The Influence of Khat on the In-vitro and In-vivo availability of Tetracycline-HCl
Farah Hamad Farah1*, Omer Ali
Attef2 and Abdul-Azim Ahmed Ali3
1Department
of Pharmaceutics, College of Pharmacy and Health Sciences, Ajman University of Science
and Technology, UAE.
2Department of Pharmaceutical Chemistry, College of Pharmacy
and Health Sciences, Ajman University of Science and Technology, UAE
3Department of Pharmaceutics, College of Pharmacy and Health
Sciences, Ajman University of Science and Technology, UAE,
*Corresponding Author E-mail: f.hamad@ajman.ac.ae.,
attef@hotmail.com, ajac.azim@ajman.ac.ae.
ABSTRACT:
The influence of khat on both in-vitro and
in-vivo availability of tetracycline-HCl has been
evaluated. The in-vitro availability data have reflected a statistically
significant interaction between khat extract and
tetracycline-HCl in two buffer solutions simulating
intestinal and gastric media (phosphate buffer-pH 7 and 0.1 HCl-pH
1.2 respectively). In the in-vivo availability studies, ten adult healthy
Yemeni volunteers participated. The obtained in-vivo availability data
indicated a statistically significant reduction in most pharmacokinetics
parameters. A statistically significant % reduction in the maximum plasma
tetracycline-HCl concentration (Cmax)
and absorption rate constant (Ka) as well as a significant
enhancement of time to reach the peak plasma concentration (Tmax),
were observed as a result of taking tetracycline-HCl
just before khat chewing (trial B) and more pronounce
during khat chewing (trial D) compared to the control
(trial C). In addition a statistically significant % reduction in area under
curve (AUC0→∞)
has been observed reflecting a reduction in the extent of in-vivo
tetracycline-HCl availability as a result of khat chewing. Statistically significant % reductions in the
biologic half-life (t½) and
elimination rate constant (Kel) were also observed.The reduction in tetracycline-HCl
concentration when mixed with khat extract, as shown
by in-vitro data, may reflect a possible formation of tetracycline-HCl complexes with one or more of khat
constituents. The reduction in the rate and extent of in-vivo tetracycline-HCl availability as a result of khat
chewing, may be due to the possible formation of non-absorbable tetracycline-HCl complexes with one or more of khat
constituents or possible delaying of gastric emptying induced by khat that may prolong the resident time of tetracycline-HCl in the stomach leading to its possible degradation into
the less antimicrobial active form epitetracycline.
KEYWORDS: availability, pharmacokinetics
parameters, tetracycline-HCl, khat.
INTRODUCTION:
There is no doubt that the habit of khat
chewing remains a major problem in
Yemeni society and the number of khat chewers seems
to be increasing[1,2]. Several reports have been published worldwide
revealing the medical effect of khat (Catha edulisForsk.) [1,3,4] ,
but little is known about the possible interactions between khat
constituents and drugs.
The most important khat leaves
constituents include more than six alkaloids that are the main determinants of
the khat CNS effects, tannins, flavonoids
and polyvalent cations, such as calcium, magnesium,
iron, cadmium, lead, copper, and zinc [3,4,5,6].On the other hand
antibiotic use in Yemen is high and the statistics of the Ministry of Public
Health and Population showed that the antibiotics group was the third group
which was imported through the years 2002 and 2003 with percentages of 13.7%
and 13.0% respectively from the total number of imported drugs [7,8 ].
Also a study performed in different general public health facilities in Yemen
showed that 61% of the prescribed drugs
were antibiotics [9]. These findings emphasize the need for
conducting research on the influence of khat chewing on the in-vivo availability of antibiotics. A
previous study on the effect of khat chewing on the
bioavailability of two β-lactam antibiotics,
namely ampicillin and amoxicillin, revealed that khat chewing significantly reduces most pharmacokinetics
parameters [10]. It has been concluded that tannins represent the
most likely component, which could be responsible for the observed effects of khat on β-lactam antibiotics
absorption [10]. The present study was carried out to investigate
the possible effect of khat on the in-vitro and in-vivo
availability of tetracycline-HCl.
MATERIALS AND METHODS:
MATERIALS:
Tetracycline-HCl capsules (Tetracaps® 500mg; Lab. Wolves N. V., Antwerb, Belgium; Batch 98E21; Exp. date 03/2015) were
kindly donated by the local agent. Tetracycline-HCl
standard reference was kindly supplied by the national quality control
laboratory of the Ministry of Public Health, Sana’a, Republic of Yemen, Jenway 6405 UV/VIS Spectrophotometer, Jenway
Ltd., England. Khat (Catha edulisForsk.) was purchased from the local
market. The type and quantities of khat taken by all volunteers throughout the trials were
kept the same. The khat used was one of the commonest
type in Yemen, locally known as “Sauty”.
Methods:
(i) In-vitro availability
studies:
Two buffer solutions (phosphate buffer pH 7 and 0.1 HClpH 1.2) were prepared to simulate the gastrointestinal
physiological fluids. 10 g ground fresh khat leaves
were separately extracted with 200ml of each buffer solution and shaken for 1
hr then filtered. Finally, activated charcoal was added to decolorize the
filtrate and then filtered again. Serial concentrations of tetracycline- HCl standard reference were prepared in both buffer
solutions in a concentration range of 0.01-2.0mg/ml. Using a spectrophotometer
(Jenway 6405 UV/VIS Spectrophotometer, Jenway Ltd., England), the absorption of tetracycline-HCl solutions was measured at the wavelengths (λ
361nm; and λ 380nm) with and without khat
extract. Calibration curves were
constructed in both buffer media and at the same wavelengths.
(ii) In-vivo availability studies:
Ten adult healthy male Yemeni volunteers participated in
the trials (average age: 24 years; average body weight: 57 kg). The trials were
conducted using a cross-over design with a wash-out period of 7 days. The
volunteers did not take any drugs a week before and during the trials. They
only drink 750 ml of water during khat chewing
session, which lasted for 4 hours. A 500mg single dose of tetracycline-HCl capsule was administered orally after an overnight fast
on an empty stomach. Tetracycline-HCl was taken on
three different occasions as follows:
Trial B: drug was taken just before khat chewing.
Trial C: drug was taken alone without khat as control.
Trial D: drug was taken halfway during the
4-hours khat chewing session.
5ml blood samples were collected at: 0.0, 0.5, 1.0, 2.0,
2.5, 3.0, 4.0, 6.0, 8.0 and 24.0 hours post tetracycline-HCl
administration. The blood samples were centrifuged immediately after collection
to separate plasma. Plasma samples were kept at 40C awaiting
analysis. The official microbiological assay according to USP XXIII [11]
has been used for sample analysis. Calibration curve for tetracycline-HCl standard reference was constructed in a concentration
range of 0.3-5 µg/ml. The mean calibration factor was 1.667. Pharmacokinetics
parameters were used to describe the rate and extent of the antibiotic
bioavailability.
Statistical analysis
Standard curves were subjected to regression analysis. The
percentage reductions ofTetracycline-HCl inboth in-vitro and in vivo availability studies were
analyzed using the Studentst-Test for Paired Data [12].
RESULTS AND DISCUSSION:
(A) Results obtained from in-vitro availability studies:
Data obtained from in-vitro availability studies are shown
in tables 1-4. Statistical analysis of the in-vitro tetracycline-HCl khat extract mixture in
phosphate buffer (pH7.0) at λ 361nm (Table 1) and at λ380nm(Table 2),
and in 0.1N HCl (pH1.2) at λ361nm (Table 3) and
at λ380nm (Table 4),all exhibit significant% reductions in tetracycline-HCl concentrations compared with the drug alone.
Table 1:
The effect of khat extract on the in-vitro availability
of tetracycline-HCl (TC) in phosphate buffer (pH 7.0)
at λ361nm.
|
Sample
Conc. [mg/ml] |
TC
without Khat
extract [mg/ml] |
TC
with Khat extract [mg/ml] |
TC
Conc. difference [mg/ml] |
TC
Conc. difference [%] |
|
0.020 |
0.0281 |
0.0161 |
0.0120 |
42.64 |
|
0.032 |
0.0336 |
0.0241 |
0.0095 |
28.31 |
|
0.050 |
0.0510 |
0.0313 |
0.0197 |
38.71 |
|
0.063 |
0.0561 |
0.0530 |
0.0031 |
5.51 |
|
0.125 |
0.0645 |
0.0593 |
0.0052 |
8.04 |
|
0.250 |
0.0649 |
0.0593 |
0.0055 |
8.53 |
|
0.500 |
0.0651 |
0.0595 |
0.0056 |
8.63 |
|
1.000 |
0.0656 |
0.0598 |
0.0057 |
8.74 |
|
|
|
|
mean: 0.0083 |
mean; 18.46% |
Variance=0.000045
t=3.057 SE=0.002 p<0.02
Table
2: The effect of khat
extract on the in-vitro availability of tetracycline-HCl
(TC) in phosphate buffer (pH=7.0) at λ380nm.
|
Sample Conc. [mg/ml] |
TC without Khat
extract [mg/ml] |
TC with Khat Extract [mg/ml] |
TC Conc. difference [mg/ml] |
TC Conc. difference [%] |
|
0.020 |
0.0256 |
0.0155 |
0.0100 |
39.20 |
|
0.032 |
0.0304 |
0.0242 |
0.0062 |
20.41 |
|
0.050 |
0.0493 |
0.0313 |
0.0180 |
36.44 |
|
0.063 |
0.0616 |
0.0600 |
0.0016 |
2.60 |
|
0.125 |
0.0914 |
0.0878 |
0.0036 |
3.89 |
|
0.250 |
0.0963 |
0.0941 |
0.0022 |
2.33 |
|
0.500 |
0.0966 |
0.0945 |
0.0021 |
2.15 |
|
1.000 |
0.0973 |
0.0951 |
0.0022 |
2.22 |
|
|
|
|
mean:
0.0057 |
mean: 13.66% |
Variance=0.000046 t=1.918
SE=0.002 p<0.05
Table 3: The effect of khat extract on the in-vitro availability of tetracycline-HCl (TC) in 0.1N HCl (pH=1.2) at
λ361nm
|
Sample Conc. [mg/ml] |
TC without Khat Extract [mg/ml] |
TC with Khat Extract [mg/ml] |
TC Conc.
difference [mg/ml] |
TC Conc.
difference [%] |
|
0.020 |
0.0254 |
0.0205 |
0.0049 |
19.40 |
|
0.032 |
0.0412 |
0.0243 |
0.0169 |
41.10 |
|
0.050 |
0.0467 |
0.0365 |
0.0102 |
21.82 |
|
0.063 |
0.0569 |
0.0511 |
0.0058 |
10.20 |
|
0.125 |
0.0602 |
0.0555 |
0.0047 |
7.81 |
|
0.250 |
0.0603 |
0.0557 |
0.0046 |
7.66 |
|
0.500 |
0.0605 |
0.0557 |
0.0048 |
7.95 |
|
1.000 |
0.0607 |
0.0558 |
0.0049 |
8.08 |
|
2.000 |
0.0612 |
0.0559 |
0.0053 |
8.62 |
|
|
|
|
mean: 0.0069 |
mean: 14.74% |
Variance=0.000020
t=4.403 SE=0.001 p<0.002
Table
4: The effect of khat
extract on the in-vitro availability of tetracycline-HCl
(TC) in 0.1N HCl (pH=1.2) at λ380nm.
|
Sample Conc. [mg/ml] |
TC without
Khat
Extract [mg/ml] |
TC with Khat Extract
[mg/ml] |
TC Conc. difference [mg/ml] |
TC Conc. difference [%] |
|
0.020 |
0.0215 |
0.0181 |
0.0034 |
15.99 |
|
0.032 |
0.0371 |
0.0211 |
0.0160 |
43.04 |
|
0.050 |
0.0430 |
0.0332 |
0.0097 |
22.68 |
|
0.063 |
0.0650 |
0.0563 |
0.0087 |
13.41 |
|
0.125 |
0.0877 |
0.0845 |
0.0032 |
3.68 |
|
0.250 |
0.0967 |
0.0934 |
0.0033 |
3.46 |
|
0.500 |
0.0972 |
0.0937 |
0.0035 |
3.58 |
|
1.000 |
0.0976 |
0.0938 |
0.0038 |
3.92 |
|
2.000 |
0.0982 |
0.0921 |
0.0061 |
6.21 |
|
|
|
|
mean: 0.0060 |
mean:12.89% |
Variance=0.000021 t=3.954
SE=0.001 p<0.01
The mean % reduction of tetracycline-HCl
concentration in phosphate buffer (pH7.0) at λ361nm and at λ 380nm were
18.64% (p<0.02) and 13.66% (p<0.05)
respectively (Tables 1 and 2). The mean
% reduction of tetracycline-HCl concentration in 0.1N
HCl (pH1.2) at λ 361nm and at λ 380nm were
14.74% (p<0.002)and 12.89% (p<0.01) respectively (Tables 3
and4). There were no clear differences in the mean %reduction of tetracycline-HCl concentration in both media. The in-vitro availability
data reflects a significant tetracycline HCl-khat
extract interaction in both media. Such tetracycline-HCl-khat extract interaction may be attributed to one or more
of the following assumptions: a-As khat contains
among its other constituents polyvalent cations
(calcium, magnesium, iron, cadmium, lead, copper, and zinc) [3,4,5,6],
then there is a possibility of tetracycline chelation
with these polyvalent cations[13,14,15]. b-Possible
complexation of tetracycline with tannic acid
available as a major constituent in khat[3,4,5]
through hydrogen bonding. c-Possible complexation of
tetracycline with cathinone, cathine,
or pseudoephedrine available in khat[1,3,4,5] via hydrogen bonding. d-Possible binding of
tetracycline with proteins available in small concentrations in khat[5]. Another important observation is that
high tetracycline-HCl concentrations in the range of
(0.06-1.0mg/ml) in both media and at both λ 361nm and λ380nm, showed
almost similar %reduction of tetracycline-HCl concentration
when mixed khat extract (Tables 1-4), indicating that
some sort of saturation process is taking place. Such saturation could be
explained on the basis that an adsorption process might also be occurring
beside complexation. The major constituent in khat that is capable of being an adsorption and is available
in high concentration of 7-14% is tannic acid [1,3,4,5]. This may
indicate that tetracycline-HCl-khat
extract interaction is not only due to complexation
but also adsorption may be involved. Further in-vitro tetracycline tannic acid
interaction experiments are needed to validate such assumptions.
(B) Results obtained from in-vivo availability studies
The effect of khat chewing on
the bioavailability of a 500mg single oral dose of tetracycline-HCl capsule was also studied in ten healthy, adult Yemeni
male volunteers with similar age and weight group to correlate it with the
in-vitro availability studies data. Reduced and % reduced tetracycline-HCl concentrations in plasma when the drug is taken just
before Khat
chewing session trial (B) and during Khat chewing
session trial (D) compared with the control trial (C) as well as
statistical analysis data of the
differences between trial (B) and (D) compared with the control trial (C) are
shown in Table 5. Pharmacokinetics parameters calculated from plasma
tetracycline-HCl concentration-time curve are shown
in table 6.
Table
5: Reduced and % reduced tetracycline-HCl (TC) concentration in plasma when the drug is taken
before (Trial B) and during Khat chewing session (Trial D) against the control (Trial
C)
|
Time [hr] |
Trial C [µg/ml] |
Trial B [µg/ml] |
Trial D [µg/ml] |
Reduced TC conc. between:
Trial C and Trial B (Trial C-Trial B)
[µg/ml] |
Reduced [%] (1) TC
conc. between: Trial C and Trial B (Trial C-Trial B) |
Reduced TC conc. between:
Trial C and Trial D (Trial C-Trial D) [µg/ml] |
Reduced [%] (2) TC
conc. between: Trial C and Trial D (Trial C-Trial D) |
|
0.0 |
0.000 |
0.000 |
0.000 |
0.000 |
0.00 |
0.000 |
0.00 |
|
0.5 |
1.984 |
1.389 |
0.574 |
0.595 |
29.97 |
1.410 |
71.06 |
|
1.0 |
2.842 |
2.284 |
1.967 |
0.558 |
19.65 |
0.875 |
30.79 |
|
2.0 |
2.927 |
2.575 |
2.184 |
0.352 |
12.03 |
0.743 |
25.38 |
|
2.5 |
3.121 |
2.667 |
2.434 |
0.454 |
14.54 |
0.687 |
22.02 |
|
3.0 |
3.092 |
2.726 |
2.484 |
0.367 |
11.86 |
0.608 |
19.68 |
|
4.0 |
3.084 |
2.559 |
2.417 |
0.525 |
17.03 |
0.667 |
21.62 |
|
6.0 |
3.017 |
2.492 |
2.317 |
0.525 |
17.40 |
0.700 |
23.20 |
|
8.0 |
2.842 |
2.317 |
2.067 |
0.525 |
18.48 |
0.775 |
27.27 |
|
24.0 |
1.875 |
1.395 |
0.574 |
0.491 |
26.17 |
1.301 |
69.38 |
|
|
|
|
|
mean:0.439 |
mean:16.71% |
mean:0.777 |
mean:31.04 |
(1)Variance=0.007
t=6.434 SE=2.60% p<0.001
(2) Variance=0.049
t=4.418 SE=7.03% p<0.002
m
Table 6: Observed
changes in pharmacokinetics (PK) parameters after a 500mg single oral dose of
tetracycline-HCl taken before (Trial B) and during Khat chewing session (Trial D) against the control (Trial
C)
|
Parameters |
Measured
PK
parameters in Trial C |
Measured
PK
parameters in Trial B |
Measured PK
parameters in Trial D |
Differences
between:
Trial C and Trial B |
[%]
differences between:
Trial C and Trial B |
Differences
Between: Trial
C and Trial D |
[%]
differences Between: Trial
C and Trial D |
|
1Cmax
[µg/ml] |
3.651 |
3.188 |
2.906 |
0.463 |
-12.68 |
0.745 |
-20.4 |
|
2Tmax
[hr] |
2.5 |
3.0 |
3.0 |
-0.5 |
+20 |
-0.5 |
+20 |
|
3Ka
[hr-1] |
1.01 |
0,847 |
0.762 |
0.164 |
-16.2 |
0.248 |
-24.6 |
|
4AUC0→24[µg
hr/ml] |
70.64 |
53.37 |
44.38 |
17..27 |
-24.5 |
26.26 |
-37.2 |
|
5AUC0→∞ [µg hr/ml] |
121.4 |
78.47 |
52.78 |
42.93 |
-35.4 |
86.62 |
-56.7 |
|
6Kel
[hr-1] |
0.0646 |
0.0432 |
0.08 |
0.02 |
-46.3 |
0.037 |
-85.2 |
|
7t½ [hr] |
16.04 |
10.73 |
8.66 |
5.31 |
-33.1 |
7.38 |
-46.0 |
1Cmax= maximum plasma tetracycline-HCl concentration. 2Tmax= time to
reach the peak plasma concentration. 3Ka = absorption
rate constant. 4AUC0→24= area under plasma drug
versus time curve from t=0 to t=24 hrs. 5AUC0→∞
= area under plasma drug versus time curve from t=0 to t=∞. 6Kel= elimination rate
constant. 7t½= the biologic half-life. Consistent with
in-vitro data, in-vivo findings of pharmacokinetics parameters indicated
statistically significant % reduction in Cmax
of 12.68% (SD=0.3274; t=14.77; p<0.05) when the drug is taken just before Khat chewing and a % reduction in Cmax
of 20.40% (SD=0.7455; t=8.795;
p<0.05)when the drug is taken during Khat chewing
compared with the control (Table 6).
There was also statistically significant enhancement of Tmax of 20% in both cases of taking the drug just before and during Khat chewing compared with the control(Table
6).Statistically significant % reduction in Ka of 16.2% and 24.6%
were also observed for taking the drug just before and during Khat chewing respectively compared with the control (Table
6).
The reduction in Cmax
and Ka and the enhancement of Tmax,
all indicate that khat Chewing significantly reduces
the rate of absorption of tetracycline-HCl. With regard to AUC0→∞ that describes
the extent of absorption of tetracycline-HCl, statistically
significant % reduction inAUC0→∞ of 35.4% and 56.7% were
observed when the drug is taken just before and during Khat
chewing respectively compared with the control (Table 6).This reflects a clear
reduction in the extent of absorption of
tetracycline-HCl. The reduction in the rate
and extent of tetracycline-HCl absorption as a result
of khat chewing, may be due to one of the following assumptions:
a- Possible interaction of tetracycline-HCl with
certain khat constituents, such as polyvalent cations (calcium, magnesium, iron, cadmium, lead, copper,
and zinc), tannic acid, cathinone, cathine and pseudoephedrine that may lead to the formation
of non-absorbable complexes. b- Possible delaying in gastric emptying induced
by khat chewing [16] may reduce the rate
of tetracycline-HCl absorption. In addition delaying gastric emptying prolongs the
resident time of the drug in the acidic pH of the stomach that may perhaps lead
to its possible degradation into the less antimicrobial active for mepitetracycline[17]. It worth mentioning that
in this in vivo study, microbiological assay procedure has been used to
determine tetracycline-HCl serum concentration. The
biologic half-life (t½) and
elimination rate constant (Kel) both
exhibit statistically significant % reduction of 33.1% and 46.3% respectively
when taking tetracycline-HCl just before khat chewing and a % reduction of 46.0% and 85.2% respectively
when the drug is taken during Khat chewing compared with the control (Table VI). t½ and Kel
are generally influenced by dose size, age, protein binding, variations in
urinary excretion and metabolic rates and co-administration of drug that influence urinary excretion and metabolic
rates as well as liver and kidney diseases [18]. The reduction in t½ and Kel of tetracycline-HCl
as a result of khat chewing may be attributed to one
or combination of the following assumptions: a-Possible enhancement of urinary
excretion of tetracycline-HCl through changing
urinary pH by khat constituents into somewhat acidic
pH that increases the concentration of the ionizable
form of the drug, hence reducing tubular reabsorption
and lowering the concentration of the drug in the blood as tetracycline-HCl is passively excreted. b-Certain Khat
constituents may induce certain microsomal enzymes
that may degrade tetracycline-HCl. These assumptions needed to be further experimentally
validated. Other factors that influence t½ and Kel
, such as age and dose size are excluded in this study, since all
volunteers were of the same age group and the same dose of the drug is used.
Concerning protein binding as one of the factor affecting t½ ,
protein binding usually leads to the displacement of a drug by another drug or
substance resulting in the prolongation not reduction in t½[18].
It should be mentioned that the calculated values of pharmacokinetics
parameters of tetracycline-HCl alone (control) i.e. Cmax,
Tmax, and AUC 0→24were
comparable with literature values, while t½ , Kel
and AUC 0→∞ values were slightly different from
literature values [19].
CONCLUSION:
Tetracycline-HCl-khat interaction has been evaluated both in-vitro and
in-vivo. In-vitro studies have indicated a statistically significant
interaction between tetracycline-HCl and khat in both phosphate buffer(pH7.0) and 0.1N HCl (pH1.2). Such interaction may be attributed to possible
complexation between tetracycline-HCl
and certain khat constituents, such as polyvalent cations, or possible complexation
and/or adsorption of tetracycline-HCl with and/or
onto tannic acid. Other possible complication may also occur with other khat constituents such as cathinone,
cathine and pseudoephedrine. In-vivo data on the
other hand was consistent with the in-vitro findings. In-vivo bioavailability
data indicated a statistically significant% reduction in Cmax,
Ka as well as a significant enhancement of Tmax,
were observed a result of taking tetracycline-HCl
just before khat chewing and more pronounce during khat chewing compared to the control. All the above three
pharmacokinetics parameters reflect reduction in the rate of tetracycline-HCl absorption. In addition a statistically significant %
reduction in AUC 0→∞has been observed, reflecting
reduction in the extent of tetracycline-HCl
absorption. Such reduction in the rate and extent of tetracycline-HCl absorption as a result of chewing khat
may be attributed to the formation of non-absorbable complexes of tetracycline HCl with one or more of khat
constituents as explained previously or possible delaying in gastric emptying
induced by khat that may prolong the resident time of
the drug in the stomach leading to its possible degradation into the less
antimicrobial active for mepitetracycline. Statistically
significant % reductions in t½ and Kel were also observed reflecting a possible enhancement of
renal excretion rate of tetracycline-HCl, perhaps by possibly
urinary pH modification by certain khat constituents ora possible enhancement of tetracycline-HCl metabolism via enzyme induction by one or more khat constituents. These assumption needed to be
experimentally validated.
ACKNOWLEDGMENT:
The authors are grateful to the national quality control
laboratory, Ministry of Public Health, Sana’a, Yemen, for donating
the tetracycline-HCl standard reference. Special
thanks are due to the ten volunteers for their effective contribution and
patient during in-vivo studies.
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Received on 21.11.2014 Modified on 15.12.2014
Accepted on 05.01.2015 ©A&V Publications All right reserved
Res. J.
Pharm. Dosage Form. & Tech. 7(1): Jan.-Mar. 2015; Page 01-06
DOI: 10.5958/0975-4377.2015.00001.4