Content uploaded by Fowad Khurshid
Author content
All content in this area was uploaded by Fowad Khurshid on Sep 24, 2019
Content may be subject to copyright.
Content uploaded by Shivli Nomani
Author content
All content in this area was uploaded by Shivli Nomani on Aug 15, 2018
Content may be subject to copyright.
Braz. J. Pharm. Sci. 2017;53(4):e17072 Page 1 / 10
Brazilian Journal of
Pharmaceutical Sciences
http://dx.doi.org/10.1590/s2175-97902017000417072
Article
*Correspondence: F. Khurshid. Department of Pharmaceutical Sciences,
SunRise University, Alwar-301030, Rajasthan, India. Tel no.: +91-9955881101.
E-mail: fowad.khurshid@gmail.com
Effect of herb-drug interactions of Bacopa monnieri Linn. (Brahmi)
formulation on the pharmacokinetics of amitriptyline in rats
Fowad Khurshid1 *, Jeyabalan Govindasamy2, Habibullah Khalilullah2, Mohammed Shivli Nomani2,
Mudassar Shahid3, Md Ruhal Ain4, Mohammad Sultan Alsultan5
1Department of Pharmaceutical Sciences, SunRise University, Alwar, Rajasthan, India, 2Department of Pharmaceutical
Chemistry, Alwar Pharmacy College, Alwar, Rajasthan, India, 3Molecular Virology Laboratory, Department of Biotechnology,
Jamia Millia Islamia, Jamia Nagar, New Delhi, India, 4School of Pharmacy, OPJS University, Rawatsar Kunjla, Rajgarh,
Churu-Rajasthan, India, 5Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Interactions between herbs and drugs may increase or decrease the pharmacological or toxicological
eects of either component. Experimental data on the pharmacokinetic interactions between herbal
products and drugs are limited. This study attempted to investigate the eect of Bacopa monnieri Linn.
(Brahmi) formulation on the pharmacokinetics of amitriptyline in rats. In this study, rats were randomly
divided into two groups (n = 6 each) which were served as a control (amitriptyline alone) and treatment
group (amitriptyline with B. monnieri), respectively. Rats in the treatment group received B. monnieri
(31 mg/kg/day) whereas the control group received normal saline by oral gavage for seven days before
a single intragastric administration of 25 mg/kg amitriptyline. Plasma concentrations of amitriptyline
were measured up to 24 h after its administration by a developed and validated high-performance liquid
chromatography method. Pretreatment with B. monnieri produced a signicant increase in the maximum
plasma concentration (Cmax), area under the curve (AUC0-t) and elimination half-life (t1/2) of amitriptyline
by 16.8%, 26.5%, and 15.5%, respectively, compared to amitriptyline alone. Moreover, oral clearance
and volume of distribution (Vss) were decreased by 26.2% and 15.5% respectively. This study concluded
that B.monnieri signicantly enhanced the oral bioavailability of amitriptyline in rats.
Keywords: Herbal drug interaction. Bacopa monnieri/eects. Amitriptyline/pharmacokinetics. CYP450.
Bioavailability.
INTRODUCTION
Amitriptyline, a tricyclic compound, has been
widely used globally for decades in the treatment of
mental illnesses, especially depression. It still serves as
one of the most commonly used antidepressants. (Leucht,
Huhn, Leucht, 2012). Although the mode of action of
amitriptyline is not evident, it is speculated to act by
inhibiting the reuptake of serotonin–norepinephrine at the
adrenergic nerve endings, thus disrupting the functions of
these chemicals (Tatsumi et al., 1997; Amin Bano, 2014).
Amitriptyline is completely but slowly absorbed from
the gastrointestinal tract after its oral uptake such that
the peak plasma concentrations are achieved within 4 to
8 h of its administration. It has a systemic bioavailability
ranging from 33 to 62% and is subjected to extensive
hepatic pre-systemic elimination (Schulz et al., 1983).
Within the liver, the drug is primarily metabolized by the
action of cytochrome P450 enzymes, namely, CYP2D6,
CYP3A4, and CYP2C19 (Rudorfer, Potter, 1999). The
significant adverse effects associated with therapeutic
concentrations of amitriptyline are manifested in terms of
a moderate therapeutic index; an overdose of it may prove
to be dangerous.
Bacopa monnieri, a perennial herb, belongs to the
family, Scrophulariaceae and is commonly known as
Brahmi in the Ayurvedic system of medicine. It has been
widely employed as a brain stimulator, antidepressant, and
memory enhancer (Stough et al., 2001). Various research
studies, performed using the standardized extracts of B.
monnieri, have demonstrated the herb to facilitate the
processes of acquisition, retention, and retrieval of learned
F. Khurshid, J. Govindasamy, H. Khalilullah, M. S. Nomani, M. Shahid, M. R. Ain, M. S. Alsultan
Braz. J. Pharm. Sci. 2017;53(4):e17072
Page 2 / 10
tasks. A study that evaluated the antidepressant potential of
B. monnieri reported it exhibit a signicant antidepressant
activity in the most commonly used behavior paradigms
in animal models of depression. These included forced
swim test and learned helplessness test (Sairam et al.,
2002). Another study that employed dierent convulsive
models in albino rats to investigate the anticonvulsant
activity of an alcoholic extract of B. monnieri reported
the presence of a broad spectrum of anticonvulsant
prole of the extract in chemical, electrical, and hypoxic
convulsions (Kaushik et al., 2009). Moreover, a clinical
trial conducted to assess the eects of administration of
B. monnieri (300 mg/day) over a period of 12 weeks on
memory performance reported the ability of the herb
to significantly improve the memory acquisition and
retention in individuals over the age of 55 years (Morgan,
Stevens, 2010). Another study that analyzed the eects
of a standardized B. monnieri (300 mg/day) on cognitive
performance, anxiety, and depression in the elderly
individuals concluded it to be a plant with a potential
to safely enhance the cognitive performance in aging
(Calabrese et al., 2008). This hypothesis was also favored
by the studies conducted in rodents where B. monnieri
provided protection against age-related oxidative stress
and some inammatory conditions (Williams et al., 2014).
It is also known to enhance the lifespan during stress
conditions (Phulara et al., 2015). The above-mentioned
ndings have been strengthened by several clinical studies
(Stough et al., 2001; Roodenrys, 2002; Calabrese et al.,
2008) that ascribed the observed antidepressant eects
to its phytochemical constituents, known as bacoside.
Bacoside is a mixture of four triglycosidic saponins,
namely, bacoside A3, bacopaside II, bacopaside X, and
bacopasaponin C (Singh et al., 1988; Dhawan, Singh,
1996; Russo, Borrelli, 2005).
B. monnieri has an extensive market both in India
and outside. The neuropharmacological properties of B.
monnieri (Russo, Borrelli, 2005; Calabrese et al., 2008)
considerably increase the chances of chronic or recurrent
usage of the herb and its associated products by patients
with mental illnesses. The prescription of therapeutic drugs
to these patients signicantly contributes to herb–drug
interactions at the physiological levels. These interactions,
in turn, may increase or decrease the pharmacological
or toxicological eects of either component (Hu et al.,
2005). Inhibition or induction of hepatic and intestinal
drug-metabolizing enzymes, particularly cytochrome
P450 (CYP) and transporters (e.g., p-glycoprotein) act
as the trigger for the interactions (Zhang et al., 2010a;
Wanwimolruk, Prachayasittikul, 2014a; Wanwimolruk,
Phopin, Prachayasittikul et al., 2014b). However, there
exists a paucity of literature on the pharmacokinetic
interactions between herbal products and drugs. The
present study attempted to investigate the effects of a
commercial formulation of B. monnieri (Brahmi) on
the pharmacokinetics of amitriptyline using rodents as
the model system. An extensive review of the literature
revealed the presence of several analytical methods, based
on high-performance liquid chromatography (HPLC), for
the estimation of amitriptyline and some of its metabolites
in biological samples (Ghahramani, Lennard, 1996;
Aymard et al., 1997; Farag et al., 2013). However, the
major drawbacks of these procedures are complexity,
time constraint, and limited sensitivity, leading to the
requirement of a rapid, specic, and sensitive analytical
method. The present study aimed to develop a highly
sensitive reversed-phase high-performance liquid
chromatography (RP-HPLC-UV) method, followed by
its validation for the quantication of amitriptyline in rat
plasma.
MATERIAL AND METHODS
Material
Amitriptyline and imipramine, as internal standards
(ISs), were purchased from Sigma-Aldrich (Germany).
High-performance liquid chromatography (HPLC)
grade LiChrosolv methanol and LiChrosolv acetonitrile
were from Merck (Darmstadt, Germany). Chemicals of
highest available commercial purity were utilized. The
commercial formulation of B. monnieri (Himalaya Drug
Company, Bangalore, India) was procured from the local
market. The HPLC-grade solvents were used for HPLC
determinations. Milli-Q plus water (Millipore, Bedford,
MA, United States) was used for all preparations. All other
chemicals were of analytical grade.
Animals and study design
The study was conducted in accordance with current
legislation on animal experiments as per Institutional
Animal Ethical Committee (CPCSEA Approval no. 962/
PO/Re/S/06/CPCSEA). Twelve male Wistar albino rats,
weighing 225 to 250 g were provided by the animal house,
Alwar Pharmacy College, Alwar, Rajasthan. The rats were
housed under standard animal conditions with alternate 12
h of light and dark cycles in an environment maintained
at a constant temperature prior to the study. Water was
supplied ad libitum. Rats were randomly divided into two
groups (n = 6 each); one served as the control (amitriptyline
alone), and the other group served as the treatment group
Eect of herb-drug interactions of Bacopa monnieri Linn. (Brahmi) formulation on the pharmacokinetics of amitriptyline in rats
Braz. J. Pharm. Sci. 2017;53(4):e17072 Page 3 / 10
(amitriptyline with B. monnieri). A randomized parallel
design study was employed to study the pharmacokinetics
of amitriptyline. Rats in the control group were provided
normal saline orally (10 mL/kg) for 7 consecutive days
followed by administration of amitriptyline (25 mg/kg bw
p.o.) on day 8, 1 h after administration of normal saline.
Amitriptyline was administered as an aqueous solution in
0.5% (w/v) sodium carboxy-methyl cellulose (CMC) at a
dose of 25 mg/kg bw p.o. Rats in the treatment group were
administered B. monnieri extract in 0.5% CMC at a dose
of 31 mg/kg/day, p.o. in saline water (Singh et al., 2013)
for 7 consecutive days. The rats were made to fast for at
least 12 h (overnight) with free access to water before the
day of the experiment. On the morning of day 8, the last
dose of B. monnieri was administered to the fasting rats.
One hour after the administration of the last dose of B.
monnieri, amitriptyline aqueous solution in 0.5% (w/v)
CMC (25 mg/kg p.o.) was administered to these rats. The
drug was administered via gastric gavage throughout the
study.
Blood collection
A total of 1 mL of blood samples were collected into
heparinized vacutainer tubes from the retroorbital plexus
of each rat using capillary tubes before and after the
administration of amitriptyline at 0, 1, 2, 4, 6, 8, 12, and
24 h. The blood samples were immediately centrifuged
at 2,500×g for 10 min to separate the plasma that was
stored at –80 ºC until analysis. To replace the uid loss,
equal volumes of normal saline were injected through the
cannula in all experiments. The same sampling scheme
was followed for rats in the treatment group to determine
the plasma concentrations of amitriptyline and evaluate
the eect of B. monnieri on amitriptyline disposition in
rats.
Amitriptyline assay by HPLC Method
Instrumentations and chromatographic conditions
The high-performance liquid chromatography
(Waters, 1525 Binary HPLC pump) employed was
equipped with the Waters-2489 UV-visible detector
and Waters-2707 Autosampler and operated by Breeze
2 service pack A (SPA) software, Waters Corporation.
The chromatographic identification was conducted at
ambient temperature. The mobile phase consisted of
a v/v ratio of acetonitrile and potassium dihydrogen
phosphate buer (KH2PO4, 38:62), which was delivered at
the isocratic condition with a ow rate of 1 mL/min. EC
150/4.6-NUCLEODUR Sphinx RP, 5 µm MACHERY-
NAGEL column was used to elute amitriptyline at a
wavelength (lmax) of 254 nm. Different combinations
of solvent systems of acetonitrile, buffer (KH2PO4):
acetonitrile, and formic acid: water, and methanol:
water: acetonitrile were tried to determine the optimum
conditions for the separation and standardization of
amitriptyline. The mobile phase consisted of acetonitrile
and 70 mM KH2PO4 buffer. Its pH was adjusted to 4.5
by 85% orthophosphoric acid in a ratio of 38:62 (v/v).
The selection of mobile phase was based on its ability to
provide high resolution for amitriptyline with minimal
tailing.
Sample preparation
The samples for analysis were prepared by the
protein precipitation method. Plasma samples stored at
around − 80˚C were thawed at room temperature and
vortexed for 30 s to ensure homogeneity. The internal
standard (8 µL, 100 µg/mL) was added to 200 µL of
plasma sample, and 592 µL of acetonitrile was added
to precipitate the protein from the sample. The samples
were mixed gently for 1.5 min followed by centrifugation
at 12,000 rpm for 10 min. After centrifugation, 700 µL
of clear supernatant was transferred into HPLC vials.
Finally, 25 µL of each sample was subjected to HPLC-
UV analysis.
Calibration and control samples
The linearity of an analytical method refers to its
ability to furnish test results that are directly proportional
to the concentration of an analyte in the samples
within a given range. In other words, linearity is the
relationship between the concentration of analyte and
assay measurement (ICH, 1996). We used correlation
coecient (R2) obtained from the linear regression to
demonstrate the linearity of the relationship between the
peak area ratio and the concentration. The experiment
was performed in triplicates with the concentration of
analyte ranging from 0.125 to 50.0 µg/mL. The relative
standard deviations were calculated for all the calibration
curve slopes. The observed straight-line equation,
y = 0.0343 × − 0.0026 (R2 = 0.9994) was used for the
calculation of plasma concentrations of amitriptyline.
Validation and stability of HPLC method
The guidelines of the international conference on
harmonization were employed to validate the HPLC
method in terms of linearity, specificity, sensitivity,
F. Khurshid, J. Govindasamy, H. Khalilullah, M. S. Nomani, M. Shahid, M. R. Ain, M. S. Alsultan
Braz. J. Pharm. Sci. 2017;53(4):e17072
Page 4 / 10
precision, and accuracy (ICH, 1996). The robustness of the
method was evaluated by intentionally introducing minor
modications in the mobile phase volume ratios. These
included mobile phase having dierent compositions of
acetonitrile-70 mM KH2PO4 (6038:62 ± 5 mL), alteration
of the pH of buer (4.5 ± 0.2), and slight changes in the
isocratic ow rate (1.0 ± 0.2 mL/min) of the mobile phase.
All the validation parameters were studied in triplicate
(n=3) at a concentration of 20 µg/mL. The stability of
amitriptyline in plasma at 4 and 20 ºC and after freeze–
thaw cycles was determined.
Pharmacokinetic analysis
We utilized the non-compartmental analysis to
determine the pharmacokinetic parameters of amitriptyline
after oral administration. The maximum observed plasma
concentration (Cmax) and the time to reach this concentration
(Tmax) following oral administration were calculated from
the observed data. The volume of distribution at steady
state (Vss) was calculated as (AUMC0-inf/AUC0-inf)*CL
and total body clearance (CL) as dose/AUC0-inf. The area
under the plasma concentration versus time curve (AUC0-t)
was calculated by the trapezoidal rule with extrapolation
to innity. The linear regression analysis of the terminal
portion of the log concentration–time data was utilized to
calculate the apparent terminal elimination rate constant
(Kel or λz) of amitriptyline. Amitriptyline apparent terminal
elimination half-life (t1/2) was calculated as (ln2)/λz,
where λz is the elimination rate constant. Pharmacokinetic
analysis was performed using the program, PKSolver for
Microsoft Excel (Zhang et al., 2010b).
Statistical data analysis
All statistical data were expressed as the mean
± standard deviation (SD). Student’s t-test on log-
transformed data was utilized for assessing the dierences
in the pharmacokinetic parameters of amitriptyline with
and without B. monnieri. Data were considered statistically
signicant at a p<0.05. All calculations were performed
using GraphPad Prism, version 3.00 for Windows (San
Diego, CA, United States).
Herbal medicine and dose calculation
The recommended adult dose of B. monnieri extract
containing 20% of total bacopasides was 300 to 400 mg
daily. A dose equivalent to human dose was used as the
animal dose of these herbs and was calculated using the
following equation.
The human dose was selected from authentic herbal
textbook resources (Reagan-Shaw, Nihal, Ahmad, 2008).
The rat dose was found to be 31 mg/kg/day (Singh et al.,
2013).
RESULTS AND DISCUSSION
HPLC method development and validation for
amitriptyline assay
The mobile phase of HPLC column was selected
on the basis of previously used and described methods
available for amitriptyline (Shen et al., 2010; Farag
et al., 2013). The KH2PO4 buffer was checked at
different concentrations (0.02, 0.05, and 0.07 M) for
determining its ecient working concentration. At lower
concentrations, amitriptyline and IS did not result in the
same retention time. At a buer concentration of 0.07
M, the analytes were found to be well separated from
plasma and hence it was chosen as the optimal buer
concentration. A range of buer pH (3.0–7.0) was assayed
to optimize the chromatographic separation. Optimal
peak separation for amitriptyline and IS was produced
using a pH value ranging between 3.0 and 7.0. However,
when the spiked matrix samples were analyzed, the peaks
from some of the plasma impurities matched with that
of IS. A pH value of 4.5 was found to be optimal for the
complete separation of amitriptyline and imipramine
(IS); at this pH value, peaks were resolved well. The nal
mobile phase consisted of acetonitrile-KH2PO4 (0.07 M),
pH 4.5 with 1.0 mL/min ow rate. This condition was
found to be outstanding in terms of sensitivity and peak
separation. The wavelengths checked in the present study
were 230, 240, and 254 nm (Olsen, Sullivan, 1995). The
wavelength of 254 nm was found to be optimal in terms
of sensitivity for all the analytes; it also avoided the
occurrence of numerous matrix impurities and exogenous
substances.
Selectivity and specificity
The selectivity of any analytical method refers
to the ability of the method to assess the analyte
unequivocally in the presence of endogenous matrix
compounds (i.e., plasma and proteins in this method).
The chromatograms of plasma samples by using HPLC
method are depicted in Figure 1. The symmetrical
peaks were observed for amitriptyline with a retention
Eect of herb-drug interactions of Bacopa monnieri Linn. (Brahmi) formulation on the pharmacokinetics of amitriptyline in rats
Braz. J. Pharm. Sci. 2017;53(4):e17072 Page 5 / 10
time of 6.15 min. There was no interference with the
amitriptyline peak, and the overall chromatographic run
time was 10 min. HPLC chromatograms of blank plasma
(Figure 1A), plasma spiked with imipramine as IS 25 ng/
mL (Figure 1B), plasma sample spiked with amitriptyline
(20 µg/mL) with retention times of 6.15 min. Retention
time of the IS was 4.52 min (Figure 1C). The plasma
samples obtained from rat, administered with 25 mg/kg
p.o. dose of amitriptyline (Figure 1D), were compared
to show the selectivity of the adopted HPLC method.
The retention times of amitriptyline and imipramine
were approximately 6.15 and 4.52 min, respectively. The
designed HPLC method was found to be considerably
selective, reected by the absence of the appearance of
any other interfering peaks around the retention times due
to endogenous matrix substances or metabolite eects
during quantication of amitriptyline and imipramine
(IS) in plasma samples.
FIGURE 1 - HPLC chromatogram of blank plasma (A), plasma spiked with imipramine as internal standard (I.S) (B), plasma
sample spiked with amitriptyline (20 µg/mL concentration) (Rt 6.15 min) and IS (Rt 4.5 min) (C), plasma samples obtained from
rat, administered with 25 mg/kg, p.o. dose of amitriptyline (D).
F. Khurshid, J. Govindasamy, H. Khalilullah, M. S. Nomani, M. Shahid, M. R. Ain, M. S. Alsultan
Braz. J. Pharm. Sci. 2017;53(4):e17072
Page 6 / 10
Precision and accuracy of developed HPLC
method
An evaluation of the concentrations of three
levels, namely, LLOQ, MQC, and ULOQ indicated
the relative standard deviation (% RSD) values to be
satisfactory. The calculated percentage recovery of
the MQC level was found to be 100.14 ± 0.15%. The
recovery values obtained from the analysis of dierent
known concentrations by the developed HPLC method
ranged from 98.31 to 99.02, indicating the method to be
highly accurate. The percentage recovery determined for
the lowest concentration (LLOQ) and MQC were 98.31
± 0.85 and 99.23 ± 0.15%, respectively, and that of the
highest concentration (ULOQ) was 99.02 ± 0.54 % during
the inter-day analysis of the samples. All the results are
listed in Table I. The reproducibility and reliability of the
method was evident by low values of % RSD for LLOQ,
MQC, and ULOQ.
Sensitivity, Limit of Detection (LOD), and Limit of
Quantification (LOQ)
The lower limit of quantitation (LLOQ) was dened
as the concentration of the lowest non-zero calibration
standard, which met the acceptance criteria for accuracy
and precision. The signal-to-noise ratios of 3.3:1 and 10:1
were considered as LOD and LOQ, respectively, and were
found to be 0.04334 and 0.13134 µg/mL, respectively.
Stability
Studies on the stability of the drug were performed
to ensure reproducibility and reliability of the method.
The amitriptyline stock solutions and its working stock
solutions were stable at 4 °C for 3 months. The stability of
the analyte in rat plasma was investigated under dierent
storage conditions. It was found to be stable under the
following conditions: at 10 °C for 24 h post-extraction,
after three freeze and thaw cycles (from −20 to 25 °C) and
at –80 °C for 90 days. The results of stability studies are
summarized in Table II.
Recovery and matrix effects
The vortexing and ultracentrifugation were used for
the extraction of the analyte and the IS. The percentage
extraction recoveries of amitriptyline were found to be
65.25, 76.75, and 74.28% in the low (LQC), medium
(MQC), and high (HQC) concentration quality control
samples (n=6), respectively. The percentage extraction
recovery of the IS was 61.65%. We did not detect any
apparent matrix eect in the determination of amitriptyline
by this method of analysis. The values for the three quality
control samples, i.e., LQC, MQC, and HQC were reported
to be 100.4, 100.13, and 100.29%, respectively.
Carryover test
The HPLC chromatograms obtained by using the
presently developed method of a blank sample (IS without
amitriptyline) were analyzed following six consecutive
analyses of ULOQ samples. The analysis revealed no
obvious carryover.
Pharmacokinetic analysis
The pharmacokinetic parameters of amitriptyline
are summarized in Table III. The mean plasma
concentrations versus time profiles of amitriptyline
after oral administration with and without B. monnieri
were characterized in rats and are presented in Figure 2.
The peak plasma concentration (Cmax) was signicantly
(p< 0.05) increased by 16.8% (2.86 ± 0.11 to
3.34 ± 0.06 µg/mL) in the presence of B. monnieri.
Similarly, the area under the plasma concentration–time
curve, AUC0–24 and AUC0-inf significantly increased by
26.5% (29.34 ± 0.90 to 37.12 ± 0.62 µg·h/mL) and 36.7%
TABLE I - Inter- and intra-day precision and recovery as accuracy of quality control samples (mean ± SD, n = 3)
Parameters Values Inter-day (µg/mL) Intra-day (µg/mL)
LLOQ (10) MQC (30) ULOQ (50) LLOQ (10) MQC (30) ULOQ (50)
Precision Mean ± SD 9.73 ± 0.03 28.96 ± 0.14 48.92 ± 0.98 9.65 ± 0.02 29.02 ± 0.16 49.06 ± 0.97
%RSD 1.65 2.98 1.85 1.52 0.85 1.45
Mean ± SD 9.95 ± 0.08 29.96 ± 0.04 49.82 ± 0.32 10.34 ± 0.02 30.02 ± 0.15 50.51 ± 0.25
Recovery % Recovery 98.31 99.23 99.02 100.49 100.13 100.29
%RSD 0.85 0.15 0.54 0.99 0.51 0.185
Eect of herb-drug interactions of Bacopa monnieri Linn. (Brahmi) formulation on the pharmacokinetics of amitriptyline in rats
Braz. J. Pharm. Sci. 2017;53(4):e17072 Page 7 / 10
(38.38 ± 1.23 to 52.46 ± 1.73), respectively, compared
to the control group. However, there was no signicant
change in the time to reach the peak plasma concentration
(Tmax) of amitriptyline in the presence of B. monnieri. On
the other hand, the calculated total oral clearance (CL)
decreased by 26.2% (0.65 ± 0.02 to 0.48 ± 0.02 mL/h;
p<0.05), while the estimated apparent oral volume of
distribution (Vss) at steady state decreased by about
15.5% (9.05 ± 0.26 to 7.65 ± 0.07 L/kg; p<0.05). Also, an
increase of elimination half-life (t1/2) from 9.63 ± 0.21 to
11.12 ± 0.39 h, which is about 15.5% as compared to the
control group, was reported.
Our study revealed the potential of B. monnieri
to signicantly alter the oral pharmacokinetic prole of
amitriptyline compared with the control. This is attributed
to the considerable enhancement in the oral bioavailability
of amitriptyline in rats. Various studies indicate the
importance of herbal drugs to alter the expression of drug-
metabolizing enzymes and membrane transporters after
administration. Cytochrome P450 (CYPs) are the primary
TABLE II - Results of stability study of QC samples at dierent conditions (mean ± SD, n = 3)
Stability test Initial concentration ± SD
(µg/mL)
Measured concentration ± SD
(µg/mL)
Stock solution (4 °C for 3 months) 250.00 249.06 ± 1.26
Working solution (4 °C for 3 months) 30.00 (HQC sample) 29.56 ± 0.56
Post-extraction (10 °C for 24 h)
a58.83 ± 0.12 58.60 ± 0.49
b29.25 ± 0.05 28.81 ± 0.45
c9.15 ± 0.09 9.04 ± 0.16
Freeze-thaw (from -20 °C to 25 °C, 3 cycles)
a59.01 ± 1.08 58.83 ± 0.42
b29.25 ± 1.12 29.11 ± 0.15
c9.88 ± 1.01 9.33 ± 0.43
Long term (-80 °C for 90 days)
a58.54 ± 0.98 58.82 ± 0.27
b29.42 ± 0.78 29.04 ± 0.22
c9.02 ± 0.29 8.94 ± 0.089
Note: avalues of ULOQ (60 µg/mL) and bvalues of MQC (30 µg/mL) samples and cvalues of LLOQ (10 µg/mL)
TABLE III - Pharmacokinetic parameters of amitriptyline control (alone) and co-administered with B.monnieri (mean ± SD, n = 6)
Pharmacokinetic parameters Amitriptyline only
(mean ± SD, n =6)
Amitriptyline with B.monnieri
(mean ± SD, n =6)
1Cmax (µg/mL) 2.86 ± 0.11 3.34 ± 0.06*
2Tmax (h) 4.0 ± 0.00 4.0 ± 0.00
3Kel (h−1)0.07 ± 0.0079 0.062 ± 0.0021*
4AUC0-24(µg·h/mL) 29.34 ± 0.90 37.12 ± 0.62*
5AUC 0-inf (µg·h/mL) 38.38 ± 1.23 52.46 ± 1.73*
6AUMC0-inf (µg·h/mL) 617.77 ± 26.04 984.13 ± 63.33*
7t1/2 (h) 9.63 ± 0.21 11.13 ± 0.39*
8Vss (L/Kg) 9.05 ± 0.26 7.65 ± 0.07*
9CL (mL/h) 0.65 ± 0.02 0.48 ± 0.02*
10MRT (h) 16.09 ±0.27 18.74 ± 0.59*
*Signicant dierence from “ Amitriptyline alone” group with t-test,*P ≤ 0.05, 1 maximum blood concentration (Cmax) , 2 time of
peak concentration(Tmax), 3elimination rate constant (kel ), 4 area under the concentration time prole curve until last observation
(AUC0-24), 5 area under the concentration time prole curve from time 0 to innity (AUC0-inf), 6 area under the rst moment curve
concentration time prole curve from time 0 to innity (AUMC0-inf) , 7 half-life (t1/2), 8 Steady‐State Volume distribution (Vss),9 Total
clearance (CL), and 10 mean residence time (MRT)
F. Khurshid, J. Govindasamy, H. Khalilullah, M. S. Nomani, M. Shahid, M. R. Ain, M. S. Alsultan
Braz. J. Pharm. Sci. 2017;53(4):e17072
Page 8 / 10
enzymes in the liver involved in the hepatic metabolism
of most drugs. These comprise CYP1A2, CYP3A4,
CYP2C9, CYP2C19, CYP2D6, and CYP2E1 (Badyal,
Dadhich, 2001). Amitriptyline, after oral administration,
is readily absorbed by the gastrointestinal tract, followed
by its metabolism, majorly on its rst pass through the
liver. CYP2D6-, CYP3A4-, and CYP2C19-mediated
N-demethylation into nortriptyline constitute the main
pathway for its metabolism (Rudorfer, Potter, 1999).
Recently, a study conducted to evaluate the eects of a
standardized extract of B. monnieri on the expression and
activity of hepatic and intestinal cytochrome P450 3A and
P-glycoprotein (Pgp) in rats revealed that administration
of B. monnieri for 7 days modulated the expression of
CYP3A and Pgp. Administration of B. monnieri led to a
reduced expression of CYP3A and concomitant decrease
in the CYP3A-dependent testosterone hydroxylase
catalytic activity in liver and intestine (Singh et al., 2013).
Another study reported similar ndings that B. monnieri
moderately inhibited CYP2C19, CYP2C9, CYP1A2, and
CYP3A4 but weakly inhibited CYP2D6 (Ramasamy,
Kiew, Chung, 2014). These findings indicated toward
the possible potential of B. monnieri to contribute to
herb–drug interactions when orally co-administered with
drugs metabolized by CYP1A2, CYP3A4, CYP2C9, and
CYP2C19. Based on this hypothesis, the plasma levels
of drug administered concomitantly could be elevated by
inhibition of drug-metabolizing enzymes to prolong the
pharmacological eects of the drug, thereby increasing
the incidence of drug-induced toxicity (Hu et al., 2005).
The enhanced bioavailability of amitriptyline observed in
the current study might result from the inhibition of the
metabolizing enzymes, CYP3A4 and CYP2C19, in the
intestinal mucosa or liver by B. monnieri. Another reason
for the inhibition of the CYP enzymes by B. monnieri
could be the presence of other constituents in the extract,
such as free aglycones, such as jujubogenin (Kawai et al.,
1974) and pseudojujubogenin (Kawai, Shibata, 1978),
which are more lipophilic. The better lipophilic property
of free aglycones enhances their binding to the CYP
isoforms through hydrogen bonding, thereby resulting
in stronger inhibitory eects (Ramasamy, Kiew, Chung,
2014). The increase in Cmax and area under curve (AUC)
and decrease in oral clearance may lead to increase in
the total bioavailability of amitriptyline, leading to toxic
eects. Hence, concomitant administration of B. monnieri
preparations with drugs that are primarily cleared through
CYP2C19-, CYP2C9-, CYP1A2-, and CYP3A4-mediated
metabolism should be administered to the patients with
increased vigilance .
CONCLUSION
The ndings of the present study suggest that the
medicinal herb, B. monnieri exhibit signicant potential
to alter pharmacokinetics of amitriptyline in rats. The
herb–drug interaction was reected by the B. monnieri-
mediated increased intestinal absorption and reduced
rst-pass metabolism of amitriptyline in the intestine and
liver through inhibition of CYP2C and CYP3A enzymes.
The results obtained in the present study indicate toward
exercising caution while administering a combination of
FIGURE 2 - Plasma concentration-time curve of amitriptyline (25 mg/kg, p.o) administered with and without B.monnieri
(31 mg/kg/day, p.o) in rats (each data is mean ± SD, n = 6)
Eect of herb-drug interactions of Bacopa monnieri Linn. (Brahmi) formulation on the pharmacokinetics of amitriptyline in rats
Braz. J. Pharm. Sci. 2017;53(4):e17072 Page 9 / 10
B. monnieri and amitriptyline. Consequently, concomitant
use of B. monnieri and amitriptyline warrants close
monitoring for possible drug interactions. Further
conrmation of these results in humans would assist in
evaluating the clinical consequences of such interactions.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest regarding the publication of this paper.
REFERENCES
Amin F, Bano B. Eect of amitriptyline an antidepressant drug
on structural and functional properties of brain cystatin. J Mol
Genet Med 2014;8(2):1000103.
Aymard G, Livi P, Pham YT, Diquet B. Sensitive and
rapid method for the simultaneous quantification of five
antidepressants with their respective metabolites in plasma
using high-performance liquid chromatography with diode-
array detection. J Chromatogr B Biomed Sci Appl 1997;700(1-
2):183-189.
Badyal DK, Dadhich AP. Cytochrome P450 and drug
interactions. Indian J Pharmacol 2001;33:248-259.
Calabrese C, Gregory WL, Leo M, Kraemer D, Bone K,
Oken B. Eects of a standardized bacopa monnieri extract on
cognitive performance, anxiety, and depression in the elderly:
a randomized, double-blind, placebo-controlled trial. J Altern
Complement Med 2008;14(6):707-713.
Dhawan BN, Singh HK. Pharmacological studies on
Bacopa monniera, an Ayurvedic nootropic agent. Eur
Neuropsychopharmacol 1996;6(Supp 3):144.
Farag RS, Darwish MZ, Fathy WM, Hammad HA. New
HPLC method to detect amitriptyline in the blood of rats on
combination treatment. Int J Chem Analyt Sci 2013;4(2):120-
124.
Ghahramani P, Lennard MS. Quantitative analysis of amitriptyline
and nortriptyline in human plasma and liver microsomal
preparations by high-performance liquid chromatography. J
Chromatogr B Biomed Appl 1996;685(2):307-313.
Hu Z, Yang X, Ho PC, Chan SY, Heng PW, Chan E, Duan W,
Koh HL, Zhou S. Herb-drug interactions: a literature review.
Drugs 2005;65(9):1239-1282.
International Conference on Harmonisation. ICH Harmonised
tripartite guideline-guidance for industry: Q2B validation of
analytical procedures: methodology. Geneva: ICH;1996. 17 p.
Kaushik D, Tripathi A, Tripathi R, Ganachari M, Khan SA.
Anticonvulsant activity of Bacopa monniera in rodents. Braz J
Pharm Sci 2009;45(4):643-649.
Kawai K-I, Akiyama T, Ogihara Y, Shibata S. A new sapogenin
in the saponins of Zizyphus jujuba, Hovenia dulcis and Bacopa
monniera. Phytochemistry 1974;13(12):2829-–2832.
Kawai K-I, Shibata S. Pseudojujubogenin, a new sapogenin
from Bacopa monniera. Phytochemistry 1978;17(2):287-–289.
Leucht C, Huhn M, Leucht S. Amitriptyline versus placebo
for major depressive disorder. Cochrane Database Syst Rev
2012;12:CD009138. issue number is missing
Morgan A, Stevens J. Does Bacopa monnieri improve memory
performance in older persons? Results of a randomized,
placebo-controlled, double-blind trial. J Altern Complement
Med 2010;16(7):753-759.
Olsen BA, Sullivan GR. Chemometric categorization of
octadecylsilyl bonded-phase silica columns using test mixtures
and confirmation of results with pharmaceutical compound
separations. J Chromatogr A 1995;692(1-2):147-159.
Phulara SC, Shukla V, Tiwari S, Pandey R. Bacopa monnieri
promotes longevity in Caenorhabditis elegans under stress
conditions. Pharmacogn Mag 2015;11(42):410-416.
Priyanka HP, Singh RV, Mishra M, ThyagaRajan S. Diverse
age-related effects of Bacopa monnieri and donepezil in
vitro on cytokine production, antioxidant enzyme activities,
and intracellular targets in splenocytes of F344 male rats. Int
Immunopharmacol 2013;15(2):260-274.
Ramasamy S, Kiew L, Chung L. Inhibition of human
cytochrome p450 enzymes by bacopa monnieri standardized
extract and constituents. Molecules 2014;19(2):2588-2601.
Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from
animal to human studies revisited. FASEB J 2008;22(3):659-
661.
Roodenrys S. Chronic eects of brahmi (Bacopa monnieri) on
human memory. Neuropsychopharmacology 2002;27(2):279-
281.
F. Khurshid, J. Govindasamy, H. Khalilullah, M. S. Nomani, M. Shahid, M. R. Ain, M. S. Alsultan
Braz. J. Pharm. Sci. 2017;53(4):e17072
Page 10 / 10
Rudorfer MV, Potter WZ. Metabolism of tricyclic
antidepressants. Cell Mol Neurobiol 1999;19(3):373-409.
Russo A, Borrelli F. Bacopa monniera, a reputed nootropic plant:
an overview. Phytomedicine 2005;12(4):305-317.
Sairam K, Dorababu M, Goel RK, Bhattacharya SK.
Antidepressant activity of standardized extract of Bacopa
monniera in experimental models of depression in rats.
Phytomedicine 2002;9(3):207–211.
Schulz P, Turner-Tamiyasu K, Smith G, Giacomini KM,
Blaschke TF. Amitriptyline disposition in young and elderly
normal men. Clin Pharmacol Ther 1983;33(3):360-366.
Shen Y, Zhu RH, Li HD, Liu YW, Xu P. Validated LC-MS
(ESI) assay for the simultaneous determination of amitriptyline
and its metabolite nortriptyline in rat plasma: application
to a pharmacokinetic comparison. J Pharm Biomed Anal
2010;53(3):735-739.
Singh HK, Rastogi RP, Srimal RC, Dhawan BN. Effect of
bacosides A and B on avoidance responses in rats. Phytother
Res 1988;2(2):70-75.
Singh R, Panduri J, Kumar D, Kumar D, Chandsana H,
Ramakrishna R, Bhatta RS. Evaluation of memory enhancing
clinically available standardized extract of Bacopa monniera on
P-glycoprotein and cytochrome P450 3A in Sprague-Dawley
rats. PLoS One 2013a;8(8):e72517.
Stough C, Lloyd J, Clarke J, Downey LA, Hutchison CW,
Rodgers T, Nathan PJ. The chronic effects of an extract of
Bacopa monniera (Brahmi) on cognitive function in healthy
human subjects. Psychopharmacology (Berl) 2001;156(4):481-
484.
Tatsumi M, Groshan K, Blakely RD, Richelson E.
Pharmacological profile of antidepressants and related
compounds at human monoamine transporters. Eur J Pharmacol
1997;340(2-3):249-258.
Wanwimolruk S, Phopin K, Prachayasittikul V. Cytochrome
P450 enzyme mediated herbal drug interactions (Part 2). Excli
J 2014b;13:869-896.
Wanwimolruk S, Prachayasittikul V. Cytochrome P450 enzyme
mediated herbal drug interactions (Part 1). Excli J 2014a;13:347-
391.
Williams R, Munch G, Gyengesi E, Bennett L. Bacopa monnieri
(L.) exerts anti-inflammatory effects on cells of the innate
immune system in vitro. Food Funct 2014;5(3):517-520.
Zhang L, Reynolds KS, Zhao P, Huang S-M. Drug interactions
evaluation: An integrated part of risk assessment of therapeutics.
Toxicol Appl Pharmacol 2010a;243(2):134-145.
Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An add-in program
for pharmacokinetic and pharmacodynamic data analysis
in Microsoft Excel. Comput Methods Programs Biomed
2010b;99(3):306-314.
Received for publication on 27th February 2017
Accepted for publication on 26th June 2017
This is an open-access article distributed under the terms of the Creative Commons Attribution License.
