Introduction
Contradicting utilisation rates of traditional medicines are reported in the scientific
literature for different countries around the globe. For example, it is frequently
stated that 80% of the population of developing countries, especially sub-Saharan
Africa, use traditional medicines [1], [2], [3], [4], which is based on data obtained in 1983 [5]. A more recent survey done in six middle-income countries (China, Ghana, India,
Mexico, Russia, and South Africa) as part of a World Health Organization (WHO) Study
on Global AGEing and adult health (SAGE) found that the use of traditional medicine
seems to be substantially lower (< 3%) [5]. Although traditional medicine use in Africa has decreased according to recent surveys,
it varies widely among countries and regions on this continent [6]. There still seems to be a relatively large demand for medicinal plant usage by
rural people, as shown by a study conducted in the Amatola region of the Eastern Cape
Province in South Africa [7]. In addition, a survey in the Southern Africa region (including Botswana, Lesotho,
South Africa, and Swaziland) found that 33.5% of participants indicated that people
living with HIV/AIDS use complementary treatments, which include traditional medicines,
to help manage their symptoms [8]. Another relatively recent survey in the eThekwini Metropolitan area of the KwaZulu
Natal Province in South Africa showed that more than 65.0% of the participants reported
use of traditional medicines before the diagnosis of HIV and 77.6% reported traditional
medicine use after their HIV diagnosis, while 4.98% of the patients used traditional
medicines concurrently with their antiretroviral medicines during the study period
[9].
Use of herbal products in conjunction with conventional medicines may lead to herb-drug
pharmacokinetic or pharmacodynamic interactions [10], [11], [12]. Pharmacokinetic interactions may occur due to changes in the absorption, distribution,
metabolism, and/or elimination (ADME) of a drug as a result of coadministration with
an herbal product [13], [14]. Mechanisms of action by which herb-drug pharmacokinetic interactions usually take
place include modulation of intestinal active efflux transporters, such as P-glycoprotein
(P-gp), as well as modulation of metabolising enzymes, specifically the cytochrome
P450 (CYP450) super family [15]. These pharmacokinetic interactions may lead to increased or decreased bioavailability
of the coadministered drug. Consequences of these bioavailability changes may either
be adverse effects and even drug toxicity due to increased drug plasma levels or lack
of pharmacological responses and treatment failure due to decreased drug plasma levels
[16].
Hypoxis hemerocallidea Fisch., C. A.Mey. & Avé-Lall. (Hypoxidaceae) is a popular traditional medicinal plant
that is characterised by star-shaped flowers, which are bright yellow in colour, and
by strap-like leaves [17]. This plant has a potato shaped tuberous rootstock (i.e., the corm), which is referred
to as the “African potato”. These corms are washed and chopped and the decoction is
administered orally after boiling [1]. H. hemerocallidea extract, which contains phytosterols as well as hypoxoside and its active analogue
rooperol, has been used for the treatment of certain types of cancers, heart failures,
nervous disorders, immune-related illnesses, and urinary tract infections [18], and has also been used for the treatment of benign prostate hyperplasia as an anti-inflammatory
agent, antioxidant, anticonvulsant, and as an antidiabetic agent [19]. In vitro studies have suggested the possibility that H. hemerocallidea can cause pharmacokinetic interactions with drugs by means of inhibiting CYP450 enzymes
as well as by modulation of efflux transporters [20]. Pharmacokinetic interactions were also observed between African potato decoctions
and efavirenz in human volunteers. However, efavirenz is only a substrate for CYP450
enzymes and is not a substrate for efflux transporters such as P-gp [21]. Due to the interplay between P-gp efflux and CYP450-related metabolism, these two
processes should ideally be considered together in terms of pharmacokinetic interactions
[22], [23]. This study was specifically conducted to identify pharmacokinetic interactions
between H. hemerocallidea materials and an antiretroviral drug (i.e., indinavir) based on efflux modulation,
since indinavir is a substrate for the efflux transporter P-gp.
Results and Discussion
The LC/MS chromatograms (chemical fingerprinting) of the H. hemerocallidea materials are shown in [Fig. 1], while the quantities of hypoxoside in each of the selected H. hemerocallidea test materials are shown in [Table 1].
Table 1 Quantity of hypoxoside in each of the selected H. hemerocallidea materials.
|
Test material
|
Hypoxoside (mg/g)
|
|
Reference dried plant material
|
13.3
|
|
Commercial product
|
0.6
|
|
Aqueous extract
|
151.7
|
Fig. 1 Chromatograms (TIC) of H. hemerocallidea materials: a reference plant material, b commercial product, c aqueous crude extract, and d chemical structure of hypoxoside [24]. Peak 1 = hypoxoside, peak 2 = dehydroxyhypoxoside, peak 3 = geraniol glycoside.
Chromatographic analysis revealed that all three of the selected H. hemerocallidea materials contained the marker molecule hypoxoside, albeit in different quantities.
The aqueous extract of the corms contained a higher quantity of hypoxoside compared
to that of the reference plant material and commercial product. The relatively low
hypoxoside content of the commercial product can possibly be explained by the formulation
composition, which caused a dilution effect due to other excipients in the dosage
form as well as possible degradation of this phytochemical during manufacture of the
product and/or storage of the raw materials.
The apparent permeability coefficient (Papp) values for indinavir in both directions across the Caco-2 cell monolayers in the
presence of each selected H. hemerocallidea material as well as the control groups are shown in [Fig. 2].
Fig. 2 Bidirectional Papp values calculated from indinavir transport across Caco-2 cell monolayers in combination
with the selected H. hemerocallidea materials (500 µg/mL) as well as the negative control group (200 µM, indinavir alone)
and positive control group (indinavir with verapamil (100 µM). AP-BL = apical-to-basolateral
direction, BL-AP = basolateral-to-apical direction, n = 3, error bars indicate standard
deviation.
[Table 2] presents the efflux ratio (ER) values calculated from the bidirectional transport
of indinavir in combination with the H. hemerocallidea materials compared to the control groups.
Table 2 ER values calculated from the bidirectional transport of indinavir across Caco-2
cell monolayers in the absence and presence of the selected H. hemerocallidea materials.
|
Experimental group
|
ER ± SD*
|
|
*SD = standard deviation
|
|
Indinavir (200 µM) alone (negative control)
|
6.27 ± 4.75
|
|
Indinavir (200 µM) with verapamil (100 µM, positive control)
|
1.10 ± 0.40
|
|
Indinavir (200 µM) with H. hemerocallidea commercial product (500 µg/mL)
|
1.42 ± 0.33
|
|
Indinavir (200 µM) with H. hemerocallidea aqueous extract (500 µg/mL)
|
2.37 ± 1.70
|
|
Indinavir (200 µM) with H. hemerocallidea reference material (500 µg/mL)
|
2.61 ± 1.02
|
Verapamil, a known P-gp inhibitor, demonstrated an increased uptake of indinavir in
the apical-to-basolateral (AP-BL) direction compared to that of the negative control
group (i.e., indinavir alone), which can be explained by an inhibition of efflux.
Inhibition of efflux was confirmed by a decreased transport in the basolateral-to-apical
(BL-AP) direction of indinavir as well as the ER value when compared to that of the
negative control group. The Caco-2 cell line therefore proved to be an acceptable
model for testing efflux modulation of indinavir. Similarly, the transport of indinavir
was increased in the AP-BL direction by all three selected H. hemerocallidea materials investigated in this study compared to that of the control group, while
indinavir transport was decreased in the BL-AP direction compared to that of the control
group. This clearly showed that active efflux transport of indinavir was inhibited
by the selected H. hemerocallidea materials, albeit not to the same extent of efflux inhibition observed for verapamil.
The effect on the efflux of indinavir in the Caco-2 model depended on the type of
H. hemerocallidea material, which contained different concentrations of hypoxoside. However, the efflux
inhibition did not correlate directly with the hypoxoside concentration and therefore
other phytoconstituents also contributed to this effect. From the in vitro transport results obtained in this study, it can be expected that coadministration
of indinavir (as well as other antiretroviral drugs that are substrates for P-gp)
with H. hemerocallidea extracts and plant materials will most probably lead to enhanced blood plasma levels
of indinavir.
Indinavir is a known substrate for CYP3A4 [25], and H. hemerocallidea extracts have been shown to inhibit CYP3A4 enzyme activity within in vitro models [26], however, this study only focussed on the effect of the modulation of efflux on
indinavir permeation and bioavailability by different H. hemerocallidea materials.
Validation of the LC/MS/MS method provided an accuracy between 94.0 and 106.9%, precision
(coefficient of variance or CV) below 7.2%, and linearity (correlation coefficient
or R2) of 0.999 for indinavir in the quality control samples. The plasma concentration
time curves for indinavir alone and in the presence of the selected H. hemerocallidea materials administered after a single administration (acute study) are shown in [Fig. 3], while the mean plasma concentrations at each time point with standard deviations
are given in Table 1S, Supporting Information.
Fig. 3 Plasma concentration time curves for indinavir alone (40 mg/kg, negative control)
and in the presence of selected H. hemerocallidea materials (15 mg/kg) as well as verapamil (9 mg/kg, positive control); n = 5, error
bars are omitted for reasons of clarity.
The bioavailability parameters for indinavir alone and in the presence of the selected
H. hemerocallidea test materials after a single administration (acute study) are listed in [Table 3]. These bioavailability parameters include the area under the curve extrapolated
to infinity (AUC0-∞) as well as maximum plasma concentration (Cmax) and relative bioavailability (Frel) values.
Table 3 Biopharmaceutical parameters for indinavir administrated to rats in the absence and
presence of the selected H. hemerocallidea materials.
|
Experimental group
|
AUC0-∞ (ng*min/mL)#
|
Cmax (ng/mL)#
|
Frel
|
|
AUC0-∞ = area under the curve, Cmax = maximum plasma concentration, Frel = relative bioavailability. #Average ± standard deviation; n = 5
|
|
Indinavir (40 mg/kg) alone (negative control)
|
1371.0 ± 389.8
|
1108.6 ± 484.3
|
1.00
|
|
Indinavir (40 mg/kg) with verapamil (9 mg/kg, positive control)
|
1501.3 ± 462.2
|
644.1 ± 488.8
|
1.10
|
|
Indinavir (40 mg/kg) with H. hemerocallidea aqueous extract (15 mg/kg)
|
1458.3 ± 512.6
|
813.6 ± 563.5
|
1.06
|
|
Indinavir (40 mg/kg) with H. hemerocallidea commercial product (15 mg/kg)
|
1564.4 ± 638.2
|
928.0 ± 493.0
|
1.14
|
|
Indinavir (40 mg/kg) with H. hemerocallidea reference plant material (15 mg/kg)
|
1456.6 ± 316.3
|
1022.0 ± 412.0
|
1.06
|
From [Table 3] it is clear that coadministration of the selected H. hemerocallidea test materials increased the bioavailability (AUC0-∞) of indinavir in rats compared to the negative control group (indinavir alone), albeit
not statistically significantly (p ≥ 0.05). In accordance with the in vitro transport results, where the H. hemerocallidea commercial product increased the AP-BL transport, the highest of all three of the
selected materials, it also exhibited the highest enhancement of indinavir bioavailability
(AUC0-∞) in the rats during the acute study. Overall, the bioavailability results from the
acute study correlated well with the in vitro transport results in terms of efflux inhibition as expressed by the efflux ratio
results (i.e., ER = 1.42 for the commercial product compared to ER = 2.37 and ER = 2.61
for the aqueous extract and reference material, respectively).
The pharmacokinetic effects of H. hemerocallidea materials on indinavir bioavailability in this study can therefore be attributed
to the inhibition of efflux, and the extent of inhibition was dependent on the type
of H. hemerocallidea material. As mentioned before, modulation of metabolism can also play a role in interactions
between H. hemerocallidea and indinavir, but this was not investigated in this study. The rat serves as a model
for certain cytochrome P450 enzymes (e.g., CYP1A2 and CYP2E1), but not for CYP3A4
[27], which is the most prevalent enzyme for indinavir metabolism in humans.
It can be deduced from this study that concurrent use of H. hemerocallidea poducts or extracts by people living with HIV/AIDS that are on antiviral treatment
(i.e., indinavir) can cause pharmacokinetic interactions that can lead to increased
blood levels of indinavir. However, in order to determine if this effect is clinically
significant, a follow-up study is needed in an animal model that expresses the CYP3A4
enzyme (e.g., pig model) in order to determine the combined effect of efflux inhibition
and metabolism inhibition on the bioavailability of indinavir.
Materials and Methods
Plant materials, chemical compounds, cell line, and growth media
The dried H. hemerocallidea bulb material (i.e., biomass reference material) was purchased from ChromaDex (sample
HHC 001 stored at the Department of Pharmaceutics, North-West University), which was
provided with a certificate of analysis as authenticated with high-performance thin-layer
chromatography. The solid oral commercial product (Hypoxis VegeCaps, batch nr. HYO001,
sample HHC 002 stored at the Department of Pharmaceutics, North-West University) containing
300 mg H. hemerocallidea plant tuber material (total mass of capsule content was 403.74 mg) was purchased
from a local health shop. The aqueous extract was prepared from H. hemerocallidea corms (voucher HH 064 retained at the Department of Pharmaceutical Sciences, Tshwane
University of Technology) by weighing approximately 5 g of dried H. hemerocallidea corm material accurately in a 50-mL Erlenmeyer flask. A volume of 10 mL of deionized
water was added and the mixture was sonicated at 45 °C for 30 min. After filtering
the mixture through a filter paper (No. 4, Whatman), the filtrate was kept aside and
the residue was returned to the flask. This process was repeated twice, where afterwards
the filtrates were combined and the residue discarded. The water filtrate was frozen
and subsequently freeze-dried overnight, and the extract was stored in a desiccator.
All three of the H. hemerocallidea materials were chemically characterised by means of liquid chromatography linked
to mass spectrometry (LC/MS) as described below in the next section.
Crixivan capsules (MSD, batch nr. w076460) containing 400 mg indinavir per capsule (total mass of capsule content was 658 mg)
was purchased from a local pharmacy (sample HHC 003 stored at the Department of Pharmaceutics,
North-West University). Verapamil was donated by Sandoz (South Africa) with a certificate
of analysis stating a purity of 99.3% (sample HHC 004 stored at the Department of
Pharmaceutics, North-West University).
The Caco-2 cell line was obtained from the European Collection of Cell Cultures (ECACC).
The high-glucose DMEM, penicillin, and streptomycin were supplied by Separations.
Whitehead Scientific supplied the nonessential amino acids, L-glutamine, and Trypsin-Versene
mixture, while foetal bovine serum and amphotericin B were obtained from The Scientific
Group.
Chemical characterisation of the Hypoxis hemerocallidea materials by means of LC/MS
Chemical fingerprinting as well as the determination of the quantity of hypoxoside
in each of the three selected H. hemerocallidea materials was done by means of LC/MS. The chromatographic analysis was performed
on a Waters Acquity UHPLC system with a photo diode array (PDA) detector (Waters).
UHPLC separation was attained on an Acquity BEH C18 column (150 mm × 2.1 mm, 1.7 µm particle size; Waters), which was maintained at 40 °C
during analysis. The mobile phase consisted of 0.1% v/v formic acid in water (solvent
A) and acetonitrile (solvent B) with a flow rate of 0.3 mL/min. A gradient elution
was employed as follows: the analysis started with a 85% A:15% B mixture, which was
changed to a mixture of 65% A:35% B in 7 min and then to 50% A:50% B in 1 min, which
was maintained for 0.5 min after which it was changed back to the initial mixture
in 0.5 min. The total running time was 11 min. The standard and samples were injected
into the mobile phase with an injection volume of 1.0 µL (full-loop injection). Mass
spectrometry (MS) was operated in the negative ion electrospray mode. The desolvation
gas was nitrogen (N2), while the desolvation temperature was set to 350 °C at a flow rate of 500 L/h and
the source temperature was set to 100 °C. The capillary and cone voltages were set
to 2500 V and 45 V, respectively. The analytical data were collected between 100 and
1000 m/z.
Bidirectional in vitro permeability across Caco-2 cell monolayers
The Caco-2 cells were cultured in high-glucose DMEM supplemented with 10% foetal bovine
serum, 1% nonessential amino acids (NEAA), 1% penicillin/streptomycin, 1% 2 mM L-glutamine,
and 1% amphotericin B (250 µg/mL). Culturing of the cells occurred at a temperature
of 37 °C with 5% carbon dioxide and 95% humidified air in a Galaxy 170R incubator
(Eppendorf). The growth medium was exchanged every second day under sterile conditions
in a laminar flow hood. The cells were examined by means of a light microscope (Nikon
Eclipse TS100/TS100F, Nikon Instruments) prior to exchange of the growth medium. The
percentage confluency was estimated and the absence of any contamination was ensured.
Subculturing by means of trypsinisation took place once the cells reached a confluency
of 50 – 60%.
Caco-2 cells (passage 50) were seeded onto Transwell 6-well membrane filters (Corning
Costar Corporation) with a surface area of 4.67 cm2 and a pore diameter of 0.4 µm. The cell suspension was obtained through trypsinisation
by adding a Trypsin-versene mixture and then incubated for 5 min. After cell detachment,
6 mL of pre-warmed growth medium were added to the flask. The cell suspension was
extensively agitated with a pipette to ensure complete cell detachment and deagglomeration
in order to form a suspension consisting of single cells. This single cell suspension
was then transferred to a 50-mL tube. A Pasteur pipette was used to agitate the cell
suspension to make sure that a homogenous cell distribution was present. A haemocytometer
was used to count the cells in the suspension after Trypan blue was added. The average
number of cells per square was calculated and this number was then multiplied with
the dilution factor (5 × 104) in order to establish the total number of cells per mL in the cell suspension. The
cell suspension was diluted to a concentration of 20 000 cells per mL, and a volume
of 2.5 mL was pipetted into each apical chamber of the membrane filter plate wells.
The growth medium was exchanged every second day and the cells were cultured for 21 – 24
days until intact epithelial monolayers were obtained.
The transepithelial electrical resistance (TEER) of each Caco-2 cell monolayer was
measured with a Millcell ERS II meter (Millipore) prior to the commencement of the
transport studies. Measurement of TEER is one method that is used to establish tight
junction integrity of Caco-2 cell monolayers and a value of ≥ 150 Ω is considered
sufficient [28]. In this study, a TEER reading of higher than 250 Ω (equivalent to 1167.5 Ω/cm2) was required before commencement of the transport studies [29]. The TEER was measured again at the end of the study to ensure cell monolayer integrity
was maintained during the transport study.
To prepare a 200-µM indinavir solution (negative control), the correct equivalent
mass of a Crixivan capsule content (i.e., 2.02 mg) was weighed and added to 10 mL
DMEM and stirred until dissolved. For the test solutions, a sufficient amount of each
of the selected H. hemerocallidea materials was added to the indinavir solution to reach a concentration of 500 µg/mL
for each of the H. hemerocallidea materials [30]. A 100-µM verapamil solution was prepared by dissolving 0.45 mg verapamil into 5 mL
in the indinavir solution. For the transport in the AP-BL direction, the growth medium
was removed from the basolateral chamber and replaced with a volume of 2.5 mL preheated
transport medium (DMEM) buffered with HEPES. The Transwell plate was then kept in
the incubator for 30 min before the growth medium was removed from the apical chambers
and replaced with 2.5 mL of the test solutions. Samples (200 µL) were withdrawn from
the basolateral chambers at time intervals of 20, 40, 60, 80, 100, and 120 min, and
were replaced with an equal volume of preheated transport buffer. Samples were analysed
by a previously described HPLC analysis method [29] to determine the concentration of indinavir that appeared in the acceptor chamber
over time.
For the transport in the BL-AP direction, the same method was used, except that the
growth medium was removed from the basolateral chambers and replaced with 2.5 mL of
each test solution. Samples (200 µL) were withdrawn from the apical chambers at the
time intervals of 20, 40, 60, 80, 100, and 120 min and immediately replaced with an
equal volume of preheated DMEM.
Indinavir alone (200 µM) served as the negative control group, while indinavir in
combination with 100 µM verapamil, a known P-gp inhibitor, served as the positive
control group. The test solutions consisting of the selected H. hemerocallidea materials (500 µg/mL) in combination with indinavir (200 µM) formed the experimental
groups. All transport experiments were conducted in triplicate.
The Papp values for the transport of indinavir was calculated according to the following equation
[29]:
Where Papp is the apparent permeability coefficient (cm/s), dQ/dt is the permeability rate (amount
permeated per minute), A is the diffusion area of the membrane (cm2), and C0 is the initial concentration of the model drug.
The ER value indicates any asymmetry in the directional transport of indinavir in
combination with the different H. hemerocallidea materials. The ER value was calculated according to the following equation:
Where Papp (B – A) is the permeability coefficient for the permeation in the basolateral-to-apical
direction and Papp (A – B) is the permeability coefficient for the permeation in the apical-to-basolateral
direction.
In vivo bioavailability studies
The in vivo pharmacokinetic study in Sprague Dawley rats [obtained from South African Vaccine
Producers (SAVP)] was approved by the Faculty of Health Sciences Animal Ethics Committee
of the University of Cape Town, South Africa (reference number: 015/041, date of approval:
17/03/2016). All animal experiments were implemented and conducted according to national
and international accepted principles and standards for humane handling of animals.
A total of 25 male Sprague Dawley rats weighing 250 – 300 g was randomly selected
and divided into 5 different groups as summarised in [Fig. 4].
Fig. 4 Layout of the in vivo pharmacokinetic study design in Sprague Dawley rats.
The test solutions consisted of the selected H. hemerocallidea materials at a concentration of 15 mg/kg coadministered with indinavir at a concentration
of 40 mg/kg (based on equivalent Crixivan capsule content), while indinavir (40 mg/kg,
based on equivalent Crixivan capsule contents) was administered alone as the negative
control group. The positive control for efflux inhibition consisted of indinavir (40 mg/kg)
with verapamil (9 mg/kg) [31]. The test solutions were administered by means of oral gavage at a volume of 500 µL
per animal.
Blood samples (200 µL) were collected from the tail veins of the animals at predetermined
time intervals of 0, 0.5, 1, 2, 4, 8, and 24 h after administration of each experimental
and control test solution. Sample tubes were sprayed with heparin beforehand, which
served as an anticoagulant. The blood samples were then centrifuged for 8 min at 14 000 rpm,
after which the plasma was recovered from each blood sample. The plasma samples were
kept at − 80 °C until the indinavir analysis occurred.
A previously described LC/MS/MS method for the analysis of indinavir was used in order
to analyse the plasma samples for indinavir concentration with slight modifications
as outlined below [32].
Briefly, the liquid chromatography method entailed using a Kinetex F5 (4.6 × 100 mm,
2.6 µm) analytical column on an Agilent 1100 series HPLC. The mobile phase consisted
of a mixture of A and B at 50 : 50 v/v, where mobile phase A was 0.1% formic acid
in water and B was 0.1% formic acid in acetonitrile. The mobile phase was delivered
at a constant flow rate of 500 µL/min. The column was kept in a column compartment
at 40 °C. An autosampler injected 10 µL onto the HPLC column. The injection needle
was rinsed with mobile phase before each injection for 30 s using the flush port wash
station. The samples were cooled to 4 °C while awaiting injection.
Detection of indinavir and the internal standard (indinavir-d
6) was performed on an AB Sciex API 3200 mass spectrometer [electrospray ionisation
(ESI) in the positive ion mode] and the settings on the apparatus are summarised in
a previously published method [31]. Furthermore, a stock solution of indinavir was prepared in DMSO at a concentration
of 1 mg/mL. Sprague-Dawley blank plasma samples were spiked with the stock solution
to obtain standard 1 (STD 1) at a concentration of 2000 ng/mL. Dilution of this solution
with blank plasma resulted in STD 2 (500 ng/mL), STD 3 (125 ng/mL), STD 4 (32 ng/mL),
STD 5 (8 ng/mL), and STD 6 (2 ng/mL). Quality control samples were also prepared in
the same pool of rat plasma at 1600 ng/mL, 400 ng/mL, 50 ng/mL, 10 ng/mL, and 2 ng/mL.
The calibration standards and quality control samples were briefly vortexed and aliquoted
into labelled polypropylene tubes, which were stored at − 80 °C until analysis with
LC/MS/MS. Samples above the upper limit of quantification were diluted 4 times with
blank plasma and reanalysed in a repeat batch [32]. The method was validated in terms of linearity, accuracy, and precision.
The extraction was done by adding 200 µL of ice-cold 0.1% formic acid in methanol
containing 20 ng/mL internal standard (indinavir-d6) to a 20-µL sample of plasma,
which was vortex mixed for 60 s. This was followed by ultrasonication for 10 min and
centrifugation at 10 000 rpm for 10 min. Then, 180 µL of the supernatant were transferred
into clean culture tubes and evaporated to dryness under nitrogen gas at 40 °C. The
residue was reconstituted with 100 µL of 0.1% formic acid in water, and vortex mixed
for 60 s. The extracts were transferred into a 96-well plate for injection. A volume
of 10 µL of the reconstituted extract solutions was injected onto the column.
WinNonlin software (Pharsight Corporation) was used in order to construct the indinavir
bioavailability profiles. Relevant pharmacokinetic parameters (i.e., Cmax and AUC0-∞) were obtained. The Frel of indinavir was calculated by using the following equation:
Where [AUC]A represents the area under the curve for indinavir in the presence of the experimental
material (i.e., each selected H. hemerocallidea material) and [AUC]B represents the area under the curve for indinavir alone.
Statistical analysis
Data analyses were performed with STATISTICA Ver 12. ANOVAʼs with Tukeyʼs honest significant
post hoc tests were performed, and statistically significant differences were accepted
when p < 0.05. All results were verified with nonparametric Kruskall-Wallis and Dunnʼs
post hoc tests.
