Fitoterapia128(2018)142–147

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Pharmacokinetic profile and oral bioavailability of Kaurenoic acid from

COPAIFERA spp. in rats
Dalyara Mendonça de Matosa, Milainy Rocha Vianab, Marcela Cristina de Oliveira Alvimb,
Lara Soares Aleixo de Carvalhoa, Laura Hora Rios Leitec, Ademar Alves Da Silva Filhoa,b,
Jorge Willian Leandro Nascimentoa,d,⁎
a
Post GRADUATION PROGRAM in PHARMACEUTICAL Sciences, FEDERAL University of Juiz de FORA, José Lourenço Kelmer Street, Juiz de FORA 36036-900, MG, BRAzil
b
DEPARTMENT of PHARMACEUTICAL Sciences, FACULTY of PHARMACY, FEDERAL University of Juiz de FORA, José Lourenço Kelmer Street, Juiz de FORA 36036-900, MG, BRAZIL
c
DEPARTMENT of Physiology, Institute of BIOLOGICAL Sciences, FEDERAL University of Juiz de FORA, José Lourenço Kelmer Street, Juiz de FORA 36036-900, MG, BRAZIL
d
DEPARTMENT of PHARMACOLOGY, Institute of BIOLOGICAL Sciences, FEDERAL University of Juiz de FORA, José Lourenço Kelmer Street, Juiz de FORA 36036-900, MG, BRAZIL

ARTICLEINFO
ABSTRACT

Keywords:
Kaurenoic acid
Kaurenoic acid (KA) is a kaurane diterpene found in several medicinal plants that displays biological
Diterpene activities, such as anti-inflammatory, smooth muscle relaxant and hypotensive response. However, there are
Pharmacokinetics no phar- macokinetic data available about this molecule. The purpose of the study was to determine the
Bioavailability pharmaco- kinetic profile and the oral bioavailability of KA in rats. Wistar rats submitted to jugular vein
Rat plasma cannulation received 50 mg/kg of KA by intravenous or oral route. The implanted cannula allowed
intravenous adminis- tration and serial blood collection along 10 h. Analytical quantification was performed
by reversed phase HPLC-UV and mobile phase composed by acetonitrile:acidified water (70:30 v/v). The
validated analytical method showed precision, accuracy, robustness, reliability and linearity between 0.75 and
100 μg/mL. KA administered intravenously showed a linear and two-compartment kinetic behavior at the
tested dose. The following pharmacokinetic parameters were determined: C max = 22.17 ± 1.65 mg/L; V =
14.53 ± 1.47 L/ kg; CL = 17.67 ± 1.50 mL/min/kg; AUC0-∞ = 2859.65 ± 278.42 mg·min/L, K = 0.073
± 0.005 h−1 and
t1/2β = 9.52 ± 0.61 h. Oral treatment did not provide detectable plasma levels of KA, avoiding the de-
termination of its bioavailability.

1. Introduction
Kaurenoic acid (KA) (ent-kaur-16-en-19-oic acid) is a kaurane-type
diterpene found in plants commonly used in folk medicine, such
as copaiba oil (COPAIFERA LANGSDORffi) [1], guaco (MIKANIA GLOMERATA
and MIKANIA LAEVIGATA ) [2] and yacon (SMALLANTHUS sonchifolius) [3].
COPAI- FERA spp. and MIKANIA spp., due to their well characterized
use in tra- ditional medicine, have been included among species of
governmental interest in Brazil to become available for population
through the Uni- fied Health System [4]. Copaiba is a large tree that
commonly grows in some states of Brazil, such as Amazonas, Pará
and Ceará. Copaiba oleoresin is popularly used to treat a variety of
conditions, for example, sore throat, urinary and pulmonary
affections, and skin diseases [1]. Guaco syrups are also widely
applied in folk medicine for treatment of
respiratory diseases and its pharmacological properties are mainly re-
lated to coumarin and kaurane-type diterpenes [2].
Kaurenoic acid is a bioactive compound that demonstrates anti-in-
flammatory and analgesic properties associated with inhibition of cy-
tokine production and activation of the NO-cyclic GMP-PKG-ATP-
sen- sitive K+ channel pathway [5]. Another important effect
of this substance includes smooth muscle relaxation by blockage of
extra- cellular Ca2+ influx and opening of ATP-sensitive potassium
channels [6,7]. In fact, several reports indicate a wide spectrum of
biological effects of KA, which also includes antimicrobial [3],
trypanocidal [8], hypotensive [9], diuretic [10], antiasthmatic [11] and
hypoglycemic
activities [12].
Even presenting a promising therapeutic potential, there are only a
few studies about the cutaneous absorption of KA [13] and the

ABBREVIATIONS: KA, kaurenoic acid; IS, internal standard; RT, retention time; HPLC, high-performance liquid chromatography; Cmax, maximum plasma concentration; Tmax, time to reach
Cmax; V, volume of distribution; CL, clearance; AUC, area under the curve; K, elimination rate constant; t1/2β, elimination half-life; CV, coefficient of variation; RE, relative error; SD,
standard deviation; i.v., intravenous; i.p., intraperitoneal
⁎
Corresponding author at: Universidade Federal de Juiz de Fora, Instituto de Ciências Biológicas, Laboratório de Farmacologia Clínica e Experimental (LaFaCE), Departamento de
Farmacologia, Rua José Lourenço Kelmer, s/n. 36036-900, Juiz de Fora, MG, Brazil.
E-MAIL ADDRESS: j o rg e . w i l l i a n @ u f j f . e du . b r (J.W.L. Nascimento).

https://doi.org/10.1016/j.fitote.2018.05.013
Received 1 December 2017; Received in revised form 4 May 2018; Accepted 13 May 2018
Availableonline14May2018
0367-326X/©2018ElsevierB.V.Allrightsreserved.
D.M.d. MATOS et AL. FitoterApiA128(
2018)142–147

bioavailability of guaco syrup metabolites after oral administration bisabolol, 1 mg/mL) were prepared in methanol and stocked at 4 °C.
[14]. The knowledge of pharmacokinetics and oral bioavailability is The cali- bration standards (0.75, 1, 2.5, 5, 10, 50, 100, μg/mL) were
crucial to select drug candidates to undergo clinical testing, besides prepared
providing basis for definition of an effective dosing regimen
associated with adequate plasma concentrations [15]. Considering that
KA is one of the major bioactive compounds found in guaco syrups
and represents a potential candidate for the development of new drugs,
the aim of this study was to determine the pharmacokinetic profile and
oral bioavail- ability of KA in rats following oral and intravenous
administration.

2. Materials and methods

2.1. CHEMICALS AND REAGENTS

KA was isolated from COPAIFERA spp. oleoresin following a previously

described methodology [16]. This substance was kindly donated by Dr.
Sergio Ricardo Ambrósio from The University of Franca (Unifran,
Franca, São Paulo, Brazil). Chemical structure of KA was confirmed by
13
C and 1H NMR analysis. The internal standard (IS), α-bisabolol, was
purchased from Sigma Aldrich (St. Louis, USA). Ortho-phosphoric acid
was obtained from Merck (Darmstadt, Germany) and the glacial acetic
acid was purchased from Exodo Cientifica (Hortolândia, Brazil). Ul-
trapure water was obtained using the Purelab Option-Q
purification system from Elga (High Wycombe, UK). Methanol and
acetonitrile (HPLC grade) were purchased from J.T. Baker
Chemicals (Xalostoc, Mexico).

2.2. ANIMAL experiments

The animal experimental protocol was approved by the Ethical

Committee for Animal Handling (CEUA number: 040/2014) of the
Federal University of Juiz de Fora and was in agreement with the
Ethical Principles in Animal Research adopted by the Brazilian
Council for Control of Animal Experimentation (CONCEA).
Male Wistar rats (200-250 g, n = 6) were used to perform the pre-
clinical pharmacokinetic study of KA. The animals were housed in
propylene cages, at 22 ± 2 °C, in groups of four, under a 12 h
light- dark cycle and with free access to food and water. Following
anesthesia achieved using a mixture of ketamine (72 mg/kg body
weight i.p.) and xylazine (3 mg/kg body weight i.p.), a SILASTIC catheter
was inserted into the right jugular vein of all animals [17,18].
Immediately after surgery, the rats received an intramuscular
prophylactic dose of antibiotics (Pentabiotic, 24,000 IU/kg body wt,
Fort Dodge) and a subcutaneous injection of analgesic medication
(Flunixin Meglumine, 1.1 mg/kg body wt, Schering-Plogh) [19]. The
animals were kept in cages individually and were allowed to recover
for at least 2 days before being submitted to the experiments. The
implanted cannula was used for intravenous administration of the
drug, while gavage was used for oral adminis- tration. Both
intravenous and oral groups received a 50 mg/Kg dose of KA prepared
in saline with 10% of Tween 80. The jugular cannula also allowed
repeated blood sampling from the right atrium in both ex-
perimental groups. Blood samples (200 μL) were collected at the
fol- lowing intervals: 0 (previous to drug administration), 0.166, 0.333,
0.5,
0.75, 1, 2, 4, 6, 8 and 10 h after administration. The blood volume
collected in each sample was replaced by donated blood from healthy
donor rats to avoid reduction in the animal's blood volume. The
samples were collected with heparinized syringes and were centrifuged
at 10000 RPM, 25 °C, for 10 min. The separated plasma was kept in
freezer (−20 °C) until HPLC analysis was carried out.

2.3. PREPARATION of STANDARD solutions, CALIBRATION AND QUALITY control

SAMPLES

Stock solutions of KA (1 mg/mL) and internal standard (α-

14
D.M.d. MATOS et AL. FitoterApiA128(
by successive dilutions, adding a known amount of KA into blank 2018)142–147

plasma. Quality control samples were prepared in plasma at low

(2.5 μg/mL), medium (50 μg/mL) and high (80 μg/mL)
concentrations of the analyte. The working solution of internal
standard (0.2 μg/mL) was obtained from dilution of stock solution in
methanol. All plasma samples were stored at −20 °C.

2.4. PLASMA SAMPLES PRETREATMENT

Sample clean-up consisted of protein precipitation with

acetonitrile [20]. For each plasma sample (100 μL) it was added 50
μL of internal standard (0.2 mg/mL) and 100 μL of acetic acid
solution (1% v/v). After acidification, the sample was mixed for 1
min on a vortex mixer. Fol- lowing this step, 200 μL of acetonitrile
were added and, once more, the sample was vigorously mixed for 1
min before centrifugation at 10000 RPM, 4 °C, for 10 min. The
supernatant was analyzed by liquid chro- matography.

2.5. CHROMATOGRAPHIC ANALYSIS

Concentrations of KA in rat plasma were determined by HPLC-

UV. The method was developed in a Waters system equipped with an
Alliance e2695 Separation Module, quaternary pump, autosampler,
degasser, column heater and double channel UV–Vis detector
(Milford, USA). The Empower 3 software was used for system
control, peak in- tegration and data analysis. Separation was carried
out in a X-Bridge C18 column (150 × 4.6 mm, 5 μm; Waters, Milford,
USA) attached to a C18 guard cartridge (20 × 4.6 mm, 5 μm; Waters,
Milford, USA). The isocratic mobile phase was composed by
acetonitrile (A) and acidified water (B) (ortho-phosphoric acid 0.1%),
A:B (70:30, v/v), at a flow rate of 1 mL/min. The column heater was
maintained at 40 °C and the UV detector was set at 200 nm. An
injection volume of 80 μL was estab- lished.

2.6. VALIDATION of the ANALYTICAL method

Method validation followed orientations of the guidance for bioa-

nalytical method validation from Food and Drug Admnistration [21].
Selectivity was assessed comparing plasma samples spiked with KA
and internal standard with blank samples. Linearity was evaluated
over the range of 0.75–100 μg/mL, using the internal standardization
method. The calibration curves were constructed plotting peak area
ratios (KA peak area/IS peak area) versus plasma concentration, at
seven con- centrations levels of KA (n = 3). A new calibration curve
was prepared for each day of analysis. Lower limit of quantification
was investigated by analyzing five replicates of the lowest
concentration of the analyte with an acceptable accuracy and
precision (< 20%), and limit of de- tection was determined as a
concentration with a signal-to-noise ratio > 5.
Precision and accuracy (intra-day and inter-day) were assessed by
quantification of five replicates prepared at low (2.5 μg/mL), medium
(50 μg/mL) and high (80 μg/mL) concentrations of analyte. Method
precision and accuracy were expressed as coefficient of variation (CV
%) and relative error (RE%), respectively. Carryover test consisted of
in- jection of a sample processed at the upper limit of quantification
(100 μg/mL) followed by injections of blank samples. Recovery was
determined comparing peak area ratio of extracted quality control
samples with standard samples of same concentrations prepared in
aqueous solution. Stability study evaluated three replicates of quality
control samples (2.5, 50 and 80 μg/mL) at room temperature, auto-
sampler and after three successive freeze and thaw cycles.

2.7. DATA ANALYSIS

Results from the HPLC analysis of the plasma samples were

plotted as drug concentration vs. time to obtain the plasma decay
curves of KA

14
Fig. 1. (A) Selectivity analysis: comparison of blank plasma chromatogram (blue line) with a plasma sample spiked with α-bisabolol (1) and kaurenoic acid (2).
(B) Calibration curve of kaurenoic acid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

[22]. Two-compartmental pharmacokinetic analysis was performed by

KA demonstrated satisfactory stability after short-term analysis
using PK Solutions 2.0 (Summit Research Services, Ashland, USA).
(room temperature), freeze and thaw cycles and post-preparative ana-
The area under the concentration-time curve was calculated from the
lysis (in autosampler). The deviation ranges are also presented in
time zero to the last data point using trapezoidal method (AUC 0-t) and
Table 1. Precision and accuracy results were expressed as coefficient
with extrapolation to infinity (AUC0-∞). The maximum plasma
of variation (CV%) and relative error (RE%), respectively. Intraday
concentration (Cmax) and the time to reach Cmax (Tmax) values were
and interday analysis of both parameters showed values of CV
observed from the experimental data. The elimination rate constant K
and RE < 15%, proving the reliability of the method to quantify KA
was determinate from the slope of the regression line, and the
in plasma samples.
elimination half-life (t1/2β) obtained by 0.693/K. All data were
The chosen method to clean-up plasma samples was protein pre-
presented as mean and standard deviation (mean ± SD).
cipitation with acetonitrile. This simple, rapid and low cost technique
results in adequate purification, allowing direct injection in HPLC
3. Results and discussion [23]. Several extraction methods for KA in plasma were tested before,
such as solid-phase extraction, protein precipitation with acetonitrile,
The analytical method proved to be simple, low cost and reliable, and li- quid-liquid extraction applying a variety of solvents.
with good performance, linearity, stability, accuracy, precision and Comparison of these techniques pointed out that protein precipitation
robustness. The addition of the internal standard, α-bisabolol, was the most efficient for recovery of KA from plasma with high
increased the method confidence, facilitating relative quantification of reproducibility [14]. However, when applying this extraction, we
KA. observed the phenomenon of co-precipitation of the analyte with
No interfering peaks were detected at the retention times of KA proteins, one of the dis- advantages of this clean-up method. To
(RT = 13.2 min) and α-bisabolol (RT = 7.5 min) (Fig. 1A). The cali- improve KA recovery, an acidification step with acetic acid was
bration curve (Fig. 1B) showed good linearity within the range of included before precipitation with acetonitrile, leading to a successful
0.75–100 μg/mL, providing the mean linear equation y = 0.0135× - extraction of KA from plasma matrix.
0.017 (r = 0.999). As mentioned before, despite being known for its great potential as
The results obtained in the validation process are presented in antispasmodic, anti-inflammatory and antihypertensive, no pharmaco-
Table 1. Limit of detection and the low limit of quantification were kinetic study of this substance has been reported following oral or i.v.
0.5 μg/mL and 0.75 μg/mL, respectively. Carryover test proved that no administration.
residual from previous elutions produced interfering peaks at the re- Pharmacokinetic characterizations of natural products are im-
tention times of analyte and internal standard. Therefore, there was no portant to provide useful data to predict biological events and sub-
risk of contamination between injections. stantiate clinical studies [24]. The kinetics of promising molecules have
been described through administration of isolated compounds like the
Table 1 clinical study of the diterpenoid glycosides, stevioside and rebaudioside
Results from analytical method validation. A, two natural sweeteners [25]. As well investigations following
oral administration of plant extracts have been carried out, for example
Parameter Result
the preclinical evaluation of ROSMARINUS offiCINALIS L. extract, known for
its
Linearity 0.75–100 μg/mL
antibacterial, antioxidant, antitumor, anti-inflammatory and anti-
Correlation Coefficient r = 0.999 nociceptive activities. After oral administration of R. offiCINALIS extract,
Limit of Quantification 0.75 μg/mL
Limit of Detection 0.5 μg/mL carnosol, rosmanol, and carnosic acid were found in rat plasma, al-
Estability: - Interday
88.92–100.80%
- Short term 94.46–105.94%
- Freeze and thaw cycles 98.89–102.42%
- Autosampler
Precision (CV%)
- Intraday 7.2
- Interday 9.3
Accuracy (RE 4.9
%) 6.5
- Intraday
lowing the have been n of KA at 1% in an oily formulation, after a 24 h exposure time,
pharmacokinetic study performed indicated that clinically relevant skin concentrations of KA were
of these diterpenes through in achieved – about 10% had been absorbed and found in the skin layers
[26]. vitro [13].
An investigation Franz-cells' Another study attempted to determine the kinetic disposition of
about the dermal system. guaco metabolites by HPLC-MS/MS in human volunteers that
absorption of KA Applicatio received
Recovery (%) 85 an oral dose of 60 mL of guaco syrup (17.6 mg of coumarin, 1.1 mg of
o-
Fig. 2. Typical chromatograms of plasma samples after kaurenoic acid administration in rat. (A) Sample collected 10 min after intravenous administration. (B)
Sample collected 1 h after oral administration.

coumaric acid and 8.9 mg of KA). However, at this dose, no

significant levels of KA were detected in plasma along 10 h (Fig. 3B),
quantifiable levels of KA were found in blood samples collected until
suggesting poor absorption or an extensive pre-systemic elimination of
10 h after administration [14].
the drug through this route. Thus, determination of its oral bioavail-
The pharmacokinetic study presented herein was conducted in rats
ability was impaired.
treated with 50 mg/kg of the drug through intravenous and oral routes.
Following intravenous administration, the KA concentration-time
The technique of jugular vein cannulation, applied for serial blood
curve can be best described as a two-compartment model, with two
collection, consists of an efficient vascular access technique suited to
distinct phases (Fig. 3A). This information was considered to calculate
monitor temporal changes in blood constituents with little animal
the pharmacokinetic parameters of the drug (Table 2). Thus, the dis-
manipulation and distress [18]. In fact, it proved to be ideal for phar-
tribution phase (α-phase) was delineated from time zero to 60 min
macokinetic studies such as this, since it guaranteed optimum re-
after administration of the drug, and the subsequent period was defined
producibility among the experimental animals, generating plasma
as the elimination phase (β-phase). This model assumes the existence
decay curves with similar profiles and small deviations. Several
of a central compartment, composed by blood and well-perfused
studies applying this technique use saline to replace the volume of
tissues that easily equilibrate with blood, and another peripheral, which
blood col- lected. However, considering that a relevant amount of
involves tissues where the drug distributes slowly [27]. Once the
blood is with- drawn from animals during the experiment (11 × 200
equilibrium between central and peripheral compartments is achieved,
μL), the blood replacement using donor rats instead of saline
the plasma concentrations decline less rapidly [28].
replacement prevents hemodilution, reducing interferences on drug's
The maximum concentration (Cmax) estimated after a single dose
pharmacokinetics.
of KA was 22.17 ± 1.65 mg/L. The estimated volume of
A typical representative chromatogram obtained from an animal
distribution (V) was 14.53 ± 1.47 L/kg. This high value of V
sample collected 10 min after i.v. administration is shown in Fig. 2A
reflects the liposolubility of KA, indicating that this molecule is
(KA retention time = 13.2 min). On the other hand, Fig. 2B
widely distributed in body tissues and fluids. The elimination half-
exemplifies a chromatogram of a sample collected 1 h after oral
life (t1/2β) is considered a dependent parameter, since its value
treatment with KA (KA not detected).
depends on clearance (CL) and volume of distribution: t1/2β = (0.693
Serial blood collections performed in intravenous group furnished
V)/CL [27]. The t1/2β of KA was
data to describe the plasma decay of KA (Fig. 3A) and determine its
9.52 ± 0.61 h, while the clearance was 17.67 ± 1.50 mL/min/kg
pharmacokinetics parameters. However, after oral administration, no
(Table 2).
Fig. 3. Comparison of kaurenoic acid plasma decay (dose = 50 mg/kg). (A) Intravenous route. (B) Oral route.
Table 2
Pharmacokinetics parameters of kaurenoic acid determined after intravenous administration (dose = 50 mg/kg).

Rat Cmax (mg/L) ASC0-t ASC0-∞ V (L/kg) CL K t1/2β (h)

(mg.min/L) (mg.min/L) (mL/min/kg) (h−1)

1 19.6 1764.1 3368.3 12.2 15 0.073 9.5

2 21.1 1412.8 2583.7 15.5 19 0.075 9.2
3 23.4 1530.9 2785.1 15.6 18 0.069 10.0
4 24.0 1702,9 2953.9 15.0 17 0.068 10.2
5 21.8 1028.3 2676.8 15.7 19 0.071 9.7
6 23.1 1685.5 2790.1 13.2 18 0.081 8.5
Mean ± SD 22.17 ± 1.65 1520.75 ± 273.42 2859.65 ± 278.42 14.53 ± 1.47 17.67 ± 1.50 0.073 ± 0.005 9.52 ± 0.61

The process from plant to medicine – which comprehends the dis-

covery, development and launch of a new drug into the market – in-
volves high investments, a long period of time and multi-disciplinary
approaches [29]. As discussed before, the knowledge about kinetics
disposition of drugs in the body brings evidence of the efficacy and
safety of active compounds, contributing to the definition of dosing
regimens [30]. The lack of preclinical pharmacokinetics studies re-
garding metabolites from natural products still represents a limitation
in the process of development of new drugs using plant active com-
pounds, therefore, the kinetic evaluation of KA performed can support
future clinical trials with this bioactive molecule.
4. Conclusion
Intravenous administration of KA follows a linear and two-com-
partment kinetic behavior at the tested dose. Oral treatment did not
provide detectable levels of KA, suggesting poor absorption or an ex-
tensive pre-systemic elimination through this route. This is the first
pharmacokinetic study of this molecule. The data presented here elu-
cidates the pharmacokinetic of KA, contributing to future clinical stu-
dies of this compound.
Author contributions
A. A. Da Silva-Filho and L. S. A. Carvalho contributed for the partial
obtainment of KA and its chemical characterization. L. H. R. Leite and
M. R. Viana performed animal experiments.
M. R. Viana and M. C. O. Alvim performed HPLC analysis. D. M.
Matos and J. W. L. Nascimento performed pharmacokinetic studies,
analyzed the data and drafted the manuscript. All authors revised the
manuscript.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
Dr. Sérgio Ricardo Ambrósio, from the University of Franca (Franca,
São Paulo, Brazil) is gratefully acknowledged for providing part of the
substance evaluated in this work. The authors are grateful to CAPES,
PIBIC/CNPq/UFJF and CNPq for the fellowships. This work was sup-
ported by FAPEMIG (Grant numbers #APQ 0171/11; APQ 02015/14;
PPM-00296-16) and CNPq (Grant number #487221/2012-5).
References
[1] L.A.F. Paiva, L.A. Gurgel, R.M. Silva, A.R. Tomé, N.V. Gramosa, E.R. Silveira,
F.A. Santos, V.S.N. Rao, Anti-inflammatory effect of kaurenoic acid, a diterpene
from COPAIFERA LANGSDORffii on acetic acid-induced colitis in rats, Vasc. Pharmacol.
39 (2002) 303–307.
[2] S.K.V. Bertolucci, A.B.D. Pereira, J.E.B.P. Pinto, J.A.A. Ribeiro, A.B. Oliveira,
F.C. Braga, Development and validation of an RP-HPLC method for quantification of
cinnamic acid derivatives and kaurane-type diterpenes in MIKANIA LAEVIGATA and
MIKANIA GLOMERATA, Planta Med. 75 (2009) 280–285.
[3] E.P. Padla, L.T. Solis, C.Y. Ragasa, Antibacterial and antifungal properties of ent-
kaurenoic acid from SMALLANTHUS sonchifolius, Chin. J. Nat. Med. 10 (2012) 408–
414.
[4] D.J. Marmitt, S. Bitencourt, A.C. Silva, C. Rempel, M.I. Goettert, Scientific pro-
duction of plant species included in the Brazilian national list of medicinal plants of interest
to the unified health system (RENISUS) from 2010 to 2013, J. Chem. Pharm. Res. 8
(2016) 123–132.
[5] S.S. Mizokami, N.S. Arakawa, S.R. Ambrosio, A.C. Zarpelon, R. Casagrande,
T.M. Cunha, S.H. Ferreira, F.Q. Cunha, W.A. Verri Jr., Kaurenoic acid from
SPHAGNETICOLA TRILOBATA inhibits inflammatory pain: effect on cytokine production
and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium
channel signaling pathway, J. Nat. Prod. 75 (2012) 896–904.
[6] K.M. de Alencar Cunha, L.A.F. Paiva, F.A. Santos, N.V. Gramosa, E.R. Silveira,
V.S.N. Rao, Smooth muscle relaxant effect of kaurenoic acid, a diterpene from
COPAIFERA LANGSDORffii on rat uterus in vitro, Phytother. Res. 17 (2003) 320–324.
[7] C.R. Tirapelli, S.R. Ambrosio, F.B. Costa, S.T. Coutinho, D.C.R. Oliveira,
A.M. Oliveira, Analysis of the mechanisms underlying the vasorelaxant action of
kaurenoic acid in the isolated rat aorta, Eur. J. Pharmacol. 492 (2004) 233–241.
[8] H.S. Vieira, J.A. Takahashi, A.B. Oliveira, E. Chiari, M.A.D. Boaventura, Novel
derivatives of kaurenoic acid: preparation and evaluation of their trypanocidal
activity, J. Braz. Chem. Soc. 13 (2002) 151–157.
[9] C.R. Tirapelli, S.R. Ambrosio, F.B. Costa, A.M. Oliveira, Diterpenes: a therapeutic
promise for cardiovascular diseases, Recent Pat. Cardiovasc. Drug Discov. 3
(2008) 1–8.
[10] L.I. Somova, F.O. Shode, K. Moodley, Y. Govender, Cardiovascular and diuretic
activity of kaurene derivatives of Xylopia aethiopica and ALEPIDEA AMATYMBICA, J.
Ethnopharmacol. 77 (2001) 165–174.
[11] J.H. Cho, J.Y. Lee, S.S. Sim, W.K. Whang, C.J. Kim, Inhibitory effects of diterpene
acids from root of ARALIA CORDATA on IgE-mediated asthma in guinea pigs, Pulm.
Pharmacol. Ther. 23 (2010) 190–199.
[12] X. Deng, Y. Shen, J. Yang, J. He, Y. Zhao, L. Peng, Y. Leng, Q. Zhao, Discovery
and structure-activity relationships of ent-Kaurene diterpenoids as potent and
selective 11β-HSD1 inhibitors: potential impact in diabetes, Eur. J. Med. Chem. 65
(2013) 403–414.
[13] J.P. Mestres, A. Usubillaga, L. Diaz, M. Larroque, L. Vian, G. Marti-Mestres,
Assessment of in vitro dermal absorption of the kaurenic acid from Coespeletia
Moriziana extracts, Fitoterapia 82 (2011) 585–590.
[14] J.C. Gasparetto, R.G. Peccinini, T.M.G. Francisco, L.B. Cerqueira, F.R. Campos,
R. Pontarolo, A kinetic study of the main guaco metabolites using syrup
formulation and the identification of an alternative route of coumarin metabolism
in humans, PLoS One 10 (2015) 1–22.
[15] B.M. Amore, J.P. Gibbs, M.G. Emery, Application of in vivo animal models to
characterize the pharmacokinetic and pharmacodynamic properties of drug can- didates
in discovery settings, Comb. Chem. High Throughput Screen. 13 (2010) 207–218.
[16] M.R. Simão, L.J. Carneiro, R.A. dos Santos, J.K. Bastos, R.C.S. Veneziani,
S.R. Ambrosio, C.S. Mizuno, In vitro cytotoxicity study of ent-kaurenoic acid deri-
vatives against human breast carcinoma cell line, Med. Chem. Res. 25 (2016) 303–
309.
[17] L.H.R. Leite, A.C.R. Lacerda, C.H. Balthazar, U. Marubayashi, C.C. Coimbra,
Central angiotensin AT1 receptors are involved in metabolic adjustments in
response to graded exercise in rats, Peptides 30 (2009) 1931–1935.
[18] K.V. Thrivikraman, R.L. Huot, P.M. Plotsky, Jugular vein catheterization for
re- peated blood sampling in the unrestrained conscious rat, Brain Res. Protocol. 10
(2002) 84–94.
[19] C.H. Balthazar, L.H.R. Leite, A.G. Rodrigues, C.C. Coimbra, Performance-
enhancing and thermoregulatory effects of intracerebroventricular dopamine in
running rats, Pharmacol. Biochem. Behav. 93 (2009) 465–469.
[20] J.W.L. Nascimento, M.J.C. Carmona, T.M.V. Strabelli, J.O.C. Auler Jr.,
S.R.C.J. Santos, Penetration of cefuroxime in subcutaneous tissue during coronary
artery bypass grafting surgery, J. Chromatogr. B 877 (2009) 3960–3964.
[21] FDA, Guidance for Industry: Bioanalytical Method Validation, United States
Department of Health and Human Service,
http://www.fda.gov/downloads/Drugs/ Guidances/ucm070107.pdf, (2001)
(accessed 29.07.2017).
[22] W.A. Ritschel, G.L. Kearns, Handbook of Basic Pharmacokinetics: Including
Clinical Applications, seventh ed., American Pharmacists Association, Washington,
2009.
[23] J. Ma, J. Shi, H. Le, R. Cho, J.C. Huang, S. Miao, B.K. Wong, A fully automated
plasma protein precipitation sample preparation method for LC–MS/MS bioana-
lysis, J. of Chromatogr. B. 862 (2008) 219–226.
[24] W. Chen, Y. Liang, L. Xie, T. Lu, X. Liu, G. Wang, Pharmacokintics of the Ginkgo B
following intravenous Administration of Ginkgo B emulsion in rats, Biol. Pharm.
 Bull. 30 (2007) 1–5.
[25] A. Wheeler, A.C. Boileau, P.C. Winkler, J.C. Compton, I. Prakash, X. Jiang,
D.A. Mandarino, Pharmacokinetics of rebaudioside A and stevioside after single
oral doses in healthy men, Food Chem. Toxicol. 46 (2008) 54–60.
[26] L. Wang, C. Gan, Z. Wang, L. Liu, M. Gao, Q. Li, C. Yang, Determination and
pharmacokinetic study of three diterpenes in rat plasma by UHPLC-ESI-MS/MS
after oral administration of Rosmarinus officinalis L. extract, Molecules 22 (2017) 1–
12.
[27]
L.A. Bauer (Ed.), Applied clinical pharmacokinetics, second ed., McGraw-Hill, New York, 2001.
[28] J.T. DiPiro, W.J. Spruill, R.A. Blouin, J.M. Pruemer (Eds.), Concepts in clinical
pharmacokinetics, fourth ed., American Society of Health-System Pharmacists,
Bethesda, 2005.
[29] S.M.K. Rates, Plants as source of drugs, Toxicon 39 (2001) 603–613.
[30] S.S. Singh, Preclinical pharmacokinetics: an approach towards safer and efficacious
drugs, Curr. Drug Metab. 7 (2006) 165–182.