominant rod opsin mutation (proline-tohistidine change at position 23) 24. Viral vectors based on
the nonpathogenic human adeno-associated virus, when
coupled with the strong rod photoreceptor specific opsin
promoter, offer an efficient and nontoxic way to deliver
and express ribozymes in photoreceptor cells for long
time. Effective ribozyme-mediated therapy also demands
careful in vitro analysis of a ribozyme’s ability to
efficiently and specifically distinguish between mutant
and wild type RNAs.
Acute Myeloid Leukemias: Acute Myeloid Leukemias
are associated with a gene, AML1/MTG8, made by
bringing together (translocation) two normal genes on
different chromosomes and fusing them. Hammerhead
ribozymes specifically targeted to split the RNA product
of the AML1/MTG8 fusion gene and preventing it from
Connective Tissue Disorders: Ribozymes can inactivate
the target RNA without relying on the host cell’s
machinery and they have the capacity to cleave more
than one copy of the target RNA by dissociating from
the cleavage products and binding to another target
molecule. Some dominant genetic disorders may also
benefit from this approach. This is the case for some
connective tissue disorders such as osteogenesis
imperfecta, Marfan syndrome and the craniosynostotic
syndromes 25.
The effects of the relocation of the wild type termini
on the folding of a cis-cleaving RNA Rz 1 that was
modified from the autolytic domains of hepatitis delta
virus (HDV) RNA were investigated26.
AIDS: Human immunodeficiency virus type 1 (HIV-1) is
the primary etiologic agent for Aquired Immune
Deficiency Syndrome (AIDS). HIV-1 is a lentivirus, a
separate genus of the Retroviridae, which are complex
Indian J.Pharm. Educ. 39(2) April - June 2005
RNA viruses that integrate into the genome of host cells
and replicate intracellularly. Ribozymes are catalytic RNA
molecules with enzyme-like cleavage properties that can
be designed to target specific RNA sequences within
the HIV-1 genome. In addition to the genomic RNA,
several RNA intermediates, including splice variants, can
be targeted by a single ribozyme. Ribozyme gene therapy
for HIV-1 infection is a therapeutic approach that offers
several potential advantages over conventional therapies
in that it can potentially impact on both viral load and
restoration of the immune system. Ribozyme gene therapy
may be used as an adjunct to chemotherapeutic drugs,
effecting viral suppression, and facilitating immune
restoration without problems of patient compliance.
Currently, an anti-HIV-1 ribozyme is being tested in two
separate Phase I clinical trials 27.
Gene Shears Pvt. Ltd is developing hammerhead
ribozyme technology for therapy against HIV infection,
targeting either the tat gene or the RNA packaging
sequence (Psi) of HIV. These ribozymes have been
expressed from constructs that were introduced into
hematopoietic cells in culture, thereby protecting the cells
against viral infection. Two phase I clinical trials are
underway to test the safety and feasibility of the
approach with the anti-tat ribozyme in human subjects 28.
Huntington Disease and Myotonic Dystrophy:
Trinucleotide Repeat Expansions (TREs) are a recently
described class of mutations characterized by a change
in the size of the genomic fragment due to amplification
of the repeated unit. A number of diseases have been
attributed to TRE, including Huntington disease and
myotonic dystrophy (DM), but attempts at gene therapy
have yet to be proved successful. A potential therapeutic
approach would be to repair the expanded repeat using
the trans-splicing ability of group I intron ribozymes 29.
Since the discovery of ribozymes and self-splicing
introns, it has been estimated that this biological property
of RNA combined with other recombinant DNA
technologies would become a tool to combat viral
diseases and control oncogenes. These goals seem like
a distinct possibility now. However, there is still a lot to
be learned about the mobility of RNA inside the cells
and the cellular factors that can impede ribozyme action
in order to capitalize fully on the targeted RNA
inactivation property of ribozymes.
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Couture, L. A. and Stinchcomb, D. T., Trends Genet.,
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Ther., 4, 2002, 154.
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Nat. Genet., 18, 1998, 378.
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J., Hum Mol Genet., 7, 1998, 1649.
20. Macpherson, J. L., Ely, J. A., Sun, L. Q. and
Symonds, G. P., Front Biosci., 1, 1999, 497.
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N., Biochemistry, 9, 1996, 124.
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Indian J.Pharm. Educ. 39(2) April - June 2005
Pharm. Chemistry
Microwave Assisted Rapid Quantitative Analysis of Aspirin,
Calcium Carbonate and Riboflavin
Shrishailappa Badami, Natasha Joseph Chelapureth, Deepa S, Sandhya Deepa E,
Elna Merilla Bose, Vijeesh Govindan and Suresh B
Dept.of Pharmaceutical Chemistry, J.S.S. College of Pharmacy, Ootacamund 643 001
Received on 25.09.2004
Modified on 31.01.2005
Accepted on 05.02.2005
In recent years microwave technology is being used in laboratories for organic synthesis. In order to utilize the
technology in Pharmaceutical Analysis the microwave methods for the quantitative analysis of aspirin, calcium carbonate
and riboflavin were developed. All the assays were carried out successfully in lesser durations and with similar percentage
purity as compared to the conventional heating. The procedures developed can be used routinely for practicals in
Pharmaceutical analysis laboratories.
Ways to minimize the consumption of energy has
received enormous attention in recent times. In the past
few years, microwave heating has been found to be a
convenient source of energy and economic by saving
energy, fuel and electricity1. A very short response time
and better yield of the products are the main advantages
of microwave heating. It finds a large number of
applications in industries and laboratories. Earlier studies
carried out in our laboratories have shown the use of
microwave technique in the synthesis of organic
molecules, analysis of oils, degradation of natural
products and qualitative analysis 2-4. In view of the
advantages of the microwave technique, in the present
work, we report quantitative analysis of aspirin, calcium
carbonate and riboflavin under microwave irradiation. The
time required for completion of reaction, purity, ease of
work up, simplicity and overall safety of procedure were
studied and compared with the conventional
Pharmacopoeial procedures 5.
Equipment and Chemicals
Microwave oven (LG Health Ware System, MG-605
AP, 900 Watts, 230 V, 50 Hz), was used. All the chemicals
used were of analytical grade.
Assay of Aspirin
The assay of aspirin was conducted as per Indian
Pharmacopoeia 5. A mixture of aspirin (1.5 g), ethanol (95%,
15 ml) and 0.05 N NaOH (50 ml) was taken in a conical
flask boiled gently for 10 min, cooled and titrated the
excess of alkali with 0.5 N HCl using phenol red as
indicator. The percentage purity of the sample was
calculated. The assay was also carried out using the
following microwave method.
A mixture of aspirin (1.5 g), ethanol (95%, 15 ml) and
0.05 N NaOH (50 ml) was taken in a conical flask and
covered with a funnel. It was placed in a domestic
Indian J.Pharm. Educ. 39(2) April - June 2005
microwave oven and subjected to microwave irradiation.
Several separate experiments were conducted at different
intensities and for various durations. Each reaction
mixture was cooled and titrated against 0.5 N HCl using
phenol red as indicator. Based on the results obtained
350 W intensity and 2 min heating duration was fixed
and the assay was repeated thrice under these conditions.
The average percentage purity was noted.
Assay of Aspirin in Aspirin - caffeine Tablets
The assay of aspirin in aspirin - caffeine tablets was
carried out as per Pharmacopoeial procedure 5. Aspirin caffeine powder equivalent to 0.7 g aspirin was taken in
a RB flask and mixed with sodium citrate (2 g) and 20 ml
water. The mixture was refluxed for 30 min, cooled and
washed the condenser with 30 ml of warm distilled water
and titrated with 0.5 N NaOH using phenolphthalein as
indicator. The percentage purity of the sample was
The assay was also carried out using the same
aspirin - caffeine powder under microwave irradiation in
a beaker covered with a petridish containing ice using
the same quantities of reagents except using 250 ml of
distilled water. The reactions were conducted at various
microwave intensities and for different durations. The
percentage purity in each case was calculated and
compared with the conventional results. At 950 W and
for 18 min microwave heating produced the same results
as that of conventional. Under these conditions the assay
was repeated thrice and the average percentage purity
was noted.
Assay of Calcium Carbonate
The assay of calcium carbonate was carried out as
per Indian Pharmacopoeia 5. Calcium carbonate (0.1 g) was
taken and dissolved in 3 ml of dilute HCl and mixed with
distilled water (10 ml), boiled gently for 10 min, cooled
and 10 ml of MgSO4 and NH3-NH4Cl buffer (10 ml) were
added. The volume was adjusted to 50 ml with distilled
water and titrated against 0.05M disodium EDTA using
mordent black – II as indicator. The percentage purity
was calculated.
The assay was also carried out under microwave
irradiation in a beaker covered with a petridish containing
ice using the same quantities of reagents except using 50
ml of distilled water along with 3 ml of dilute HCl. The
assay was carried out at various intensities and for
different durations in separate experiments. Based on
the results obtained 750 W intensity and 4 min heating
was fixed. Using these conditions the experiment was
repeated thrice and the average percentage purity was
Assay of Riboflavin
The assay was also carried out in subdued light as
per Indian Pharmacopoeia 5. To a weighed quantity of
riboflavin (75 mg), distilled water (150 ml) and glacial acetic
acid (2 ml) were added and heated on a water bath for 1 h
with frequent agitation until riboflavin was dissolved. It
was cooled and diluted with distilled water to 1000 ml.
To 10 ml of this solution, 3.5 ml of 0.1 M sodium acetate
was added and diluted with distilled water to 50 ml. The
extinction of the resulting solution was measured at 444
nm. The content of riboflavin was calculated taking 323
as the E1%, 1 cm at the maximum at 444 nm.
The assay was also carried out in subdued light
under microwave irradiation. A weighed quantity of
riboflavin (75 mg), distilled water (150 ml) and glacial acetic
acid (2 ml) were taken in a 500 ml beaker and heated in a
microwave oven. The solution was cooled and diluted
with distilled water to 1000 ml. To 10 ml of this solution,
3.5 ml of 0.1 M sodium acetate was added and diluted
with distilled water to 50 ml. The extinction of the resulting
solution was measured at 444 nm. The content of
riboflavin was calculated taking 323 as the E1%, 1 cm at
the maximum at 444 nm. Several experiments were
conducted at various intensities and for various
durations. Based on the results, 100% intensity and 4
min duration was fixed. Using these conditions, the
experiment was repeated thrice and the average
percentage purity was noted.
Assay of aspirin by the conventional procedure
involving 10 min heating gave a percentage purity of
98.23. The assay under microwave heating at 350 W
intensity for 2 min gave 99.63% purity. Assay of aspirin
in aspirin - caffeine tablets by the conventional heating
for 30 min gave 98.14% purity and by the microwave
method at 950 W for 18 min showed 99.05% purity. In
the assay of calcium carbonate, the conventional method
involving 10 min heating showed 98.8% purity and the
microwave method at 750 W for 4 min resulted in 99.39%
purity. The percentage purity of riboflavin obtained by
the conventional method of 1 h heating was found to be
99.60 and the same by the microwave method at 100%
intensity for 4 min heating time was found to be 99.68.
All these readings are an average of three readings.
In conclusion, we have developed rapid quantitative
estimation procedures for the assays of aspirin, calcium
carbonate and riboflavin under microwave irradiation. The
conventional kitchen microwave can be used without any
modification in the laboratories. The other advantage of
this technique is several students simultaneously can
perform reactions in one microwave. Hence, the technique
is found to be a fast, cheap, reliable and ecofriendly that
can be used routinely in Pharmaceutical analysis
laboratories. The technique is presently being practiced
for B.Pharm practicals in our institution.
Table 1: Duration and percentage purity of quantitative estimations by conventional and microwave methods
Sl. No.
Assay of aspirin
Assay of aspirin in
aspirin-caffeine tablets
Assay of riboflavin
*Average of three readings
Duration by method, min
The present work is a part of B. Pharm project work
and the authors acknowledge His Holiness Jagadguru
% Purity by method*
Sri Shivarathri Deshikendra Maha Swamigalawaru of
Suttur Mutt, Mysore, for providing the facilities and
Indian J.Pharm. Educ. 39(2) April - June 2005
Suresh B., Determination of Saponification value
using microwave irradiation, Indian Drugs, 39, 2002,
Sharma S.V., Rama Sharma G.V.S. and Suresh B.,
More Chemistry: An eco-friendly technology, Indian
Journal of Pharmaceutical Sciences, 64, 2002, 337344.
Sharma S.V., Badami S., Venkateshwarlu L. and
Suresh B., Microwave technology in Pharmaceutical
chemistry practical: synthesis of organic drugs,
Indian drugs, 40, 2003, 450-454.
Badami S., Manohara Reddy S.A., Sharma S.V., and
Badami S…. et al., Use of Microwave Technique in
Pharmaceutical Organic Chemistry Practical, Indian
Journal of Pharmaceutical Education, 37, 2003, 199203.
Indian Pharmacopoeia, Vol. 1, 4th Ed., Delhi:Controller
of Publications, Govt. of India, 1996.
Indian J.Pharm. Educ. 39(2) April - June 2005
Pharm. Education
Institutional Excellence – An Approach to
Contend in Global World
A. M. Godbole and S. A. Sreenivas
Department of Pharmaceutics, K.L.E.S’s College of Pharmacy, Hubli 580 031
Received on 28.10.2004
Modified on 06.01.2005
Accepted on 02.02.2005
A. M. Godbole
S. A. Sreenivas
Education must undergo a paradigm shift in old norms and beliefs must be challenged. Colleges must learn to work
with fewer resources. Education professionals must help students develop the skills they need to compete in the global
world. Quality is a single most important issue in education which can bring a revolutionary change. The knowledge
needed to improve our education system already exists within the education community. The major difficulty educational
professional’s face today is their inability to deal with the ‘system failures’, which are preventing them, from developing
new educational processes that will improve the quality of education. Every institution must have a strong desire,
hunger to achieve more and more. In the present scenario of globalization which has even entered the field of education,
the words like Reform or Perish and Change is the key word. The graduates coming out must be complete individuals
with dynamism, good ability, problem solving skills, hard working graduates. To have a good product, pharmaceutical
institution must gear up by adapting a systematic work plan and its effective execution. One can bring in a systematic
transformation in style of working of an institution. Excellence and its achievements is not one man’s job but is purely
work of a strong force of tough, determined individuals and the force must be headed by an able, dynamic, professional
and above all a visionary.
Pharmaceutical education in India is now a mass
system - it has increased in size and heterogeneity. As a
means of raising productivity and making the system
focus upon outputs, pharmaceutical organizations have
fostered diversification, choice, and competition. In a mass
system, more graduates in particular areas of knowledge
are produced: in other words, a competitive market for
graduates is created. The process of valuing a graduate
in the employment market is fraught with difficulties.
Employers in the graduate market have little more to make
their decision upon than the reputation of the degree.
While they may have some information about the
technical competence of a graduate in a field-specific area,
they do not have information about employment-related
skills or about the attainments of individual graduates in
areas related to employment.
To produce quality graduates by an organization/
institution needs to undergo whole process through
quality. Quality in education can be incorporated by
taking measures in all the corners, right from student
admission to examination results. Nowadays
pharmaceutical institutions are moving towards
Accreditation and ISO certification for giving quality
education and distinguish from the other institutions.
Overall in India, there are only thirteen NBA accredited
colleges and Karnataka has a good contribution of five
colleges. One can blindly say that these accredited
colleges give quality education but only to an extent.
Hence now all the institutions who have been accredited
are moving towards ISO certification.
Institutions now well understand how to maintain
and develop their market position under such
circumstances. The principal means of operation is to
influence demand by providing subsidies to students—
that is, to provide more services, better teaching, better
research facilities, better libraries/on-line resources and
the like for each rupee that a student spends at that
institution. Vision and mission plays an important role in
modifying the institution.
Vision and Mission
The organization’s vision is a statement that
describes what the organization wishes to be in the future
– an expression of the organization’s aspirations, the
touch stone against which all actions, or proposed
actions, can be judged, and is long-term. The mission is
a statement of what the organization wants to achieve.
The typical content is:
• The role or contribution of the organization
The definition of the institution
Specific, distinctive competencies
Indications of future direction
Achievement of quality and excellence
Before embarking on this journey, one must have
clear motives and the level of commitment that is present
within an organization, particularly at the senior
management level.
There are two basic approaches to quality
management implementation:
The “blitz” approach, where the whole organization
is exposed very rapidly to the concepts and
education started. This can lead to many problems
Indian J.Pharm. Educ. 39(2) April - June 2005
associated with not knowing what to do next (or
first), leading to an detrimental situation that is
neither the implementation of quality nor achieving
best to the organization.
The slow, planned purposeful approach that causes
a gradual change to take place, so that “organization” becomes “the realization of quality and excellence” in a near seamless transition. An important
first stage is for the organization’s leaders to be
role models of a culture of quality and excellence,
so that these ideas permeate the organization with
support from the top.
The three basic principles will never change and are
very important:
1. Focus on the customer (Student)
The customer should be mainly concentrated in the
institutions, without which nothing can be achieved and
if at all achieved, will go waste.
What are graduate qualities/attributes/capabilities/
Graduates should be enterprising and employable
and should exhibit skills that global capital recognizes.
They must have the attributes of worker flexibility (the
ability to work as an individual or in teams based
situations and hold attitudes of self responsibility towards
up-skilling and re-skilling); have personal and transferable
skills (highly developed communication skills and problem
solving, be computer literate). In addition, workers should
possess international perspectives so that they are able
to understand the cultural forces that interact in their
globalized work-place. While there are elements of this
employability agenda in any set of abilities of graduates
this paper takes the view that a university graduate should
develop abilities that go beyond those related to
employability. Indeed, the mix of skills, attributes,
capabilities and qualities of a graduate are shaped by a
complex balance of stakeholder interests. Inevitably, the
value that is attached to such skills, attributes, capabilities
or qualities depends on the interests of each stakeholder,
and, at a system level, such interests may be represented
by peak bodies who talk generally about the quality of
There are wide-ranging differences in the ways that
universities employ the notion of graduate qualities or
skills. A major difference is the scope of the graduate
qualities or skills and their public specification. Some
universities prefer to limit their attention to employmentrelated skills and place lesser importance upon social and
cultural areas such as ethical professional behavior, social
responsibility and cultural plurality and sensitivity.
Further, in some cases, the specification of the quality is
limited to the cognitive (or other) skill, without any
attention to the context of application of the skill. In such
Indian J.Pharm. Educ. 39(2) April - June 2005
cases, it is left open whether the skill may apply to the
context of personal action, behavior in a professional
group, or actions as a socially responsible citizen.
Whatever the level of specification, there are two key
characteristics which set the parameters for the degree
of practical commitment that an institution has in applying
their concept of graduate qualities.
These are:
the extent to which curriculum design for courses is
directed towards achieving a particular set of
qualities, and
the extent to which formal assessment and reporting
(both formative and summative) are shaped by the
2. Measures of institutional commitment to graduate
The two key characteristics mentioned at the end of
the previous section, curriculum intention and assessment
and reporting, can work together to provide a signal to
the market about the extent to which a university is
prepared to assure the market (and institutional
stakeholders) of its commitment to delivering graduates
who have the particular qualities or attributes that the
institution claims.
In attempting to provide such assurances,
universities may take a variety of positions.
For the purpose of describing of a continuum of
positions, a strong position is where:
the course and the teaching is designed to develop
graduate qualities, and
the summative assessment tasks and techniques
have been changed to assess the level of
development of each quality in students and this is
reported upon by the institution.
A weak position is where there is neither conscious
attempt to design or teach for graduate qualities nor any
formal student assessment for such qualities. There is,
however, a further dimension to add to the above
positions. A strong position is where the course of study
is designed to develop graduate qualities, and, as a part
of the delivery of the course, students are encouraged to
compile a portfolio of experiences or work completed.
Such a folio would provide evidence of their attainments
in either the complete set of qualities expected in a
graduate or in those areas/qualities thought to be
employment related. Institutions might further support
this activity by providing a statement of their intentions
that courses develop a set of graduate qualities and
supply course outlines that demonstrate those intentions.
3. All staff members are committed to quality
and excellence
Staff members should be totally dedicated to the
improvement of the institution and students. The role of
faculty carries lot of importance in building a quality
institution. Faculty members must have an unending urge
to excel by constant upgradation of their knowledge. They
must be provided with all necessary assistance to
carryout research and publications. The faculty members
should have an unending courage for learning and selfrenewing. Self-renewing faculty will contribute more for
the development of the institution by their following
efforts. There are at least ten basic qualities shared by
self-renewing teachers who seek to be their best in all
seasons of their lives for the development.
1. They are value-driven
Self-renewing faculties are committed to values and
purpose. They know what they prefer. Their primary
anchors are within themselves. For them, renewal is not
mere responsiveness to change; it is the repeated revival
of the central concerns of their lives within the changing
contexts in which they find themselves. Something is
always at stake, something matters, and time gets
organized around those critical priorities. They are
determined to make a difference.
2. They are connected to the world around them
Self-renewing faculty stay connected to the world
around them. They are not loners. They seek out friends
who can and will talk about whatever needs to be talked
about the whole of life experience, up and down and all
around. They listen and empathize with life everywhere.
They care and communicate. They stay in contact with
their children and/or parents, students and colleagues,
and take initiative in sustaining relationships. They may
not be joiners, but they feel that the world is there for
them to enjoy, to grab on to, and to learn from. They
network information, contacts, and resources. They
support causes and take stands.
3. They require solitude and quiet
Self-renewing faculty requires times of solitude and
quiet. They know how to refill their cups before they get
emptied. They plan time for introspection as well as for
interaction and decision-making. They have private lives
that they nurture and love. They have regularly
scheduled times when they withdraw from routines to
spend time alone. They retreat to some “secret garden”
where renewal is predictable, simple, spontaneous, and
wonderful. In solitude, they look, listen, meditate, and
nurture themselves. They honor their inner life and outer
sustain the self-renewal process. Renewal must be built
into the ordinary, chaotic, ongoing rhythms of our lifestyles and work-styles. Self-renewing faculty pace
themselves, they schedule episodic breaks from their
routine time, such as travel, holidays, vacations, retreats,
seminars, and ruthless situation. They are not trying to
sustain optimal performance at everything they do; rather,
they seek to be fully present and available for all the
occasions of their life course. They are more interested
in quality time than in busy schedules, more concerned
with effective lives than with efficient actions, more
committed to integrity and style than to short-term results
and applause.
5. They learn from downtime
Self-renewing faculty learns from their
disappointments, necessary losses, and down times. Like
the lives of most people, their lives are sometimes full of
funk and disorientation. They do not live lives without
stress, failures, mistakes, loss and tragedy. Their lives
are not sweet or perfect. They know that they have
unresolved conflicts, limited perspectives, and impulses
that sometimes overpower them. They do not deny the
dilemmas of their lives. They accept the loose ends and
unfinished business of their lives as part of their own
future agenda.
In this paper we have tried to give brief details with
which the institution can march towards excellence. As
the trends are changing, the graduates who are the
outcome of the institution should be built up with all the
character to compete in the global market and can protract
the challenge in the employment market. By implementing
quality in the education, the system can be transformed
to meet the on going global challenges.
Madhavan, M., TQM in Educational Institutions,
Indian. J. of Tech. Edu., 20(3), 1997, 24-27.
Jorgensen, N. E. and Weil, T.P., Regulating Managed
Care Plans, Managed Care Quarterly, 6 (3), 1998, 716.
National Association of Boards of Pharmacy Home
Page. Available from:
April 14, 2000.
Kale, Shantanu, Pharmacy Education: Current
Problems and Suggested Solutions, Indian. J. Pharm.
Educ., 38(3) 2004, 154-160.
4. They pace themselves
Our current practice for renewal is to indulge in
rigorous work schedules throughout each week,
punctuated by “time-off” on weekends and vacations.
In a chaotic world, occasional breaks are not enough to
Shukla, Ravi N, A Quality Journey, Indian. J. of Tech.
Edu., 25(1), 2002, 66-68.
Indian J.Pharm. Educ. 39(2) April - June 2005
Pharm. Chemistry
CoMFA-A 3D QSAR Approach And Its Applications
Sanmati K. Jain
SLT Institute of Pharm. Sciences, Guru Ghasidas University, Bilaspur-495009
Received on 29.10.2004
Accepted on 29.12.2004
Comparative Molecular Field Analysis (CoMFA) is the most frequently used receptor-independent (RI) 3D-QSAR
approach, reflecting a novel, conceptually satisfying scientific approach. In this method a relationship is established
between the biological activities of a set of compounds and their steric and electrostatic properties. CoMFA has shown
its practical value in drug design.
Comparative Molecular Field Analysis (CoMFA) is
a three-dimensional quantitative structure activity
relationship (3D-QSAR) approach, introduced in 1988 by
Cramer1,2. In 1979, Cramer and Milne made a first attempt
to compare molecules by aligning them in space and by
mapping their molecular fields to a 3D grid 3. CoMFA
approach was an application of the dynamic lattice
oriented molecular modeling system (DYLOMMS), as it
was called till 1987. A real advance resulted in 1987, the
method was still named DYLOMMS but now it used grids
including several thousands of points, partial least
squares (PLS) analysis and most important, a crossvalidation procedure to check the predictive ability of
different models. In 1988, a key publication appeared2
and the method was called comparative molecular field
analysis (CoMFA) from then and on.
CoMFA is a receptor-independent (RI) 3D-QSAR
approach, reflecting a novel, conceptually satisfying
scientific approach reduced to practice as a well-written
and versatile software package. In this method a
relationship is established between the biological
activities of a set of compounds and their steric and
electronic properties 4.
Steps of a CoMFA
There are several important and critical steps in a
CoMFA study. CoMFA describe 3D structure activity
relationships in a quantitative manner. First, a group of
compounds which have a common pharmacophore is
selected to be included in the analysis. As a most
important precondition, all molecules have to interact with
the same kind of receptor (or enzyme, ion channel,
transporter) in the same manner. Then three-dimensional
structures of reasonable conformations must be generated
from the 2D-structures. Several 2D/3D-conversion
procedures are in use or have been described in literature,
e.g.CONCORD 5-7 , AIMB8 , WIZARD & COBRA 9-12 .
Alternatively, 3D- structures derived from crystallographic
Indian J.Pharm. Educ. 39(2) April - June 2005
analysis or 2D- NMR studies may be used. The energyminimized structures are stored in a database and fitted
to each other according to their chemical similarity by
using a pharmacophore hypothesis and postulating
orientation rules.
In the next step, a certain subgroup of molecules is
selected which constitutes a training set to derive the
CoMFA model. The residual molecules are considered to
be a test set which independently proves the validity of
the derived model(s). A sufficiently large box is
positioned around the molecules and a grid distance is
defined. Different atomic probes, e.g., a carbon atom, a
positively or negatively charged atom, a hydrogen bond
donor or acceptor, or a lipophilic probe, are used to
calculate field values in each grid point, i.e., the energy
values which the probe would experience in the
corresponding position of the regular 3D lattice. These
‘fields’ correspond to tables, most often including several
thousands of columns, which must be correlated with
the binding affinities or with other biological activity
values. PLS analysis is the most appropriate method for
this purpose.
The result of the analysis corresponds to a regression
equation with thousands of coefficients. Most often it is
presented as a set of contour maps. These contour maps
show favorable and unfavorable regions for
electropositive or electronegative substituents in certain
positions. Predictions for the test set (compounds not
included in the analysis) and for other compounds can
be made, either by a qualitative inspection of these
contour maps or, in a quantitative manner, by calculating
the fields of these molecules and by inserting the grid
values into the PLS model.
Molecular Alignment
The molecular alignment, i.e. the selection and
relative orientation of a certain 3D- structures out of
several conformers of each molecule, is the most
important determinant in a CoMFA study. A field fit
procedure has been proposed to improve the alignment,
the objective of the field is to minimize the residual mean
square differences between a fixed template field
(consisting of a steric and electrostatic field) and the
fields of the molecules to be aligned. It is claimed that
most often better results are obtained by using this field
fit, after a first, preliminary alignment of molecules.
Rational methods for the alignment of congeneric
molecules are e.g. the active analog approach and the
distance geometry method13-15. In the active analog
approach, large numbers of different conformations of
the molecules are superimposed; often only a few
conformations are allowed for all molecules, which lead
to a restricted conformational space for their common
pharmacophore. Distance geometry as the name implies,
approaches geometric problems by focusing on the
distances between points, rather than on coordinates or
angles. In 3D-QSAR applications, the points are atoms,
and the geometric problems can include conformational
analysis, molecular alignment and ligand- receptor
Several other computer assisted or computersautomated procedures have been developed for the
alignment of molecules. ALADDIN16 calculates the
location of points, which may be considered for the
superposition of the molecules for all low-energy
conformers of a series of compounds. Such points are,
e.g. atoms, ring centers, and projections from the molecule
to hydrogen bond donor, acceptor, and charged groups
at the binding site. The molecule with the smallest number
of possible conformations forms the template. Disco1 7
uses a clique–detection method to find superposition of
the molecules that contain at least one conformation of
each compound in the user–defined three-dimensional
arrangement of site points. The program CATALYST18
claims to use a corresponding strategy to derive 3Dpharmacophore from sets of active molecules and to
search 3D-databases for hits that are ordered according
to the quality of fit of the molecules to the hypothetical
Grid Size
Once the molecules are aligned a grid or lattice is
established which surrounds the set of analogs in
potential receptor space. Current CoMFA studies seldom
use grid resolution less then 1 Ao and, most often, 2 Ao.
The choice of grid resolution represents a compromise
between computational practicality and detailing of the
fields. If the grid resolution is too small, the number of
field–points (cells) becomes too large to perform a timely
analysis. Moreover spatial information on field preference
can be lost, through a ‘smearing out’ effect, if the cells
become too small. The grid resolution in the 1 to 2 Ao
range corresponds to, at best, differentiating single
carbon-carbon (1.54 Ao) from one another.
Any chemical unit can be used as a field probe in a
CoMFA analysis. Also, the number of field probes used
to construct a 3D-QSAR is not limited. In practice, three
(default) probes are usually employed: a steric probe, a
positive–charge probe, and a negative–probe. It is
assumed that the selected set of field probes will at the
least identify the major receptor field characteristics in
biological activity for the training set of ligands.
CoMFA Interaction Energy
The fields that a certain probe atom would experience
at every grid point are calculated for each molecule,
leading to thousands of columns in the X-block. For steric
field a (a/r12- b/r6) Lennard -Jones potential is calculated,
the electrostatic field is 1/r Coulomb potential. The
commercial version of CoMFA uses a (Îr) -1 distance
dependant coulomb potential to evaluate electrostatic
fields, where r is the probe-ligand atom distance and Î
is the molecular dielectric. In the large majority of
applications Î is set to the default value of unity. Large
positive energy values, i.e. grid points inside the
molecules are set constant at certain cut-off valued to
avoid unreasonably large energy values. The default
value of 30 kcal/mole is used as the maximum electrostatic
and steric energy cutoff. Normally, the steric and
electrostatic fields are kept separate for ease of
interpretation of the results. Grid points without variance
(e.g. in the corners of the box, far away from the van der
Waals spheres of the molecules) are eliminated before
the analysis is performed.
Other fields than those implemented in the CoMFA
program have been proposed for 3D-QSAR analyses, e.g.,
different interaction fields calculated by the program
GRID19,20 or hydrophobic fields derived from HINT21-23.
In addition, any other parameters, e.g. physico-chemical
properties like log P or quantum-chemical indices, may
be added to the X-block, if they are properly weighted.
Partial Least Squares (PLS) and Cross-Validation in
The last step in a CoMFA is a partial least square
analysis to determine the minimal set of grid points which
Indian J.Pharm. Educ. 39(2) April - June 2005
is necessary to explain the biological activities of the
Partial least–squares is an iterative procedure that
applies two criteria to produce its solution. First, to extract
a new component, the criterion is to maximize the degree
of commonality between all of the structural parameter
columns (independent variable) collectively and the
experimental data (dependent variable). Second, in the
evaluation phase of a PLS iteration, the criterion for
acceptance of the principal component just generated is
an improvement in the ability to predict, not to reproduce,
the dependent variable.
The technique used in PLS to assess the predictive
ability of a QSAR is cross-validation24. Cross-validation
is based on the idea that the best way to assess predictive
performance is to predict. When cross-validating, one
pretends that one or more of the unknown experimental
value is, in fact, unknown. The analysis being crossvalidated is repeated, excluding the temporarily
‘unknown’ compounds and then using the resulting
equation to predict the experimental measurement of the
omitted compound(s). The cross-validation cycle is
repeated until each compound has been excluded and
predicted exactly once. The results of cross-validation
are the sum of the squared prediction errors, sometimes
called the predicted residual sum of squares (PRESS).
For evaluation of the overall analysis, the PRESS is
commonly expressed as a cross–validated correlation
coefficient r2, or xv - r2, value.
In CoMFA two r2 values are encountered in addition
to the xv-r2. The second r2 is the conventional, and is a
measure of how well a particular model reproduces or fits
the input data. It is calculated in the same way as xv-r2,
except that PRESS is replaced by the sum of the squares
of differences between the least-squares fit and the
experimental observations. In PLS both values are
obtained the xv-r2 in the cross-validation step and
conventional r2 in describing the final best-fit model. The
third r2 value, the predictive r2, arises after a QSAR has
been used to predict the target property of structures
not included in the training set.
r2cv = 1- PRESS / (Y - Ymean )2
Where, PRESS = S (Y - Ypred)2
Predictive r2
r2pred = (SD-PRESS) / SD
Where, SD = sum of squared deviations between
biological activities of the test set and mean activity of
the training set molecules.
Indian J.Pharm. Educ. 39(2) April - June 2005
PRESS = sum of squared deviations between
predicted and actual activity values for every molecule
in the test set.
The PLS variant, generated optimal linear PLS
estimations (GOLPE) 25,26 seems to be better suited than
ordinary PLS analysis, because it eliminates variables not
contributing to prediction in a stepwise procedure. Some
recent applications in CoMFA studies confirm that the
predictive power of the CoMFA model increases after
reduction of the number of variables according to the
GOLPE procedure.
The risk of chance correlation seems to be low in
CoMFA studies if arbitrary orientations of the molecules
are chosen instead of a reasonable alignment or if series
of random numbers are correlated with biological
activities 27.
Most often the results of a CoMFA study are
presented in graphical form. Typically, there will be two
contour levels displayed for each type of CoMFA field,
highlighting the regions of greatest association; the most
positive and the most negative. These contour maps are
helpful in suggesting new compounds likely to have
higher property values.
The CoMFA QSAR equation can also be used to
predict property values, providing a vehicle for ranking
potential compounds for synthesis. Difference maps are
proposed as tools to analyze and identify areas of interest
with respect to activity and selectivity, if two different
types of biological activities are compared. CoMFA offer
many different options resulting from slightly different
alignments of the molecules or certain side chains,
different positions or sizes of the grid boxes, the use of
different fields and additional variables, different values
for the cut-off of large positive energy values, etc. This
flexibility of the CoMFA method makes it a powerful tool
to perform a QSAR study.
Biological activities of new compounds can be
predicted by transforming the PLS result into a multiple
regression equation28,29. For a comparison of classical
QSAR analyses with 3D-QSAR studies the PLS results
have been presented in the form of multiple regression
equations, including the PLS components as the
independent variables. One can not expect reliable
predictions of biological activity values for analogs
having additional side chains or groups with significantly
different electronic properties. The use of additional or
other fields than the default steric and electronic fields
of the original CoMFA method, together with PLS
analysis or GOLPE, is quite common as a valuable
extension of the CoMFA program.
The first application of CoMFA was to a series of
steroid molecules. It correlates the binding affinities of
21 steroids to human corticosteroid globulins (CBG) and
testosterone binding globulins2, (TBG). There are many
reports in the literature of successful application of
CoMFA that have not only led to predictive models within
an analogue series of biologically active molecules 30, but
also to insightful information on the general requirements
for the expression of the activity.
CoMFA and related 3D–QSAR approaches have
been applied to correlate various physico-chemical
properties. 3D-QSAR studies that correlate s, inductive
and resonance parameters of benzoic acids31 as well as
pKa value of clonidine analogs32 show that a H+ field
precisely describes such electronic parameters, e.g. s m, p
of benzoic acids. Steric parameters of benzoic acids, like
surface area and van der Waals volume can be described
by a steric field alone, while Es values of acetic acid methyl
esters need a combination of both steric and electrostatic
fields. CoMFA fields were also proposed and used to
derive new steric and electronic parameters for classical
QSAR studies.
The CoMFA methodology can also be used to
describe non-linear lipophilicity activity relationship, e.g.
the inhibitory activities of quaternary alkylbenzyl dimethyl
ammonium compounds vs. Clostridium welchii33, other
antibacterial and hemolytic activities, and toxic activities
of alkanes in mice, only homologous series of compounds
can be investigated34,35.
CoMFA a ligand interaction based model deals with
the quantitative description of ligand- protein
interactions, like enzymes, e.g., angiotensin converting
enzyme inhibition36, prostaglandin synthetase inhibition,
renin inhibition 37, carbonic anhydrase inhibition 38 ,
interleukin 1-b converting enzyme (ICE) inhibition39 ,
arylalkylamine N-acetyl transferase (AANAT)
inhibition40, selective COX-2 inhibition41, receptor binding,
e.g., binding of a 1-adrenergic agonists, benzodiazepine
receptor binding42-45, H2 receptor binding46, endothelin
receptor antagonism47, adenosine A 2A receptor binding48,
ion channels, e.g., hydantoin binding of neuronal
voltage–sensitive channel 49 , calcium ion channel
inhibition50 and other ligand –protein interactions, e.g.,
the binding of steroids to various carrier proteins51-53,
GABA-uptake inhibition, ligand binding to viral coat
proteins54 etc.
CoMFA also applied to correlate various other
biological activities, e.g., response of tumor cells to
estrogens 55, diuretic activities of sulfonamides in the
isolated perfused tubulus of rabbit kidney, various
biological activities of prostaglandin analogs56 and 3DQSAR
However, despite the simplicity of the analysis and
the good correlation obtained in these studies, a ligandinteractions based model like the CoMFA method should
not be used to model non-linear effects arising from
transport and distribution, no reasonable results can be
expected for sets of compounds which are no
homologous series.
Cramer, R.D., Patterson, D.E and Bunce, J.D., J Am
Chem Soc, 110, 1988, 5959.
Clark, M…. et al., Tetrahedron Comput Methodology,
3, 1990, 47.
Cramer III, R.D. and Milne, M., Abstracts of Papers,
Am Chem. Soc., April 1979, Computer Chemistry
Section, no. 44.
Charifson, P.S. (ed), Practical application of
computer-aided drug design, New York: Marcel
Dekker, 1997, 109.
Pearlman, R.S., Chem. Design Automation News,
2(1), 1987, 1.
Rusinko, A…. et al., CONCORD, Tripos Associates
Inc., St. Louis, MO, USA.
Kubinyi, H. (ed.), 3D-QSAR in drug design, theory,
methods, and applications, Leiden: ESCOM Science
Publishers, 1993.
Wipke, W.T. and Hahn, M.A., Tetrahedron Comp.
Methodology, 1, 1988, 141.
Dolata, D.P., Leach, A.R. and Prout, K., J Comput
Aided Mol Des, 1, 1987, 73.
10. Leach, A.R., Prout, K. and Dolata, D.P., J Comput
Aided Mol Des, 2, 1988, 107.
11. Leach, A.R., Prout, K. and Dolata, D.P., J Comput
Chem, 11, 1990, 680.
12. Leach, A.R. and Prout, K., J Comput Chem, 11, 1990,
Indian J.Pharm. Educ. 39(2) April - June 2005
13. Humblet, C. and Marshall, G.R., Drug Dev Res, 1,
1981, 409.
35. Kim, K.H., J Comput Aided Mol Des, 7, 1993, 71-82.
14. Mayer, D., Naylor, C.B., Motoc, I. and Marshall, G.R.,
J Comput Aided Mol Des, 1, 1987, 3.
37. Klebe, G. and Abraham, U., J Med Chem, 36, 1993,
15. Dammkoehler, R.A…. et al., J Comput Aided Mol
Des, 3, 1989, 3.
38. Baldwin, J.J…. et al., J Med Chem, 32, 1989, 2510.
16. Van Drie, J.H., Weininger, D. and Martin, Y.C., J
Comput Aided Mol Des, 3, 1989, 225.
17. Martin, Y.C… et al., J Comput Aided Mol Des, 7,
1993, 83.
18. CATALST, Bio CAD Corporation, Mountain View,
19. Goodford, P. J., J Med Chem, 28, 1985, 849.
20. GRID Program, V7, Molecular Discovery Ltd. West
Way House, Elms Parade, Oxford, England.
21. Wireko, F.C., Kellog, G.E. and Abraham, D.J., J Med
Chem, 34, 1991, 758.
22. Kellog, G.E., Semus, S.F. and Abraham, D.J., J
Comput Aided Mol Des, 5, 1991, 545.
23. Kellog, G.E. and Abraham, D.J., J Mol Graphics, 10,
1992, 212.
24. Cramer, R.D., Bunce, J.D. and Pattersonm D.E., Quant
Struct Act Relat, 7, 1988, 18.
25. Baroni, M…. et al., J Chemometrics, 6, 1992, 347.
26. Baroni, M…. et al., Quant Struct Act Relat, 12, 1993,
27. Clark, M. and Cramer, R.D., Quant Struct Act Relat,
12, 1993, 137.
36. Marshall, G.R., Eur J Pharmacol, 183, 1990, 15.
39. Kulkarni, S.S. and Kulkarni, V.M., J Med Chem, 42,
1999, 373.
40. Chavatte, P… et al., Quant Struct Act Relat, 20, 2002,
41. Marot, C., Chavatte, P. and Lesieur, D., Quant Struct
Act Relat, 19 (2), 2000, 127-34.
42. Allen, M.S…. et al., J Med Chem, 33, 1990, 2343.
43. Diaz-Arauzo, H., Koehler, K.F., Hagen, T.J. and
Cook, J.M., Life Sci, 49, 1991, 207.
44. Allen, M.S…. et al, J Med Chem, 35, 1992, 4001.
45. Greco, G., Novellino, E., Silipo, C. and Vittoria, A.,
Quant Struct Act Relat, 11, 1992, 461.
46. Dove, S. and Buschauer, A., Quant Struct Act Relat,
18, 1999, 329.
47. Chen, Q… et al., Quant Struct Act Relat, 18, 1999,
48. Dotychinova, I., Valkova, I. and Natcheva, R., Quant
Struct Act Relat, 20 (2), 2001, 124.
49. Brown, M.L….. et al, J Med Chem, 42, 1999, 1537.
50. Schleifer, K.J. and Tot, E., Quant Struct Act Relat,
21 (3), 2002, 239.
51. Norinder, U., J Comput Aided Mol Des, 5, 1991, 419.
28. Norden, B., Edlund, U., Johnels, D. and Wold, S.,
Quant Struct Act Relat, 2, 1983, 73.
52. Opera, T.I., Ciubotariu, D., Sulea, T.I. and Simon, Z.,
Quant Struct Act Relat, 12, 1993, 21.
29. Norinder, U., J Comput Aided Mol Des, 4, 1990, 381.
53. Loughney, D.A. and Schwender, C.F., J Comput
Aided Mol Des, 6, 1992, 569.
30. Thibuat, U., ‘Application of CoMFA and related 3DQSAR approaches’ in 3D-QSAR in drug design;
theory, methods and applications (Kubinyi, H., ed.),
Leiden: ESCOM Science, 1993, 661.
31. Kim, K.H. and Martin, Y.C., J Org Chem, 56, 1991,
32. Kim, K.H. and Martin, Y.C., J Med Chem, 34, 1991,
33. Kim, K.H., Med Chem Res, 2, 1992, 22.
54. Diana, G.D…. et al, J Med Chem, 35, 1992, 1002.
55. Wiese, T.E., Palomino, E., Horwitz, J.P. and Brooks,
S.C., Abstracts of Papers, Am Chem Soc, April 1991,
Medicinal Chemistry Section, no. 150.
56. Koehler, K.F…. et al, Abstracts of Papers, Am Chem
Soc, August 1990, Medicinal Chemistry Section, no.
57. Kulkarni, S.K., Gediya, L.K. and Kulkarni, V.M.,
Bioorg Med Chem, 7, 1999, 1475.
34. Kim, K.H., Quant Struct Act Relat, 11, 1992, 309.
Indian J.Pharm. Educ. 39(2) April - June 2005
Pharm. Chemistry
Spectrometric Method for Determination of Carbocysteine
P. N. Sanjay Pai, Gopalkrishna Rao, B. Balakrishna, Hamid Khan and K. Pasha
Department of Quality Assurance, Al-Ameen College of Pharmacy, Bangalore 560 027
Received on 17.8.2004
Modified on 28.1.2005
Accepted on 24.3.2005
A simple, colorimetric method is developed for the estimation of carbocysteine. The method is based on the chemical
reaction of primary amino group present in carbocysteine with carbon disulphide to form dithiocarbamic acid, which on
further reaction with cupric chloride forms a colored copper chelate. The yellow chromophore has absorption maxima of
413 nm and obeys Beer’s law in the concentration range of 100-800 µg/10ml. Results of the analysis were statistically
validated by recovery studies. The proposed method was found to be suitable for routine determination of carbocysteine
as active pharmaceutical ingredient.
Chemically carbocysteine is S-carboxymethyl Lcysteine. Carbocysteine has a significant importance as
a potent expectorant and is used as mucolytic agent1.
Literature survey reveals that the drug is determined
mainly by HPLC2-5 methods. The main objective of the
present study was to develop a sensitive and selective
colorimetric method for routine estimation of
carbocysteine by exploiting its amino group using
dithiocarbamic acid method 6 . A Shimadzu UV/Vis
Spectrophotometer (UV 1601) with 1 cm matched quartz
cell was used.
A standard solution of carbocysteine was prepared
by dissolving 100 mg of carbocysteine in 10 ml of 10%w/
v sodium hydroxide. The volume was made up to 100 ml
with dilute sodium hydroxide in a 100 ml volumetric flask
to get a solution of strength 1 mg/ml. A 10% v/v aqueous
acetic acid solution was prepared by diluting 10 ml of
glacial acetic acid to 100 ml with distilled water. Carbon
disulphide-pyridine- isopropyl alcohol reagent (CPI
reagent) was prepared by mixing 35 ml of carbon
disulphide, 25 ml of pyridine and 65 ml of isopropyl
alcohol in accurate quantities. Cupric chloride solution
was prepared by dissolving 0.12 g of cupric chloride in
250 ml of water and further diluted to 500 ml with pyridine.
To 1 ml of the standard carbocysteine solution (1
mg/ml) taken in a separating funnel, 4 ml of CPI reagent
and 2 ml of cupric chloride reagent were added. The
mixture was then allowed to stand for 10 min, and then
vigorously agitated. Further 3 ml of aqueous acetic acid
solution and 3 ml of benzene were added. The mixture
was agitated for 5 more min and the two liquid phases
were allowed to separate. The upper organic layer was
collected in a 10 ml volumetric flask, the volume made up
with isopropyl alcohol solvent and allowed to stand for
20 min for complete development of colour. The solution
was scanned in the wavelength region of 400-760 nm
against reagent blank. The absorption maxima of the
yellow chromogen was found to be 413 nm. The method
was validated for fixing optimum concentration and
volume required for reagents to show maximum
absorbance, stability of color and order of mixing. The
colour was stable up to 2 hours. The standard curve was
then plotted for linearity (concentration vs absorbance)
and was found to be in the range of 100-800 µg/10ml.
The molar absorptivity was determined to be 3.27×104 l.
mol-1cm-1 and Sandell’s sensitivity 10.186 x 10-2 µg/cm20.001 absorption units. A regression equation (y=a+bx)
was obtained by linear least squares treatment of the
results, established slope(b) of 0.009816 and intercept(a)
0.0001, with relative standard deviation of 0.2308191 and
coefficient of variation 0.05327. Percentage range of error
was calculated as 0.23%. The accuracy and reliability of
the method was proved through recovery studies.
The method was extended for determination of
carbocysteine in bulk drug samples obtained from three
different manufacturers. A solution of 1 mg/ ml of the
sample carbocysteine was prepared. The absorbance
values obtained for the sample solution were correlated
with standard calibration curve to obtain the
concentration. The recovery studies were carried out at
three levels by adding 0.1, 0.2 and 0.3 ml of standard
solutions of carbocysteine to 0.2 ml each of previously
analyzed samples in three 10 ml volumetric flasks. The
mixtures were analysed by the proposed method. The
results are presented in Table 1.
The proposed colorimetric method is based on the
principle of chemical reaction of primary amines in
carbocysteine with carbon disulphide to form
dithiocarbamic acid, which on further reaction with cupric
chloride forms a yellow colored copper chelate, which is
estimated colorimetrically. The scheme of this novel
reaction is given in Figure 1.
Indian J.Pharm. Educ. 39(2) April - June 2005
Table 1 : Recovery Study
from the std.
graph (mg)*
* Average of 3 readings
Figure 1: Dithiocarbamic acid – carbocysteine - copper complex
The proposed method was found to be sensitive,
accurate, precise and reproducible and can be used for
the routine determination of carbocysteine bulk drug.
The authors thank Astra Zeneca Pharmaceuticals
Limited, Bangalore for providing Reference standard
Carbocysteine and also bulk samples of Carbocysteine
supplied from different sources. The research team
acknowledges Prof. B. G. Shivananda, Principal, Al –
Ameen College of Pharmacy for providing the facility to
carry out the work.
Pentari, J. G., Efstathiou, C. E. and Koupparis, M. A.,
Kinetic determination of carbocysteine in syrup based
on its reaction with 1-fluoro-2,4-dinitrobenzene
monitored with a fluoride-selective electrode, Int. J.
Pharm., 77(31), 1991, 41-46.
De-Schutter, J.A., Vander, W. G., Vanden, B. W. and
Demoerloose, P., Determination of Scarboxymethylcysteine in serum by reversed phase
ion pair liquid chromatography with column switching
following pre-column derivatization with ophthalaldehyde, J. Chromatogr., 428(15), 1988, 301310.
Dubruc, C… et al., Determination of S-carboxymethylL-cysteine in plasma by high performance liquid
chromatography with column switching following
precolumn derivatization, J. Chromatogr., 417(5),
1987, 208-215.
Siggia, S. and Hanna, J.G., Quantitative Organic
Analysis, 30 th Ed., New York: InterScience
Publication, 1979, 615.
Parfitt K., Ed, Martindale – The complete drug
reference, 32nd Ed., London: Pharmaceutical press,
1999, 1056.
Staffeldt. B., Brockmoller, J. and Roots, I.,
Determination of S-carboxymethyl-L-cysteine and
some of its metabolites in urine and serum by high
performance liquid chromatography using fluorescent
pre-column labeling, J. Chromatogr. Biomed. Appl.,
571(15), 1991, 133-147.
Indian J.Pharm. Educ. 39(2) April - June 2005
Ph.D Thesis
Study of anti-ulcer activity of some herbal drugs
Siddhraj Singh Sisodia
B.N. College of Pharmacy, Udaipur (Rajasthan) 313 002
Mohanlal Sukhadia University, Udaipur
Dr. M. Bhatnagar, Prof., Cellular and Neurobiology Lab.,
M.L.S. University, Udaipur (Raj.)
Dr. C.P. Jain, Assistant Prof., Dept. of Pharmacy,
M.L.S. University, Udaipur (Raj.)
15 th February 2005
Received & accepted on: 14.03.2005
Peptic ulcer disease is one of the common
gastrointestinal disorder affecting millions of people, it
kills few but troubles many. The etiology of gastric ulcer
is not known in most cases, but it is generally accepted
that it results from an imbalance between aggressive
factors (Gastric HCl, Pepsin secretion, H. Pylori, Alcohol,
NSAIDs, Malignancy etc.) and defensive factors (Gastric
mucosal barrier, Endogenous prostaglandin’s, Secretin,
Somatostatin, Epidermal growth factors, blood flow etc.).
Many herbal drugs are being used as Ayurvedic
preparations or in Indian traditional medicinal system for
the management of peptic ulcer. Withania somnifera
Dunal is commonly known as “Ashwagandha” which
means it has strength and odour of horse. Asparagus
racemosus Willd. is commonly known as “Shatavari”.
Shatavari is used for sexual debility and infertility in both
Aim of the present investigation was to compare that
anti-secretory, anti-ulcer and antioxidant activity of
Asparagus racemosus willd. and Withania somnifera
Dunal root extract with standard drug Rantidine in
various models of gastric ulcer in rats. Ulcer was induced
by the indomethacin (NSAID’s) and swim (restraint)
stress treatment. Adult albino rats (Wistar strain) of the
either sex were used for the present study. Dried roots
(Asparagus racemosus and Withania somnifera) were
purified using absorption method. After purification, the
roots were packed in high quality filter paper and the
successive solvent extract (methanol) was prepared by
continuous extraction method with the help of soxhlet
extractor. After vacuoevaporation crude extract was
suspended in 0.5% Carboxy Methyl Cellulose (CMC).
Acute administration of NSAIDs like indomethacin
and pyloric ligation produced severe gastric lesion in
stomach of rats. In the present study, Idomethacin
treatment and pylorus ligation of animals significantly
increased the ulcer index, volume of gastric content, free
acidity and total acidity when compared with control
untreated group. In our study, methanolic extract of both
drugs Asparagus racemosus (100 mg/ kg/ day, bw, p.o.)
and Withania somnifera (100 mg/ kg/ day, bw, p.o.)
reduced the incidence of gastric ulcer, volume of gastric
content, free acidity and total acidity significantly in
indomethacin induced gastric ulcerative rats. Decreased
ulcer index and gastric content are implicated with the
gastroprotective effects of drugs. Significant reduction
in free and total acidity is also implicated in the anti-ulcer
effect of these drugs. Administration of 50% reduced
combined dose of Asparagus racemosus (50 mg/ kg/ day,
bw, p.o.) plus Withania somnifera (50 mg/ kg/ day, bw,
p.o.) also produced significant reduction in ulcer index,
volume of gastric content, free acidity and total acidity
as compared to control untreated group but their effects
on these parameters were less as compared to
independent administration of Asparagus racemosus (100
mg/ kg/ day, bw, p.o.) and Withania somnifera (100 mg /
kg/ day, bw, p.o.). In the present study, experimental
group showed significant decrease in total carbohydrate
(TC) and decrease in TC/ Total protein (TP) ratio.
Administration of Asparagus racemosus, Withania
somnifera and 50% mix dose of Asparagus racemosus
and Withania somnifera showed significant increase in
total carbohydrate (TC) and TC/ Total protein (TP) ratio
of the gastric juice. However, they did not have any effect
on total protein of the gastric juice. Administration of 30
mg/ kg/ day, bw, p.o. ranitidine for 15 days produced
significant decrease in ulcer index, volume of gastric
content, free acidity and total acidity it also significantly
increased the total carbohydrate (TC), Total Protein (TP)
and TC/TP ratio. In the present study, important
antioxidant status of indomethacin treated, indomethacin
plus Asparagus racemosus, indomethacin plus Withania
somnifera, 50% mix dose of Asparagus racemosus and
Indian J.Pharm. Educ. 39(2) April - June 2005
Withania somnifera, only Asparagus racemosus and only
Withania somnifera were also observed. All the treatment
schedules for both the drugs significantly increased the
level of catalase and superoxide dismutase (SOD) in the
stomach, liver, lungs and kidney. In our study a
significant increase in the antioxidant activity of
gastromucosal, liver homogenate, lungs and kidney
homogenate was evident as there was significant decrease
in lipid peroxidation. In indomethacin treated group the
level of malondialdehyde (MDA) was significantly
increased where as ascorbic acid content decreased
Stress ulceration in the stomach is associated with
clinical conditions like trauma, head injury, burns, shock,
sepsis and neurological disorders and thus now regarded
as a multifactorial phenomenon. In the present
investigation water immersion stress was used to produce
severe gastric ulceration and hemorrhagic spots. The
ulcer index, volume of gastric content, free acidity and
total acidity were significantly increased after swim stress
regimen in the present study. The result of the present
study demonstrate the anti-ulcer activity of both root
extract of Asparagus racemosus and Withania somnifera
against stress induced gastric ulcerative rats which was
evident from a significant decrease in the ulcer index,
volume of gastric content, free acidity and total acidity.
An increase in gastric acid secretion and reduction of
gastric mucus (gastric content) play a major role in the
pathogenesis of stress induced gastric ulcers. Total
carbohydrate (TC) and TC/ Total protein (TP) ratio was
significantly increased after the administration of
Indian J.Pharm. Educ. 39(2) April - June 2005
Asparagus racemosus and Withania somnifera in stress
induced gastric ulcerative rats. Defensive mucin
secretion was quantified in term of TC/TP ratio in the
gastric juice. TC/TP ratio is taken as a reliable marker for
mucin secretion. The TC/TP ratio of stress plus Withania
somnifera is more as compared to stress plus Asparagus
racemosus and stress plus Ranitidine.
Study also showed increase in antioxidant defense
enzymes wiz superoxide dismutase, catalase and ascorbic
acid increased significantly while a significant decrease
in lipid peroxidation was observed after A. raemosus and
W. somnifera treatment in stress induced gastric ulcerative
It is thus concluded that both drugs Asparagus
racemosus and Withania somnifera at dose 100 mg/ kg/
day, bw, p.o., is effective against gastric ulcerative rats.
50:50% mix dose of Asparagus racemosus (50 mg/ kg/
day, bw, p.o.) plus Withania somnifera (50 mg/ kg/ day,
bw, p.o.) also showed significant of protection against
gastric ulceration.
In indomethacin (NSAID) induced gastric ulcerative
model, the Asparagus racemosus was more effective as
compared to only Withania somnifera extract and 50:50%
mix dose of both Asparagus racemosus and Withania
somnifera. Interestingly, the antiulcerogenic effect of
Asparagus racemosus was closely similar to that of
standard anti-ulcer drug ranitidine. Withania somnifera
(100 mg mg/ kg/ day, bw, p.o.) was more effective in the
ulcer produced by swim stress as compared to Asparagus
Book Review
Title of the book : Pharmaceutical Microbiology
: N. K. Jain
: Vallabh Prakashan, Delhi
: Rs.150
Microbiology is one of the most applied of all the
biological sciences. It is one of the most essential subjects
in the Pharmacy curriculum. Even though a number of
books are available, there is a demand for better versions.
The present book is an attempt to provide overall
information to the readers interested in pharmaceutical
microbiology. Although relatively concise (at 344 pages)
the coverage of the subject matter is comprehensive, and
provides a valuable reference source for students at
undergraduate level.
There are 17 Chapters in the book.
Chapter 1 deals with history of microbiology. The
chapter is very brief, but it covers most of the important
Chapter 2 deals with Microbial world. It is very simple
and comprehensive Bergey’s Manual is also included.
Chapter 3 deals with Microbiological Methods.
Staining, culture, biochemical identification and counting
techniques are explained in brief.
Chapter 4 on Bacteria deals with structure,
classification, growth requirements and reproduction of
Chapter 5 on Fungi includes structure, reproduction
and diseases caused by Fungi.
Chapter 6 on virus includes general characteristics,
structure, multiplication, and identification and cultivation
Chapter 7 deals with microbial metabolism.
Chapter 8 is on Sterilization. The Chapter covers all
the important methods of sterilization including their
validation methods and sterility testing methods. More
details could have been included.
Chapter 9 is on Disinfections. The chapter includes
properties, types, classification and evaluation methods.
brief. Section on Emerging diseases is given. A list of all
the micro organisms with the various diseases they cause
could have been included.
Chapter 11 on Immunology includes brief information
on types of lmmunity, antigens, antibodies and antigenantibody reactions. A table explaining the Serological
tests is also presented which is very simple.
Chapter 12 on Immunization explains briefly about
the various vaccines.
Chapter 13 deals with Industrial Microbiology. The
chapter includes Fermentation and production of
antibiotics, vitamins and other products. The flow
diagrams are quite well explanatory in nature. A section
on enzymes their extraction and immobilization technique
is also presented.
Chapter 14 deals with Microbial transformation. It
includes most of the important microbial conversion
Chapter 15 deals with Pharmaceutical Biotechnology.
Recombinant technology and Monoclonal Antibodies and
applications of genetic engineering are explained in brief.
Chapter 16 deals with Biotechnology Drugs. A brief
introduction to various products is included.
Chapter 17 deals with Microbiological Assays. The
chapter is very comprehensive and includes assays of
antibiotics and vitamins.
Overall, the book is well written, interesting and clear
with useful tables and figures. I would recommend this
book for undergraduates for reference.
Reviewed by
Prof. Salma Khanam
Al-Ameen College of Pharmacy
Bangalore 560 027
Chapter 10 deals with Infection. The chapter is very
Indian J.Pharm. Educ. 39(2) April - June 2005