Laszczyk2009 HTSH

Review 1549
Pentacyclic Triterpenes of the Lupane, Oleanane and

Ursane Group as Tools in Cancer Therapy
Author Melanie N. Laszczyk 1, 2
1
Affiliations Betulin Institut, Darmstadt, Germany
2
Carl Gustav Carus-Institut, Niefern-Öschelbronn, Germany
Downloaded by: National Dong Hwa University. Copyrighted material.

Key words Abstract lished using these triterpenes in cancer therapy.
l
" pentacyclic triterpenes
! They provide a multitarget potential for coping
l
" multifunctional agent
Today cancer treatment is not only a question of with new cancer strategies. Whether this is an ef-
l
" cancer treatment
eliminating cancer cells by induction of cell death. fective approach for cancer treatment has to be
New therapeutic strategies also include targeting proven. Because various triterpenes are an in-
the tumour microenvironment, avoiding angio- creasingly promising group of plant metabolites,
genesis, modulating the immune response or the the utilisation of different plants as their sources
chronic inflammation that is often associated is of interest. Parts of plants, for example birch
with cancer. Furthermore, the induction of redif- bark, rosemary leaves, apple peel and mistletoe
ferentiation of dedifferentiated cancer cells is an shoots are rich in triterpenes and provide differ-
interesting aspect in developing new therapy ent triterpene compositions.
strategies. Plants provide a broad spectrum of po-
tential drug substances for cancer therapy with
multifaceted effects and targets. Pentacyclic tri- Abbreviations
terpenes are one group of promising secondary !
plant metabolites. This review summarizes the ACC: antigen-dependent complement-
potential of triterpenes belonging to the lupane, mediated cytotoxicity
oleanane or ursane group, to treat cancer by dif- ADCC: antibody-dependent cell mediated
ferent modes of action. Since Pisha et al. reported cytotoxicity
in 1995 that betulinic acid is a highly promising APN: aminopeptidase N
anticancer drug after inducing apoptosis in mela- ARE: antioxidant response element
noma cell lines in vitro and in vivo, experimental BAEC: bovine aortic endothelial cell
work focused on the apoptosis inducing mecha- CAM: chick embryo chorioallantoic mem-
nisms of betulinic acid and other triterpenes. The brane
antitumour effects were subsequently confirmed CAT: catalase
in a series of cancer cell lines from other origins, COX-2: cyclooxygenase-2
received March 28, 2009
revised July 27, 2009 for example breast, colon, lung and neuroblasto- DMBA: 7,12-dimethylbenz(a)anthracene
accepted August 5, 2009 ma. In addition, in the last decade many studies DMSO: dimethylsulfoxide
have shown further effects that justify the expec- ECM: extracellular matrix
Bibliography
tation that triterpenes are useful to treat cancer FGF: fibroblast growth factor
DOI 10.1055/s-0029-1186102
Published online September 9, by several modes of action. Thus, triterpene acids GM‑CSF: granulocyte-macrophage colony-
2009 are known mainly for their antiangiogenic effects stimulating factor
Planta Med 2009; 75: as well as their differentiation inducing effects. In GSH: glutathione
1549–1560 © Georg Thieme
particular, lupane-type triterpenes, such as betu- HUVEC: human umbilical vein endothelial cell
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943 lin, betulinic acid and lupeol, display anti-inflam- ID: inhibition dose
matory activities which often accompany im- IL: interleukin
Correspondence mune modulation. Triterpene acids as well as tri- iNOS: inducible NO-synthase
Dr. Melanie N. Laszczyk
Betulin-Institut terpene monoalcohols and diols also show an LD: lethal dose
Blumenstr. 25 antioxidative potential. The pharmacological po- NADP+: nicotinamide adenine dinucleotide
64297 Darmstadt tential of triterpenes of the lupane, oleanane or phosphate
Germany
Phone: + 49 72 33 82 93 71 ursane type for cancer treatment seems high; NF-κB: nuclear factor-kappaB
[email protected] although up to now no clinical trial has been pub- NO: nitric oxide
Laszczyk MN. Pentacyclic Triterpenes of … Planta Med 2009; 75: 1549–1560

1550 Review
Nrf2: nuclear factor E2-related factor 2 Sp: specificity protein

PCNA: proliferating cell nuclear antigen TE: triterpene dry extract
PGE2: prostaglandin E2 TNF: tumour necrosis factor
PLA2: phospholipase A2 TPA: 12-O-tetradecanoylphorbol-13-acetate
PPAR: peroxisome proliferator activated receptor TRPC6: transient receptor potential channel 6
ROS: reactive oxygen species VEGF: vascular endothelial growth factor
SOD: superoxide dismutase
Introduction the outer bark of birch, leaves of rosemary and olive, mistletoe as
! well as plane tree bark and apple peelings contain more than 1 %
Today solid tumours are no longer considered as a mere accumu- (w/w) of these pentacyclic triterpenes (l " Table 1). These plants
lation of abnormal, malignant cancer cells. The tumour environ- can be used to obtain triterpene dry extracts consisting of 50–
ment, the tumour stroma, is becoming more and more impor- 90 % (w/w) triterpenes [5]. Depending on the plant material, lu-
tant. Therefore, treatment strategies have to be changed. Most peol, betulin, betulinic acid, oleanolic acid, ursolic acid or an
therapies now try to eliminate cancer cells by inducing apoptosis equal mixture of these substances are the main components of
or necrosis. New therapy strategies include the treatment of the such dry extracts [6] (l " Fig. 1). This kind of triterpene extract
tumour environment, avoiding angiogenesis and modulating the may be used as starting material for further pharmaceutical de-
immune response or the chronic inflammation that is often asso- velopment.
ciated with cancer promotion and progression. Another approach In the last 15 years hundreds of publications have highlighted the

is to redifferentiate proliferating tumour cells. broad spectrum of biological activities of lupane, oleanane and
In addition, chemoprevention is important to avoid cancer pro- ursane triterpenes. The literature search for this review is based
motion. Cancer results from a multistage carcinogenesis process: on an actual PubMed search focused on the last two years. On ac-
initiation, promotion and progression. Because reducing the ini- count of the low water solubility of triterpenes, special attention
tiation phase to a zero level is impossible, the most efficient inter- was given to the concentrations used in in vitro experiments.
vention would be at the promotion phase to eliminate premalig- Concentrations above 100 µM often bias the results, because of
nant cells before they become malignant [1]. Therefore, the con- an insoluble fraction. In case of in vivo data, we included effects
cept of delaying or preventing this transformation is worth test- that are described by almost all different work groups.
ing in future studies [2]. Because of their cytotoxicity against various cancer cell lines the
Pentacyclic triterpenes are secondary plant metabolites which group of lupane, oleanane and ursane triterpenes are considered
arise from cyclization of squalene [3]. This article focused on tri- as promising anticancer drugs. Nevertheless, due to their various
terpenes of the lupeol, oleanane and ursane type. They are found pharmacological activities including antiangiogenic, anti-in-
in different plant organs, e.g., in bark, cork, or in the wax covering flammatory as well as antioxidant effects and the ability to en-
leaves or peel. Low amounts (< 0.1 % of the dry weight of a plant hance cell differentiation, they are more than a simple cytotoxic
organ) are ubiquitously present in plants. However, there are a anticancer drug and are suitable for modern cancer strategies
few species that display a high amount of these pentacyclic triter- (l" Fig. 2). Moreover, they are regarded as essential parts of hu-
penes (> 1 % of the dry weight of the plant organ). The highest tri- man nutrition because of their chemopreventive potential to
terpene amount has been found in the outer bark of white birch. fend off cancer promotion [7, 8].
The white outer bark contains up to 34% (w/w) betulin [4]. Beside
Plant Part Triterpene Amount Reference Table 1 Plants which display a

Betula alba L., Betulaceae bark lupeol 1–2 % [5] high amount of pentacyclic triter-
betulin 10–34 % [4, 5] penes.
betulinic acid 0.5–1.5% [5]
oleanolic acid 0–1.5 % [5]
Rosmarinus officinalis L., Lamiaceae leaves betulinic acid 1.5% [6]
oleanolic acid 1.2% [6]
ursolic acid 3.0% [6]
Malus domestica Mill., Rosaceae fruit ursolic acid 2.0% [6]
peel
Platanus L., Platanaceae bark betulinic acid 2.4% [6]
Viscum album L., Viscaceae sapling oleanolic acid 1.0% [6]
Olea europaea L., Oleaceae leaves oleanolic acid 3.1% [6]
Nerium oleander L., Apocynaceae leaves ursolic acid 1.2% [6]
Arctostaphylos uva-ursi L., Ericaceae leaves ursolic acid 1.2% [6]
Coffea L., Rubiaceae leaves ursolic acid 1.8% [6]
Eucalyptus LʼHér., Myrtaceae leaves ursolic acid 1.2% [6]
Lavandula angustifolia L., Lamiaceae leaves ursolic acid 1.6% [6]
Salvia officinalis L., Lamiaceae leaves ursolic acid 1.8% [6]
Syzygium aromaticum L., Myrtaceae flowers oleanolic acid 1.6% [6]
Thymus vulgaris L., Lamiaceae leaves ursolic acid 1.0% [6]

Review 1551

Fig. 1 Chemical structures of (A) lupeol, (B) betulin, (C) betulinic acid, (D) oleanan structure with five six-rings. This is quite similar to the ursan group,
erythrodiol, (E) oleanolic acid and (F) ursolic acid. Lupeol, betulin and betu- which is represented by ursolic acid. The difference between the oleanan and
linic acid belong to the lupan type pentacyclic triterpenes consisting of four the ursan type is the methyl-group localization of the E-ring.
six-rings and one five-ring, whereas erythrodiol and oleanolic acid exhibit an
Apoptosis
!
Induction of apoptosis by pro-apoptotic agents is one important
part of cancer therapy. But apoptosis in cancer cells is often im-
paired or even blocked by mutated genes regulating the cell cycle
or an imbalanced ratio of pro- and antiapoptotic proteins. There-
fore it is necessary to target different steps of the apoptotic pro-
cess to bypass such blocks with respect to the type of cancer. This
review highlights only a few aspects of the knowledge about tri-
terpenes and apoptosis. But it should give an impression of the
diversity of mechanisms triggered by these triterpenes and with
it the chance to overcome apoptosis resistance in cancer cells.
Triterpenes trigger apoptosis by different modes of action, as ex-
tensively described in a series of reviews, especially for betulinic
acid [7–9]. First it was assumed that betulinic acid is a selective
cytotoxic compound against melanoma cells. However, up to
now a large panel of cancer cell lines have proven to be sensitive
to betulinic acid and other pentacyclic triterpenes. It is also as-
sumed by some authors that there is a selective sensitivity
against malignant cells. Nevertheless, cytotoxicity against pri-
mary cells such as fibroblasts, melanocytes, keratinocytes, neuro-
Fig. 2 Pentacyclic triterpenes such as lupeol, betulin, betulinic acid, nal cells and peripheral blood lymphocytes is reported, but they
erythrodiol, oleanolic acid and ursolic acid exhibit various pharmacological seem to tolerate higher triterpene concentrations than cancer
activities. Besides targeting tumour cells by induction of apoptosis, they cells of the same origin [10–14]. Whether this may result in a
also modulate the tumour environment displaying antiangiogenic, anti-in- positive effect in vivo, when cancer cells are in a united cell struc-
flammatory as well as antioxidant effects and enhancing cell differentia- ture is questionable. But in the case of triterpene acids another
tion. The efficacy of each single compound might differ with respect to the possibility to enhance their activity in cancer tissue was ob-
various activities, so the combination of these substances might have a
served. In vitro the activity of betulinic acid was increased by de-
benefit in order to treat cancer from different from different angles in
modern cancer strategies. creasing the pH [15]. And interestingly in athymic mice carrying
human melanoma xenografts, its highest concentration after in-

1552 Review
traperitoneal injection (500 mg/kg) compared with other tissues neuroblastoma (GOTO, NB-1) [34]. However, in nonmalignant,
like liver, lung, and kidney was found in the tumour tissue, which immortalised HaCaT keratinocytes induction of caspase-depen-
often exhibits a lower pH, caused by a changed metabolism. This dent apoptosis has been observed [5] and recently, Pyo et al. re-
could be an explanation of the triterpene acid accumulation in vealed anticancer activity of betulin (20 µM) against a human lung
the melanoma tissue. Up to now, only a few investigations in- cancer cell line (A549) by induction of apoptosis [35]. Erythrodiol,
clude pH variations [15, 16], but this fact could be an important the closely related diol of the oleanane group, has not been inves-
factor for increasing the activity of triterpene acids in cancer tigated very thoroughly either, but in 2008 apoptotic activity in
treatment and should be investigated in more detail. HT-29 human adenocarcinoma cells at concentrations of 50–
The apoptosis mechanism of betulinic acid has been investigated 150 µM was reported [36]. In the case of betulin and erythrodiol,
quite well and was reviewed in 2009 by Fulda [9]. In short, betu- it is difficult to evaluate their pro-apoptotic potential compared to
linic acid induces apoptosis via the intrinsic pathway by affecting betulinic acid, because of the low amount of published data.
the mitochondrial membrane potential [17] and initiates reactive Sometimes only moderate pro-apoptotic effects of triterpenes are
oxygen species (ROS) generation linked to an activation of pro- observed in vitro, as reported for immortalised HaCaT keratino-
apoptotic p38 MAPK and SAP/JNK kinases [18, 19]. A similar in- cytes or human epidermoid carcinoma cells (A431) treated with a
crease of ROS was also observed for oleanolic acid (25 µM) in as- triterpene dry extract from birch bark containing 80 % betulin and
trocytoma cell lines [20]. While recently published data reported up to 4 % betulinic acid and smaller amounts of lupeol and olea-
Bax/Bak-independent apoptosis induction by betulinic acid in nolic acid [5]. It was only able to induce a twofold higher apoptosis
various cancer cell lines [21], a number of publications show a rate in HaCaT keratinocytes. At first these results seem to have no
modulation of anti- and pro-apoptotic proteins of the Bcl-2 fam- relevance for therapeutic treatment. Nevertheless the triterpene
ily [13, 22–25]. The modulation of pro- and antiapoptotic factors extract was successful in vivo treating actinic keratosis [37].

is complex and probably cell-type-dependent. It is likely that In summary, the apoptotic pathway for betulinic acid is well
context dependency also plays a role with respect to nuclear fac- known. Triggering the intrinsic pathway via destruction of the
tor kappa-B (NF-κB) modulation. While NF-κB is activated by be- mitochondrial membrane potential and including MAP kinase
tulinic acid (20 µM) in a variety of cancer cell lines resulting in in- and PI3K/Akt pathways, seems to be the mode of action. In re-
duction of apoptosis [26], NF-κB inhibition is observed in chemo- spect of the antioxidative activity discussed later, the induction
resistant androgen-refractory prostate cancer cells exhibiting of ROS species in the case of apoptosis is highly interesting. Po-
constitutive Rel/NF-κB activation [27]. Similar effects of triter- tentially the concentration is the critical parameter causing
penes on NF-κB related to inflammation have been observed apoptosis or an antioxidative effect. Oleanolic acid may act in a
and are discussed later. One important detail to overcome some way similar to betulinic acid by activating caspase-9.
types of apoptosis resistance is the independence of betulinic ac- Unlike triterpene acids, lupeol triggers the extrinsic pathway via
id induced apoptosis of p53 that is frequently mutated in cancer the Fas-receptor. There is still a lack of data for the diols betulin
cells [13, 22, 24]. and erythrodiol, thus a prediction of their mechanism is not yet
While apoptosis induced by betulinic acid seems to be indepen- possible.
dent of the Fas receptor [22], lupeol targets this receptor and con- Due to the different mechanisms triggered, the use of different
sequently activates the extrinsic pathway via caspase 8. For ex- triterpenes, also in a mixture may increase the chance of over-
ample, lupeol (20 µM) significantly increased the expression of coming the chemoresistence of tumour cells. At the moment, this
the FADD protein and the Fas receptor in androgen sensitive is the conclusion drawn only from the results of various re-
prostate cancer cells [28]. Furthermore, lupeol sensitises chemo- searches carried out independently. Only well organised wide-
resistant human pancreatic cancer cells (PaC), to undergo apop- spread analysis of a cancer cell panel treated with different triter-
tosis by recombinant TRAIL via suppression of cFLIP [29]. Besides, penes under standardised parameters could generate really com-
various targets of lupeol are reported to overcome apoptosis re- parable data for determining which triterpene or triterpene mix-
sistance by inhibition of oncogenes and activation of tumour sup- tures exhibit the best chance of being active against a particular
pressor genes. At a concentration of 30 µM, lupeol reduces the ex- cancer cell. But this could be an opportunity for tapping the full
pression of commonly overexpressed Ras oncoprotein resulting potential of triterpenes that induce apoptosis.
in the inhibition of the PI3K/Akt pathway that is known for pro-
moting cell growth [30]. Coincidentally, the expression of phos-
pho-p38 MAPK, which triggers an antiapoptotic response to tu- Antiangiogenic Effects
mour cells, was decreased together with NF-κB occurrence. These !
modulations were accompanied by induction of apoptosis in the Angiogenesis is a key process for the outgrowth of cancer cells and
otherwise resistant pancreatic cells [30]. their spread into other tissues. Therefore, suppressing this process
Also ursolic acid and oleanolic acid exhibit pro-apoptotic activity, is one important pillar of cancer treatment. There are four key
as reviewed by Ovesna and colleagues in 2004 [8]. Recent results steps in angiogenesis which are potential therapeutic targets:
indicated a modulation of the Bcl-2 protein family due to a sup- degradation of extracellular matrix, migration and proliferation
pression of NF-κB by ursolic acid (50 µM) in B16.F10 mouse mel- of aortic endothelial cells and the formation of new blood vessels.
anoma cells. Induction of apoptosis was accompanied by activa- The initial results in 1995 of Sohn and colleagues provided an in-
tion of p53 and caspase-3 gene expression [31]. Oleanolic acid dication that ursolic acid and oleanolic acid have antiangiogenic
(80 µM) showed apoptosis induction in leukaemia cells (HL60) effects on bovine aortic endothelial cells (BAEC) in the CAM
via activation of caspase-9 and caspase-3 accompanied by the (chick embryo chorioallantoic membrane) assay [38]. Here, ur-
cleavage of poly(ADP-ribose) polymerase (PARP) [32]. solic acid (ID50: 4 µM) was more effective than oleanolic acid
Betulin has often been found to be inactive or weakly active (ID50: 40 µM). However, the key steps of angiogenesis targeted
against several cancer cell lines such as melanoma (MEL-2), epi- by triterpene acids had not been identified at this time. Further
dermoid carcinoma (KB) [33], leukaemia (HL60, U937, K562) or investigations focussing on the different key steps disclosed ef-

Review 1553
fects on the angiogenic process but these proved controversial. In be due to activation of selective proteasome-dependent degrada-
serum free cultures of human umbilical vein endothelial cells tion of the transcription factors specificity protein 1 (Sp1), Sp3,
(HUVECs), ursolic acid (10–100 µM) increases expression of ad- and Sp4 which regulate the VEGF expression and that are mostly
hesion molecules that support angiogenesis, such as ICAM-1 and overexpressed in tumours, as was shown by Chintharlapalli and
CD31, and the expression of angiogenic growth factors, particu- colleagues [47]. The concentration-dependent effect on the tran-
larly vascular endothelial growth factor (VEGF) and fibroblast scription factors and the expression of VEGF by betulinic acid
growth factor-2 (FGF-2) [39]. This suggests a possible support of could be fully blocked by using a proteasome inhibitor.
the migration step and the structure formation process. Further- Only one publication reports an antiangiogenic effect of lupeol.
more, 4–20 µM ursolic acid failed to produce a significant inhibi- You and colleagues found that lupeol also inhibits HUVEC tube
tion of the invasion capability of BAEC through matrigel. In addi- formation [48].
tion, the degradation of extracellular matrix (ECM) by ECM deg- Based on the current literature, primarily triterpene acids seem
radation proteins as MMP-2 and urokinase was assumed to be to have an antiangiogenic effect. However, to clarify the exact
stimulated by ursolic acid (4 µM) due to an increased expression mechanisms by which they exert this effect more experimental
shown by gelatinase and urokinase zymography of these en- work is still needed, even if there are some doubts in the case of
zymes in BAEC [40]. But this should be investigated in more de- ursolic acid because of the upregulation of pro-angiogenic factors
tail, considering that two years later Jedinak found a strong inhi- such as MMP-2 or VEGF in vitro. It is necessary to interpret these
bition of urokinase activity by ursolic acid in a cell free system pro-angiogenic data very carefully. There are four key steps in an-
[41]. Despite the enhancement of pro-angiogenic factors, in giogenesis. These include degradation of the extracellular matrix,
2004 Cardenas confirmed Sohnʼs (1995) observation of the anti- migration and proliferation of aortic endothelial cells and the for-
angiogenic effect of ursolic acid in the CAM assay. Furthermore, mation of new blood vessels. All four steps are necessary for suc-

ursolic acid treatment of HUVECs and rat aortic rings that were cessful angiogenesis. Most in vitro experiments focus only on
stimulated by cultivation in medium supplemented with serum parts of the process. This does not enable a prediction of full an-
in contrast to serum free medium surprisingly caused inhibition giogenesis. Instead, in vivo models, such as the CAM assay, con-
of the angiogenic phenotype, including the formation of a capil- sider the whole process. Thus they provide information about
lary network-like structure by HUVECs and a greater extent of the efficacy of the substances on the end result, namely forming
endothelial sprouting in rat aortic rings [39]. Due to the some- new blood vessels, i.e., not just on one essential factor in a com-
times contradictory effects of ursolic acid on different steps of an- plex network, such as VEGF.
giogenesis, one must be cautious concerning its antiangiogenic Nevertheless, the in vitro data give hints for understanding the
potential. The generation of in vivo data is necessary in order to mechanism. In respect of the differentiation inducing activity of
include the influence of the tissue milieu and thus appropriately triterpenes discussed later, the most interesting result is that be-
evaluate its effects. sides the regulation of endothelial cell proliferation by modula-
For betulinic acid, older studies revealed that it inhibits enzy- tion of growth factors such as VEGF, the aspect of inducing differ-
matic activity of aminopeptidase N (APN) in a cell free system entiation to stop proliferation may also play a role in the angio-
[42]. APN is a widely distributed, membrane-bound, zinc-depen- genic efficacy of triterpenes.
dent metalloproteinase that is known to play an important role in
tumour-vasculogenesis and is essential for the endothelial cell
tube formation [43, 44]. Betulinic acid (2 µM) potently inhibits Anti-inflammatory Effects
basic fibroblast growth factor (bFGF)-induced invasion and tube !
formation of BAECs [45]. Initially it was assumed that APN could Recent studies have revealed a clear role for inflammation in the
be the target of betulinic acid. Kwon et al. confirmed that betu- development and progression of cancer and in the immune re-
linic acid strongly inhibits enzymatic activity of APN in a cell free sponse against it by orchestrating the tumour supporting envi-
system, but not when enzymatic activity was measured in betu- ronment [49].
linic acid treated endothelial cells. He provided evidence that the Lupanes, oleananes and ursanes applied orally or topically exhib-
antiangiogenic activity of betulinic acid was accompanied by it significant anti-inflammatory activity in vivo. This was demon-
modulation of the mitochondrial membrane function by decreas- strated in 12-O-tetradecanoylphorbol-13-acetate (TPA), carra-
ing the mitochondrial redox potential. This effect could be geenan, serotonin or croton oil induced paw/ear oedema tests,
blocked by different mitochondrial permeability transition in- as well as in arthritic animal models [50–56]. Efforts to work out
hibitors such as cyclosporine A or bongkrekic acid. In view of the the underlying mechanism in vitro are in progress (reviewed in
effects of betulinic acid on the mitochondrial membrane, this [7, 8, 57–59]) and several potential targets have been discovered.
seems to be a target structure worth considering. But in this con- Besides direct effects on the morphology or the activity of im-
text, the modulation of the mitochondrial membrane does not mune cells, such as macrophages, dendritic cells, T cells or other
cause the release of apoptogenic factors that directly trigger cell leukocytes, which may suppress the immune response [60–63],
death. Betulinic acid at up to 9 µM did not affect the endothelial an influence on pro-inflammatory cytokines, e.g., TNF-α, INFγ,
cell viability in the formed tubes. Kwon hypothesised that betu- IL-1β, IL-6, IL-2, IL-4, IL-5, IL-8, or IL-13 [31, 50, 60, 64–66] has
linic acid had a specific effect on the angiogenic differentiation been reported.
of endothelial cells, rather than an antiproliferative activity [45]. The expression of these cytokines is regulated by the transcrip-
It is known, that a modulation of the mitochondrial oxidative tion factor NF-κB, which is therefore a pivotal target. Further-
phosphorylation can enhance angiogenic differentiation of endo- more, NF-κB is commonly overexpressed in cancer cells. On the
thelial cells, stopping their proliferative activity [46]. However, in one hand this may support the maintenance of a chronically in-
a human prostate cancer cell line (LNCaP) and in vivo, betulinic flamed microenvironment and on the other hand it often sup-
acid acts via decreasing expression of VEGF [47]. Thus there presses apoptosis of the tumour cells [67]. In the last few years,
might be also an antiproliferative effect. The mechanism might several groups have published controversial data concerning the

1554 Review
activity of betulinic acid and ursolic acid on NF-κB [26, 68]. Kas- ing beneficial physiological effects, for example in cellular re-
perczyk et al. [26] postulated an NF- κB activating effect of betu- sponses to noxia, or in the regulation of immune responses. Over-
linic acid (13–22 µM) on various cancer cell lines (neuroblastoma, production results in oxidative stress that can be an important
melanoma, glioblastoma). In contrast, in 2003 Takada and Aggar- mediator of damage to cell structures [78]. Initially increased lev-
wal described an inhibition of NF-κB regulated cyclooxygenase-2 els of ROS disrupt cell membrane integrity by oxidation of unsat-
(COX-2) expression and determined a maximal suppressive effect urated membrane lipids. Lipid peroxidation is commonly related
of betulinic acid at a concentration of 30 µM on NF-κB in colon to cardiovascular diseases [79], autoimmune diseases or chronic
carcinoma cells [68]. Similarly NF-κB in melanoma cells was in- inflammation [80]. Furthermore, free radicals cause DNA damage
hibited by ursolic acid (50 µM) accompanied by downregulation which may result in tumour initiation and promotion [81]. Thus
of pro-inflammatory cytokines such as TNF-α; IL-1β, IL-6, and regulation of the ROS level may be an important preventive
GM‑CSF and apoptosis occurred after 48 h [31]. Also, carcino- measure and may also support the anticancer therapies, by
gen-induced NF-κB expression is decreased by ursolic acid [69]. avoiding oxidative stress.
However, in contrast to this, in resting macrophages ursolic acid The organism uses two antioxidative mechanisms to regulate the
and also oleanolic acid activate NF-κB causing increased expres- level of free radicals, first an enzymatic and second a non-enzy-
sion of pro-inflammatory mediators such as TNF-α at concentra- matic system [82, 83]. The enzymatic system concerns enzymes
tions of 5 µM and 4 µM, respectively [70, 71]. such as superoxide dismutase (SOD) or catalase (CAT) that are
At first these results seem to be contradictory, but the different oxidised and reduced within a cascade to eliminate the free radi-
observations could be based on concentration-dependent effects, cals. The non-enzymatic system deals with antioxidants. One of
as observed for oleanolic acid with respect to the TNF-α produc- the bodyʼs own antioxidants is glutathione (GSH). It exists as a
tion of human mononuclear cells [65] or for betulinic acid monomeric tripeptide (GSH); when oxidised a GSSG dimer is

concerning TNF-α and IL-1β production in non-stimulated generated. In order to use the reducing power of glutathione to
RAW264.7 macrophages [63]. Another conceivable reason is the catalyze disulfide reductions in the presence of NADPH and glu-
influence of the milieu or the cell status that may crucially mod- tathione reductase, enzymes such as glutathione-S-transferase
ulate triterpene effects. Their investigation on TNF-α or nitric ox- and glutathione peroxidase are necessary [84].
ide (NO) production via inducible NO-synthase (iNOS) indicate Triterpenes, particularly lupeol, but also betulin and ursolic acid
that [63, 71, 72]. Using a stimulated cell system (e.g,. activated are known for their antioxidative potential [85–90]. They do not
macrophages) it was possible to observe an inhibition of pro-in- act as a classical antioxidant; however, triterpenes activate the
flammatory mediators by triterpene treatment [63, 72]. How- enzymatic system by increasing the activity of SOD as well as
ever, treatment of non-stimulated cells, such as resting macro- CAT and glutathione S-transferase and glutathione peroxidase
phages, with triterpenes led to an increase of pro-inflammatory [90–92].
factors such as TNF-α or IL-1β [64, 71]. Certainly, these are in vitro In detail, lupeol especially displays convincing effects particularly
data and up to now it is not clear whether these findings are on chronic inflammatory diseases such as chronic arthritis, but
meaningful for in vivo models or in therapeutic use. But triter- also as a chemoprotective agent. When arthritic rats were treated
pene-induced effects seem to be critically affected by environ- orally with lupeol (50 mg/kg body weight daily for 8 days), a sig-
mental conditions. nificant decrease of the inflammatory symptoms was observed,
Phospholipase A2 (PLA2) provides substrate for cyclooxygenase while the activity of the antioxidative enzymes SOD and CAT
and 5-lipoxygenase. These pathways are major pathways of the were elevated [93]. Another positive effect of lupeol is docu-
inflammation process. Betulin and betulinic acid [73], as well as mented in the case of hyperoxaluria in rats [94]. The excess of ox-
oleanolic acid [74], can inhibit PLA2. Downstream, COX-2 and its alate causes a high oxidative stress on the renal tissue. Similar to
product prostaglandin E2 (PGE2) are also repressed by lupeol, be- lupeol, betulin (35 mg/kg body weight daily for 21 days) normal-
tulin, betulinic, and ursolic acid [61, 63, 68, 75, 76]. Again COX-2 ises the glutathione status, increases the SOD and CAT activity
expression is regulated by NF-κB, suggesting an inhibitory effect [90] and decreases the peroxidation of erythrocyte membrane
of triterpenes on this transcription factor [68, 77]. lipids as well as normalises the activity of membrane bound AT-
Unfortunately, the use of different cell systems, with a diverse Pases [92]. A third indication that has been investigated is hyper-
metabolic background, plus the usage of different triterpenes, in cholesterolemia. Lupeol (50 mg/kg body weight daily) normalises
various concentrations, precludes suggesting an exact mode of ac- the lipid profile and activates the bodyʼs own antioxidative sys-
tion for the anti-inflammatory effect of these substances. How- tem followed by a decrease of oxidative stress in rats. This results
ever, the abundance of data, and especially the in vivo observa- in a protection of renal tissue. In this case the antioxidative effect
tions, evidenced the anti-inflammatory potential of the listed of lupeol is called cardioprotective or renalprotective [87, 95, 96].
pentacyclic triterpenes. A promising target for the triterpenes pre- In the majority of in vivo studies lupeol and also betulin were ad-
sented seems to be NF-κB. A number of proteins modulated by tri- ministered orally daily without any adverse effects at a dosage of
terpenes, such as TNF-α, IL-8 or COX-2 are under control of this 35–50 mg/kg body weight, which is a high dose. Considering an
transcription factor. However, the milieu, such as the cell status, average human body weight of 60 kg, the application of 3 g of
has to be considered because it seems to have a strong influence the drug per day would be necessary.
on the triterpene effects and should receive special attention. The diseases mentioned are not directly related to the develop-
ment of cancer, but they illustrate the antioxidative activity of lu-
peol or betulin. This seems to be not only a central part of their
Antioxidative Effects biological activity but also the basic mechanism of their chemo-
! preventive effects which may avoid cancer development.
ROS are well recognised as playing a dual role as both deleterious Substances that cause cell damage, particularly DNA damage or
and beneficial species. ROS are normally generated by tightly induce chronic inflammations are potential carcinogens. Agents
regulated enzymes to maintain moderate concentrations, provid- that shield the organism from these attacks are called chemopre-

Review 1555
ventive. Recently lupeol was termed as a chemopreventive agent, Redifferentiation

reviewed by Chaturvedi and colleagues [7]. This includes hepato- !
protective effects protecting liver cells from cadmium, 7,12-di- Differentiation and proliferation of a cell are mutually exclusive.
methylbenz(a)anthracene (DMBA) or hepatotoxic aflatoxins Differentiation takes place in the G0 state of a cell. In order to pro-
[97–100] or cardioprotective activity shielding cardiac tissue liferate, reentering the cell cycle is necessary. Thus, achieving dif-
from cyclophosphamide induced cardiotoxicity [88] by oral ad- ferentiation of a still proliferating cancer cell is one possible ap-
ministration of lupeol. Furthermore, a chemoprotective effect of proach of cancer treatment. In this respect, compounds that in-
topically applied lupeol was observed when skin was treated fluence and enhance differentiation processes in cells are prom-
with benzoylperoxides [89, 101] or DMBA [102]. All these poten- ising candidates for cancer therapy. In case of triterpenes, very in-
tial carcinogenic agents cause oxidative stress, deplete gluta- teresting data reveal the promotion of differentiation of healthy
thione and decrease the activity of antioxidant enzymes. Lupeol cells such as keratinocytes and also of the redifferentiation of tu-
regenerates the glutathione pool along with an elevation in the mour cells.
activities of the antioxidising enzymes and anti-oxidants. With regard to differentiation of healthy cells, it was shown that
Further cytoprotective effects are also known for betulin and tri- betulinic acid induces differentiation in normal keratinocytes in
terpene acids. In vitro pretreatment with 2 and 22 µM ursolic ac- vitro at concentrations of 9–18 µM, including an upregulation of
id protects human lymphocytes against UVB-induced lipid per- filaggrin and involucrin [11]. Likewise, oleanolic acid, but not ur-
oxidation and DNA-damage concentration-dependently [86]. solic acid, increases the expression of these two differentiation
Various in vitro studies revealed hepatoprotective effects of betu- markers in vivo in mouse skin disrupted by tape stripping. Olea-
lin, betulinic, ursolic and oleanolic acid against cadmium or etha- nolic acid not only upregulated structural proteins localised in
nol-induced toxicity in HepG2 cells (2–11 µM) [103, 104]. It the spinous/granular layers in the epidermis, but also the corni-

should be mentioned that these effects occur using subtoxic tri- fied envelope formation as the final product of terminal differen-
terpene concentrations depending on the cell type. Recently pub- tiation was increased and the barrier function of the epidermis
lished data showed chemopreventive activity of oleanolic and ur- was regenerated, as measured by a decreased transepidermal
solic acid also in vivo. Rats treated orally with 1,2-dimethylhydra- water loss [108]. Similar results were obtained with a highly pu-
zine developed colon associated carcinogenic dysplasia caused by rified triterpene dry extract from birch bark consisting of betulin
agent-induced oxidative stress. Simultaneous oral administration (80%), betulinic acid, oleanolic acid, and lupeol (1–4 %). Normal
of 25 mg/kg body weight oleanolic or ursolic acid decreases the human keratinocytes and human skin explants were treated with
appearance of cell damage [105]. 10 µg/mL of the extract. Besides the upregulation of early and late
Further experiments focussed on hepatoprotective effects have differentiation markers such as loricrin, filaggrin, involucrin,
identified a possible key target for the antioxidative effect of tri- transglutaminase, and keratin 10, as well as increased Notch2 ex-
terpenes. Oleanolic acid treatment dramatically increased expression, also a typical degradation of DNA strictly limited to the
pression of the transcription factor nuclear factor E2-related fac- distal stratum granulosum cells was observed [109].
tor 2 (Nrf2) [106]. Regulatory regions of the genes for cytoprotec- One possible signalling cascade activated by triterpenes is the
tive enzymes such as glutathione S-transferase or SOD contain peroxisome proliferator activated receptor-α (PPAR-α) pathway,
the antioxidant response element (ARE), which is activated upon which is involved in the regulation of lipid metabolism in the ep-
binding of Nrf2. Nrf2 has been shown to be essential in the upre- idermis [110]. Oleanolic acid and ursolic acid topically applied on
gulation of these genes in response to oxidative stress [107]. The disrupted mouse skin improved the recovery of permeability bar-
mechanism of increasing Nrf2 expression by oleanolic acid has rier functions and stimulated epidermal differentiation via PPAR-
not been clarified so far. One hypothesis might be the generation α expression [108, 111]. Another hypothesis is the selective upre-
of ROS that triggers the antioxidative cascade including Nrf2 ex- gulation of the transient receptor potential channel 6 (TRPC6) by
pression. For oleanolic acid treatment at concentrations of 25 µM, triterpenes in primary keratinocytes [109]. TRPC6 is a calcium
accumulation of ROS in an astrocytoma cell line resulting in channel mediating differentiation by regulating the Ca2+ influx
apoptosis has been reported [20], but it was interpreted only in into the keratinocytes [112].
the context of apoptosis induction. Also betulinic acid (20– Besides the upregulation of differentiation markers, or the acti-
100 µM) showed ROS generation at concentrations resulting in vation of corresponding signalling pathways, morphological
apoptosis [18, 24]. Therefore the question arises, whether gener- changes are also an important part of the differentiation of many
ation of ROS could be induced by triterpenes or especially by tri- cell types and, therefore, a useful additional marker to recognise
terpene acids used in a subtoxic concentration, and if this could differentiation. Thus, the morphological change of primary kera-
lead to an activation of the antioxidative system. tinocytes into a typical hexagonal corneocyte shape under triter-
Taken together, lupeol and betulin, as well as triterpene acids, pene acid treatment encourages the suggestion of their differenti-
such as oleanolic acid and ursolic acid, display an antioxidative ation-inducing efficacy [11, 108]. Also mammary epithelial cells of
activity. The underlying mechanism is the modulation of the rats as well as tumour cells, such as tetracarcinoma stem cells
bodyʼs own enzymatic antioxidative system including enzymes [113], showed morphological changes when treated with olea-
such as SOD and CAT, as well as glutathione S-transferase, per- nolic acid or ursolic acid [114]. Not only triterpene acids induce
haps triggered by the activation of Nrf2 and leading to a generally such morphological changes, but also treatment with lupeol re-
elevated antioxidant status of the organism. The antioxidative ef- sults in modifications of the cell shape, e.g., of the mouse melano-
fect of triterpenes seems to be a central part of their biological ma cell line B16 2F2, and an inhibition of the ability of the cells to
activity and may also be useful as a preventive strategy in the migrate, thus reducing their malignant potential [115]. The mor-
case of cancer. phological changes mediated by alterations of the cytoskeleton
are accompanied by the degradation and reconstruction of micro-
filaments consisting of actin, whereas the actin content of the cells
remains constant [20, 115, 116]. Up to now it is not clear whether

1556 Review
these changes are directly induced by triterpenoids or are a result growth arrest in melanoma tumours by interfering with the cy-
of the activated differentiation process. But it should be men- clin/cdk2/p21 complex activity [124].
tioned that morphological changes could also be observed in the Modern cancer treatment also includes modulation of the im-
apoptosis process induced by triterpenes [20]. Therefore the inter- mune system. As noted above, in vitro a broad spectrum of im-
pretation of this phenomenon must be carefully evaluated. mune modulations by triterpenes is observed. Indeed, this could
Moreover, differentiation is also indicated by regaining or devel- be confirmed in some in vivo studies, e.g., in a study using mela-
oping special cell type specific properties. For example, myeloid noma (B16.F10) bearing mice (C57BL/6). I. p. injected ursolic or
leukaemia cells (HL-60) amplify 1-alpha,25-dihydroxyvitamin oleanolic acid (50 µmol/kg body weight, for 5 days) was found to
D3 induced monocytic marker expression such as CD11b or CD14 produce enhanced natural killer cell activity and increased the
when treated additionally with betulinic acid [117]. Oleanolic ac- cytokine IL-2 that promotes the lytic activity of NK cells. In addi-
id decreases the proliferation rate of M1 mouse carcinoma cells tion, antibody-dependent cell mediated cytotoxicity (ADCC) as
and human leukaemia cells (HL-60) while phagocytotic activity well as antibody-dependent complement-mediated cytotoxicity
is increased. And lupeol as well as betulin and betulinic acid in- (ACC) were enhanced. According to the expected anticancer ef-
duce melanogenesis in B16 2F2 mouse melanoma cells [118]. fect, the elevated levels of GM‑CSF and IL-6 in tumour-bearing
The ability to induce differentiation or redifferentiation, instead control animals were also reduced by the treatment with ursolic
of acting as a cytotoxic substance, is always a question of the acid [125]. Another study showed lupeol efficacy in a TPA in-
available triterpene concentration and depends also on cell type duced mouse skin tumourigenesis model (CD1) by its anti-in-
and cell status [109]. Until now some mechanistic investigations flammatory activity. Prior topical application of 1–2 mg/animal
have been carried out only in keratinocytes, revealing two possi- lupeol resulted in the inhibition of the TPA induced activation of
ble targets: PPAR-α and TRPC6. For other cell types, the observa- PI3K and NF-κB and in an inhibition of COX-2 and iNOS protein

tions were only of the differentiation process or the outcome, for expression. The mice showed significant reduced tumour inci-
example, morphological changes, developing special abilities dence, lower tumour burden and a delay in the latency period
such as phagocytotic activity or expressing differentiation for tumour appearance [76].
markers such as CD11b or CD14 in the case of myeloid leukaemia While the antiangiogenic effect of ursolic acid in vitro was still
cells. The differentiation data are still fragmentary, but here the being discussed, Lee et al. (2001) proposed this kind of anticancer
potential of triterpenes seems to be worth investigating in detail. effect based on in vivo studies. Reduced oxygen consumption
after treatment as well as a significant decreased tumour intersti-
tial fluid and blood pressure were obtained after i. p. application
Anticancer Activity in vivo of 100 mg/kg body weight ursolic acid. This was accompanied by
! an inhibited tumour growth of a murine fibrosarcoma (FSaII)
The relevance of in vitro results can only be judged against subse- [126]. The inhibitory action of ursolic acid on urokinase as ob-
quent in vivo studies. Because of this, the strong interest on pen- served in vitro was assumed to be important with respect not on-
tacyclic triterpenes as anticancer agents did not start until betu- ly to the antiangiogenic effect but also to the suppression of tu-
linic acid was found to be effective in vivo against melanoma by mour-invasion and metastasis. An in vivo study on B16 mouse
Pisha et al. in 1995 [119]. melanoma treated C57BL/6 mice showed the complete inhibition
Bioavailability is the precondition of in vivo effects. Only a few of lung colonisation after 50 mg/kg ursolic acid administered i. p.
pharmacokinetic studies have been published. BA was found in daily, immediately after tumour injection during 16 consecutive
various tissues 24 h after i. p. administration (500 mg/kg; mouse) days. The authors proposed that besides the in vitro observed
and reached its highest concentration in perirenal fat. Peak se- urokinase inhibition activity of ursolic acid, an inhibition of ca-
rum concentration of 4.0 µg/mL was observed at 0.23 h after ap- thepsin B, which represents another possible drug target for the
plication [120]. BE reaches a saturation concentration of 138 ng/ suppression of tumour invasion and metastasis, may also play a
mL within 4 h after i. p. administration to rats [121]. These rela- crucial role [41]. Comparable results with respect to tumour me-
tively low serum levels can be explained by the low solubility in tastasis were found for betulinic acid (10 mg/kg body weight per
water (BA, OA: 0.02 µg/mL and BE < 0.1 µg/mL) [62, 122]. It is day) when used in a similar experimental setup [127]. As already
known that OA is able to bind to plasmin and albumin [17, 21], mentioned above, betulinic acid targets VEGF expression in a
so binding phenomena could support the bioavailability. Thus prostate cancer mouse model and induces the selective protea-
the prediction of in vivo effects and the concentrations necessary some-mediated degradation of transcription factor specificity
for them may be unreliable if based on in vitro results. protein Sp1, Sp3 and Sp4 [47].
For instance, cytotoxicity was shown for lupeol combating testos- Another approach for the usage of triterpenes in cancer-therapy
terone-induced prostate enlargement in mice, by inducing apop- is their combination with established chemotherapeutics. In vivo
tosis in the hypodiploid regions, and in tumours with human studies using an orthotopic metastatic nude mouse model of oral
prostate origin in a xenograft model [28, 123]. Recent findings tongue squamous cell carcinoma showed that lupeol (2 mg/kg
showed that lupeol (40 mg/kg body weight thrice a week) also in- body weight) dramatically decreased the tumour volume and
hibits the growth of highly aggressive human metastatic melano- suppressed local metastasis without side effects. It was 3-fold
ma cells (451Lu) in an athymic nude mouse xenograft model. Im- more effective than cisplatin, a commonly used chemotherapeu-
munohistochemical analysis of tumour tissue revealed that ani- tic agent with severe side effects. Surprisingly, lupeol in combina-
mals receiving lupeol exhibit decreased Ki67 and PCNA-positive tion with low-dose cisplatin was 13-fold more potent than lupeol
cells, suggesting an antiproliferative effect of lupeol. This corre- alone and up to 40-fold more than cisplatin alone, certainly with-
lated with a decreased number of cyclin D1, cyclin D2 and Cdk2 out side effects in the animal model used [128]. In another study
positive cells and an elevated level of p21 protein compared to betulinic acid augmented the inhibitory effect of vincristine, the
the control mice. The latter result indicates that lupeol causes major chemotherapeutic agent used for the treatment of melano-
ma [127]. Combination with triterpenes allowed reduction of the

Review 1557
concentration of the chemotherapeutic agent, without loosing Another possible application is combining triterpenes with al-
the effectiveness of treatment, while side effects were decreased. ready commonly used chemotherapeutic agents. This may allow
As mentioned previously, up to now there has been only very lowering the chemotherapeutic dose without loss of efficacy but
limited experience in the treatment of human cancer patients hopefully brings with it less adverse effects and may even give
with the listed triterpenoids. It should be mentioned, that in ani- synergistic effects.
mal studies dosages of between 10 and 100 mg/kg body weight The pharmacological potential of triterpenes for cancer treat-
are applied which is between 0.6 and 6 g per patient per applica- ment seems to be high, although up to now no clinical trial has
tion (60 kg body weight assumed). This is an unusually high been published using triterpenes in cancer therapy. This may be
amount for a drug, and the feasibility and the relevance of this explained by their almost complete insolubility in water [5, 122].
dose is questionable. Nevertheless, betulinic acid is currently But this galenic problem can be solved by derivatisation, com-
undergoing a phase II clinical trial for dysplastic melanocytic nae- plexation [132, 133], or liposomal formulation [134]. Another
vus (web site: ClinicalTrials.gov). Beside this, recent data show problem might be that triterpenes often provide only moderate
the successful topical treatment of precancerous lesions, namely effects in vitro, perhaps due to their poor solubility [122] and
actinic keratoses, with a triterpene dry extract (TE) of the outer the use of solvents such as dimethylsulfoxide (DMSO) that are
bark of birch as mentioned above [37]. Immunohistochemical in- not inert. In our experience, using 1 % DMSO in cell culture me-
vestigations of biopsies before and after a three month treatment dium, a maximum concentration between 20 and 40 µg/ml triter-
with TE showed a downstaging of the actinic keratosis and a re- penes is possible without crystallisation but it has to be carefully
organised epidermal structure. checked in each case. Nonetheless they exhibit convincing effects
It has been possible to confirm in vivo all the general effects of the when applied in vivo, as seen in the case of the birch bark TE ex-
triterpenes discussed that were predicted based on in vitro data tract. Further, the high dose that is used in animal tests might be

(inducing apoptosis, anti-inflammatory, antiangiogenic and anti- an obstacle to transferring it to a therapeutic dose used in human
oxidative effects), except for the redifferentiation effect. In this treatment. Up to now, only two clinical trials have been carried
case, the observation of the downstaging of actinic keratosis le- out with these substances, one treating actinic keratosis and an-
sions after a three month treatment with triterpenes in a clinical other dysplastic melanocytic naevus. In each case topical oint-
trial has been the only observation available until now, but there ments were used. Internal application, such as s. c. or i. v., has
is no experimental work. Thus it is too soon to claim a re-differ- not yet been tested in human cancer treatment.
entiation effect for the triterpenes in cancer treatment; however Looking into future, there remains some tasks to do in order to
it is a promising field of triterpene research. tap the full potential of these triterpenes. A lot of effects are not
Administration of drugs also raises the question of their toxicity fully understood like the antioxidative efficacy or the differentia-
in vivo. Pentacyclic triterpenes of the lupane, oleanane and ur- tion effects. Another point is that the strength of each single com-
sane group are considered as relatively nontoxic drugs. A recently pound is not defined detailed enough, to find their optimal do-
published subchronic toxicity study showed that intraperitoneal main or to compose them. The differences between the experi-
and subcutaneous administration of a triterpene mixture (80 % mental settings, the used concentrations, time courses or the var-
betulin, betulinic acid, lupeol, oleanolic acid, erythrodiol 1–4 %) ious cell lines, with different metabolic background and varying
produces no toxic effects [121]. This is in line with previously cell culture parameters, makes it difficult to compare and com-
published data for single triterpenes. For example, i. p. adminis- bine results to get an idea of the underlying mechanisms. There-
tered oleanolic acid has a LD50 of 1500 mg/mL in mice [129] and fore, parallel-testing of triterpenes in a standardised setting
a single s. c. dose of 1000 mg/mL caused no toxic effects in rats would be preferable. Due to their insolubility in water the bio-
[130]. Also i. p. administration of 500 mg/kg body weight betu- availability is not given in an optimal way, nevertheless, in vivo
linic acid in suspension caused no toxicity [131]. As summarised effects are observed. This may be improved by optimising the ga-
previously, administration of betulin or lupeol at concentrations lenic form or the application route.
of 35–50 mg/kg body weight also produces no toxic effects. Their chemopreventive activity makes triterpenes interesting in
another field. As secondary plant metabolites they are present
in food. For example, due to the common use of olives and herbs
Conclusion such as rosemary of the Lamiaceae family, the Mediterranean di-
! et is high in triterpenes. It is not proven yet, whether they are re-
Cancer is a disease with multiple etiological factors and multiple sponsible for the beneficial effects of this nutrition on health.
oncogenes are involved in its pathogenesis. Therefore, beside a In conclusion pentacyclic triterpenes are a rich natural pool of
combination of different treatment strategies, multifunctional promising anticancer drugs as well as chemopreventive agents.
agents with multiple targets also offer a more rational approach Further, they are available in high amounts also in industrial
than single ones to both its prevention and therapy. waste products like birch bark or the pulpy residue from apples.
The literature survey reveals that lupeol, betulin, betulinic acid, This and their promising pharmacological effects warrants fur-
oleanolic and ursolic acid are multitarget agents (l " Fig. 2). They ther pharmaceutical development and clinical investigations to
fit to the concept of modern cancer therapy, by treating cancer conclude the puzzle of their biological activities.
from different sides, including the tumour environment and the
immune system.
But parallel testing of these compounds revealed differences in Acknowledgements
their efficacy in several assays. Therefore, the combination of dif- !
ferent triterpenes may be a way to improve their potential as The author thanks Stefan F. Martin, Sebastian Jäger, Irmgard Mer-
multitarget drugs. Plants like white birch, rosemary, or mistletoe fort and David J. Heaf for expert assistance and critical reading in
offer different natural compositions of triterpenes, and their tri- preparation of the manuscript.
terpene extracts may be used as starting material for further
pharmaceutical development.

1558 Review
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Review 1549

Pentacyclic Triterpenes of the Lupane, Oleanane and

Author Melanie N. Laszczyk 1, 2

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Laszczyk MN. Pentacyclic Triterpenes of … Planta Med 2009; 75: 1549–1560

Nrf2: nuclear factor E2-related factor 2 Sp: specificity protein

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Plant Part Triterpene Amount Reference Table 1 Plants which display a

Laszczyk MN. Pentacyclic Triterpenes of … Planta Med 2009; 75: 1549–1560

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ventive. Recently lupeol was termed as a chemopreventive agent, Redifferentiation

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Laszczyk MN. Pentacyclic Triterpenes of … Planta Med 2009; 75: 1549–1560

References 25 Rzeski W, Stepulak A, Szymanski M, Sifringer M, Kaczor J, Wejksza K,

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