Mini-reviewGarcinol: Current status of its anti-oxidative, anti-inammatory andanti-cancer effectsChaoqun Liua,b, Paul Chi-Lui Hob, Fang Cheng Wonga, Gautam Sethic, Ling Zhi Wanga,c,*,Boon Cher Goha,c,d,**aCancer Science Institute of Singapore, National University of Singapore, Singapore 117599bDepartment of Pharmacy, National University of Singapore, Singapore 117543cDepartment of Pharmacology, National University Health System, Singapore 117597dDepartment of Haematology-Oncology, National University Health System, Singapore 119228AR T I C L E I NF OArticle history:Received 16 January 2015Received in revised form 12 March 2015Accepted 12 March 2015Keywords:Garcinia indicaGarcinolPharmacodynamicsPharmacokineticsToxicologyA B S T R AC TGarcinol is the main medicinal component of the dried fruit rind of Garcinia indica (G. indica), which hastraditionally been extensively used to treat gastric ailments and skin irritation. In vitro studies of garcinolrevealed its potential therapeutic effects, such as its anti-oxidative, anti-inammatory and anti-cancerproperties. Similarly, in vivo studies in animal models also demonstrated the ecacy of garcinol for thetreatment of various inammatory and cancerous conditions. Despite being well tolerated in preclini-cal studies, thetoxicological proleof garcinol remainselusive. Moreimportantly, systematicpharmacokinetics (PK) studies of garcinol to establish an appropriate route of administration and its ef-fective concentration range under physiological conditions have not yet been performed. PK studies playan essential role in translating the preclinical ndings of garcinol from cell line models and animal speciesto humans, thereby facilitating dose selection, the characterization of the therapeutic index, identica-tion of a metabolic pathway, and the determination of garcinols potency and tolerability. This paper reviewsthe current studies of garcinol as a potential anti-oxidant, anti-inammatory and anti-cancer agent andhighlights the importance of performing preclinical PK and toxicological studies on garcinol for its de-velopment pipeline. 2015 Elsevier Ireland Ltd. All rights reserved.IntroductionGarcinia indica (G. indica, also known as kokum) is a small trop-ical evergreen tree that was rst discovered in the Western Ghatsof India [1]. Natural compounds that have been isolated from therind of the fruit of garcinol include hydroxycitric acid, hydroxycitricacid lactone, citric acid, oxalic acid, malic acid, ascorbic acid, poly-phenols and anthocyanin [2,3]. The development of various analyticaltechniqueshasrecentlyfacilitatedtheextractionofgarcinol, apolyisoprenylated benzophenone derivative, from the rinds [4]. Theextraction of pure garcinol from G. indica enables its use in in vitroexperiments to delineate its potential therapeutic indications [5].By subjecting multiple cell lines to garcinol, molecular targets werecapturedandmechanisticexplanationsforitspharmacologicalactions could be inferred. The effects of garcinol observed in animalstudies provide evidence to justify or nullify its potential as a ther-apeutic agent. According to the drug development pipeline, garcinolis now at the stage of preclinical development, at which point phar-macodynamics (PD), toxicology and pharmacokinetic (PK) studiesshould be performed before the drug can proceed to the clinicaltesting phase. However, the absence of information on its absorp-tion, distribution, metabolism and elimination (ADME) as well asthe absence of toxicity proles impedes the use of garcinol in clin-ical studies. This current research gap in the toxicological and PKstudies warrants further investigations to rationalize and acceler-ate the development of garcinol as a drug candidate for clinical trials.The objectives of the present revieware to summarize the data fromAbbreviations: G. indica, Garcinia indica; PD, pharmacodynamics; PK, pharma-cokinetics;ADME, absorption, distribution, metabolismandelimination;OH,hydroxyl radical; ROS, reactive oxygen species; DPPH, 1,1-diphenyl-2-picrylhydrazyl;NFB, nuclear factor-k B; iNOS, inducible nitric oxide synthase; NO, nitric oxide;ERK1/2, extracellular signal-regulated kinase; MAPK, P38/Mitogen-activated proteinkinase; cPLA2, cytosolic phospholipases A2; PGs, prostaglandins; COX-2,cyclooxygenase-2; 5-LOX, 5-Lipoxygenase; LTs, leukotrienes; PI3K, phosphoinositide3-kinase; Akt, serine/threonine-specic protein kinase; CDKIs, cyclin-dependentkinases inhibitors; HAT, histone acetyltransferase; p300/PCAF, P300/CBP-associatedfactor; miRNAs, micro RNAs; MMPs, metalloproteinases; VEGFs, vascular endothe-lial growth factors; ACF, aberrant crypt foci; Cmax, maximum concentration; Tmax,time to maximum concentration; t1/2, elimination half-life; HCC, hepatocarcinoma;HNSCC, head and neck squamous cell carcinoma).* Corresponding author. Tel.: +6565168925; fax: +6568739664.E-mail addresses: csiwl@nus.edu.sg (L.Z. Wang).** Corresponding author. Tel.: +6565168925; fax: +6568739664.E-mail addresses: phcgbc@nus.edu.sg (B.C. Goh).http://dx.doi.org/10.1016/j.canlet.2015.03.0190304-3835/ 2015 Elsevier Ireland Ltd. All rights reserved.Cancer Letters 362 (2015) 814Contents lists available at ScienceDirectCancer Lettersj our nal homepage: www. el sevi er. com/ l ocat e/ canl etin vitro and in vivo studies of garcinol as a potential therapeutic agentand highlight the importance of performing preclinical toxicologi-cal and PK studies that could be the basis for future ecacy andsafety investigations.Uses of G. indicaMany uses have been reported for G. indica since historical times.In the culinary eld, the dried fruit rind can be used as a garnishto impart an acidic avor to curries, while extracts from the fruitcan be ground, steeped in syrup and consumed as a refreshing bev-erage [6]. In addition to the fruit rind, the oil-rich seed of G. indica,known as kokum butter, have also been used in the cosmetics andconfectionery industries [6]. In the Indian traditional systemof med-icine, Ayurveda, G. indicahas long been established as a multi-purpose herbal medicine in treating ailments. The leaves and fruitsare ground and administered to patients to treat indigestion. Therinds are used for inammatory conditions, and the fruit extract ispurported to be a natural antacid used to relieve gastric reux [7].Chemical structure and properties of garcinolGarcinol (molecular mass: 602.39) was rst extracted in 1981from the dried fruit rind of G. indica using hexane [4]. It was pre-cipitated as yellow needle-like crystals with a concentration of 1.5%in hexane [4]. A physical analysis of garcinol identied a meltingpoint of122 C and optical rotation of[]22D 143 (1% chloro-form). Themolecularformula, C38H50O6, wassuggestedtohavestructural similarities to xanthochymol (molecular mass: 602.82 and[]22D +141 (1% chloroform), a prenylated chalconoid previouslydiscovered from Garcinia xanthochymus [4]. However, based on itsoptical rotation sign, garcinol may be more closely associated withcambogin (molecular mass: 602.27 and []22D 132.9 (1% chlo-roform) [8,9], an isoprenylated benzophenone derivative isolatedfromG. cambogia and characterized by Venkatsuamy et al. [4,5]. Thehighly similar physical properties between cambogin and garcinolspurredresearcherstostructurallydistinguishthetwocom-pounds. The structural analysis conrmed the structure of garcinolas an isoprenylated benzophenone with an enolizable 1,3-diketonegroup conjugated to a 3,4-dihydroxybenzoyl moiety and two -unsaturated carbonyl groups. Hence, the molecular masses of garcinoland cambogin are the same, but their chemical structures differ[4,5,10], as depicted in Fig. 1, which also shows the structure ofxanthochymol.Current studies on PD actionsNumerous in vitro cell-based and in vivo animal model studieshave been carried out to elucidate the mechanisms ofaction ofgarcinol. A summary of these studies will be given later.In vitro studies on anti-oxidant propertiesGiventhe growing interest inthe potential healthbenets of con-suming dietary antioxidants, numerous studies have examined theuse of traditional herbs for the treatment of human disease [11,12].In general, phytochemicals may act as antioxidants by either eradi-cating free radicals or inhibiting pro-oxidants. They mitigate theinteraction between reactive oxygen species (ROS) with deoxyri-bonucleic acid (DNA) bases, lipids, proteins, and other biomolecules[13] and hence retard the onset and progression ofvarious dis-eases, including neurodegenerative diseases andcancer [1416]. Thefree radical, superoxide anion (O2) scavenging activity of garcinolwas shown to be as potent as that of gallic acid and stronger thanthat of (+)-catechin [16]. Furthermore, garcinol scavenges the freeradical DPPH(1,1-diphenyl-2-picrylhydrazyl) withthree times greaterpotency than DL--tocopherol, a lipid-soluble natural anti-oxidant[15]. In addition, garcinol was shown to be able to quench OHatan IC50of 0.32 M[16], thus preventing OH-induced DNAdamage.Similarly, 100 Mperoxynitrite was quenched with 0.2 Mgarcinol[17]. Thefreeradical quenchingactivities displayedbygarcinol suggestthat it may play a role in the protection against neurodegenerativediseases that are associated with a surplus of ROS [18].Despite promising in vitro results, the determination of garcinolsin vivo concentration is crucial because the anti-oxidative effect dis-played by garcinol was demonstrated using cell-free assays. Althoughgarcinol was found to be more potent than DL--tocopherol in anaqueous ethanol solution, it displayed poorer anti-oxidative prop-ertiesthantocopherolwhentestedinamicellarsystem[15].Therefore, the cellular uptake of garcinol through the amphiphilicphospholipid bilayer that surrounds the cell requires investiga-tion. The observed differences in the anti-oxidant ecacy may bereconciled by performing a PK analysis of garcinol in animal studiesto account for its tissue uptake and consequently, its physiologicalanti-oxidative signicance.In vitro studies on anti-inammatory effectsThe anti-inammatory effects of certain herbs have been studiedin detail; the most prominent example is aspirin, which is derivedFig. 1. A: Chemical structure of garcinol extracted from Garcinia indica. B: Structure of cambogin extracted from Garcinia cambogia. C: Structure of xanthochymol extractedfrom Garcinia xanthochymus [10].9 C. Liu et al./Cancer Letters 362 (2015) 814fromthe bark of the willowtree and one of the most important anti-inammatory drugs in clinical use. Current studies have shown thatgarcinol exhibits an anti-inammatory effect by interfering withvarious inammatory cascades. First, garcinol was found to affectthe nuclear factor NFkB signaling pathway. A study showed thatgarcinol suppressed inducible nitric oxide synthase (iNOS) synthe-sis most effectively at 5 M by inhibiting NFkB activation, leadingto reduced nitric oxide (NO) generation [19], a known physiologicfree radical believed to be elevated in neurodegenerative diseases,diabetes and rheumatoid arthritis [20]. Moreover, extracellular signal-regulated kinase 1/2 (ERK1/2), a protein kinase of the P38/Mitogen-activatedproteinkinase(MAPK)family, canalsomediatetheinammationprocess[21]. Undernormalphysiologicalcondi-tions, phosphorylated ERK1/2 activates cytosolic phospholipases A2(cPLA2) and leads to the formation of COX-2, an enzyme that pro-duces prostaglandins (PGs) [22]. In lipopolysaccharide-activatedmacrophages, 1 M garcinol inhibited the production of COX-2 andPGs. The proposed mechanism of action involves interference withthe LPS-mediated phosphorylation of ERK1/2, which reduces thelevel of COX-2 products in LPS-stimulated cells [23]. The third anti-inammatorypathwayofgarcinolwasfoundtoberelatedto5-lipoxygenase (5-LOX) activation, which is responsible for pro-ducing inammatory molecules, such as leukotrienes (LTs) [22].Garcinol inhibited the activation of 5-LOX at IC50 values of 0.1 Min in vitro cell-free assays and 1.9 M in cellular studies [24]. Thisdifference could be due to the lower activity of garcinol on intra-cellular 5-LOX [24]. Furthermore, garcinol inhibited isolated COX-1activation with an IC50 of 12 M, but this effect was not observedin human whole blood [24]. The loss of potency in human wholeblood could be due to the strong albumin-binding of garcinol [24].Together, these results suggest the urgent need to determine theconcentration of garcinol attainable in in vivo systems.Chronic inammation has been shown to be involved in thepathogenesisofvariouscancermodels[25]. Theelucidationofgarcinols anti-inammatory action has in turn inspired studies ofinammation-related cancers, which will be discussed in detail inSection Anti-inammation.Anti-cancer activitiesVarious PD studies of garcinol and different cancer types havebeen conducted, and the results are summarized in Table 1. In ad-dition to reducing inammation, garcinol displayed in vitro and invivo anti-cancer effects in various cancers. The mechanisms that un-derlie garcinols anti-cancer effect include the induction of apoptosis,inhibition of cell growth and proliferation, stimulation of cell cyclearrest and prevention of cancer cell metastasis.In vitro studiesAnti-inammation. Garcinolsanti-cancereffectsweretestedincancer cell models whose carcinogenesis is known to be affectedby inammation. The pathways and major targets of garcinol in theinammatory cascade are exemplied in Fig. 2. The up-regulationof COX-2 expression has been observed in lung and colon cancers,while increases in 5-LOX, COX-2 and PGE2are observed in early pan-creatic cancer [4850]. In human lung carcinoma cell lines andhuman pancreatic BxPC-3 cell lines, garcinol inhibited COX-2 ex-pression and the downstream prostanoid synthesis at IC50 valuesof 10 M and 20 M, respectively [24,41], resulting in reduced tu-morigenic cell proliferation.Table 1Summary of results from in vitro and in vivo studies to investigate mechanisms of action of garcinol in different cancer types.Cancer type Study type Mechanisms In vitro concentration(s)/M in vivo dose/mg/kg ReferenceBreast In vitro Apoptosis MDA-MB-231: 35 (IC50) [26]Breast In vitro Apoptosis and reduced angiogenesis MDA-MB-231: 10MCF-7: 20 (IC50)[27]In vivo (p.o.) Reduced cell proliferation Mice: 285.71Breast In vitro HAT inhibition MCF7: 10 [28]Breast In vitro Cell cycle arrest MDA-MB-231: 20 [29]Colon In vitro Apoptosis HT-29: 11.4 (IC50)HCT-116: 12 (IC50)[30]Colon In vitro Apoptosis HT-29: 20 [31]Colon In vivo (p.o.) Anti-inammatory Mice: 500 ppma[32]Colon In vivo (p.o.) Anti-inammatory Rat: 6.53 [33]Colon In vitro Apoptosis HCT116: 15 [34]Leukaemia In vitro Apoptosis U937, and K562: 20 (IC50) [35]Leukaemia In vitro Apoptosis HL-60: 9.42 (IC50) [36]Leukaemia In vitro Apoptosis HL-60: 16 (IC50) [10]Liver In vitro Apoptosis Hep3B: 20 (IC50) [37]Liver In vitro Apoptosis and cell cycle arrest C3A: 25 [38]In vivo (i.p.) Apoptosis Mice: 1Lung In vitro Cell cycle arrest H460: 7.5 (IC50)H1299: 7.5 (IC50)[39]Lung In vitro HAT inhibition A549: 12 (IC50) [40]Pancreatic In vitro Apoptosis and reduced angiogenesis BxPC-3: 20 (IC50)Panc-1: 10 (IC50)[41]Pancreatic In vitro Apoptosis, cell cycle arrest and reduced angiogenesis BxPC-3: 15 (IC50)Panc-1: 7 (IC50)[42]Pancreatic In vitro Apoptosis and gene modulation BxPC-3: 5Panc-1: 5[43]Breast, prostate pancreatic In vitro Cell cycle arrest and gene modulation MDA-MB-231: 10 [44]In vivo (p.o.) Cell cycle arrest and gene modulation Mice: 285.71Oral In vivo (topical) Anti-inammatory Hamster: 0.5 mM [45]Tongue In vivo (p.o.) Cell cycle arrest and anti-inammatory Rat: 26.05 [46]Head and neck In vitro Apoptosis, cell cycle arrest, and reduced angiogenesis CAL27 and UMSCC1: 10 [47]In vivo (i.p.) Reduced cell proliferation Mice: 1IC50 concentrations are given if reported in the respective paper. When not reported, the lowest concentration at which garcinol demonstrated observed activities in therespective cell lines are reported.aOral dose given to each animal could not be determined due to insucient information on dietary intake of garcinol.i.p.: intraperitoneal injection; p.o.: oral gavage.10 C. Liu et al./Cancer Letters 362 (2015) 814Induction of apoptosis. Garcinol interferes with multiple signalingcascades to induce apoptosis in several cancer cell lines. The knownmechanisms of actionof garcinol inapoptosis involve theinactivation of the signal transducer and activator of transcription3(STAT-3), NFBandphosphoinositide3-kinase(PI3K)/serine/threonine-specic protein kinase (Akt) signaling pathways[31,38,44]. This inactivation increases the expression of pro-apoptoticgene products (Bax, Bak) over pro-survival gene products (Bcl-xL,Bcl-2, Mcl-1, survivin), the release of cytochrome c into the cytosol,the activation of caspase-3, and thus, the induction of apoptosis [51].In vitro studies of human liver cancer cells showed that garcinolinhibitedthephosphorylationanddimerizationofSTAT-3inadose-dependent manner, thus interfering with the pSTAT-3 nucleartranslocation that mediates the transcription of STAT-3-responsivegenes, including Bcl-xL, Mcl-1 and survivin [38]. Furthermore, NFB,a molecule constitutively active in pancreatic adenocarcinoma cells[52], waseffectivelydepletedbygarcinolinadose-dependentmanner, leading to cancer cell apoptosis [41]. Based on its abilityto down-regulate NF-B, garcinol has been studied in human breastcancer cell lines that are known to demonstrate NF-kB activation[53]. Garcinol was more potent against oestrogen-receptor positive(MCF-7 cells) than triple-negative breast cancer cell lines (MDA-MB-231 cells), with IC50 values of 20 M and 35 M, respectively[26]. Garcinol also affects focal adhesion kinase, which utilizes theSrc, ERKandthePI3K/Akt axis toactivatecell survival andproliferation in colorectal carcinoma cells [31,34]. Garcinol was foundto induce apoptosis in the human colorectal cancer cell line HT-29at a relatively low IC50 of 10 M [31,34].Inhibition of cell cycle. Garcinol also exerts anti-cancer effects by in-hibiting the cell cycle, which is dysregulated in many cancers. Thecyclin-dependent kinase inhibitors (CDKIs) p21Waf1/Cip1and p27KIP1target the key cell cycle checkpoint molecules CDKs, specically theCDK2 and CDK4/cyclin complex, to induce G1 cell cycle arrest andinhibit cell division [54,55]. In p53-null H1299 lung cancer cells,10 M garcinol was shown to cause cell cycle arrest at the G1 phaseby up-regulating p21Waf1/Cip1gene expression [39]. Interestingly, p53wild-type A549 cells treated with less than 4 M garcinol contin-ued to divide, suggesting that garcinol may not be able to inducecell cycle arrest in p53 wild-type cells or that the dose was insuf-cient to induce cell cycle arrest in this cell type [39]. A PK evaluationof garcinol is critical to determine its effective dose for both in vitroand/or in vivo studies.Modulationof gene expression. Growinginterest inhistoneacetyltransferase (HAT) inhibitors as potential agents to treat cancerhasalsoledtoanti-cancerstudiesofhistoneacetylationinre-sponse to garcinol [56]. Several members of the HAT family havebeen implicated in hematological malignancies [57]. Specically,P300/CBP-associated factor (p300/PCAF) dysfunction in the HATfamily is associated with the onset of acute leukemia [58]. In vitrostudies showed that p300/PCAF is one of the molecular targets ofgarcinol (IC507 Mfor p300 and IC505 Mfor PCAF) [8]. Recent func-tional studies ofmicro RNAs (miRNAs) revealed that the highlyconserved and non-protein-coding short RNA products may par-ticipateincarcinogenesisbyactingastumorsuppressorsoroncogenes [59]. For instance, preclinical studies of breast cancertissues conrm the function of miRNA let-7 as a tumor suppres-sorgene[60]. InMDA-MB-231andBT-549breastcancercells,garcinol up-regulated the expression levels of let-7, further eluci-datingthemechanismbywhichitinhibitsbreastcancer[27].Moreover, elevatedoncogenicmiRNA-21levelsareobservedinbreast, colon and pancreatic cancers [6163]. Garcinol also down-regulated the expression level of miRNA-21 in the human pancreaticFig. 2. Pharmacodynamic effects and mechanisms of action of garcinol reported in in vitro and in vivo studies.11 C. Liu et al./Cancer Letters 362 (2015) 814cancer cell lines BxPC-3 and Panc-1 [42]. Taken together, these noveldiscoveries ofgarcinols ability to inhibit p300/PCAF and down-regulate oncogenic miRNA make it an attractive agent to modulategene expression and affect downstream PD events in various cancertypes. Thus, these studies may serve as a motivation to study thePK prole of garcinol in order to explore its effective tumor uptakeconcentration in xenografted mouse models.Reduction in tumor angiogenesis and metastasis. The role of matrixmetalloproteinases (MMPs) and vascular endothelial growth factors(VEGFs) in promoting the metastatic potential of cancer cells is wellestablishedinbreast, pancreaticandcoloncancer[64,65]. Incolorectal cancer, the expression levels of VEGF and MMP-7 stronglycorrelate with metastatic potential [66]. Garcinol reduces the MMP-7levels in HT-29 cells [45]. In addition to MMP-7, MMP-9 has re-cently been identied as a key regulator ofangiogenesis in themetastatic pancreatic islets of transgenic mice [57]. The ability ofgarcinol to reduce MMP-9 levels in the pancreatic cancer cell modelsBxPC-3 and Panc-1 indicates the potential effect of garcinol on thefunction of MMPs [43].Overall, the concentrations used in different cell culture assaysvaried dramatically, from 5 M in pancreatic cancer cells to 35 Min breast cancer cells [26,43]. Despite these differences, micromo-lar concentrations of garcinol were effective in almost all studiesconducted thus far [67]. However, this phenomenon is common formost natural products, which require higher consumption in orderto observe benecial effects [68]. Based on the reported IC50 values,garcinol appears to be more effective in inducing apoptosis and cellcycle arrest in pancreatic cancer, lung cancer and leukaemia cellsthan in colon, liver and breast cancer cells. Thus, the use of garcinolfor the former malignancies should be explored further.In vivo studiesCurrent in vivo anti-cancer studies ofgarcinol focused on itschemopreventionandchemotherapeutic actions. First, thechemopreventive effect of garcinol against inammation-inducedcancer was explored in 4 in vivo studies, which examined varieddoses, administration frequencies and administration routes forgarcinol. The dietary administration of 6.53 mg/kg of garcinol to arat with inammation-induced colonic aberrant crypt foci (ACF) (pre-cursor lesions for colon carcinoma) for 5 weeks was found to reducethe incidence of colon carcinogenesis by 40.2%. This effect could beattributed to an increase in glutathione S-transferase and quinonereductase detoxifying enzymes observed in the rat livers [33]. In ad-dition, 22 weeks of dietary intake equivalent to 26.05 mg/kg garcinolfor a rat resulted in COX-2 suppression and cell cycle arrest at theG1 phase. These effects decreased the incidence of inammation-induced neoplastic tongue carcinoma by 44.4% [46]. In the thirdstudy, a thrice weekly topical application of 0.5 mM garcinol for 25weeks to the chemically treated cheeks of hamsters decreased theoccurrence of inammation-induced oral carcinogenesis by 19.0%,likely by down-regulating the 5-LOX enzyme and LTB4 [45]. Anotherstudy showed a 44.4% reduction of inammation-induced colorectaltumorformationafterfeedingmicefoodsupplementedwith500 ppm garcinol for 24 weeks. Garcinol was shown to interruptthe MAPK/ERK, Pl3K/Akt and Wnt pathways in this study [32]. Theseresults reect the benecial effect of garcinol against inammation-induced colon cancer.Other in vivo evidence suggested that garcinol could reduce tumorgrowth and metastasis. The oral administration of285.71 mg/kggarcinol to mice bearing MDA-MB-231 breast cancer xenografts ata dosing frequency of 5 days per week for 4 weeks reduced breasttumor weight by 42.9%. Garcinol was shown to interrupt the STAT-3signaling cascade, which was also demonstrated in vitro by the samegroup [26,44]. Moreover, the group used the same mouse model andgarcinol dose (285.71 mg/kg) but increased the frequency to 6 daysper week for 4 weeks. This regiment signicantly decreased the levelof the tumor proliferation biomarker Ki-67, which indicated a re-duction in breast tumor proliferation in garcinol-treated mice [44].Furthermore, the relative level of miRNA let-7 was also found to be1.11 times higher in the garcinol-treated group compared with thecontrol[27]. Thesestudiesfurthersupporttheinvitroexperi-mentsconductedwithMDA-MB-231cellsdescribedinSectionModulation of gene expression, which indicated that garcinol couldinhibit breast tumor proliferation. A 1 mg/kg dose of garcinol ad-ministered intraperitoneally (i.p.) 5 days per week for 3 weeks tomice carrying hepatocarcinoma (HCC) xenografts reduced the livertumor volume by 200 mm3, at least in part by interrupting STAT-3phosphorylation, reducing the level of the pro-survival protein Bcl-2and activating cleaved-caspase-3 [38]. In another head and necksquamous cell carcinoma (HNSCC) xenograft mouse model, 1 mg/kgof garcinol administered i.p. 5 days per week for 4 weeks reducedthe tumor volume by 100 mm3[47]. Garcinol also suppressed STAT-3phosphorylation in mice in this study [47]. These results suggestthat garcinol inhibits cancers, such as HCC and HNSCC, via a STAT-3-mediated pathway [69].However, the lack of PK studies may call the reliability of datacollected fromboth in vitro and in vivo studies into question. There-fore, theeffectiveconcentrationrangeofgarcinolneedstobedeterminedwithaPKanalysistocomplementexistingecacystudies of garcinol against different types of cancer.Importance of PK and toxicological studiesAlthough accumulating evidence fromin vitro and in vivo studiesindicated that garcinol exerts multiple pharmacological effects, suchas anti-oxidant, anti-inammatory and anti-cancer effects, the de-velopment of garcinol into a drug candidate has progressed slowly.PK and toxicological studies are needed to accelerate this processand bridge the current research gap between in vitro and in vivondings. Interpretation based only on in vitro data has failed to con-sider the PK properties of garcinol, the presence of active metabolitesand the safety of dosage ranges to be employed in in vivo studies.These considerations are important for garcinol because many naturalproducts and their derivatives are pro-drugs that must undergo met-abolic conversion either by the intestinal microora or mammalianphase I and/or II metabolism before becoming an active pharma-cological agent (metabolite) [70]. For instance, CPT-11 (Irinotecan),a derivative of camptothecin, is a prodrug of SN-38, which is ap-proximately 100- to 1000-fold more cytotoxic than the parent drug.CPT-11 has shown a broad spectrum of clinical antitumor activi-ties against many cancer types, including colorectal, lung, cervical,and ovarian cancers [71].Oral bioavailability is another key factor to ensure an effectivedrug concentration is physiologically achievable. PK studies canindicate the bioavailability ofa drug, which can in turn help tooptimize the administrationroute andschedule toachievetherapeutic ecacy [72]. Although curcumin, a dietary polyphenolwith diverse pharmacological effects, has been demonstrated assubstantially ecacious and safe, its poor bioavailability (1% in rats)[73] restricts its clinical application. Therefore, extensive formulationstrategies have been employed to improve its bioavailability [74].Garcinol, likecurcumin, isanaturalcompoundthathasbeenextensively studied and suggested to have benecial therapeuticeffects [28]. Thus, lessons must be learned from previous examplesin order to progress studies ofgarcinol to the clinical phase. PKstudies performed with robust and sensitive bio-analytical methodsallow us to identify ADME constraints and better understand thebioavailability and other important PK parameters of garcinol, suchas the Cmax(maximumpeak concentration), t1/2(half-life), clearance,volume of distribution and linearity [75].12 C. Liu et al./Cancer Letters 362 (2015) 814WhilePKisanintegralpartofthedrugdevelopment, thetoxicologicalaspectsofgarcinolareequallyimportantbecauseecacy and safety are not mutually exclusive [76]. A previous studyshowed that feeding 285.71 mg/kg garcinol did not result in systemictoxicity and mortality in a mouse model inammation-inducedcoloncancer [27,44]. Despite the lack of apparent toxicitydemonstratedinanimal studies, thesystemicexposurewithrepeated doses ofgarcinol should be thoroughly investigated toidentify the maximum tolerated dose (MTD) and non-observedadverse effect limit. The lack of toxicological studies of garcinol hasprecluded the identication of the toxicity proles of garcinol as atherapeuticagent. Nevertheless, thedevelopmentofanaturalcompound into a clinically employed therapy is not an unrealisticexpectation. For example, galantamine, an alkaloid extracted fromGalanthus nivalis, is now being used as the rst-line clinical therapyfor Alzheimers disease. It has been shown to have favorable PKfeatures, includinglineareliminationkinetics, anappropriatehalf-life and high oral bioavailability, in addition to its desirableecacy and safety prole [77]. Overall, PK and toxicological studiesplay a vital role in elucidating the ecacy and safety of garcinol andtranslating the research ndings fromthe preclinical to clinical stage.Without this information, the signicance ofcurrent preclinicalndings is elusive and can be misleading.Conclusion and future perspectivesAdvances in analytical sciences have accelerated the commer-cializationof puriedgarcinol, whichhasgreatlyimprovedtheevaluation of the various properties of garcinol, especially its anti-oxidant, anti-inammatoryandanti-canceractivities. Theanti-oxidant natureof garcinol largelydependsonitspolyphenolicstructure, while its anti-inammatory effects could be attributedto its inhibition of COX-2, 5-LOX and iNOS synthesis. In addition,theanti-canceractivitiesof garcinol involveanti-inammation,the induction ofapoptosis and cell cycle arrest, the inhibition ofangiogenesisandthemodulationof geneexpression. However,the development of garcinol has lacked systematic PK evaluationto determine the effects of the concentration on its pharmacody-namics characteristics. To the best ofour knowledge, a sensitivebio-analyticalmethodtodeterminetheeffectiveplasmaandin-tracellular concentrations of garcinol is currently lacking, which isthemainobstacletounderstandinggarcinolsbiologicallyrele-vant concentrations in animal models. The PK parameters of garcinolmust beevaluatedinordertopreciselydeneitsPKlinearity,effectivedose, routeof administration, administrationschedule,and ADME. Because the extracts derived from G. indica have beenusedextensivelyfor centuries, thesafetyof theseproducts isgenerally not an issue ofconcern. However, a systematic toxico-logical studyof puriedgarcinol has not yet beenconducted.Clearly, further examining the pharmacological prole of garcinolcan help to reconcile the results gathered from in vitro and in vivostudies in order to elucidate the benet of garcinol for the treat-ment of specic diseases and ultimately advance the use of garcinolfromempirical studies toevidence-based, clinicallyapplicablepharmacotherapy.AcknowledgementsThe study was sponsored by the National Research Foundationof Singapore (Experimental Therapeutics Program/R-713-001-011-271)andtheNationalMedicalResearchCouncilofSingapore(NMRC/CSA/021/2010). TheauthorsthanktheNUHSMedicalPublications Support Unit, Singapore, for assistance inthepreparation of this manuscript.Conict of interest statementNone declared.References[1] H.D. Ramachandran, Plant prole, phytochemistry and pharmacology of garciniaindica: a review, Int. J. Pharm. Sci. Rev. Res. 27 (2) (2014) 376381.[2] G.K. Jayaprakasha, K.K. 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