Xanthorrhizol: a review of its pharmacological activities and anticancer properties

Previous studies evaluated that XNT has antimicrobial 15], 16], 19], 22]–25], anti-inflammatory 10], 17], 26], 27], antioxidant 5], 17], antihyperglycemic 6], antihypertensive 21], 28], antiplatelet 29], nephroprotective and hepatoprotective 30]–32], estrogenic and antiestrogenic properties 20], 33]. These pharmacological activities are summarized in Table 1.

Table 1. Historical application of XNT

Antimicrobial properties

XNT is considered active against a variety of pathogenic microorganisms. Antimicrobial
effects of XNT included antibacterial 15], 16], 22], anticandidal 19], 23] and antifungal activities 24], 25]. There have been evaluated by in vitro susceptibility tests such as minimum inhibitory
concentration (MIC), minimum bactericidal concentration (MBC), minimum fungicidal
concentration (MFC), NCCLS (M38-A) standard method and biofilm quantification.

Earlier study by Hwang and colleagues reported that XNT showed the highest antibacterial
activity against dental caries causing bacteria (Streptococcus species) followed by periodontitis causing bacteria (Actinomyces viscosus and Porphyromona gingialis) 16]. XNT also strongly inhibited Gram-positive bacteria Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative bacteria Escherichia coli34] and acne-causing bacteria Propionibacterium acnes35].

Moreover, the ability of XNT in preventing dental plaque and removing oral bacterial
biofilms has been demonstrated on the oral Streptococcus mutans biofilms in vitro 22]. Biofilms removal activities were affected by XNT concentration, exposure time and
the biofilm phase growth. For example, XNT (5 µM) completely inhibited the formation
of S. mutans biofilms at adherent growth phase, whilst XNT (50 µM) removed 76 % of biofilm at
plateau accumulated phase after 60 min exposure. XNT killed S. mutans at planktonic growth due to its direct contact with biofilm outer layer cells 15], 22], 36]. The antimicrobial activities were induced by the capability of the hydrophobic chains
of XNT to penetrate and reduce the viability of dental plaque biofilm 37].

For anticandidal activity, XNT inhibited planktonic cells of Candida albicans at MICs range of 1–15 µg/mL 19]. This finding is controversial with the previous result 16], where Candida albicans were found to be resistant to XNT. Since there is lack of information on the XNT’s
condition used in the previous work 16], we infer that XNT dissolved in dimethyl sulfoxide (DMSO) 19] may enhance anticandidal activity towards C. albicans. The ability of XNT to prevent and kill C. albicans was further supported by Rukayadi and Hwang, where XNT at 8 µg/mL completely reduced
C. albicans biofilms at adherent phase, whilst 32 µg/mL reduced 88 and 67.5 % of biofilm at intermediate
and mature phase, respectively 23]. It was also active against pathogenic non-Candida albicans species such as C. glabrata, C. guilliermondii and C. parapsilosis biofilms in vitro 19], 38]. These results indicated that XNT might be used to cure biofilm-related candidal
infections and treat candidiasis.

On the other hand, XNT performed antifungal activity against planktonic fungal cells
such as Malassezia species 24] and opportunistic filamentous fungi 25]. Anti-Malassezia activity of XNT was reported in M. furfur and M. pachydermatis24]. XNT also inhibited the conidial germination of all six filamentous fungi species
such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Rhizopus oryzae and Trichophyton mentagrophtes based on NCCLS (M38-A) standard method. Its effect was comparable to amphotericin
B 25].

Although antimicrobial mechanisms of XNT are not well understood, we believe that
XNT may suppress nuclear factor kappaB (NF-kB) and mitogen-activated protein kinase
(MAPK) induced by microbial infection. XNT has been demonstrated to inactivate both
of them in skin cancer 26]. According to Wilken et al., infectious antigens could induce the activation of NF-kB
39]. For example, exposure of epithelial cells to C. albicans hyphae stimulates pro-inflammatory immune responses via NF-kB and MAPK pathways 40], which are also involved in the carcinogenesis 41], 42].

Based on epidemiologic studies, it has been estimated about 15 % of the worldwide
cancer incidence is considerably related with microbial infection 43]. Chronic infection of human papilloma virus in immunocompetent hosts causes cervical
carcinoma, whilst hepatitis B and C virus infection leads to hepatocellular carcinoma.
Mirobes may also induce cancer incidence through opportunistic infection such as human
herpes virus (HHV)-8 infection leading to Kaposi’s sarcoma 44]–46]. In addition, gastric cancer secondary to Helicobacter pylori colonization or colon cancer may occur in certain people due to abnormal immune responses
to microbes contributed by chronic inflammatory bowel disease precipitated by the
intestinal microflora 44]–46]. Since XNT has anticancer and antimicrobial properties, we suggest that its antimicrobial
mechanism studies should be conducted not only to develop XNT as a potent antimicrobial
agent, but also provides new insight on the suppression of microbes-induced cancer
in the future.

Anti-inflammatory properties

First in vitro anti-inflammatory report of XNT has been shown in lipopolysaccharide-activated
mouse leukaemic monocyte macrophage cell RAW 264.7 27]. XNT reduced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)
activity by inhibiting the production of prostaglandin E2 (PGE2) and nitric oxide
(NO) respectively in lipopolysaccharide-activated mouse macrophage cell RAW 264.7.
These results indicated XNT may be a potent COX-2 and iNOS inhibitors 27], which is suggested by another anti-inflammatory assay of XNT performed in activated
primary cultured microglial cells induced by lipopolysaccharide 17]. It was found to inhibit COX-2, iNOS, proinflammatory cytokine interleukin-6 (IL-6)
and tumor necrosis factor-? (TNF-?) in activated microglial cells. It is clear that
XNT is capable to inhibit COX-2 and iNOS as consistent with several findings 26], 27], 30], whilst IL-6 and TNF-? as consistent with recent report 6].

Further in vivo anti-inflammatory studies of XNT have been conducted in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse acute inflammation model 26]. XNT has been reported to counteract the effect of TPA-induced ornithine decarboxylase
(ODC), COX-2 and iNOS activation in mouse skin. Since pro-inflammatory proteins COX-2
and iNOS are highly associated with cutaneous inflammation, cell proliferation and
skin tumor promotion 26], 47], their suppression are important to alleviate inflammation and prevent cancer 26], 41], 42], 48]. The expression of COX-2 and iNOS might be regulated by transcription factor, NF-kB,
as reported in cultured cell lines and TPA-induced cutaneous inflammation in mouse
skin 17], 26]. When nuclear translocation and DNA binding of NF-kB increase in response to external
stimuli, NF-kB stimulates COX-2 and iNOS transcription 26], 48]. Thus, NF-kB plays a pivotal role in inflammation and tumorigenesis.

Another study postulated that XNT may exert anti-inflammatory activity by blocking
the neurogenic and inflammatory pain response in the formalin induced pain test in
rats 10]. It may partly contribute to the analgesic effects or antinociceptive activity. However,
the detailed mechanisms have not been worked out. From the integration of findings
10], 17], 26], 27], we summarize that anti-inflammatory mechanism of XNT involved inhibition of IL-6
and TNF-?, and suppression of COX-2 and iNOS expression via NF-kB pathway resulting
PGE2 and NO reduction.

Antioxidant properties

Antioxidant properties of XNT contribute to its neuroprotective 17] and LDL oxidation inhibitory effects. XNT has been known to possess in vitro antioxidant
activity against murine hippocampal neuronal HT22 cell line 17] and copper-mediated isolated human low-density lipoprotein (LDL) oxidation 5]. In murine hippocampal neuronal HT22 cell line, XNT reduced the free radical-mediated
oxidative damage 17]. Its antioxidant properties exerted potent neuroprotective effects by suppressing
hydrogen peroxide (H
₂
O
₂
)-induced lipid peroxidation in rat brain homogenates, glutamate-induced neurotoxicity
and reactive oxygen species (ROS) production in HT22 cells. These results indicated
that XNT could be a potent agent to treat Alzheimer’s disease and ROS associated neurological
disease 17].

On the other hand, the inhibition of copper-catalysed LDL oxidation was evaluated
employing thiobarbituric acid reactive substances (TBARSs) assay with human LDL as
the oxidation substrate 5]. XNT strongly inhibited human LDL peroxidation in a dose-dependent manner. The presence
of phenolic hydroxyl group (sesquiterpene phenol) on the bisabolene skeleton of XNT,
has most probably contributed to its strong antioxidant properties by chelating Cu
²⁺
. This in turns may suppress the initiation of LDL oxidation and generation of free
radicals at the lipoprotein 5]. We suggest that XNT might be subjected to further investigation in cardiovascular
disorders because high LDL antioxidant activity could reduce the risk of heart attack.
In vivo antioxidant assay could also be conducted in the future.

Antihyperglycemic properties

In vivo antihyperglycemic effects of XNT have been demonstrated in the high-fat diet
(HFD)-induced obese mice 6]. XNT and C. xanthorrhiza extract with standardized XNT reduced the levels of insulin, glucose, free fatty
acid (FFA), and triglyceride (TG) in their serum. XNT also reduced the size of epididymal
fat pad and adipocyte and decreased the production of inflammatory cytokines such
as TNF-?, IL-6, interleukin-1ß (IL-1ß), and C-reactive protein (CRP) in adipose tissue,
liver and muscle of HFD-induced obese mice. Thus, XNT may prevent fatty liver disease
(accumulation of liver fat) and chronic inflammation 6].

These results showed that XNT’s antihyperglycemic and anti-inflammatory activities
may restrict and treat type 2 diabetes, which is mainly caused by obesity-induced
insulin resistance 6]. Insulin resistance is related to chronic low-grade inflammation states such as increased
proinflmmatory cytokine levels. The inflammation process is initiated by the activation
of TNF-?, IL-6, IL-1ß and CRP, which are known to disrupt the transduction of insulin
signalling causing insulin resistance 6]. Based on this study, we reveal that XNT could suppress HFD-induced metabolic disorders
including hyperglycemia, inflammation and hepatic injury by inhibiting fatty acid
release from adipose tissue. We suggest that anti-obesity effects of XNT and its related
mechanisms of action could be studied in the future.

Antihypertensive properties

XNT extracted from Iostephane heterophylla has shown potential antihypertensive activities 28]. A preliminary study demonstrated that XNT effectively inhibited precontractions
induced by calcium chloride, potassium chloride and noradrenaline in rat thoracic
aorta rings. The vasorelaxation effect of XNT indicated that it may act as a calcium
antagonist by reducing calcium influx into vascular smooth muscle cells in rat aorta.
In fact, its calcium antagonistic activity has been illustrated earlier in isolated
rat uterine smooth muscle 21]. XNT attenuated the effect of rat uterus’ tonic contraction stimulated by calcium
chloride, potassium and calcium channel agonist in a dose-dependent manner. This might
be due to the ability of XNT to block the voltage operated calcium influx in myometrial
cells. According to Grossman and Messerli, calcium antagonists reduce blood pressure
via vasodilation and decreased peripheral resistance 49]. Since calcium antagonists have been well established as basic antihypertensive drugs
50], we believe that XNT may have blood pressure-lowering effect. However, detailed antihypertensive
activities and mechanisms of XNT are yet to be elucidated.

Antiplatelet properties

In vitro antiplatelet activity of XNT (100 µg/mL) showed a strong inhibition towards
platelet aggregation stimulated by arachidonic acid (100 %), collagen (81.3 %) and
adenosine diphosphate (ADP) (78.6 %) in human whole blood 29]. Although previous studies reported that the antiplatelet activity of curcumin was
higher than XNT 29], the potential of XNT as an antiplatelet compound should not be neglected. We suggest
that its antiplatelet mechanism requires further investigation.

Nephroprotective and hepatoprotective properties: cisplastin-induced toxicity

Nephroprotective and hepatoprotective effects of XNT have been performed in male ICR
mice treated with cisplatin 30]–32]. Cisplatin is a potent chemotherapeutic drug 31], 51], but the occurrence of nephrotoxicity has become the main limitation of using cisplatin-based
chemotherapy 32], 52]. XNT exhibited nephroprotective effect by attenuating the increased specific gravity
of kidney induced by cisplatin 32]. Cisplatin-induced kidney injury was reported as increased kidney weight, enhanced
lipid peroxidation in kidney tissues, weakened filtration and excretion process of
kidney, and subsequently increased blood urea nitrogen and serum creatinine levels.
Pretreatment of XNT obviously restored the kidney weight to the base level and attenuated
the elevated levels of blood urea nitrogen and serum creatinine. Although DNA-binding
activity of NF-kB and activator protein 1 (AP-1) did not contribute to the nephroprotective
effect 32], the exact mechanism has not yet been identified.

High dose of cisplatin also induces hepatotoxicity 30], 31]. Cisplatin increased DNA-binding activity of NF-kB but suppressed DNA-binding activity
of AP-1. The function of NF-kB is to stimulate COX-2 and iNOS, which are associated
with inflammation and toxicity. XNT pretreatment has been shown to abrogate these
effects. XNT elicited hepatoprotective effects by reducing blood glutamate-pyruvate
transaminase (GPT) and glutamate–oxaloacetate transaminase (GOT) levels caused by
cisplatin 30]. The mechanism involved XNT’s dose-dependent attenuation of c-Jun N-terminal kinases
(JNKs) phosphorylation in MAPK signaling, especially JNK1 31]. This action may inhibit the transcription of COX-2, iNOS and transcription factor
subunits (c-fos and p50). When XNT suppressed cisplatin-induced c-Fos protein expression,
it may modulate the DNA-binding activity of NF-kB and AP-1, which in turns regulate
COX-2 and iNOS expression. Mitochondrial apoptosis was excluded since the expression
of both cytochrome c and caspase-9 was not changed 31]. Thus, it has been concluded that XNT minimized side effects of cisplatin-induced
hepatotoxicity by regulating the DNA-binding activities of transcription factors NF-kB
and AP-1 30] via blocking the phosphorylation of JNK(s) 31].

It was believed that XNT exerted better suppressing effect towards cisplatin-induced
nephrotoxicity 32] and hepatotoxicity than curcumin 30], 31]. At the same dose, curcumin was less effective in attenuating the elevated levels
of blood urea nitrogen and serum creatinine 32]. XNT downregulated COX-2 and iNOS gene expression, but curcumin suppressed only COX-2
gene 30]. Moreover, XNT abrogated the expression of NF-kB subunit, p50 and AP-1 subunit, c-fos,
but not curcumin 31]. Combined with the findings of both nephroprotective and hepatoprotective effects,
we assume that XNT could be clinically applied as a suppressant of toxicity for patients
administrated with high dose cisplatin to prevent kidney and liver damage.

Estrogenic and anti-estrogenic properties

XNT has been known to possess estrogenic activity in estrogen receptor (ER)-positive
MCF-7 cells during the state of hormone starvation 20], 33]. It has been reported that XNT treatment upregulated ER target gene expression, trefoil
factor 1 (pS2) and promoted the interaction of ER-estrogen response elements (EREs)
in MCF-7 cells. Since XNT has been proven to possess estrogenic activity in negligible
estrogen level 20], we suggest that XNT could be further explored in the treatment of estrogen deficiency-induced
menopausal symptoms, cardiovascular disease and osteoporosis.

In contrast, XNT was revealed as partial estrogen antagonist in T47D breast cancer
cells 12]. In molecular docking simulation, the binding interaction between XNT and human estrogen
receptor-? (hER?) indicated that XNT might be able to compete with estradiol. Both
XNT and estradiol showed almost similar binding free-energy. Also, a strong hydrophobic
interaction found between XNT and hER? may be due to the presence of hydroxyl group
(1-OH) and alkyl chains, leading to its potential as partial antagonist hER?. The
postulation was confirmed by pharmacophore modeling, which identified that 1-OH and
alkyl chain were two important chemical features of XNT as partial antagonist hER?
to strongly inhibit T47D cells. This molecular interaction with hER? also involved
aromatic ring of XNT 12].

Based on the estrogenic 20], 33] and anti-estrogenic activities 12] reported, we suggest that XNT may act as a potent phytoestrogen with beneficial therapeutic
potential. According to Tham et al., the partial estrogenic/anti-estrogenic behaviour
is a common characteristic of phytoestrogens 53]. The estrogenic activity of phytoestrogens is 100 to 1000-fold weaker than 17?-estradiol,
but its concentrations may be 100-fold higher than endogenous estrogens in the body
53]. Hence, we believe that abundant XNT molecules might act as competitive inhibitors
of endogenous 17?-estradiol. XNT may block the actions of estradiol from binding to
ERs of breast cancer cells, thus inhibiting tumor growth. Seeing that tumorigenesis
of ER-positive luminal A cell lines (MCF-7 and T47D) can be suppressed by anti-estrogen
therapy 54], XNT could be developed as a potential anti-estrogen agent.

To further study the effects of XNT as phytoestrogens in vitro, estrogen should not
be excluded in experimental condition because circulating estradiol exists at all
stages of the life cycle 53]. XNT may exert both estrogenic and anti-estrogenic effects on human metabolism, depending
on XNT and endogenous estrogens concentration, gender and menopausal status.