Fitoterapia 128 (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 T

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

A R T I C LE I N FO A B S T R A C T

Keywords: Kaurenoic acid (KA) is a kaurane diterpene found in several medicinal plants that displays biological activities,
Kaurenoic acid such as anti-inflammatory, smooth muscle relaxant and hypotensive response. However, there are no phar-
Diterpene macokinetic data available about this molecule. The purpose of the study was to determine the pharmaco-
Pharmacokinetics kinetic profile and the oral bioavailability of KA in rats. Wistar rats submitted to jugular vein cannulation
Bioavailability
received 50 mg/kg of KA by intravenous or oral route. The implanted cannula allowed intravenous adminis-
Rat plasma
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: Cmax = 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: jorge.willian@ufjf.edu.br (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
Available online 14 May 2018
0367-326X/ © 2018 Elsevier B.V. All rights reserved.
D.M.d. Matos et al.
bioavailability of guaco syrup metabolites after oral administration
[14]. The knowledge of pharmacokinetics and oral bioavailability is
crucial to select drug candidates to undergo clinical testing, besides
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-bisabolol,
1 mg/mL) were prepared in methanol and stocked at 4 °C. The cali-
bration standards (0.75, 1, 2.5, 5, 10, 50, 100, μg/mL) were prepared
143
Fitoterapia 128 (2018) 142–147
by successive dilutions, adding a known amount of KA into blank
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
D.M.d. Matos et al. Fitoterapia 128 (2018) 142–147

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). The (room temperature), freeze and thaw cycles and post-preparative ana-
area under the concentration-time curve was calculated from the time lysis (in autosampler). The deviation ranges are also presented in
zero to the last data point using trapezoidal method (AUC0-t) and with Table 1. Precision and accuracy results were expressed as coefficient of
extrapolation to infinity (AUC0-∞). The maximum plasma concentration variation (CV%) and relative error (RE%), respectively. Intraday and
(Cmax) and the time to reach Cmax (Tmax) values were observed from the interday analysis of both parameters showed values of CV and
experimental data. The elimination rate constant K was determinate RE < 15%, proving the reliability of the method to quantify KA in
from the slope of the regression line, and the elimination half-life (t1/2β) plasma samples.
obtained by 0.693/K. All data were presented as mean and standard The chosen method to clean-up plasma samples was protein pre-
deviation (mean ± SD). cipitation with acetonitrile. This simple, rapid and low cost technique
results in adequate purification, allowing direct injection in HPLC [23].
Several extraction methods for KA in plasma were tested before, such as
3. Results and discussion solid-phase extraction, protein precipitation with acetonitrile, and li-
quid-liquid extraction applying a variety of solvents. Comparison of
The analytical method proved to be simple, low cost and reliable, these techniques pointed out that protein precipitation was the most
with good performance, linearity, stability, accuracy, precision and efficient for recovery of KA from plasma with high reproducibility [14].
robustness. The addition of the internal standard, α-bisabolol, increased However, when applying this extraction, we observed the phenomenon
the method confidence, facilitating relative quantification of KA. of co-precipitation of the analyte with proteins, one of the dis-
No interfering peaks were detected at the retention times of KA advantages of this clean-up method. To improve KA recovery, an
(RT = 13.2 min) and α-bisabolol (RT = 7.5 min) (Fig. 1A). The cali- acidification step with acetic acid was included before precipitation
bration curve (Fig. 1B) showed good linearity within the range of with acetonitrile, leading to a successful extraction of KA from plasma
0.75–100 μg/mL, providing the mean linear equation y = 0.0135× - 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
Parameter Result administration of plant extracts have been carried out, for example 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
Limit of Quantification 0.75 μg/mL
nociceptive activities. After oral administration of R. officinalis extract,
Limit of Detection 0.5 μg/mL carnosol, rosmanol, and carnosic acid were found in rat plasma, al-
Estability: 88.92–100.80% lowing the pharmacokinetic study of these diterpenes [26].
- Short term 94.46–105.94% An investigation about the dermal absorption of KA have been
- Freeze and thaw cycles 98.89–102.42%
performed through in vitro Franz-cells' system. Application of KA at 1%
- Autosampler
Precision (CV%) in an oily formulation, after a 24 h exposure time, indicated that
- Intraday 7.2 clinically relevant skin concentrations of KA were achieved – about
- Interday 9.3 10% had been absorbed and found in the skin layers [13].
Accuracy (RE%) 4.9
Another study attempted to determine the kinetic disposition of
- Intraday 6.5
- Interday
guaco metabolites by HPLC-MS/MS in human volunteers that received
Recovery (%) 85 an oral dose of 60 mL of guaco syrup (17.6 mg of coumarin, 1.1 mg of o-

144
D.M.d. Matos et al.
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 quantifiable
levels of KA were found in blood samples collected until 10 h after
administration [14].
The pharmacokinetic study presented herein was conducted in rats
treated with 50 mg/kg of the drug through intravenous and oral routes.
The technique of jugular vein cannulation, applied for serial blood
collection, consists of an efficient vascular access technique suited to
monitor temporal changes in blood constituents with little animal
manipulation and distress [18]. In fact, it proved to be ideal for phar-
macokinetic studies such as this, since it guaranteed optimum re-
producibility among the experimental animals, generating plasma
decay curves with similar profiles and small deviations. Several studies
applying this technique use saline to replace the volume of blood col-
lected. However, considering that a relevant amount of blood is with-
drawn from animals during the experiment (11 × 200 μL), the blood
replacement using donor rats instead of saline replacement prevents
hemodilution, reducing interferences on drug's pharmacokinetics.
A typical representative chromatogram obtained from an animal
sample collected 10 min after i.v. administration is shown in Fig. 2A
(KA retention time = 13.2 min). On the other hand, Fig. 2B exemplifies
a chromatogram of a sample collected 1 h after oral treatment with KA
(KA not detected).
Serial blood collections performed in intravenous group furnished
data to describe the plasma decay of KA (Fig. 3A) and determine its
pharmacokinetics parameters. However, after oral administration, no
Fig. 3. Comparison of kaurenoic acid plasma decay (dose = 50 mg/kg). (A) Intravenous route. (B) Oral route.
145
Fitoterapia 128 (2018) 142–147
significant levels of KA were detected in plasma along 10 h (Fig. 3B),
suggesting poor absorption or an extensive pre-systemic elimination of
the drug through this route. Thus, determination of its oral bioavail-
ability was impaired.
Following intravenous administration, the KA concentration-time
curve can be best described as a two-compartment model, with two
distinct phases (Fig. 3A). This information was considered to calculate
the pharmacokinetic parameters of the drug (Table 2). Thus, the dis-
tribution phase (α-phase) was delineated from time zero to 60 min after
administration of the drug, and the subsequent period was defined as
the elimination phase (β-phase). This model assumes the existence of a
central compartment, composed by blood and well-perfused tissues that
easily equilibrate with blood, and another peripheral, which involves
tissues where the drug distributes slowly [27]. Once the equilibrium
between central and peripheral compartments is achieved, the plasma
concentrations decline less rapidly [28].
The maximum concentration (Cmax) estimated after a single dose of
KA was 22.17 ± 1.65 mg/L. The estimated volume of distribution (V)
was 14.53 ± 1.47 L/kg. This high value of V reflects the liposolubility
of KA, indicating that this molecule is widely distributed in body tissues
and fluids. The elimination half-life (t1/2β) is considered a dependent
parameter, since its value depends on clearance (CL) and volume of
distribution: t1/2β = (0.693 V)/CL [27]. The t1/2β of KA was
9.52 ± 0.61 h, while the clearance was 17.67 ± 1.50 mL/min/kg
(Table 2).
D.M.d. Matos et al.
Table 2
Pharmacokinetics parameters of kaurenoic acid determined after intravenous administration (dose = 50 mg/kg).
Rat Cmax (mg/L) ASC0-t
(mg.min/L)
1 19.6 1764.1
2 21.1 1412.8
3 23.4 1530.9
4 24.0 1702,9
5 21.8 1028.3
6 23.1 1685.5
Mean ± SD 22.17 ± 1.65 1520.75 ± 273.42
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).
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