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Bioavailability of Pentacyclic Triterpene
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189
Pentacyclic triterpenes’ water lipophilicity and insolubility have a
signicant impact on their interactions with components of the gastrointestinal
tract’s absorptive surface. The partition coefcient between water (P
) and
ow
octanol can be used to determine a compound’s lipophilicity, with octanol
having lipophilicity similar to that of cell membranes. The coefcient can
be utilized as one of the predictors of drug absorption by passive diffusion
(Artursson et al., 2001).
Intestinal permeability rises with log P
(Martin, 1981). Conversely, for log P
with log P
because of low aqueous solubility compounds, separation to
ow
ow
until it reaches a plateau at two
ow
of four further, permeability reduces
the extracellular uids from the cell membrane at a lower rate (transcellular
route). Table 6.1 shows the partition coefcients of pentacyclic triterpenes.
Table 6.1: Water/Octanol Partition Coefcients (Pow; Shows as a Log Pow) of
Pentacyclic Triterpenes
Compound Log Pow
Lupeol 7.45
Betulin 6.17
Betulinic acid
Oleanolic acid
Maslinic acid
Ursolic acid
Asiatic acid
Corosolic acid
β-boswellic acid
6.73
6.47
5.52
6.43
5.80
5.51
6.58
Other physicochemical characteristics of the compounds to be
absorbed should also be taken into account like intramolecular H-bonding,
polymorphic forms, H-bonding with the solvent, intermolecular H-bonding,
rate of dissolution, salt form and ionic charge status, crystallinity, molecular
weight, and intermolecular H-bonding (Bérangère et al., 2004; Buckley
et al., 2012). Furthermore, because bioactive compounds are found
in medicinal plants and foods product, a better understanding of their
physicochemical properties could aid in understanding their interactions
with complex matrices. It is widely acknowledged that a molecule should
have no more than ve hydrogen donors’ bond and 10 hydrogen acceptor
bonds, a molecular mass of lower than 500, and a log P of lower than
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Natural Compounds: An Introduction
ve for oral absorption. Triterpenes, like many natural products, are an
exception to the “rule of ve” (Lipinski, 2000). The absorption of bioactive
compounds is undoubtedly inuenced by the activities of gut microbiota
and of the digestive enzymes. In most cases, the small intestine can only
absorb aglycones. Before absorption, bacterial enzymes and intestinal
enzymes in the large intestine will most likely have to hydrolyze glycosides
of pentacyclic triterpenes (Rafat et al., 2008).
The surface area over which absorption takes place and the time
the compound requires to pass in connection with that area are both
determinants that affect the extent of compound absorption. Other factors
that can have an impact include the degree of absorption of a compound,
the pH of the medium from which absorption occurs, the rate of dissolution
of the compound, and host factors, e.g., physiological state, age, infectious
disease state, body secretions, genotype, and nutrient status, are regarded as
extremely important (Xu et al., 2005; Benet et al., 2016).
The metabolism of a drug, which comprises of its biotransformation
into other molecules that are normally more easily excreted in the urine and
more water-soluble, affects its oral bioavailability. These biotransformations
are mostly found in the liver, although they can also be found in lung, brain,
kidney, blood, and gastrointestinal tissue. Drug metabolizing enzymes like
phase I and II are used for catalysis during the biotransformation process in
the liver. Additionally, phase III carriers are in charge of removing rened
and unprocessed materials from cells. These proteins work together to form
a barrier to drug entry and play critical roles in drug distribution, excretion,
and absorption (Yang et al., 2010).
Since determining the bioavailability of pentacyclic triterpene in a
dynamic matrix is fraught with difculties, bioaccessibility is frequently
overlooked in studies on pentacyclic triterpene bioavailability. The
majority of experiments utilize pure compounds (chemically synthesized
or extracted from foods, medical plants), which prevents extrapolation of
the ndings to more realistic contexts in which the important substances are
utilized in a complex matrix. Furthermore, the terms “bioavailability” and
“absorption” are mostly used interchangeably in these experiments, even
though absorption is merely one of the measures included in the transfer of
a substance from its administration site into the circulatory system (Berger
et al., 2003).
In vitro assays, humans, and animal models have been used to
investigate the bioavailability of pentacyclic triterpene. Various methods
Bioavailability of Pentacyclic Triterpene
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have been used to enhance the bioavailability of these substances, including
increasing absorption site afnity or solubility by chemical or technological
modications of the compounds, as well as the design of nanoparticles,
liposomes, and micelles (Cháirez-Ramírez et al., 2015). The results of these
studies are listed in subsections.
191
6.3.1. In Vitro Studies Conducted with Pentacyclic Triterpenes
to Foretell the In Vivo Bioavailability
The intestinal barrier should be crossed by released compounds to enter the
bloodstream. Passive transcellular diffusion, Passive paracellular diffusion,
facilitated transfer by membrane proteins, and exo-, endo-, or trans-cytosis,
and active (carrier-mediated) transport are some of the different transport
ways through which this can happen (Shaik et al., 2012).
To research, the absorption processes of substances given orally, in vitro
models have been created. In vitro non-cellular-based and cellular-based
models, as well as in vivo ndings, show a strong correlation, according to the
studies. In vitro methods for evaluating the permeability of inappropriately
soluble materials have since been optimized to maintain a high degree of
accuracy (Yee, 1997).
Lupeol found in pineapple, green pepper, and strawberries, is a
lupane-type triterpene. Despite its well-known bioactivities, just one
manuscript reported on a lupeol in vitro permeability analysis (Yee et al.,
1997). Permeability tests were performed using a Caco-2 cell monolayer
developed in a bicameral environment in this research. The cell monolayer
is enabled to establish on a permeable membrane that separates two
compartments termed “basolateral” and “apical” in this type of experiment.
The two compartments become physically isolated as these cells polarize
throughout the differentiation process that proceeds conuency and form
close junctions. The intestinal lumen is represented on the apical line, while
the systemic circulation is represented upon on the basolateral side. The
material of concern should then be placed in the apical compartment, and
the system is allowed to handle the substance’s transport for a set amount
of time, usually about one-three hours. Following the incubation period, the
medium on either line and also the cells themselves, are gathered and sent
to be extracted for further quantication of the substance of about concern
(Rajendran et al., 2008; Patlolla and Rao, 2012).
Drug delivery across various pathways through the intestinal epithelium
can be predicted using Caco-2 cell monolayers. Equation (2) is used
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Natural Compounds: An Introduction
to quantify the apparent coefcient of permeability (P
), which is a
app
quantication of a compound’s capacity to reach and cross the intestinal
resistance. CD is the original concentration applied in the apical side (µg/
3
cm
), A is the surface area of the porous membrane (cm2) and Q is the sum
of compounds (µg) transferred over time t (s). Material with P
−6
than 1 × 10
cm/s are considered as poor (0%–20%), between 1 and 1 × 10−6
cm/s are moderately (20%–70%), and greater than 1 × 10
values less
app
−6
cm/s are well
(70%–100%) orally consumed in humans (Jeong et al., 2007).
Chairez-Ramrez et al. (2015) investigated the permeability of lupeol
present in nanoparticles comprising 16% (w/v) lupeol and found that just a
small proportion of lupeol was transferred from its apical to the basolateral
side after 8 hours of incubation. The authors determined that, while there was
no evidence of transport through the Caco-2 cell model, the strongest anti-
inammatory consequences were seen at the strongest dose of actually pure
lupeol (20 µM) and the poorest dosage of the nanonutraceutical substance
(5 M), meaning that this distinction may be due to improved bio-availability
of encapsulated lupeol (Skarke et al., 2012).
One more lupane-type pentacyclic triterpene betulinic acid has an
anticancer and anti-HIV effect. But, because of its poor aqueous solubility,
its effective concentration is lower, and reduced absorption in the
gastrointestinal tract, severely restricting its therapeutic usage. To attenuate
the solubility of the betulinic acid moiety in water, betulinic acid-based
derivatives have been produced. These derivatives were studied using a
non-cellular model labeled the parallel articial membrane permeability
assay (USA, PAMPA™, Watertown, Millipore, MA) to forecast the passive
diffusion of betulinic acid derivatives (Figure 6.4) through a lipid sheet
that mimicked the intestinal lipid bi-layer. The betulinic acid derivatives
had permeabilities ranging from 4.9% to 32.7%. Regarding the threshold of
quantitation (3 g/mL), betulinic acid was not observed utilizing this assay. In
comparison to existing drugs, the authors found that derivatives of betulinic
acid fell into the category of “moderate” to “weak” permeable derivatives
(Figure 6.4) (Sterk et al., 2004; Tong et al., 2011).
Bioavailability of Pentacyclic Triterpene
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Figure 6.4: Rajendran et al. analyzed the chemical compositions of betulinic
acid derivatives.
Source: https://pubmed.ncbi.nlm.nih.gov/17851638/.
193
Utilizing in vitro Caco-2 cells, permeability experiments with oleanolic
acid were also conducted by researchers (Tong et al., 2007). According to
Jeong et al. (2007), the Papp of oleanolic acid at 10 and 20 µM in the apical
−6
to the basolateral direction (1.1–1.3 × 10
low permeability norm atenolol (0.25 × 10
cm/s)) was close to that of the
−6
cm/s), implying that oleanolic
acid is improperly absorbed. Furthermore, Jeong et al. (2007) discovered
that the P
for the basolateral to the apical direction and the Papp for the
app
apical to basolateral direction was not signicantly different, implying that
oleanolic acid is transported through the intestinal membrane by passive
diffusion rather than being efuxed by the transporter (Kaeidi et al., 2011;
Wang et al., 2011).
In vitro transport experiments by utilizing monolayer cell of Caco2 tested an oral stable dispersion of oleanolic acid formulated through
spray freeze drying technology in various formulations. The inclusion of
sodium caprate working as a permeation enhancer in the formulation and
wetting agent. It improved oleanolic acid permeation through the Caco-2
cell monolayer by 2.76 times in 2 hours (p < 0.05). Increased permeability
was accompanied by a decrease in transepithelial electrical resistance, a
commonly used quantitative technique for assessing the integrity of cell
monolayers developed on membrane implants in cell culture models. As a
result, the authors hypothesized that this indicates improved transport via
the paracellular path due to the widening of cellular closed junctions (Cao
et al., 2013).
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Natural Compounds: An Introduction
In Caco-2 permeability tests, oleanolic acid prodrugs (Figure 6.5) were
also tested, and each prodrug demonstrated the subsequent variations in
permeability via the Caco-2 cell monolayer: 9d (1.39-fold), 9a (2.26-fold),
9b (3.31-fold), 7c (2.03-fold), 9c (1.87-fold), 7b (2.10-fold), and 7a (5.27fold). The authors declared that the transepithelial electrical resistance was
changed a small, showing that the close junctions were not widened (Cao et
al., 2013).
Figure 6.5: Cao et al. analyzed the chemical properties of oleanolic acid prodrugs.
Source: https://pubs.acs.org/doi/abs/10.1021/mp300647m.
Ursolic acid, both as a free compound or extract as ethanol from Salvia
ofcinalis L., was utilized in one in vitro permeability study. That study
utilizes intestinal epithelial Caco-2 cell monolayers of a human being.
The ethanol extract of S. ofcinalis contained 2.6 ± 0.4 g/L of ursolic acid,
according to HPLC testing. S. Ofcinalis extract and Ursolic acid were
applied to the bicameral systems of the apical chamber at concentrations
of 2, 5, 10, and 20 µM (non-cytotoxic concentrations), and basolateral
solutions were obtained after 0.5, 1, 2, and 4 hours. The authors looked
at the cellular and apical and compartments after 4 hours. Ursolic acid
absorption as a free substance and in S. Ofcinalis extract improved linearly
from 0.03 ± 0.01 to –0.2 ± 0.04 µM/h/cm2. It was not saturable at any of the
tested concentrations (5–20 M) or time points (0.5–4 h), implying passive
diffusion uptake. Since the permeability coefcients for the ursolic acid-
free compound are 2.8 ± 0.1 × 10
−6
is 2.5 ± 0.4 × 10
cm/s, therefore it is found that no signicant difference
−6
cm/s and for ursolic acid in plant extract
exists between them (Srinivasan et al., 2015).
Yuan et al. (2015) investigated Asiatic acid absorption in an in vitro
model utilizing the Caco-2 cell line. The permeabilities of Asiatic acid were
estimated to be greater than 1 × 10
−5
cm/s after 2 µM transportation of Asiatic
Bioavailability of Pentacyclic Triterpene
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195
acid through the Caco-2 cell monolayer from basolateral to apical and from
apical to basolateral sides, indicating strong absorption. The permeabilities
−5
were higher than 1 × 10
cm·s−1 in a rat intestinal perfusion model,
verifying the absorption ndings in the absorption model of the Caco-2 cell
absorption model (Qiang et al., 2011).
Boswellic acid absorption in human Caco-2 cell lines has been
investigated in a variety of studies. Krüger et al. (2009) investigated the
permeability of 3-acetyl-11-keto-boswellic acid and 11-keto-boswellic
acid, and also their interactions with the following three transporters:
P-glycoprotein, the organic anion transporter polypeptides family member
1B3 (OATP1B3), the multidrug resistance-associated protein 2 (MRP2).
A B. Serrata extract was also tested. The tests were conducted using 9.5
µM of 3-acetyl-11-keto-β-boswellic acid and 10 µM of 11-keto-β-boswellic
acid in the extract and as free compounds. This compound can be graded
as moderately absorbed based on the coefcient of apparent permeability
achieved for totally isolated 11-keto—boswellic acid having a permeability
of 1.69 x 10
6
cm/s. Permeability of 2.14 × 10−6 cm/s was calculated as the
Papp value for this compound in the crude extract. When permeability
experiments were conducted using the extracted compound and the complex
sample, the isolated 3-acetyl-11-keto-boswellic acid was not identied.
The authors stated as well that a substantial depletion of mass balance was
observed with both compounds after absorptive transport trials, but since they
performed control experiments without the Caco-2 monolayer, they claimed
that these compounds were primarily accumulated in and/or adsorbed by the
cell monolayer. These compounds inuenced the behavior of the MRP2 and
OATP1B3 transporters, implying that therapeutic associations with other
anionic drugs are possible. According to the researchers, these compounds
are not P-glycoprotein substrates.
Buckley et al. (2012) conducted permeability experiments on Caco2 cell lines using 3-acetyl-11-keto-boswellic acid and 11-keto-boswellic
acid. These researchers created formulations that improved 54-times the
solubility of boswellic acids. In contrast to an un-formulated extract, one
of the formulations (extract-phospholipid complex) enhanced the mass ux
of 3-acetyl-11-keto-boswellic and 11-keto-boswellicacids respectively by 8
and 15 times (Juan et al., 2008; Yin et al., 2012).
Lipinski (2000) carry out permeability tests of other boswellic acids in
Caco-2, and the resulting coefcients of apparent permeability are seen in
Table 6.2. The permeabilities of 3-acetyl-11-keto-boswellic and 11-keto-
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Natural Compounds: An Introduction
boswellic acids had been underrated in previous studies, according to these
scientists, and their utilized Caco-2 model. The bovine serum albumin
incorporation to the basolateral side and the utilization of modied fasted
condition simulated the apical side of intestinal uid brought it nearer to the
physiological environment, allowing for a greater estimation of absorption
in vivo. Depending on the coefcient values of apparent permeability
stated by Yee et al. (1997) substances are classied as poorly, fairly, or well
absorbable. As per the ndings of researchers, 3-acetyl-11-keto-boswellic
and 11-keto-boswellic acids are well absorbable almost 70–100% (Tausch
et al., 2009).
Table 6.2: Gerbeth et al. Published the Mean of Apparent Permeability Coef-
cient Values for Boswellic Acids
Compound
11-keto-β-boswellic acid
3-acetyl-11-keto-β-boswellic acid
β-boswellic acid
3-acetyl-β-boswellic acid
α-boswellic acid
3-acetyl-α-boswellic acid 4.72
Papp Value
cm/s
29.54
17.83
4.47
6.18
5.52
× 10
−6
6.3.2. In Vivo Bioavailability of Bioactive Pentacyclic
Triterpenes
Rajendran et al. (2008) have documented the bioavailability of betulinic acid
in vivo studies. After even a 500-mg/kg intraperitoneal injection to mice,
researchers conducted a pharmacokinetic analysis on betulinic acid and
found that it is broadly spread in many tissues. Serum specimens were tested
after a betulinic acid dosage of 250 or 500 mg/kg was given intraperitoneally.
For pharmacokinetic modeling, a two-compartment, first-order model
was used. When betulinic acid was tested in an improved formulation in
spray-dried mucoadhesive microparticles, researchers observed an increase
in pharmacokinetic parameters (Razboršek et al., 2008). When tested in
metastatic tumor models and mice orthotopic lung cancer A549 models,
these authors found a substantial improvement in the betulinic acid oral
bioavailability (Table 6.3), which culminated in a greater anticancer impact.
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197
The weights and volumes of lung tumors were substantially decreased after
three weeks of oral administration of this formulation at a dosage of 100 mg/
kg (Siddiqui et al., 2007).
Many studies have focused on various formulation methods for betulinic
acid, such as complexation with beta-cyclodextrin, gamma-cyclodextrin,
and nanoemulsion, but the authors only reported an increase in the anticancer
activity in in vitro experiments on tumors and in vivo tests on experimental
models of animals in these manuscripts (Raval et al., 2015).
Various betulinic acid derivatives of the pharmacokinetics have also
been investigated, and their chemical compositions are shown in Figure 6.4.
Dependent on in vitro ndings, Rajendran et al. (2008) chose a dihydro-
betulinic acid derivative altered at the C-3 position (4-nitrobenzyl-oximino)
to conduct an in vivo assay utilizing male Wistar rats (Derivative 1 in Figure
6.2). pharmacokinetic modeling based on non-compartmental analysis
was chosen, and the recorded results are summarized in Table 6.3. In the
xenograft model of human colon cancer, the investigators discovered that
Derivative 1 had greater pharmacokinetic properties and in vivo antitumor
efcacy than betulinic acid (Figure 6.6).
Table 6.3: Pharmacokinetic Parameters of Betulinic Acid and Betulinic Acid
Derivatives in Various Formulations
Compound Species
Betulinic
acid
Betulinic
acid
Betulinic
acid
Betulinic
acid
BA-SD Rat (plasma) 100 Oral 53.86 ± 7.79 4.54 ±
23-Hydroxybetulinic acid
Bevirimat Human
Derivative 1 Rat (plasma) 10 Intravenous 43.6 ± 6.3 101.5 ±
(Sample)
Mice (serum)
Mice (serum)
Mice (skin) 500 Intraperitoneal 3504.0 300.9 3.90
Rat (plasma) 100 Oral 7.26 ± 1.65 1.16 ±
Mouse
(plasma)
(plasma)
Dose (mg/
kg)
500 Intraperitoneal 39.9 4.00 0.22
250 Intraperitoneal 18.4 2.21 0.15
200 Intragastric 24.9 3.1 2
200 Oral 1113.7 ±
Route of Administration
AUC
→∞
0
(µg·h/mL)
216.7
C
(µg/
max
mL)
0.22
0.25
58.0 ±
10.83
21.7
T
max
2.36 ±
0.38
3.17 ±
0.85
1.50
(0.8–3.0)
0.05 ±
0.0
(h)
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Natural Compounds: An Introduction
Note: BA-SD: betulinic acid in spray-dried mucoadhesive microparticles;
0,30
bevirimat: 3-O-(3
(AUC
innity time; C
∞): area under the plasma concentration-time curve from zero to
0→
max
-dimethylsuccinyl)-betulinic acid; Derivative 1;
: maximum plasma concentration; T
: time to maximum
max
concentration. Values are expressed as the mean ± standard error of the
mean when available.
Figure 6.6: Chemical structure of Bevirimat and 23-hydroxy botulinic.
Source: https://pubmed.ncbi.nlm.nih.gov/15915449/.
Quantitative study after intragastric administration of 23-hydroxy
botulinic acid in mouse plasma, Yang et al. (2010) established a new assay
dependent on mass spectrometry/liquid chromatography. In modeling
pharmacokinetic, a two-compartment, rst-order model was chosen,
and the recorded data are also shown in Table 6.3. The removal half-life
of 23-hydroxy botulinic acid was reported to be 25.6 hours, with low
bioavailability of 2.3% (Judy et al., 2003).
Derivative of betulinic acid called Bevirimat [3-O-(30,
30-dimethylsuccinyl)-betulinic acid] presents a successful class of antiHIV doers with a unique mechanism of action. It prevents the HIV-1
aging process by preventing the conversion from p25 to p24 functional,
ending HIV-1 particles in noninfectious form. Martin (1981) measured
AUCp.o for various oral doses of bevirimat and found that the compound
has pharmacokinetics that is dose-proportional after 10 days of repetitive
dosing. Non-compartmental approaches were used to estimate the parameter
of pharmacokinetic for bevirimat, and the observed results for Day 10 are
shown in Table 6.3. As per the literature, betulinic acid is 99.99% attached to
serum proteins in rat, dog, and mouse plasma (Jeong et al., 2007).
Cao et al. (2013) recorded pharmacokinetic characteristics of oleanolic
acid having two amino acid ester prodrugs and oleanolic acid after oral
administration to rats who obtained oleanolic acid of 300 mg/kg and its
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