Key words
Boswellia serrata
- Burseraceae - boswellic acids - derivatization - pharmacokinetics - analysis methods
- new formula
Abbreviations
AKBA:
3-acetyl-11-keto-β -boswellic acid
Akt:
serine/threonine kinase
α -ABA:
3-acetyl-α -boswellic acid
β -ABA:
3-acetyl-β -boswellic acid
APCI:
atmospheric pressure chemical ionization
API:
atmospheric pressure ionization
ATM:
ataxia-telangiectasia mutated
BA:
boswellic acid
α -BA:
α -boswellic acid
β -BA:
β -boswellic acid
BA145:
3-O-n-butyryl-11-keto-b-boswellic acid
BA-PC:
a complex of boswellic acid prepared with phosphatidyl-choline
BCDD:
butyl2-cyano-3,11-dioxours-1,12-dien-24-oate
BKBA:
butyryloxy-11-keto-β -boswellic acid
BOBA:
3-α-butyryloxy-β -boswellic acid
BSE:
Boswellia serrata extract
catG:
cathepsin G
C–Kβ BA:
3-cinnamoyl-11-keto-β -boswellic acid
Cmax
:
maximum concentration
CEMB:
2-cyano, 3-enone of methyl boswellates
COX-1:
cyclooxygenase-1
COX-2:
cyclooxygenase-2
CXCR4:
CXC chemokine receptor type 4
CYP3A4:
cytochrome P450, family 3, subfamily A, polypeptide 4
DNA:
deoxyribonucleic acid
EGFR:
epidermal growth factor receptor
ERK2:
signal-regulated kinase 2
ESI:
electrospray ionization
GC-NICI/MS:
gas chromatography-negative ion chemical ionization-mass
HL-60 cell:
human promyelocytic leukemia cells
HLE:
human leukocyte elastase
HLM:
human liver microsomes
HKBA:
hexanoyloxy-11-keto-β -boswellic acid
HMEC:
human microvascular cell
HPTLC:
high-performance thin-layer chromatographic
Hsp:
heat shock protein-90
IBD:
inflammatory bowel disease
KBA:
11-keto-β -boswellic acid
5-LOX:
5-lipoxygenase
LCMS:
liquid chromatography-tandem mass spectrometry
LPS:
lipopolysaccharide
LTB4
:
leukotriene B4
LT:
leukotriene
MAPK:
mitogen-activated protein kinase
MeSH:
medical subject headings
miRNA:
microRNA
MMP:
matrix metalloproteinase
mPGES1:
microsomal prostaglandin E2 synthase-1
MRP2:
multidrug resistant protein
mTOR:
mammalian target of rapamycin
NF-κ B:
nuclear factor κ B
NO:
nitric oxide
OA:
osteoarthritis
OATP1B3:
organic anion transporting polypeptides, member 1B3
PARP:
poly ADP-ribose polymerase
PBCEC:
porcine brain capillary endothelial cells
PGE2:
prostaglandin E2
Pgp:
P-glycoprotein
PI3K:
phosphoinositide-3-kinase
PKBA:
propionyloxy-11-keto-β -boswellic acid
p12-LO:
platelet-type 12-lipoxygenase
PMNL:
polymorphonuclear leukocytes
POBA:
3-α -propionyloxy-β -boswellic acid
PUMA:
p53 upregulated modulator of apoptosis
RLM:
rat liver microsomes
RNA:
ribonucleic acid
ROS:
reactive oxygen species
SAR:
structure-activity relationship
STAT:
signal transducer and activator of transcription protein
tid:
ter in die (Latin: three times a day)
2TNF-α
:
tumor necrosis factor-α
TPD:
triterpenediol
VCAM-1:
vascular cell adhesion molecule-1
VEGF:
vascular endothelial growth factor
VLB:
human lymphocytic leukaemia cell line
Wnt:
Wingless-Int
Introduction
Frankincense has been considered throughout the ages to have a wealth of medicinal
supporting properties. It has been widely used in India, China, and African countries,
and has become popular in Western countries in the last two decades. Frankincense
is the common name given to oleogum resins of Boswellia species (Burseraceae: approximately 20 species) [1 ], [2 ]. In fact, the most important frankincense-producing species are Boswellia serrata in Northwestern India and Boswellia carterii in Africa (Northern Somalia, Sudan, Eritrea, and Ethiopia). Large amounts of experimental
data from in vitro studies and animal models support the potential of different extracts or constituents
from frankincense for the treatment of various diseases, and BAs are considered the
most bioactive constituents of frankincense. Thus, BAs and their bioactivities, including
their principal activities, i.e., anti-inflammatory and antitumor properties, as well
as many other pharmacological activities, such as antiulcer, immunomodulatory, hypolipidemic,
antibacterial, and antiobesity effects [3 ], [4 ], [5 ], [6 ], [7 ], [8 ], [9 ], [10 ], [11 ], have become major research priorities. Based on this background, extracts from
frankincense and BAs have been considered a promising alternative based on several
clinical trials, including trials for the treatment of IBD, Crohnʼs disease, ulcerative
colitis [12 ], [13 ], osteoarthritis [14 ], [15 ], [16 ], rheumatoid arthritis [17 ], asthma [18 ], photoaged skin [19 ], and peritumoral brain edema [20 ]. In addition, BAs-rich extracts from B. serrata have been introduced into medicinal commodities, such as H15 [20 ], 5-Loxin [14 ], and Aflapin [15 ], [21 ]. Moreover, BSE was assigned orphan drug status in 2002 by the European Medicines
Agency for the treatment of peritumoral edema.
The anti-inflammatory and antitumor activities of BAs are the most extensively researched
activities in experimental and clinical studies [4 ], [22 ], and different functional groups have the potential to influence the pharmacological
mechanisms of BAs [23 ], [24 ]. Multiple mechanisms underlying their anti-inflammatory, antitumor, and other biological
activities mainly contribute to AKBA, thereby resulting in the enrichment of AKBA
in medicinal commodities, such as 5-Loxin (BSE enriched with 30 % AKBA) [14 ] and Aflapin [21 ] (BSE enriched with at least 20 % AKBA). Thus, many research studies have attempted
to increase the diversity of the biological activities of other BAs, such as KBA and
β -BA, which also have strong anti-inflammatory but poor antitumor activity. However,
various pharmacokinetic studies using different standard analytical methods ([Table 1 ]) have revealed the low bioavailability of BAs, particularly AKBA and KBA, in human
and animals, which results in questions regarding their pharmacological relevance
for in vivo bioactivities. Despite the widespread use of preparations from frankincense, only
limited studies have attempted to resolve the poor bioavailability of BAs. Nevertheless,
the sparse approaches that have addressed this issue indicate that certain improvements
have been achieved, particularly in the application of new formulas to enhance the
pharmacological activities by increasing the availability of BAs in the plasma and
brain [25 ], [26 ].
Table 1 Plasma and brain concentrations of boswellic acids determined in different studies.
Dosage of BSE
Methods
Concentrations of BAs determined in plasma (µM) or brain* (ng/g)
Ref.
β -BA
α -BA
β -ABA
α -ABA
KBA
AKBA
H = human; R = rats; NA = not analyzed; ND = not detectable; a = approximation; b = mean ± SE;
c = absolute contents; d = [range]; e = steady state concentration; f = mean ± SD;
g = mean; h = Cmax
1600 mg/day (n = 1)H
HPLCa,h
NA
NA
NA
NA
1.7
NA
[80 ]
333 mg/day for 7 d (n = 12)H
HPLCb,h
NA
NA
NA
NA
2.72 ± 0.18
ND
[81 ]
4 × 786 mg/day for 10 d (n = 1)H
HPLCc
10.1
3.5
2.4
4.0
0.3
0.1
[82 ]
3 × 800 mg/day for 4 wk (n = 3)H
HPLC-ESI/MSb,e
6.35 ± 0.52
NA
4.9 ± 0.5
NA
0.3 ± 0.1
0.04 ± 0.01
[41 ]
4200 mg/day (n = 14)H
HPLC-ESI/MSd
[0.19–26.20]
[0.08–10.59]
[0.26–12.31]
[0.14–5.99]
[0.01–0.52]
[0–0.03]
[83 ]
240 mg/kg (n = 3)R
HPLC-ESI/MSf,h
2.33 ± 1.02
1.19 ± 0.63
1.65 ± 0.11
0.48 ± 0.06
0.38 ± 0.15
0.33 ± 0.16
[84 ]
240 mg/kg (n = 6)R *
HPLC-ESI/MSg
1066.6
485.1
163.7
43.0
11.6
37.5
240 mg/kg (n = 9)R *
HPLC-APCI/MSh
NA
NA
NA
NA
0.4
0.2
[85 ]
240 mg/kg (n = 9)R *
HPLC-APCI/MSh
NA
NA
NA
NA
99
95
500 mg/day (n = 6)H
HPTLCd
NA
NA
NA
NA
NA
[0.05–0.13]
[87 ]
In this review, we provide the first summary of the more active derivatives from different
BAs. In addition, the available pharmacokinetic studies and approaches for enhancing
the bioavailability of BAs are summarized exhaustively and systematically.
Methods
Data sources and search strategy
Primary studies were identified by searching electronic databases (PubMed, Web of
Science, Wiley, and Medline), and the search terms (frankincense, boswellic acids,
Boswellia extract, derivatives, pharmacokineticsm and systematic reviews) were mainly determined
using a combination of MeSH and free-word terms in the individual databases. The results
were initially screened by title to exclude any obviously irrelevant articles, and
the potential hits were downloaded into NoteExpress 2 files. No language restrictions
were applied. Publications up to the end of April 2014 were included in the review.
Selection criteria and data collection
The abstracts of the selected articles were examined independently by two reviewers
who applied the selection criteria. All possible articles were retained, and their
full text was critically reviewed. Abstracts reporting incomplete data for evaluation
were excluded. Publications with complete experiments, credible data, and a certain
reputation were selected. The inclusion articles relevant to our subject (bioactive
molecular targets, synthetic derivatives, pharmacokinetics, and the new technologies
of preparation of BAs) were further selected.
Structure-Activity Relationships of Boswellic Acids and Synthetic Derivatives
Structure-Activity Relationships of Boswellic Acids and Synthetic Derivatives
Interaction of boswellic acids with molecular targets
BAs are generally defined as a bioactive mixture of four major beta- and two alpha-pentacyclic
triterpene acids: β -BA (1 ), α -BA (2 ), β -ABA (3 ), α -ABA (4 ), KBA (5 ), and AKBA (6 ). Their structures are shown in [Fig. 1 ]. BAs ([Fig. 1 ]) exist as ursane (α ) and oleanane (β ) types depending on the position of two methyl groups on C-19/C-20 (α -types: geminal groups on C-20; β -types: vicinal groups on C-19/C-20). Further structural variety can be achieved by
the presence or absence of a carbonyl group on C-11 and an acetoxy group or a hydroxyl
group on C-3. A series of pharmacological studies indicated that β -types exert stronger bioactivities compared with the corresponding α -types [27 ]; moreover, different functional groups have a potential impact on the pharmacological
activities of BAs.
Fig. 1 Chemical structures of the main boswellic acids. The characteristic structure of
α -types is marked in blue. (Color figure available online only.)
BAs are pharmacologically active ingredients of frankincense with anti-inflammatory
properties. Through a series of in vitro experiments, KBA and AKBA, possessing an active carbonyl group at C-11, have long
been considered the major contributors to the anti-inflammatory effect of frankincense.
KBA and AKBA are selective and non-redox inhibitors of 5-LOX, and AKBA was the most
efficient BA tested, with IC50 values of 1.5–50 µM (concentration required for 50 % inhibition of 1.5 µM). In addition,
BAs that lack the 11-keto function inhibited 5-LOX incompletely and exhibited maximal
inhibitory activity against 5-LOX (60 % inhibition) at concentrations of approximately
15 µM, and this maximal activity could not be augmented even with an increase in concentration
up to 50 µM [8 ], [23 ], [28 ], [29 ], [30 ], [31 ], [32 ]. SARs have shown that the 11-keto function is essential for 5-LOX activity, whereas
the acetoxy group on position C-3 increases the affinity of AKBA to its effector site
[23 ]. In addition to the intervention with LTs via the inhibition of 5-LOX, AKBA and
KBA possess potential cyclooxygenase-1 inhibitory activity (AKBA: IC50 = 6 µM; KBA: IC50 = 14 µM), but β -BA and β -ABA showed weak inhibitory activity (IC50 > 100 µM) [33 ]. AKBA and KBA are able to activate p38 MAPK and p42/44 MAPK and stimulate Ca2+ mobilization in PMNL [34 ], [35 ]. In addition, AKBA is able to decrease the activity of HLE (IC50 = 15 µM) [36 ] and inhibit p12-LO (IC50 = 15 µM) and is directly bound to this enzyme in vitro
[37 ]. 5-Loxin (standardized BSE enriched with 30 % AKBA) was found to prevent MMPs and
the inducible expression of mediators of apoptosis of TNF-α -inducible genes, resulting in anti-inflammatory effects, and completely abrogated
the TNF-α -induced VCAM-1 expression in HMECs [38 ]. Obviously, compared with KBA, AKBA has an acetoxy group at C-3, which may increase
the above activities.
The analysis of the relevant anti-inflammatory activity showed that, in comparison
to AKBA and KBA, β -BA possessing a 3-hydroxyl function not only evoked moderate Ca2+ mobilization and activated p38 MAPK but also induced the phosphorylation of ERK2
and Akt [39 ]. β -BA acts selectively in part via the inhibition of PGE2 by directly and functionally
interfering with mPGES1 in human whole blood (IC50 = 3 µM), but AKBA and KBA fail to suppress PGE2 synthesis [40 ]. β -BA is able to suppress LPS activity (IC50 = 1.8 µM) directly, whereas KBA and AKBA, possessing an active C-11 carbonyl group,
and β -ABA, lacking a C-11 carbonyl group but possessing a C-3 acetoxy group, failed in
this regard. The SAR indicated that the C-3 acetoxy group and the C-11-keto moiety
may hamper the bioactivity, and the C-3 hydroxyl function may be a determinant for
the inhibition of LPS [24 ]. Interestingly, β -ABA (IC50 = 1.2 µM) was shown to be equipotent to β -BA (IC50 = 0.8 µM) for the reduction of catG activities, whereas KBA (IC50 = 3.7 µM) was less potent, and catG was suppressed by AKBA at the lowest concentration
(IC50 = 0.6 µM) [41 ].
AKBA possesses antiproliferative and apoptotic effects in various tumor cells by increasing
caspase-8, caspase-9, and caspase-3 activities accompanied by the cleavage of PARP
[42 ], [43 ], [44 ], activation of the PI3K/Akt pathway, and inhibition of the PI3K pathway [45 ]. AKBA is able to enhance apoptosis and inhibit the growth, metastasis, and angiogenesis
of tumors. It was also correlated with the potential inhibition of the NF-κ B system [46 ], [47 ], [48 ], downregulation of the expression of COX-2, MMPs, CXCR4, and VEGF [47 ], [48 ], [49 ], [50 ], [51 ], and the modulation of the Wnt/β -catenin [52 ], EGFR, and ATM/P53 signaling pathways [53 ]. AKBA has also been found to bind and inhibit topoisomerases I and Iiα
[54 ]. With the application of gene chip technology, a panel of genes from diverse functional
groups was significantly associated with sensitivity to β -BA and α -BA, as well as transcription factors and signal transduction [55 ]. β -BA, β -ABA, KBA, and AKBA were examined for their antitumor activity in vitro , and it was found that these inhibit the synthesis of DNA, RNA, and protein in human
leukemia HL-60 cells in a dose-dependent manner with IC50 values ranging from 0.6 to 7.1 µM [56 ], [57 ]. In particular, AKBA induces the most pronounced inhibitory effects on DNA, RNA,
and protein synthesis with IC50 values of 0.6, 0.5, and 4.1 µM, respectively [56 ]. In addition, AKBA exerts its antitumor effect in CRC cells by modulating specific
miRNAs within the let-7 and miR-200 miRNA families [58 ] and demethylating and reactivating methylation-silenced tumor suppressor genes [59 ]. Moreover, AKBA also possesses potential antimicrobial [10 ], [11 ], [59 ], [60 ], antiulcer [50 ], [61 ], and lipolysis-inducing activities [9 ]. The other BAs exhibit weaker effects or even fail to present most of the aforementioned
activities.
New synthetic boswellic acids analogues/derivatives
Despite KBA and β -BA possessing multiple mechanisms of anti-inflammatory activity, other pharmacological
activities, especially the antitumor effect, were unsatisfactory. With the purpose
of enhancing and extending more extensive activities of BAs, especially as KBA, β -BA, and the α -type of BAs, applications of chemical modification and biotransformation have been
considered as a possible mean of transformation of BAs for bioactive derivatives.
Structures of selected screened maternal and relevant derivatives are shown in [Fig. 2 ] (analogues/derivatives of α - or β -BA), [Fig. 3 ] (analogues/derivatives of KBA), and [Fig. 4 ] (analogues/derivatives of α -ABA and AKBA).
Fig. 2 Chemical structures of α - and β -boswellic acid analogues/derivatives. The characteristic structure of α -types is marked in blue. The introduced functional groups and their corresponding
positions are marked in red. (Color figure available online only.)
Fig. 3 Chemical structures of 11-keto-β -boswellic acid analogues/derivatives. The introduced functional groups and their
corresponding positions are marked in red. (Color figure available online only.)
Fig. 4 Chemical structures of 3-acetyl-α -boswellic acid and 3-acetyl-11-keto-β -boswellic acid analogues/derivatives. The characteristic structure of α -types is marked in blue. The introduced functional groups and their corresponding
positions are marked in red. (Color figure available online only.)
α - and β -Boswellic acid analogues/derivatives
β -CEMB (7 ) and α -CEMB (2-cyano, 3-enone of methyl boswellates) (8 ) are modified from β -BA and α -BA and show potent cytotoxic (a number of cancer cell lines with IC50 s ranging from 0.2 to 0.6 µM), growth inhibition, anti-inflammatory, and pro-differentiative
activities. CEMB inhibited DNA synthesis and induced apoptosis in A549 cells (lung
carcinoma cell line) at concentrations of 0.25 µM and 1 µM, respectively, and this
action is mediated by caspase-8 [62 ].
POBA (9 ) is the propionyloxy derivative of β -BA and exhibits significant cytotoxic activity on a wide range of human cancer cell
lines via potential suppression of the PI3K pathway [63 ].
BOBA (10 ) is the butyryloxy derivative of β -BA and provides evidence of PI3K-mediated apoptosis. This novel compound was developed
in an attempt to discover anticancer therapeutics and has the potential to target
the mitochondria-dependent pathway, NF-κ B, and the DNA repair system in apoptosis [64 ].
3β ,24-dihydroxyolean-12-ene (11 ) and 3α ,24-dihydroxyurs-12-ene (12 ) are obtained by the reduction reaction from β -BA and α -BA. TPD, which is a mixture composed of 11 and 12 , produced oxidative stress in prostate cancer cells that triggers self-demise by
the ROS- and NO-regulated activation of both the intrinsic and extrinsic signaling
cascades [65 ].
The rhein-boswellic acid conjugate 13 is synthesized chemically by two molecules of α -BA and one molecule of rhein through a bioreversible ester linkage [66 ]. The results of the evaluation of the disease-modifying effects of the conjugate
in collagenase (type-II)-induced OA in Wister rats revealed significant antiarthritic
and antiulcer activities [67 ].
4-Amino analogues 14 and 15 were screened and prepared from β -BA, wherein the carboxyl group in the ursane nucleus was replaced by an amino function
via the Curtius reaction. Compound 14 showed cytotoxicity (80–96 %) on prostate (DU-145) and colon (SW-620 and 502 713)
cell lines, and compound 15 showed cytotoxicity (73–99 %) against prostate (DU-145) and colon (SW-620, 502 713,
HT-29) cell lines [68 ].
11-Keto-β -boswellic acid analogues/derivatives
4-Amino analogues 16 and 17 were screened and prepared from KBA using the method mentioned above [68 ]. Compound 16 showed cytotoxicity (72 %) against colon cell lines (SW-620), and compound 17 showed cytotoxicity (71–94 %) against prostate (DU-145) and colon (SW-620, 502 713,
and HT-29) cell lines. Furthermore, the results of the analysis of the cytotoxicity
of analogues 14, 15 , and 17 at a concentration of 1 µM indicated that compound 17 is the most promising analogue that displays significant activity. Compounds 14 to 17 display improved cytotoxicity compared with the parent compound and also exhibit
apoptotic activity by inducing DNA fragmentation.
An endoperoxide (18 ) was synthesized from KBA. It has been shown that this compound is an initiator of
programmed cell death and has shown high antitumor activity against a panel of 15
human cancer cell lines. In fact, compound 18 induces apoptosis with an average IC50 value of 0.4–4.5 µM [69 ].
BCDD (19 ) is a cyano derivative of KBA and has been found to be highly cytotoxic against human
papillomavirus (HPV)-positive cervical cancer cells, HeLa (HPV18) and SiHa (HPV16)
cells. The detailed mechanism of the cytotoxicity in HeLa cells suggests that BCDD
causes apoptosis, regulates the expression of p53/PUMA/p21 and affects other cell
survival signaling events [70 ]. Moreover, it inhibits PI3K/AKT activity and enhances NF-κ B and Hsp-90 in HL-60 cells, which may increase the sensitivity of cells to apoptosis
[71 ].
HKBA (20 ), BKBA (21 ), and PKBA (22 ) are active derivatives synthesized from KBA [72 ], [73 ]. The IC50 values obtained for these compounds in the induction of apoptosis in HL-60 cells
have been found to be 4.5, 7.7, and 8.7 µg/mL, respectively. In particular, HKBA presents
better antiproliferative and proapoptotic potential against the HL-60 cell line than
the previously known boswellic acids due to the inhibition of topoisomerases I and
II [72 ].
BA145 (23 ) was synthesized from KBA and was screened as a promising lead that exhibited cytotoxicity
against various human cancer cell lines by inhibiting the NF-κ B and STAT family of proteins [74 ]. A further study indicated that apoptosis induced by BA145 in HL-60 cells is completely
dependent on caspases, and the MEK1/2 inhibitor PD98059 and the PI3K inhibitor LY294002
protect HL-60 cells by diminishing the activation of pre-executioner caspases-8 and
-9 by BA145 [75 ].
C–Kβ BA (24 ) was synthesized from KBA and exhibited specific antiproliferative and proapoptotic
effects in cancer cell lines in vitro and in PC-3 prostate cancer xenografts in vivo , even at low concentrations and after a single, rather short exposure. These effects
have been attributed to its inhibition of the mTOR signaling pathway [76 ].
7-β -hydroxy-11-keto-β -boswellic acid (25 ) and 7β ,22-β -dihydroxy-11-keto-β -boswellic acid (26 ) were screened from metabolites of the biotransformation of KBA by Cunninghamella blakesleana and exhibited IC50 values of 25.8 and 12.5 µM, respectively, by the stronger inhibition of NO production
induced by LPS without decreasing cell viability in RAW 264.7 macrophages [77 ].
3-Acetyl-α -boswellic acid analogues/derivatives
3α -Acetyl-11-keto-α -boswellic acid (27 ) was synthesized by a radical-type reaction using bromine and α -ABA in an α -configuration in contrast with AKBA. 3α -Acetyl-11-keto-α -boswellic acid triggers apoptosis in chemoresistant and androgen-independent cancer
cells in vitro and in vivo by the activation of caspase 3 and the induction of DNA fragmentation [78 ].
3-Acetyl-11-keto-β -boswellic acid analogues/derivatives
7-β -Hydroxyl-3-acety-11-keto-β -boswellic acid (28 ) and 7-β -16α -dihydroxy-3-acety-11-keto-β -boswellic acid (29 ) were screened from metabolites of the biotransformation of AKBA by C. blakesleana and exhibited IC50 values of 1.5 and 10.5 µM, respectively, compared with the IC50 value of 15.6 obtained for AKBA, due to the stronger inhibition of NO production
induced by LPS and cytotoxicity in RAW 264.7 macrophages [79 ].
The above-described derivatives demonstrate considerable bioactivities, particularly
antitumor activity, and are even stronger than their parents. As shown in [Fig. 2 ] to [4 ], the addition of functional groups has mainly focused on the transformation of the
hydroxyl group at C-3 and the carboxyl group at C-24, and the other nonfunctional
groups of the carbon atoms were added to a hydroxyl group. Other advantages are that
the simultaneous screening of derivatives can provide the active sites and selectable
bioactive target structures. Investigations of the safety, pharmacokinetics, and other
bioactivities of the derivatives remain to be conducted in the future.
Available Pharmacokinetic Studies of Boswellic Acids
Available Pharmacokinetic Studies of Boswellic Acids
Despite the widespread use of preparations from frankincense and BAs, it is important
to emphasize that the pharmacokinetic properties of a drug are very important for
exerting an effective role in vivo . Pharmacokinetics is a subject that studies the kinetic process of absorption, distribution,
metabolism, and excretion of drugs in the body. The concentration of the drug in the
plasma and the target organ lesions is a very important pharmacokinetic parameter
that can be used to measure whether the drug can achieve an effective concentration.
The detection of the concentrations of BAs in the plasma and brain has been published
in previous articles, and different methods have different sensitivity, accuracy,
precision, and concentration ranges. In this review, we summarize the different methods
used for measuring the concentration of BAs by administering different oral doses
in animals (plasma and brain) and humans (plasma), and discuss the sensitivity and
measurement ranges for the determination of different BAs. The determined concentrations
of BAs in the plasma and brain, the subjects (animals or humans), dosage, and relational
analysis methods are shown in [Table 1 ].
High-performance liquid chromatography analysis
The concentrations of KBA in the plasma of a volunteer who was administered a single
dose of 1600 mg of a BSE formulated as a hard gelatin capsule were determined by HPLC,
and a Cmax of approximately 1.7 µM was obtained after 1 h. A linear quantification range from
0.1 and 2.0 mg/mL for KBA in the plasma was established, and the detection limit of
the method for KBA was approximately 0.040 µ/mL [80 ]. Twelve healthy adult men volunteers were administered the WokVel™ capsule (333 mg
BSE) containing 18.51 % β -BA, 6.93 % α -BA, 8.58 % β -ABA, 1.85 % α -ABA, 6.44 % KBA, and 2 % AKBA for seven days, and the Cmax of KBA determined by HPLC reached 2.72 × 10−3 ± 0.18 µM after 4.55 ± 0.55 h [81 ]. The plasma levels of a patient with a brain tumor treated for ten days with a dose
of BSE (4 × 786 mg/day) containing 19.22 % β -BA, 13.78 % α -BA, 10.04 % β -ABA, 3.37 % α -ABA, 6.66 % KBA, and 3.81 % AKBA were analyzed. The absolute contents of β -BA, α -BA, β -ABA, α -ABA, KBA, and AKBA were determined by HPLC to be 10.1 µM, 3.5 µM, 2.4 µM, 4.0 µM,
0.34 µM, and 0.1 µM, respectively. Linear quantification ranges of plasma levels of
0.02 to 1.1 µg/ml and 0.16 to 9 µg/ml were established for the six main BAs and the
other six pentacyclic triterpenic acids [82 ].
Liquid chromatography-mass spectrometry analysis
LC-MS is considered a more sensitive detection method for the determination of trace
amounts of BAs than HPLC. This is due to API, a type of soft ionization technique
that includes APCI and ESI, which provides efficient ionization for very different
molecules, including polar, labile, and high-molecular mass drugs and metabolites.
The plasma concentrations of patients with Crohnʼs disease were measured for a period
of four weeks after the administration of three soft gelatin capsules containing 800 mg
of BSE by triple quadrupole LC-ESI/MS. β -ABA, β -BA, KBA, and AKBA were found at concentrations of 4.9 ± 0.5 µM, 6.35 ± 0.5 µM, 0.33 ± 0.1 µM,
and 0.04 ± 0.01 µM, respectively. The calibration standards were used with the following
concentrations ranges: β -BA, 0.125 to 1250 ng/mL; and Aβ -BA, KBA, and AKBA, 0.013 to 125 ng/mL [41 ]. Fourteen patients were administered a high dose of 4200 mg of BSE each day, and
the presence of β -BA, α -BA, β -ABA, α -ABA, KBA, and AKBA was confirmed by HPLC-ESI/MS at the steady plasma concentrations
(ng/mL) of 87.0–11 948.5, 36.7–4830.1, 131.4–6131.3, 73.4–2985.8, 6.4–247.5, and 0–15.5,
respectively. The detection range of KBA and AKBA was from 5.0 to 3000 ng/mL, and
that of β -BA, β -ABA, α -BA, and α -ABA was from 0.5 to 12 000 ng/mL [83 ]. As determined by HPLC-ESI/MS [83 ], the concentrations of KBA, AKBA, β -BA, β -ABA, α -BA, and α -ABA in the rat brain 8 h after the oral administration of 240 mg/kg of BSE were 11.6 ng/g,
37.5 ng/g, 1066.6 ng/g, 163.7 ng/g, 485.1 ng/g, and 43.0 ng/g, respectively [84 ]. The determinations of KBA and AKBA in the rat plasma and brain following the administration
of a single oral dose of 240 mg/kg BSE were performed by triple quadrupole LC-APCI/MS.
In the rat plasma, KBA and AKBA were detected to reach concentrations of 0.4 µM and
0.2 µM. A concentration of 0.3 µM was detected in the brain, corresponding to 99 KBA
and 95 ng/g AKBA in the brain. The linearity was obtained for the entire calibration
range of KBA and AKBA from 5 to 1500 ng/mL in the plasma, and of KBA and AKBA from
5 to 1000 ng/mL in the brain [85 ].
Gas chromatography-mass spectrometry analysis
The GC-NICI/MS analytical method was applied for the quantitative determination of
KBA in human plasma, and the method revealed a validated lower limit of quantification
of 0.0100 mg/mL using 1 mL of plasma. The calibration curves were linear in the measured
range of 10.0 to 2000 ng/mL plasma, but the plasma concentrations obtained in humans
after oral administration were not analyzed [86 ].
High-performance thin-layer chromatography analysis
A rapid and sensitive HPTLC method was developed and validated for the quantitative
estimation of BAs in formulations containing BSE and AKBA in human plasma. The limits
of detection and quantification for AKBA in the human plasma were found to be 8.75
and 29.15 ng/mL, respectively, and the plasma levels of AKBA in six human volunteers
2 h after the administration of a single oral dose of a Boswell® tablet containing
500 mg of BSE were in the range of 24.02 to 67.04 ng/mL plasma [87 ].
Questioning the pharmacological relevance
KBA and AKBA are direct 5-LOX inhibitors that efficiently suppress 5-LOX product synthesis
in isolated neutrophils (IC50 = 2.8 to 8.8 µM) but fail to inhibit 5-LOX product synthesis in human whole blood.
Meanwhile, a single dose (800 mg) oral administration of BSE (H15) to human healthy
volunteers failed to suppress the LTB4 plasma levels [32 ]. Similarly, KBA and AKBA were found to significantly inhibit mPGES1 in the cell-free
assay (AKBA: IC50 = 3 µM; KBA: IC50 = 10 µM), but they all failed to suppress PGE2 formation in human whole blood and in rats [40 ]. Moreover, fairly low plasma levels of KBA (0.01–0.52 µM) and AKBA (0–0.03 µM) were
obtained even after a high oral administration of frankincense (4200 mg). The poor
bioavailabilities of KBA and AKBA have limited their pharmacological efficacy in vivo and result in questions of their pharmacologically relevance. Frankincense usually
contains much more β -BA and β -ABA than AKBA and KBA, which correlates to the considerable plasma levels of β -BA and β -ABA ([Table 1 ]) [82 ], [88 ], and both of them presented IC50 values greater than those determined for catG inhibition (β -BA: IC50 = 0.8 µM; β -ABA: IC50 = 1.2 µM) [41 ]. β -BA results in the greatest inhibition of PGE2 (IC50 = 3 µM) and LPS (IC50 = 1.8 µM), which implies the interaction of β -BA with catG and indicates that PGE2 and LPS may be reasonable molecular targets related to anti-inflammatory activity.
Exploration of poor bioavailability
The possible factors responsible for the poor pharmacokinetics of BAs have also been
investigated in several studies. A previous study revealed that the very low bioavailabilities
of KBA and AKBA after the oral intake of Boswellia extract may be attributed to an altered absorption of these acids because they are
substrates for Pgp in VLB and PBCEC cells, whereas β -BA, β -ABA, α -BA and α -ABA without a keto group have been found to not be subject to the efflux activity
of Pgp [89 ]. The study of the metabolism of BAs in RLM, hepatocytes, and HLM showed that KBA
induces the cells to strongly undergo extensive phase I metabolism, whereas AKBA does
not [90 ]. In addition, β -BA also induces extensive phase I metabolism in HLM and RLM, but α -BA is extensively phase I metabolized in HLM but not in RLM, whereas the acetylated
α -ABA, β -ABA, and AKBA are rather metabolically stable. The acetyl group at position 3 appears
to impede phase I metabolism. All six BAs are not subjected to phase II metabolism;
hence, the extensive phase I metabolism of KBA may be the main reason for its low
plasma concentration [84 ]. The observed extensive phase I metabolism of KBA strongly contributes to its low
bioavailability. The study of the permeation of KBA and AKBA in the Caco-2 model showed
the poor permeability of AKBA and the moderate absorption of KBA and revealed that
the poor absorption of AKBA from the gastrointestinal tract may contribute to its
low bioavailability. In addition, this study suggested that the Pgp inhibition may
be attributed to KBA and AKBA being allosteric inhibitors [91 ], rather than the Pgp inhibition of KBA and AKBA, as mentioned above, which is due
to Pgp substrates [89 ]. Because the Caco-2 model is adapted to physiological conditions, the permeability
of AKBA is improved compared with the results described above, and the explanation
for this is assumed to be that the greater distribution of AKBA in different compartments
may contribute to its low bioavailability [84 ]. Further investigations of the permeability-related hurdles in the oral delivery
of KBA include the gastrointestinal instability, the CYP3A4-mediated intestinal metabolism,
the accumulation within the enterocytes and saturable kinetics [92 ].
It is remarkable that their poor bioavailability has limited their practical and effective
role in vivo because the most bioactive interactions of BAs are observed at relatively high concentrations
in vitro .
Bioavailability Enhancement of Boswellic Acids
Bioavailability Enhancement of Boswellic Acids
For a long time, AKBA and KBA have been believed to be responsible for the pharmacological
activities of frankincense. However, in view of their fairly low plasma and brain
levels and in consideration of their failure to inhibit 5-LOX in whole blood, the
suppression of LTB4 synthesis in vivo by administered frankincense extracts is questionable. Only β -BA, as the best inhibitor of PGE2 and LPS, has considerable bioavailability with
a high content in frankincense. To make better use of all the potential pharmacological
properties of different BAs based on the presented background, we summarize some of
the approaches that have been used to address this problem.
Administering with a standardized meal
Some scholars have attempted to research how to resolve the problem of the poor bioavailability
of BAs. The effect of food intake on bioavailability was analyzed, and the maximum
concentrations of BAs in human plasma are presented in [Table 2 ].
Table 2 Maximum plasma concentrations of boswellic acids determined in human plasma under
fasted and fed conditions.
Dosage
Methods
Condition
Cmax of BAs determined in human plasma (µM)
Ref.
β -BA
α -BA
β -ABA
α -ABA
KBA
AKBA
NA = not analyzed; ND = not detectable; a = mean and [range]; b = mean ± SD
3 × BSE-018 capsules/day (n = 12)
HPLCa
fasted
0.41 [0.09–3.96]
ND
ND
ND
0.17 [0.05–0.52]
0.01 [0.00–0.08]
[93 ]
fed
2.45 [0.90–4.67]
0.69 [0.09–2.93]
ND
0.23 [0.09–0.80]
0.48 [0.21–0.88]
0.05 [0.02–0.51]
2 × Boswelan capsules/day(n = 15)
HPLC-MSb
fasted
NA
NA
NA
NA
0.33 ± 0.13
0.05 ± 0.03
[94 ]
fed
NA
NA
NA
NA
0.43 ± 0.20
0.06 ± 0.05
The plasma concentrations of BAs in 12 male volunteers who were administered a single
dose of three BSE-018 capsules (262 mg of dry BSE containing 18.2 % β -BA, 13.2 % α -BA, 10.5 β -ABA, 3.3 % α -ABA, 6.1 % KBA, and 3.7 % AKBA/capsule) in the fasted state and in a standardized
high-fat meal (fed state) were determined by HPLC. The concomitant food intake significantly
increased the mean area under the concentration-time curve and enhanced the plasma
concentrations of β -BA, KBA, and AKBA in comparison to the starved volunteers. The pharmacokinetic profile
of BSE-018 greatly varied with food intake, an effect that is likely due to the improved
absorption of BAs in the presence of bile acids [93 ]. The plasma concentrations of KBA and AKBA in 15 male volunteers who were administered
a single dose of two Boswelan capsules (400 mg of BSE containing 2.77 % KBA and 2.64 %
AKBA/capsule) in the fasted and fed conditions were determined by HPLC-MS. This study
confirmed the results of the study described above [93 ], which showed that food interacts with the bioavailability of KBA but not of AKBA.
Moreover, the magnitude of the increase in the KBA plasma concentrations with food
was much less pronounced. This study indicated that the in vivo KBA and AKBA plasma concentrations would start to increase with in vitro IC50 s for catG and 5-LOX after a significant but clinically infeasible dose increase of
10- to 15-fold Boswelan tid [94 ].
Although the plasma concentrations of BAs were increased when administered with a
high-fat meal compared with the fasted state, as shown in [Table 2 ], the problem of the low bioavailability of BAs has not been fundamentally resolved.
Administering with anionic drugs
The transporter-mediated uptake of drugs into and out of cells is another important
determinant of drug disposition and bioavailability. A previous study revealed that
KBA and AKBA are able to modulate the activity of organic anion transporters (OATP1B3)
and the multidrug resistant protein MRP2, demonstrating that the combined administration
of BAs with anionic drugs is a possible solution [91 ].
New formula compatibility
The application of new formulations and formula technology to BAs has achieved some
initial success in several in vitro and rat studies. The improvements in the pharmacological activity and investigatory
parameters by the new formulations compared with non-formulations are displayed in
[Table 3 ].
Table 3 Improvements in the bioactivities and investigatory parameters by the new formulations
compared with non-formulations.
Formulation
Enhanced activities
Indicators
Conditions
Cplasma
# (µM), Cbrain * (ng/g), Jss
& (mg/cm2 per h), and Kp
× 10− 2△ (cm/h) of BAs
Ref.
β -BA
α -BA
β -ABA
α -ABA
KBA
AKBA
R = rats; F = formulation; NF: non-formulation; NA = not analyzed; \ = not investigated;
C plasma
# = concentrations of BAs in plasma; C brain * = concentrations of BAs in brain; Jss
& = permeation rate at steady state; Kp
× 10− 2△ = permeability coefficient; a = mean ± SD; b = the relevant literature provided only
multiple comparison, there was no accurate numerical reference; c = [range]; d = C
max
BAs-Phosphatidyl-
Anti-inflammatory, hypolipidemic
\R
\
\
\
\
\
\
\
[95 ]
AKBA-Polymeric nanomicelles
Anti-inflammatory, anti-arthritis
Skin permeationR
NF&
NA
NA
NA
NA
NA
0.161 ± 0.035
[96 ]
F&
NA
NA
NA
NA
NA
0.488 ± 0.036
NF△
NA
NA
NA
NA
NA
0.341 ± 0.098
F△
NA
NA
NA
NA
NA
1.036 ± 0.151
BSE-Phospholipid and pluronic f127
\
PharmacokineticR
NF*,a
1066.6 ± 781.6
485.1 ± 363.8
163.7 ± 248.9
43.0 ± 55.7
11.6 ± 12.6
37.5 ± 56.8
[25 ]
F*,a
5908.2 ± 2275.6
2443.9 ± 914.4
156.1 ± 82.7
60.1 ± 25.4
13.6 ± 4.2
203.8 ± 86.9
F vs. NF#,b
6-fold
6-fold
6-fold
6-fold
26-fold
14-fold
BSE-Phytosome® (Soy lecithin)
\
PharmacokineticR
NF*,c
[0.0–98.2]
[241.3–380.6]
[35.5–318.9]
[39.9–93.1]
[0.0–44.9]
[0.0–45.0]
[26 ]
F*,c
[0.0–1376.8]
[219.93–850.0]
4.5–317.7
10.9–100.9
[22.4–477.8]
[0.0–586.1]
NF#,a,d
2.2 ± 0.3
1.1 ± 0.4
5.0 ± 2.1
1.0 ± 0.2
0.6 ± 0.3
0.6 ± 0.1
F#,a,d
6.6 ± 4.1
2.2 ± 1.3
4.2 ± 2.5
1.45 ± 0.7
1.6 ± 0.6
1.2 ± 0.6
BA-PC is a complex of BA prepared with phosphatidyl-choline. The complex shows better
anti-inflammatory and hypolipidemic activity compared with BA alone, and this effect
is attributed to the amphiphilic nature of BA-PC, which greatly enhances the water
and lipid solubility of BAs. The combination of phospholipids with BAs provides a
potential bioavailability enhancement and faster and improved absorption in the intestinal
tract [95 ].
AKBA is loaded in the polymeric nanomicelles of N-isopropylacrylamide, N-vinylpyrrolidone,
and acrylic acid, which offers the possibility of a controlled release, the protection
of active substances, and nanometer sizes. In aqueous buffer (pH 7.4), the release
of AKBA from the polymeric nanomicelles is 23 and 55 % after 2 and 8 h, respectively.
The AKBA polymeric nanomicelle gel shows significantly enhanced anti-inflammatory
and antiarthritic activities and a 3-fold increase compared with the AKBA gel containing
the same amount of AKBA in in vitro skin permeation studies, which shows its great potential for transdermal drug delivery
[96 ].
A chosen formulation composed of BSE/ phospholipid/ pluronic f127 (1 : 1 : 1 w/w/w) has been considered a basis for the production of
a dispensable free-flowing powder that can be further processed into tablets or capsules.
The increased solubility of KBA, AKBA, α -BA, β -BA, α -ABA, and β -ABA is approximately up to 54-fold greater compared with the non-formulated extract,
and these exhibited the highest mass net flux in the permeability of the physiological
Caco-2 cell model. As determined by the LC-MS method, the oral administration of this
formulation to rats (240 mg/kg) resulted in 26- and 14-fold higher plasma levels of
KBA and AKBA, respectively. In the brain, 5-fold higher levels of AKBA compared with
the non-formulated extract were determined 8 h after oral administration [25 ].
Casperome™ is a formulation of BSE and Phytosome® (soy lecithin) at a 1 : 1 ratio.
Compared with the non-formulated extract, Casperome™ provides significantly higher
plasma levels, i.e., up to 7-fold for KBA and 3-fold for β -BA, expressed as the area under the plasma concentration-time curve. In the brain,
there were increases in the concentrations of KBA and AKBA (35-fold) and β -BA (3-fold). It is noteworthy that up to 17-fold higher BA levels were observed in
poorly vascularized organs [26 ].
The evidence described above led us to hypothesize that BAs will be further developed
in the near future. Preparations have been designed to improve the bioavailability
of BAs by enhancing absorption, and preliminary studies are positive.
Conclusions
The reputation of BAs as potential pharmaceutical candidates has found support in
numerous studies. In particular, AKBA, as the most bioactive and representative BA,
has been used as an enriched key component for the evaluation of the quality of frankincense
used for the treatment of peritumoral edema [20 ] and therapeutic interventions against inflammatory diseases [15 ]. Based on potential pharmacological activities with multiple targets of BAs, we
summarized various studies on the application of chemical modification and bio-derivatization
approaches to different BAs for the purpose of obtaining more valuable active derivatives.
The available pharmacokinetic investigations using different analysis methods have
revealed the poor bioavailability of BAs, particularly KBA and AKBA. Their plasma
and brain concentrations have been found to be very low following the oral administration
of even a high dose. Bioavailability has been a major hurdle in the translation of
the preclinical potential of frankincense extracts and BAs into therapeutic effects.
Therefore, we summarized some approaches for enhancing pharmacological activities
by increasing the availability of BAs.
Above all, we anticipate further research on BAs and accurate pharmacological mechanisms
for explaining the potential medicinal uses of frankincense. We also anticipate more
novel approaches for enhancing the bioavailability and bioactivity of BAs in humans
to increase the range of clinical applications and the development of more bioactive
and bioavailable derivatives in the near future.
Acknowledgements
This study was financially supported by the National Science and Technology Major
Project of the Ministry of Science and Technology of China (Project No. 2009ZX09 103–314),
the Major State Basic Research Development Program of China, 973 Program (Project
No. 2012CB518 401), the National Natural Science Foundation of China (Project No. 81 470 177),
and the Central Research Institutes of Basic Research and Public Service Special Operations
(Project Nos. YZ-1412 and YZ-1317).
