Wayne State University Wayne State University Associated BioMed Central Scholarship 2009 Emerging role of Garcinol, the antioxidant chalcone from Garcinia indica Choisy and its synthetic analogs Subhash Padhye Wayne State University School of Medicine, [email protected] Aamir Ahmad Wayne State University School of Medicine, [email protected] Nikhil Oswal D.Y. Patil Institute of Pharmacy and Pharmaceutical Sciences and Research, India, [email protected] Fazlul H. Sarkar Wayne State University School of Medicine, [email protected] Recommended Citation Padhyeet al. Journal of Hematology & Oncology2009,2:38 doi:10.1186/1756-8722-2-38 Available at: http://digitalcommons.wayne.edu/biomedcentral/118 This Article is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Associated BioMed Central Scholarship by an authorized administrator of DigitalCommons@WayneState. Journal of Hematology & Oncology BioMed Central Review Open Access Emerging role of Garcinol, the antioxidant chalcone from Garcinia indica Choisy and its synthetic analogs Subhash Padhye1,2, Aamir Ahmad1, Nikhil Oswal3 and Fazlul H Sarkar*1 Address: 1Department of Pathology, Barbara Ann Karmanos Cancer Center and Wayne State University School of Medicine, Detroit, MI 48201, USA, 2D.Y. Patil University of Pharmaceutical Sciences and Research, Pune 411018, India and 3D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pune 411018, India Email: [email protected]; [email protected]; [email protected]; FazlulHSarkar*[email protected] * Corresponding author Published: 2 September 2009 Received: 1 August 2009 Accepted: 2 September 2009 Journal of Hematology & Oncology 2009, 2:38 doi:10.1186/1756-8722-2-38 This article is available from: http://www.jhoonline.org/content/2/1/38 © 2009 Padhye et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Garcinol, harvested from Garcinia indica, has traditionally been used in tropical regions and appreciated for centuries; however its biological properties are only beginning to be elucidated. There is ample data to suggest potent antioxidant properties of this compound which have been used to explain most of its observed biological activities. However, emerging evidence suggests that garcinol could be useful as an anti-cancer agent, and it is increasingly being realized that garcinol is a pleiotropic agent capable of modulating key regulatory cell signaling pathways. Here we have summarized the progress of our current research knowledge on garcinol and its observed biological activities. We have also provided an explanation of observed properties based on its chemical structure and provided an insight into the structure and properties of chalcones, the precursors of garcinol. The available data is promising but more detailed investigations into the various properties of this compound, particularly its anti-cancer activity are urgently needed, and it is our hope that this review will stimulate further research for elucidating and appreciating the value of this nature's wonder agent. Introduction Panarpuli, Ratamba or Amsool. In English language, it is It is difficult to imagine that the pink sweet smelling drink also known by various names such as Mangosteen, wild that is served to the world travelers spending summer hol- Mangosteen, or Red Mango. According to botanical classi- idays on the beautiful beaches of Goa in India, upon their fication the tree is classified as Garcinia indica (Family: arrival at the hotel, could one day end up on the labora- Clusiaceae; Genus: Garcinia)which has many culinary, tory tables of Cancer Institutes around the world. The wel- pharmaceutical and industrial uses. The genus Garcinia come drink happens to be made from the syrup includes some 200 species found in the tropics, especially formulated from the fruits locally known as 'Kokum' Asia and Africa. Out of 35 species found in India, 17 are which is steeped in sugar syrup to make a drink which is endemic. Of these, seven are endemic to the region of used to avoid skin damages and allergies from the sun and Western Ghats including the state of Goa, six in the Anda- tropical climate. The plant grows extensively on the west- man and Nicobar Islands and four in the North-Eastern ern coast of India and is known by various names across region of India. India including Bindin, Biran, Bhirand, Bhinda, Katambi, Page 1 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 The Garcinia indica seed contains 23-26% oil, which remains solid at room temperature and is used in the preparation of chocolates, medicines and cosmetics. It is used as a slightly bitter spice in recipes from the state of Maharashtra in India and as a souring agent and a substi- tute for tamarind paste in Indian curries. Recently, some industries have started extracting hydroxycitric acid (HCA) from the rind of the fruit which is an important constituent used as a hypocholesterolaemic agent. HCA is also a potential anti-obesity agent [1]. It suppresses fatty acid synthesis, lipogenesis, and food intake, and thus induces weight loss. Kokum Butter is an excellent emol- lient used by the cosmetic industry for preparations of lotions, creams, lip-balms and soaps. It has relatively high melting point and is considered as one of the most stable exotic butter which dose not need any refrigeration. It is extracted from the Kokum seed and is supposed to reduce SfFrtiorgumuc rtGeuar 1rec ionfia G inadriccianol, Curcumin and compounds extracted degeneration of the skin cells and restore elasticity. The Structure of Garcinol, Curcumin and compounds extract of the plant finds place in the specialty cuisine of extracted from Garcinia indica. West Coast of India as an appetizer while decoction of the bark is used for treating paralysis. The antioxidant activity of aqueous extract of the plant has been reported, which detector and electrospray ionization mass spectra. is higher than other reported spices and fruits thus pro- Absorption at 276 nm was chosen as the measuring wave- moting its use in cooking, home remedies and as a soft length at which resolution of both compounds could be drink [2]. Garcinia indica extract has also been shown to obtained [6-9]. More recently, these workers have devel- inhibit Aspergillus flavus and aflatoxin B production oped a rapid, sensitive and simple reverse-phase high-per- 1 thereby demonstrating its putative bio-preservative prop- formance liquid chromatography-electrospray ionization erties [3]. Addition of Garcinia extract to fresh skipjack mass spectrometric method for the identification and (dark muscle fish) has been demonstrated to prevent his- quantification of two isomeric polyisoprenylated benzo- tamine formation by lowering the pH to 3.2-3.6 [4]. Since phenones, isoxanthochymol and camboginol, in the histamine is known to give rise to allergic reactions, Gar- extracts of the stem bark, seeds and seed pericarps of Gar- cinia extracts can potentially find use in anti-allergy med- cinia indica and in the fruit rinds of Garcinia cambogia [10]. ications. The major organic acid in leaves and rinds of Garcinia indica is reported to be (-)-hydroxycitric acid, present to Isolation and characterization of chemical the extent of 4.1-4.6 and 10.3-12.7% respectively, as constituents determined by HPLC [11,12]. Garcinia indica extracts, especially from its rind, are rich in polyisoprenylated benzophenone derivatives such as Gar- Garcinol, with a molecular weight of 602, is the active cinol and its colorless isomer, Isogarcinol. The rind also principle of Garcinia indica, which is crystallized out as contains hydroxycitric acid (HCA), hydroxycitric acid lac- yellow needles (1.5%) from the hexane extract of the fruit tone, citric acid and oxalic acid. The structures of these rind. The molecular formula and the absorption spectral compounds are shown in Figure 1. The fruit also contains data indicate that the compound is possibly related to the other compounds including malic acid, polyphenols, car- isomeric Xanthochymol and more appropriately, in view bohydrates, anthocyanin, pigments and ascorbic acid. of the sign of optical rotation, to Cambogin. The presence Garcinol shows strong antioxidant activity since it con- of an enolisable 1, 3-diketone system in the molecule is tains both phenolic hydroxyl groups as well as a β-dike- confirmed by the formation of two isomeric trimethyl tone moiety, and in this respect it resembles with the well- ethers, hydrolysable to single dimethyl ether with dilute known antioxidant of plant origin, viz. Curcumin [5] (Fig- alkali. Alkali degradation of the methyl ether under ure 1). stronger conditions (20% ethanolic KOH, reflux) yields veratric acid indicating the presence of a 3,4-dihydroxy- A reverse-phase high-performance liquid chromato- benzoyl unit. The UV spectrum of garcinol suggests that graphic method has been developed by Chattopadhyay the 1, 3-diketone system is conjugated to the 3, 4-dihy- and Kumar for qualitative and quantitative analysis of droxybenzoyl moiety. The IR spectrum of the trimethyl Xanthochymol and Isoxanthochymol in the fruit rinds, ether shows the presence of a saturated carbonyl group leaves and seed pericarps of Garcinia indica using PDA (1727 cm-1) and two α, β-unsaturated carbonyl groups Page 2 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 (1668 and 1642 cm-1), accounting for all the oxygen rase (AChE) and butylcholinesterase. The IC value of 50 atoms. garcinol (0.66 μM) against AChE is comparable to that of the reference compound Galanthamine (0.50 μM) [26]. The PMR spectrum of garcinol in CDC1 shows the pres- 3 ence of two saturated tertiary methyls (singlets at δ l.01 Isogarcinol also shows biological activities similar to that and 1.17) and seven = C-CH groups (signals at δ 1.54 for of garcinol and has been claimed to be an anti-inflamma- 3 two methyls and at 1.60, 1.67, 1.70, 1.74 and 1.84 for one tory and antitumor compound, a lipase inhibitor, an anti- methyl each). It also shows signals for a vinylic methylene obesity agent as well as an antiulcer agent [18]. Sang et al. (δ 4.38, 2 H, broad singlet) and three other olefinic pro- have studied the interaction of garcinol with peroxyl rad- tons (δ 5.0 m) in addition to three aromatic protons (ABX icals generated by thermolysis of the initiator 2, 2'-azobis- pattern around δ 6.60 and 6.95) and a hydrogen bonded isobutyronitrile (AIBN) and have succeeded in isolating phenolic hydroxyl at δ 18.0. The mass spectrum of garci- and characterizing reaction products of garcinol in a nol is very similar to that of Xanthochymol exhibiting homogeneous acetone system. The resulting compounds major peaks at m/e 602(M+), 465(M+ -C H , base peak), were found capable of inducing apoptosis in human 10 17 341 (465-C H ) and 137 (Dihydroxybenzoyl). These fea- leukemia HL-60 cells and inhibit NO radical generation as 9 16 tures clearly indicate that the structure of garcinol is bio- well as LPS-induced iNOS gene expression, respectively genetically derivable from Maclurin (2,4,6,3',4'- [18,19]. Garcinol showed good antitumor activity against pentahydroxybenzophenone) and five isoprenyl units human leukemia HL-60 cells, being more effective than [13,14]. curcumin, which was used as a reference compounds in these studies. In addition to HL-60 cells, the chemothera- Chemistry of garcinol peutic potential of garcinol has been examined on other The principle antioxidant substance of Garcinia indica and cell lines as well such as murine macrophage RAW 264.7 other species is Garcinol (Figure 1) also called as Cam- cells and cyclin D1-positive cells showing similar results. boginol, which is a tri-isoprenylated chalcone [15,16]. Additionally garcinol also inhibits histone acetyltrans- This compound is extracted from the dried fruit rind of ferases (HATs, IC = 7 μM) and p300/CPB-associated fac- 50 the plant. It scavenges 1, 1-diphenyl-2-picrylhydrazyl tor (PCAF, IC = 5 μM), both of which are known to 50 (DPPH) free radical (3 times more effectively than DL-R- modulate gene expression [27]. tocopherol), hydroxyl radical (more effectively than DL- R-tocopherol), methyl radical, and superoxide anion [17]. Biological activities of garcinol Sang et al. have reported the structure of some oxidation a. Antioxidant Activity products of garcinol and have proposed mechanisms for Garcinol has been shown to possess antioxidant activity the formation of these products [18,19]. Their results sug- in the H O -NaOH-DMSO system as well as the radical 2 2 gest that garcinol can play an important role in the treat- scavenging activity against superoxide anion, hydroxyl ment of gastric ulcers caused by the hydroxyl radicals or radical and methyl radical respectively. The emulsified chronic infection with Helicobacter pylori, which, together garcinol suppresses superoxide anion to almost same with cells from gastric mucous membrane, produces extent as DL-α tocopherol by weight, while it exhibits hydroxyl radicals and superoxide anions. Presently, treat- nearly three times greater free radical scavenging activity ment with Clarithromycin antibiotic is the therapy of against 2, 2, diphenyl-1-picrylhydrazyl (DPPH) radicals choice for treating H. pylori infection, which, however, suf- than DL-α tocopherol by weight [28]. The following par- fers from side effects and emergence of rapid resistance agraphs describe the known mechanism of antioxidant [20,21]. Garcinol may be a viable alternative to conven- activity of garcinol. tional antibiotics. Garcinol shows antibacterial activity against Methicillin-resistant Staphylococcus aureus [22] Hong et al. have investigated possible mechanisms of which is comparable to that of the antibiotic Vancomycin antioxidant action of garcinol and its derivatives on ara- (MIC - 3-12 μg/mL for garcinol Vs. 6 μg/mL for Vancomy- chidonic acid metabolism and NO radical synthesis at cin) [23]. It also inhibits topoisomerases I and II (IC = concentrations (>1 μM) that may be achievable under in 50 43 and 55 μg/mL respectively) at concentrations compa- vivo conditions. The preliminary results indicate that peak rable to that of Etoposide (IC = 70 μg/mL for topoi- plasma and urine plasma concentration levels of garcinol 50 somerases II) [24]. Although this compound has been in CD-1 female mice were 12 and 2.7 μM respectively, shown to exhibit therapeutic activity against gram-posi- after oral gavage of garcinol (10 mg dose per mouse) [29]. tive and gram-negative cocci, mycobacteria and fungi, it Sang et al. also proposed the antioxidant mechanism of has been found to be inactive against gram-negative garcinol according to which the compound reacts with enteric bacilli, yeasts and viruses [25]. Garcinol exerts peroxyl radicals by a single electron transfer followed by anti-cholinesterase properties towards acetyl cholineste- deprotonation of the hydroxyl group from the enolized 1, Page 3 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 3-diketone to form a resonance pair. Depending on the (iNOS), respectively. The enzyme iNOS is stimulated by position of hydroxyl group (C-3 or C-5) which initiates inflammatory cytokines for NO production by macro- the reaction, different compounds are formed [18,19]. phages and by many other cell types. It has been reported that garcinol inhibits the expression of iNOS and COX-2 The neuroprotective effects of garcinol were examined by in lipopolysaccharide (LPS)-activated macrophages [31]. Liao et al who found that at 5 μM concentration it pre- It was observed that garcinol strongly blocks the LPS- vented NO radical accumulation in LPS-treated astrocytes induced activation of eukaryotic transcription factor NF- and significantly reduced the expression of LPS-induced κB [31]. This inhibition of NF-κB activation takes place inflammatory mediators, such as iNOS and COX-2 [30]. through the suppression of phosphorylation of IκB-α and These results suggest that the neuroprotective effects of p38 Mitogen-Activated Protein Kinases (MAPK). Garcinol garcinol are associated with its antioxidant nature involv- lowers the LPS-induced increase of intracellular reactive ing inhibition of iNOS induction in astrocytes. It has been oxygen species (ROS), which contribute to the activation suggested that the compound may be neuroprotective of NF-κB [31]. against brain injury through similar mechanism [30]. Yamaguchi et al. studied various pharmacological activi- Recently Koeberle et al. have shown that garcinol signifi- ties of garcinol including antioxidant activity, chelating cantly interferes with two enzymes that play crucial roles activity, free radical scavenging activity and anti-glycation in inflammation and tumorigenesis, viz. 5-lipoxygenase activity. They observed that garcinol exhibited reasonable and microsomal prostaglandin PGE synthase (mPGES)-1 2 antioxidant activity in the micellar linoleic acid peroxida- [32]. In cell-free assays garcinol inhibits the activity of tion system and exhibited chelating activity at almost the purified 5-lipoxygenase and blocks the mPGES-1-medi- same level as citrates. In a phenazine methosulfate/ ated conversion of PGH to PGE with IC values of 0.1 2 2 50 NADH-nitro blue tetrazolium system garcinol exhibited and 0.3 μM respectively. Garcinol was found to suppress superoxide anion scavenging activity and suppressed pro- 5-lipoxygenase product formations in intact human neu- tein glycation in a bovine serum albumin/fructose system. trophils and reduced PGE formation in interleukin-1β- 2 Thus, the compound may be useful as a glycation inhibi- stimulated A549 human lung carcinoma cells as well as in tor under specified conditions [17]. human whole blood stimulated by lipopolysaccharide. Garcinol also interfered with isolated COX-1 enzyme b. Anti-inflammatory activity (IC = 12 μM) and with the formation of COX-1-derived 50 Aberrant arachidonic acid metabolism and generation of 12(S)-hydroxy-5-cis-8, 10-trans-heptadecatrienoic acid as nitric oxide radicals (NO) have been shown to be well as thromboxane B in human platelets. The high 2 involved in inflammation and carcinogenesis [29]. Ara- potency of garcinol in selectively suppressing PGE syn- 2 chidonic acid is released by phospholipase A (cPLA2) thesis and 5-lipoxygenase product formations provides 2 from membrane phospholipids and is further metabo- the molecular basis for its anti-inflammatory and anti-car- lized by cyclooxygenase (COX), lipooxygenase (LOX) cinogenic effects and rationalizes its therapeutic use [32]. enzymes and Cytochrome P450 pathways. Modulation of arachidonic acid metabolism by inhibiting COX and LOX c. Anticancer activity enzymes has been considered as an effective approach for The effects of garcinol and its oxidative derivatives have treating inflammation and for cancer chemoprevention been investigated on the growth of HT-29 and HCT-116 [29]. Garcinol and its derivatives modulate arachidonic colon cancer cells, as well as IEC-6 and INT-407 which are acid metabolism by retarding the phosphorylation of the normal immortalized intestinal cells [33]. Garcinol cytosolic PLA2 (cPLA ) through the inhibition of extracel- and its derivatives showed potent growth-inhibitory 2 lular ERK1/2 kinase activation and suppression of iNOS effects on all intestinal cells, with IC values in the range 50 expression through modulation of the Janus kinase (JAK)/ of 3.2-21.4 μM after 72 hr treatment. Garcinol was found STAT-1 signaling pathway. When added prior to LPS, gar- to be more effective in inhibiting growth of cancer cells cinol suppressed NF-κB activation and COX-2 expression than that of normal immortalized cells. These results indi- through the interruption of LPS binding to toll-like recep- cate that garcinol and its derivatives can inhibit intestinal tors [29]. cancer cell growth without affecting normal cells. How- ever, it should be pointed out that at low concentrations The nitric oxide radical moiety is involved in various phys- garcinol can stimulate cell growth [33]. An earlier study iological processes, including vasodilation, inhibition of investigated the modifying effects of dietary feeding of the platelet function, synaptic neurotransmission as well as compound on the development of azoxymethane host defense. The formation of NO radicals from arginine (AOM)-induced colonic aberrant crypt foci (ACF) in male in the biological system is catalyzed by three different F344 rats [34]. The study also assessed the effects of garci- types of nitric oxide synthase (NOS) enzymes, viz. nol on proliferating cell nuclear antigen (PCNA) index in endothelial NOS, neuronal NOS and inducible NOS ACF and activities of detoxifying enzymes such as glutath- Page 4 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 ione S-transferase (GST) and quinone reductase (QR) in the BrdU-labeling index and cyclin D1-positive cell ratio, liver. It was observed that garcinol administration signifi- suggesting reduction in cell proliferation activity in the cantly lowers PCNA index in ACF and significantly ele- tongue. The COX-2 expression in the tongue lesions was vates liver GST and QR activities. In addition, garcinol was also suppressed. They concluded that dietary administra- also found to suppress O(2)(-) and NO generation and tion of garcinol inhibits 4-NQO-induced tongue carcino- expression of iNOS and COX-2 proteins. These observa- genesis through suppression of increased cell tions suggest possible chemopreventive role of garcinol proliferation activity in the target tissues and/or COX-2 [34]. In yet another report on the suppression of ACF for- expression in the tongue lesions [38]. mation in rats by garcinol [35], the beneficial effects of garcinol against tumor prevention in human colorectal The potent cytotoxic activity for the methanol extract of cancer cell line, HT-29 were investigated. Matrigel analysis the fruit rinds of Garcinia indica against three human can- showed that exposure of HT-29 cells to 10 μM garcinol cer cell lines, viz. colon (COLO-320-DM), breast (MCF-7) inhibited cell invasion and decreased the dose-dependent and liver (WRL-68) has been reported [39]. Fractionation tyrosine phosphorylation of focal adhesion kinase (FAK). of the methanol extract into hexane-, chloroform- and Western blot analysis demonstrated that garcinol inhibits ethyl acetate-soluble portions was performed and their activation of the Src, MAPK/ERK, and PI3K/Akt signaling cytotoxic activity was evaluated. The ethyl acetate fraction pathways. Additionally, these studies demonstrated that was found to be the most effective as compared to the two decreased MMP-7 protein levels in HT-29 cells result in other fractions. Thus, current results provide evidence for sensitization to garcinol and that the compound signifi- the potential of garcinol as a chemopreventive agent in cantly inhibits the expression of MMP-7 in IL-1beta- carcinogenesis. Additionally, feeding garcinol-containing induced HT-29 cells. Thus, garcinol reduces cell invasion diets does not cause retardation of body weight gain and and survival through the inhibition of FAK's downstream pathological alterations in liver and other organs includ- signaling [35]. ing kidney, lung, heart, and esophagus, which is indica- tive of the low toxicity of the compound, which is a very In human leukemia HL-60 cells, garcinol has been attractive feature of any anti-cancer agent. reported to display strong growth inhibitory activity (IC 50 = 9.42 μM) through induction of caspase-3/CPP32 activ- d. Anti-HIV Activity ity in a dose- and time-dependent manner and inducing Histone acetylation is a diagnostic feature of transcrip- degradation of poly (ADP-ribose) polymerase (PARP) tionally active genes [40]. The proper recruitment and protein [5]. This induction of apoptosis provides a pivotal function of histone acetyltransferases (HATs) and histone mechanism for its cancer chemopreventive action. In a deacetylases (HDACs) are key regulatory steps for gene study comprising four human leukemia cells lines, loss of expression and cell cycle. Functional defects of either of mitochondrial membrane potential was observed during these enzymes may lead to several diseases, including can- garcinol-induced apoptosis [36]. Garcinol also modulates cer. It has been reported that garcinol is a potent non-spe- arachidonic acid metabolism by blocking the phosphor- cific inhibitor of histone acetyltransferases p300 (IC50 = ylation of cPLA2 and by decreasing iNOS protein level 7 μM) which strongly inhibited HAT activity-dependent mediated via inhibition of STAT-1 activation. These activ- chromatin transcription, whereas transcription from DNA ities may contribute to the anti-inflammatory and anti- template was not affected [40]. In order to find out more cancer properties of garcinol and its derivatives [29]. potent, specific, and less toxic inhibitors, Mantelingu et al. [41] synthesized and characterized several derivatives of Two new benzophenones corresponding to the 13-O- isogarcinol (IG), a product of intramolecular cyclization methyl ethers of garcinol and isogarcinol were tested for of garcinol, by controlled modification and mono-substi- their inhibitory effects on Epstein-Barr virus early antigen tution at C-14 position. In this way they were able to syn- activation induced by 12-O-tetradecanoylphorbol-13-ace- thesize 14-isopropoxy IG (LTK-13) and 14-methoxy IG tate (TPA) in Raji cells and their radical-scavenging ability (LTK-14) derivatives of isogarcinol. The di-substitution against 1,1-diphenyl-2-picrylhydrazyl (DPPH) was dem- yielded 13, 14 di-isopropoxy IG (LTK-13A), 13, 14 di- onstrated [37]. The cyclized polyprenylbenzophenones methoxy IG (LTK-14A), 13, 14 di-acetoxy IG (LTK-15) and showed comparable or stronger potential cancer chemo- 13, 14 di-sulfoxy (LTK-19) isogarcinol compounds, preventive activity when compared to glycyrrhetic acid, a respectively. It was found that the mono-substituted isog- known anti-tumor promoter. Yoshida and coworkers arcinol derivatives like LTK-13, -14, and di-substituted have demonstrated that dietary garcinol significantly LTK-19 derivative could inhibit the p300-HAT activity but decreases the incidence and multiplicity of 4-NQO- not the PCAF-HAT activity, although the parent isogarci- induced tongue neoplasms and pre-neoplasms as com- nol compound inhibited HAT activities of both p300 and pared to the control diet [38]. It also significantly reduced PCAF non-specifically. Page 5 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 Interestingly, one of the derivatives, LTK-15, seemed to hydroxyl radical has been found to be stronger than that loose the HAT inhibition activity: it could inhibit the of α-Tocopherol [17] while its other scavenging activities p300-mediated acetylation less than 10% and had no were found to be slightly weaker. Since hydroxyl radical is effect on PCAF-HAT activity. Furthermore, the other di- regarded as the most damaging Reactive Oxygen Species substituted isogarcinol derivatives, LTK-13A and LTK- (ROS), garcinol is expected to be useful for preventing dis- 14A, also lost their activity completely. The IC values, of eases caused by the hydroxyl radical damages such as 50 LTK-13, -14, and -19, to inhibit p300- HAT activity were stress-induced gastric ulcer [44,45] and NSAID drug- found to be 5-7 μM, which is comparable to isogarcinol. induced gastric ulcers [46,47]. In the water immersion In order to visualize the inhibition pattern of histone stress model, Yamaguchi et al. have shown that garcinol acetylation, HAT assay products were analyzed by fluorog- suppressed gastric injury formation to almost same extent raphy followed by autoradiography. In agreement with as cetraxate hydrochloride as a positive control [28]. It filter-binding data, it was found that in the presence of 10 also prevented indomethacin-induced gastric injury. μM of LTK-13, -14, and -19, the p300-mediated acetyla- These results suggest that garcinol may have potential as tion of histones H3 and H4 were equally inhibited up to an anti-ulcer drug. Although mechanism of its anti-ulcer 85%-90% as compared to DMSO solvent control. The his- activity is not yet understood, it may be speculated that tone acetylation by PCAF (predominantly at histone H3) the compound may scavenge reactive oxygen species on was not affected by LTK-13, -14, and -19. As expected, the the surface of gastric mucosa, thus protecting cells from presence of 10-μM concentration of isogarcinol efficiently injury [28]. inhibited histone acetylation by both p300 and PCAF. However, dose-dependent inhibition of p300- HAT activ- Structure-activity considerations for garcinol ity was observed in the presence of LTK-14. Significantly, It has been clearly established that the C-3 kenotic group HAT-activity of PCAF remained unchanged even in the and the phenolic ring bearing hydroxyl group are the prin- presence of 50 μM LTK14 and these chromatin modifying cipal oxidation sites of garcinol generating its oxidized enzyme activities were not affected by the presence of iso- products during metabolic transformations some of garcinol and its derivatives. Taken together, the data sug- which are also biologically active [18,19]. It has also been gests that the isogarcinol derivatives are specific inhibitors found that the 1, 2 carbon-carbon double bond of the α, of p300-HAT activity [41,42]. β-unsaturated ketone is important for apoptosis-inducing activity and cytotoxicity of garcinol [5]. The double bond Since reversible acetylation of histone and non-histone of the isoprenyl group is also a principal site of the anti- proteins plays pivotal role in cellular homeostasis [43], oxidant reaction of garcinol; however, compounds with- dysfunction of histone acetyltransferases (HATs) is known out having such substitution and bearing structural to cause several diseases including cancer, neurodegenara- resemblance to garcinol, like curcumin, have been found tion, asthma, diabetes, AIDS, and cardiac hypertrophy. to be potent antioxidants [48]. The isoprenyl chain of gar- Moreover, since p300 protein plays a critical role in cell cinol consists of hydrophobic faces, which are important growth, differentiation, and death, several of these func- for its binding to biological targets [49]. tions require intrinsic HAT activity of p300-HAT; how- ever, the molecular basis of p300 contribution toward Chalcones as a garcinol analoges diverse cellular processes is still unresolved [43]. Mante- Kostanecki, who pioneered work in the synthesis of natu- lingu et al. [41] have described the synthesis and charac- ral coloring compounds, first coined the term 'chalcone'. terization of a set of p300-HAT-specific small-molecule An interesting feature of chalcones is that they serve as inhibitors derived from garcinol that are highly toxic to starting materials for another class of naturally occurring cells. They have shown that these specific inhibitors selec- and widely distributed pigments, flavones [50]. They are tively block the p300-mediated acetylation of p53 in vivo. considered to be precursors of flavonoids and isoflavo- Furthermore, inhibition of p300-HAT down-regulates noids, which are abundant in edible plants. Chalcones are several genes but, significantly, few important genes are intermediates in the synthesis of flavones. Chemically also up regulated. Remarkably, these inhibitors were they are open-chain flavonoids in which the two aromatic found to be non-toxic to T cells, while inhibiting histone rings are joined by a three-carbon α, β-unsaturated carbo- acetylation of HIV infected cells and consequently inhib- nyl system (1, 3-diphenyl-2-propen-1-one). Chalcones iting the multiplication of HIV. Hence, garcinol holds tre- exhibit many pharmacological activities including anti- mendous therapeutic potential for different diseases leishmanial [51], anti-inflammatory [52,53], anti-mitotic including AIDS and cancer. [54], anti-invasive [55], anti-tuberculosis [56], anti-fungal [57], cysteinyl leukotriene receptor-1 antagonist [58], e. Anti-ulcer activity anti-malarial [59,60], anti-plasmodial, antitumor, immu- Garcinol has potent free radical scavenging activity as nosuppressive, antioxidant [61], anti-fibrogenic and judged from its interactions in three types of free radical modulation of P-glycoprotein-mediated multi-drug resist- generating systems. Its scavenging activity against ance [62]. Recent studies have shown that chalcones Page 6 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 inhibit cancer cell proliferation in vivo and are effective priate ketone in a polar solvent like methanol or ethanol agents against skin cancers [63,64]. They also induce (Figure 3) [61]. The synthesis of hydroxylated chalcones apoptosis in various cell types, including breast cancers by the Claisen-Schimdt method requires protection of the [65]. Several oxygenated chalcones; hydroxyl chalcones, phenolic hydroxyl groups on aldehyde and ketone (except bis-chalcones and quinolinyl chalcone analogs exhibit ortho-hydroxyl groups), generally as tetrahydropyranyl anti-malarial activity [66,67]. Some chalcones also dem- (THP), methoxymethyl (MOM), or methoxyethoxyme- onstrate their ability to block voltage-dependent potas- thyl (MEM) ethers. The MOM and MEM ethers are cleaved sium channels [68]. These limited yet interesting studies in the presence of acid, under such conditions; and hence clearly suggest the beneficial effects of chalcones and the side reactions compromise the yield of the final prod- other derivatives in human health and diseases. uct [72]. The Cα-Cβ double bond in the 'enone' moiety of chalcones can adopt Z or E configuration. The E-isomer is a. Structural chemistry of chalcones thermodynamically more stable and almost all chalcones Chalcones consist of two aromatic rings in trans configu- are isolated in this form. Iwata and co-workers have ration, separated by three carbons, of which two are con- reported isomerization of E-chalcone to the Z form by nected by double bond while the third is a carbonyl group exposing the methanolic solution of the chalcone to nor- [69]. Garcinol is an example of prenylated chalcones, con- mal visible light [73]. Interestingly, the Z isomer showed taining two aromatic rings separated by carbonyl group more potent antitumor activity than the original E form. (Figure 2), which is structurally similar to curcumin that Photoisomerization of the predominant E isomer to the Z resembles chalcones when opened [5]. Genealogical stud- isomer may cause change in biological activity and the ies have shown that chalcones have evolved prior to garci- ease with which the reaction occurs suggest that it is pru- nol, and chalcones are derived from three acetates and dent to protect solution of chalcones from light. cinnamic acid as shown in Figure 2. Since chalcones are efficient precursors of isoflavonoids, the required aryl Ducki et al. have noted that the two bonds were posi- migration of ring B from the beta position to the alpha tioned cis with respect to each other in several X-ray crystal position of the phenylpropanoid precursor must take structures of chalcones [54]. The s-cis conformer was more place after formation of the basic C skeleton [70]. A vast stable than the s-trans conformer by, at least, 3.9 kJ/mol. 15 number of naturally occurring chalcones are polyhydrox- On the other hand, when a methyl group was introduced ylated in the aryl rings. The radical quenching properties at the Cα position, the disposition of the carbonyl and of the phenolic groups present in many chalcones have Cα-Cβ double bonds altered to the trans orientation. For raised interest in using these compounds as therapeutic these α-methyl chalcones, molecular mechanics calcula- agents or food preservatives [71]. tions showed that the minimum energy conformers were s-trans and no s-cis conformation was evident within a 10- Chalcones are readily synthesized by the base-catalyzed kJ/mol range of the global energy minimum. The α- Claisen-Schimdt condensation of an aldehyde and appro- methyl group also caused significant loss of planarity between ring A and the enone (θ1 56-88°). The α-methyl- chalcones are found to have greater cytotoxic activity against a human leukemia cell line than the unsubstanti- ated analogues. Their unique geometrical features were AtFiei.g sSutrruec 2tural similarity between chalcone and garcinol moie- AFi.g Sucrhee m3e of synthesis of chalcones A. Structural similarity between chalcone and garci- A. Scheme of synthesis of chalcones. B. s-cis and s-trans nol moieties. B. Formation of chalcone and migration of conformation of chalcones. ring B. Page 7 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 cited as a possible factor contributing to the enhanced [86] and these reports, taken together, suggest that some biological activity. hydroxy chalcones might be promising antithrombotic or anti-inflammatory agents. b. Biological Activities of chalcones Xia and coworkers were the first to demonstrate improved Saxena and coworkers grafted chalcone derivatives on anti-proliferative activity of chalcones with substituted estradiol framework some of which showed potent anti- amino groups [74]. LeBlanc et al. have shown that meth- cancer activity against some human cancer cell lines [87]. oxylated chalcones with a 3'-amino group had sub-micro- Thus, compounds B and C in Figure 4 show potent activ- molar IC values against murine melanoma B16 cells ity against estrogen receptor-positive and hormone- 50 [75]. Dimmock and coworkers proposed that the presence dependent human breast cancer cell lines, MCF-7. Chal- of amino function increases the reactivity of chalcones as cone A was further modified to yield corresponding Michael acceptors and subsequently their anti-prolifera- indanone derivative (C) using the Nazarov reaction, tive activity [76]. They postulated that the amino function which showed better activity than the parent compound would be protonated at low pH environment normally against MCF-7 breast cancer cell line. Active anticancer encountered in tumors. The electron withdrawing effect of derivatives were also evaluated for osmotic hemolysis the protonated ammonium function would enhance the using the erythrocyte as a model system. It was observed electrophilicity of the β-carbon in the enone linkage, that chalcone derivatives showing cytotoxicity against hence increasing its reactivity as a Michael acceptor [77]. cancer cell lines did not affect the fragility of erythrocytes Liquorice has been used in China for the treatment of gas- and hence may be considered as non-toxic to normal cells; tric and duodenal ulcers, bronchial asthma, Addison's dis- however, further research in this area is urgently needed. ease, poisoning by food and drugs and skin disease such Nitric oxide production by trimethoxy chalcone deriva- as eczema and urticaria [78]. It still finds medicinal appli- tives, with various patterns of fluorination, has also been cation because of its wide-ranging therapeutic properties, evaluated [88]. One of this compounds, 2, 4, 6-trimeth- including relief from rheumatic and other types of pain oxy-20-trifluoromethylchalcone, inhibited the produc- and healing effect on ulcers. The crude extract of Liquorice tion of NO and prostaglandin E2 in lipopolysaccharide- has also found commercial use as a food additive in Japan stimulated RAW 264.7 macrophage cells. The inhibition since it contains the sweetening principle glycyrrhizin. (76.3% inhibition at 10 μM concentration) was dose- The Liquorice extracts contains a chalcone, viz. Isoliqurit- dependent without any evidence of a cytotoxic effect. It igenin, which is currently in use as a phosphodiesterase III was suggested that NO reduction was a consequence of inhibitor for the treatment of cardiovascular diseases [79]. inhibition of the expression PGE accumulation. The 2 In the Far East countries such as Korea, Japan, and China, fluorinated chalcones tested by Nakamura et al. showed 5- another chalcone compound called 'Butein' has also been lipoxygenase inhibition on rat basophilic leukemia-1 traditionally used for treatment of pain, thrombotic dis- (RBL-1) cells and inhibitory action on Fe3+-ADP induced ease, stomach cancer, and parasitic infection as well as a NADPH-dependent lipid peroxidation in rat liver micro- food additive [80]. somes [89]. The potencies were comparable or better than those of the lead compound, viz. 3,4-dihydroxychalcone. Anti-angiogenic effect of xanthochymol and Isoxantho- The structure of fluorinated chalcone is presented in Fig- chymol, the chalcones isolated from the hop, has been ure 5. reported [81]. A dose-dependant reduction of newly formed capillary growth by xanthochymol was observed, Why Chalcones are good analogs of Garcinol? at a concentration range of 0.5-10 μM (IC value of 2.2 1. Structural Similarity 50 μM) under in vitro conditions. Later, it was shown that Chemically, garcinol is a polyisoprenylated chalcone con- xanthochymol repressed both the NF-κB and Akt path- taining two aromatic rings separated by a carbonyl group. ways in the endothelial cells, indicating that components The α,β-unsaturated ketone system important for the of these pathways are major targets in the molecular apoptosis-inducing activity is present between the two mechanism of this compound [82]. Xanthochymol also rings in case of chalcones but within the ring B in case of reduced VEGF secretion, decreased cell invasion and met- garcinol (Figure 2). Garcinol differs from chalcones with alloprotease production in acute and chronic myeloge- the presence of isoprenyl groups, which makes its struc- nous leukemia cell lines [83]. 2'-hydroxychalcones, ture more complex and adds to its antioxidant activity [5]. 4'hydroxychalcones and 2', 4'-dihydroxychalcones inhibit 12-Lipoxygenase and cyclooxygenase enzymes in the 2. Reaction Similarity mouse epidermis [84] and two synthetic 2'-hydroxychal- The reducibility of the carbonyl function in chalcones and cones that exert topical anti-inflammatory effects in mice its relationship to biological activity has been investigated have also been reported [85]. The good selective inhibi- [90]. In quantitative structure-activity relationships tory effects of 2', 5' dihydroxychalcones on arachidonic (QSAR), the reducibility of the carbonyl function serves as acid-induced platelet aggregation have been suggested an indirect indicator of the electron density on the carbo- Page 8 of 13 (page number not for citation purposes) Journal of Hematology & Oncology 2009, 2:38 http://www.jhoonline.org/content/2/1/38 FStirguucrteur 4es of some therapeutically active chalcone compounds Structures of some therapeutically active chalcone compounds. nyl function. A readily reducible carbonyl group would the reducibility of the carbonyl bond (for example, meas- imply that the carbonyl carbon is electron-deficient. Elec- ured in terms of reduction peak potentials in cyclic volt- tron delocalization along the α, β-unsaturated chain ametry) and the susceptibility to nucleophilic attack at the would render the β-carbon electron deficient and, accord- β-carbon. On the other hand, in vivo reduction of the car- ingly, more susceptible to attack by thiols and other nucle- bonyl group to an alcohol is unlikely to predominate, as ophiles. Thus, one would expect a relationship between seen from the in vitro biotransformation of 4-dimethyl- amino-4' (imidazol-1-yl) chalcone [91]. In case of garci- nol no reactions has been reported but presence of carbonyl group may suggest that such reactions could occur. 3. Similarity in Biological Activities Chalcones as well as garcinol are reported as potent anti- oxidants and have been screened for their anti-inflamma- tory, anti-cancer, anti-HIV, anti-biotic, anti-fungal and anti-tumor activities. Structurally, chalcones are more eas- ily amenable for structural modification and optimization for some selective biological activity than garcinol. 4. Metal Complexation The synthesis and structural studies of complexes of Co FFliugourrinea 5ted chalcone: as anti-inflammatory agent (II), Ni (II), Cu (II), Zn (II) and Cd (II) with substituted Fluorinated chalcone: as anti-inflammatory agent. chalcones has been reported [92]. In general, for metal Page 9 of 13 (page number not for citation purposes)