Chemical and Biological Aspects of Garcinol and Isogarcinol: Recent Developments

The natural polyisoprenylated benzophenone derivatives garcinol and isogarcinol are secondary plant metabolites isolated from various Garcinia species including Garcinia indica. This review takes stock of the recent chemical and biological research into these interesting natural compounds over the last five years. New biological sources and chemical syntheses are discussed followed by new insights into the activity of garcinol and isogarcinol against cancer, pathogenic bacteria, parasite infections and various inflammatory diseases.


Introduction
Natural products and their semi-synthetic derivatives occupy a salient position concerning activity and percentage share of new investigational and eventually approved drugs for the clinic. [1] The traditional folk medicine of East Asia (Traditional Chinese Medicine, Kampo), South Asia (Ayurveda, Unani), the Middle-East (traditional Greco-Arab and Islamic Medicine) and other Asian regions, in particular, proved to be a valuable source of drug discovery based on natural products. [2,3] In India, for example, plants or plant products from Boswellia, Curcuma, Plumbago, Lawsonia, and Garcinia among others, whose manifold activities were confirmed by modern laboratories meanwhile, have been applied by traditional healers until today. [4,5] Garcinia species produce the biologically active benzophenones/polycyclic polyprenylated acylphloroglucinols (PPAPs) garcinol and isogarcinol which have raised the interest of chemists and molecular biologists alike ( Figure 1). [6,7] Garcinol is easily available in large amounts and high purity by extraction of dried kokum plums (Garcinia indica) followed by chromatographic purification and/or crystallization. [6,8] Isogarcinol is easily prepared from garcinol by treatment with diluted hydrochloric acid. [9] The activity spectra of garcinol and isogarcinol include anticancer, antibiotic, antioxidant and anti-inflammatory effects. [10] Their modes of action are diverse and include the inhibition of NF-κB and STAT as well as of histone acetyl transferases (HATs). [11] Several reviews and book chapters covered the extensive chemistry and biological activities of garcinol and isogarcinol over the last decade. [4,6,7,10,11] The present review highlights the most recent proceedings in this field.

An Update on The Isolation, Analysis, and Synthesis of Garcinol and Isogarcinol
Garcinol and isogarcinol were isolated from various Garcinia species. High yields of garcinol can be obtained from dried kokum (Garcinia indica) plums while garcinol can be easily converted to isogarcinol under acidic conditions. [6,8] High yields of garcinol (up to 5 g from 500 g dried kokum plums) were achieved by extraction of chopped dried kokum plums with methanol, evaporation of the solvent, redissolution of the methanol extract in ethyl acetate, washing of the latter with water, column chromatography of the concentrated ethyl acetate extract (silica gel 60, ethyl acetate/hexane 1:2) followed by crystallization of the concentrated eluate from hexane to give yellow needles. [6] Isogarcinol can be easily prepared by treatment of garcinol with aqueous HCl in toluene at room temperature. [6] Another suitable large scale isolation method initially removed hydroxycitric acid from Garcinia indica fruits by washing with water, followed by methanol extraction of the fruits, adsorption of the concentrated methanol extract on Celite and extraction of the methanol extract loaded Celite with hexane followed by column chromatography of the hexane extract. [8] Recently, garcinol and isogarcinol were isolated from fruits of Garcinia multiflora, a medicinal plant of South China known for its antioxidant activity. [10,12] The authors obtained 500 mg garcinol and 101 mg isogarcinol from 5.2 kg dried G. multiflora fruits (powdered dried G. multiflora fruits were extracted with 95 % ethanol and the ethanol extract was extracted with petroleum ether followed by column chromatography on silica gel and recrystallization to obtain garcinol). [12] Garcinol was also detected in and isolated from plants of the Garcinia species G. morella, G.
yunnanensis, G. xanthochymus and G. travancorica. [13][14][15][16] In addition, garcinol and isogarcinol were recently found in the stem bark of the Garcinia species G. buchananii. [17] Isogarcinol was also isolated from G. punctata and G. ovalifolia. [18,19] Quantitative analyses of garcinol contents in biological material are usually carried out by LC/MS and HPLC methods. [20][21][22] Recently, the garcinol contents of Garcinia indica samples and of the commercially available formulations Tryodashang Guggul and Slimmerz capsules was determined by High Performance Thin Layer Chromatography (HPTLC). It was shown that the dried fruit rinds of Garcinia indica contained 2.5 % garcinol while Tryodashang Guggul had 0.701 % and Slimmerz capsules 0.760 % garcinol. [23] With aqueous two-phase systems (ATPS) containing ethanol and ammonium sulfate garcinol and isogarcinol accumulated in the ethanol phase while anthocyanin and hydroxycitrate built up in the salt-rich phase. [24] Gold nanoparticles (AuNPs), prepared from G. indica fruit rind extract, showed distinct catalytic activities such as a reduction of toxic 4-nitrophenol to 4-aminophenol when combined with NaBH 4 . [25] Distinct progress has also been achieved in the field of the total synthesis of garcinol and isogarcinol. Socolsky and Plietker described a concise total synthesis of racemic garcinol and isogarcinol in 13 steps starting from acetylacetone, which was α-prenylated and reacted with formaldehyde via deacetylating aldol-type condensation to give enone 1 (Scheme 1). The latter was submitted to a domino Michael addition-Knoevenagel condensation with dimethyl 1,3-acetonedicarboxylate to afford the cyclohexanone hub of intermediates 2 and 3. Allylation of the keto ester 3 with 4-acetoxyprenyl chloride gave transselectively 4, which was β-methylated and O-alloc protected to furnish enol allyl carbonates 5a and 5b. Both regioisomers 5 underwent a carboxylative, diastereoselective, Pd-catalyzed Tsuji-Trost allylation yielding the same cyclohexanone 6. Its subsequent Pdcatalyzed allyl-allyl cross-coupling with allylpinacolborane afforded exclusively the 1,5-dienyl derivative 7, which was submitted to a Dieckmann condensation Bernhard  leaving bicyclo[3.3.1]nonatrione 8. Its cross-metathesis with amylene to introduce the prenyl residues, followed by benzoylation and deprotection finally yielded the natural product garcinol. [26] 3

. An Update on The Activities of Garcinol and Isogarcinol against Tumor Models
The significant activity of garcinol and isogarcinol against various tumor models is largely attributed to the inhibition of histone acetyl transferases (HATs), NF-κB signaling, and STAT-signaling. Meanwhile, new promising results were disclosed ( Table 1).
Lung cancer is one of the most lethal cancers and leads annually to ca. 1.2 million deaths worldwide. [27] Non-small cell lung cancer cells were sensitized to cisplatin and erlotinib treatment by garcinol. This effect was mediated by miRNAs and garcinol was able to upregulate EMT-modulating miRNAs such as let-7c and miR-200b. [28] Lung cancer stem cells (LCSCs) were also targeted by garcinol and repression of the Wnt/βcatenin/STAT3 signaling pathway by garcinol treatment suppressed the ability of NSCLC cells to form spheres and reduced the tumor growth in the H441 LCSC mouse xenograft model. [29] In addition, garcinol activated DDIT3 (DNA damage-inducible transcript 3) and suppressed the cancer stem cell marker ALDH1 A1 (aldehyde dehydrogenase 1 family member A1) in A549 NSCLC cells. [30] Colorectal cancer (CRC) also belongs to the most lethal cancer diseases worldwide. [31] The emergence of CRC is closely connected with genetic factors (ca. 25 % of CRC appears in people with close relatives suffering from CRC), as well as obesity and dietary factors. [32] HT-29 CRC cells treated with garcinol showed in-creased apoptosis rates, reduced cell growth, and a less pronounced tendency to angiogenesis and invasion due to downregulation of mPGES1, HIF-1α, VEGF and MMP expression. [33] In HCT-116 CRC cells, the HAT inhibitory activity of garcinol reduced 8-oxoG damage repair by suppression of base excision repair (BER). [34] Breast cancer is the most prevalent cancer type of women. [35] Resistance to chemotherapy is a big problem. Garcinol was able to sensitize breast tumors to treatment with taxol in vitro and in vivo (orthotopic 4T1 mammary carcinoma). Synergistic tumor growth inhibition and anti-metastatic activity were observed Table 1. Recently discovered antitumor effects of garcinol and isogarcinol.

. An Update on The Activities of Garcinol against Viruses, Bacteria, Yeasts, and Protozoal Parasites
Garcinol has shown promising activity against causing agents and consequences of various infectious diseases. Table 2 summarizes a few of the more interesting findings.
As early as 2007, the suppression of viral transcription by HIV was documented for isogarcinol derivatives as well as their p300 inhibitory activity. [60] Recently, a similar mode of action against the influenza virus was reported of garcinol. Garcinol inhibited both PCAF (p300/CBP-associated factor) and GCN5 in influenza A leading to a reduced acetylation of the viral nucleoprotein and to the regulation of the viral polymerase function. Interestingly, both HATs target different lysines of the nucleoprotein: while PCAF acetylate Lys-31, GCN5 acetylates Lys 90. Both lysines regulate opposite effects: deacetylated Lys-31 (by suppression of PCAF) enhanced viral polymerase activities while deacetylated Lys-90 (by suppression of GCN5) decreased the viral polymerase function. [61] The lethal toxin (LT) of the Gram-positive bacterium Bacillus anthracis induces actin stress fiber formation in infected cells by suppression of HDAC expression and, thus, this process is controlled by histone acetylation. In contrast to LT, the HAT inhibitor garcinol reduced stress fiber formation in LT-treated HUVEC cells and so might act as an antidote for LT intoxication. [62] The activity of garcinol against cells of the pathogenic fungus Candida albicans was studied as well. Garcinol and xanthochymol induced apoptosis in C. albicans hyphae and inhibited biofilm production by this fungus. [15] The viability of Toxoplasma gondii, which is a protozoal parasite causing toxoplasmosis, is regulated by lysine acetylation. The GCN5 family KAT TgGCN5b of T. gondii is crucial for replication of the T. gondii tachyzoites. Garcinol treatment led to decreased levels of acetylated histone H3 of TgGCN5b and to an inhibition of replication (IC 50 = 1.7 μM). Similar replication inhibitory effects were observed for the malaria parasite Plasmodium falciparum (IC 50 = 1.69 μM for chloroquine-sensitive HB3 strains, and IC 50 = 2.05 μM for chloroquine-resistant Dd2 strains; inhibition of erythrocytic asexual replication). [63]

New Effects on Inflammation Processes and Neurodegenerative Diseases
The antioxidant and anti-inflammatory properties of garcinol and isogarcinol are well described ( Table 3). [10] Only recently, isogarcinol was reported to have significant antioxidant effects rescuing human cells from oxidative stress while the genotoxicity of isogarcinol was negligible. [64] Topically applied garcinol efficiently blocked skin inflammation and tumorigenesis induced by 12-O-tetradecanoylphorbol 13-acetate in mice by suppression of NF-κB, ERK, JNK, p38 MAPK, PI3 K and Akt. [65] Intimal hyperplasia is based on vascular inflammation and NFk B activation and the HAT PCAF promoted NF-κB-regulated inflammation. Garcinol suppressed CCL2 and TNF-α expression in leukocytes and vascular smooth muscle cells and Table 3. Recently discovered effects of garcinol and isogarcinol on inflammation and neurodegenerative diseases.

Epilepsy
Decrease of mortality and of seizure scores in vivo [86] Suppression of BDNF and TrkB and upregulation of GABA A and GAD65 [86] Cocaine abuse Support of drug abstinence [87,88] Inhibition of reinstatement by reconsolidationbased modes [85,88] reduced arterial adherence and infiltration by leukocytes and macrophages in vivo when administered in a Pluronic gel enabling a slow garcinol drug release. [66] In contrast to these and other previous findings about the considerable anti-inflammatory activities of garcinol, a newer study described the enhancement of lipopolysaccharide(LPS)-induced inflammation by intraperitoneally administered garcinol (injection of 10 mg/kg in DMSO) both in vitro and in vivo. [6,67] The expression of TNF-α and IL-6 was markedly increased by garcinol in this case, which was correlated with reduced acetylation of NF-κB. Hence, more research is necessary into the positive and negative effects of garcinol on various forms of inflammation processes. Orally administered isogarcinol (100 mg/kg) showed significant activity against collagen-induced arthritis (CIA) and xylene-induced ear edema in mice. Reduced bone and cartilage damage as well as low concentrations of inflammatory cytokines were observed upon isogarcinol treatment of CIA mice. Suppression of the expression of NF-κB, iNOS, COX-2, NFAT and IL-2 was observed after treatment with isogarcinol from in vitro experiments. [68] The same group investigated the activity of isogarcinol against systemic lupus erythematosus (SLE). Mice with chronic graft-versus-host disease (cGVHD, an SLE in vivo model) were treated with isogarcinol (60 mg/kg) leading to reduced renal histopathology and proteinuria as well as normalized serum biochemical indicator. [69] Isogarcinol (100 mg/kg, orally) also amended skin lesions (similar to psoriasis) induced by exposure to imiquimod in mice. Suppression of IL-23/Th17 axis genes of isogarcinol-treated mice was observed while isogarcinol was less toxic to liver and kidneys than cyclosporine A. [70] A diet of garcinol (5 mg/kg per day) in groundnut oil was given to male Wistar rats in order to investigate any drug effects on peritoneal macrophages of garcinol-fed rats. It showed positive effects and reduced excretion of lysosomal enzymes such as collagenase, elastase and hyaluronidase. [71] In addition, isogarcinol exerted its immune modulatory effects by direct binding and inhibition of calcineurin. [72] Histone acetylation was also correlated with liver inflammation and acute liver failure in mice and treatment with garcinol (20 mg/kg/day, i. p.) suppressed histone acetylation and prolonged survival of mice suffering from acute liver failure. [73] In endometriosis models, garcinol (0.2 μg/g/day, i. p.) could replace the function of the transcription factor KLF11, a repressor of scar-tissue collagen (COL1 A1/Col1a1), leading to fibrosis reversal in Klf11 À /À animals. [74] Obesity-related inflammation was regulated by garcinol via increase of levels of intestinal commensal bacteria Akkermansia. Garcinol blocked the formation of high fat diet (HFD)-induced obesity and suppressed glutamate pyruvate transaminase, cholesterol and triacylglycerol in the plasma of animals fed with garcinol. [75] Pretreatment of leptinstimulated cells with garcinol (1 μM) also inhibited leptin-associated cPLA 2 expression via inhibition of p300 HAT. [76] In addition, garcinol displayed beneficial effects on diabetic Wistar rats and oral application of garcinol (10 mg/kg and 20 mg/kg) normalized diabetic parameters in the rats similar to the antidiabetic drug glibenclamide. [77] Osteolysis is often based on activated osteoclasts which are bone-resorbing osteoimmune cells. [78] Osteoclastogenesis relies on RANKL (receptor activator of NF-κB) and treatment of BMM cells (bone marrow monocytes) with garcinol in vitro and of C57BL/6 mice in vivo (mouse calvarial osteolysis model. sub-cutaneous injection of 5 mg/kg in) suppressed osteoclastogenesis by downregulation of PI3 K/Akt, MAPK and NF-κB signaling. [79] Effects of garcinol and isogarcinol on various neural inflammation and neural degenerative disease models were disclosed. Experimental autoimmune encephalomyelitis (EAE) is a preclinical murine model for the investigation of multiple sclerosis (MS). EAE mice treated with isogarcinol (100 mg/kg/day, orally) showed lower degrees of intracranial lesions and demyelination of the spinal cord due to drug interference with the JAK/STAT signaling pathway. [80] Thus, isogarcinol can be a little toxic alternative to currently applied drugs for the treatment of MS. Intrathecal injections of garcinol in rats (L 5 spinal nerve ligation/ SNL model) prolonged thermal withdrawal latency (TWL) and, thus, reduced neuropathic pain likely via suppression of acetyl-p65. [81] Neuroinflammation of microglia was suppressed by treatment with garcinol and downregulation of NF-κB signaling. Garcinol also reduced the expression of COX-2/PGE2, iNOS and interleukins (IL-1b, IL-6) in the SNL rat spinal cord. [82] Monoamine oxidase-B (MAOÀ B) metabolizes dopamine and, thus, represents a suitable target for the treatment of Parkinson's disease (PD) based on dopamine depletion in certain parts of the brain. Garcinol exhibited a distinct MAOÀ B inhibition similar to known MAOÀ B inhibitors and together with its neuroprotective effects, this compound appears particularly promising for the treatment of PD. [83] Garcinol also reduced the side-effects of dopamine replacement therapy by L-DOPA. Dyskinesia induced by L-DOPA in 6-hydroxydopamine (6-OHDA)-lesioned mice was reduced by co-treatment with garcinol (5 mg/kg, orally). [84] Garcinol also inhibited catechol-O-methyltransferase leading to an increased L-DOPA bioavailability and it reduced hyperhomocysteinemia associated with L-DOPA treatment. This indicates the great potential of garcinol for the treatment of PD in combination with L-DOPA. [85] It was observed that garcinol-pretreatment (50, 100 or 200 mg/kg, i. p.) of mice with pentylenetetrazole (PTZ)-induced epilepsy led to a marked decrease of mortality and of seizure scores. In addition, the memory and cognition of these garcinol-treated mice improved distinctly. Suppression of BDNF and TrkB by garcinol as well as garcinolinduced upregulation of GABA A and GAD65 were identified as anti-epileptic modes of action of garcinol. [86] Garcinol (10 mg/kg, i. p.) also showed beneficial effects in rats exposed to cocaine and it inhibited reinstatement by reconsolidation-based modes following cocaine reactivation. [87,88] Reactivated memories were affected by garcinol which displayed long-lasting effects and, thus, garcinol can support drug abstinence and be a suitable therapy for psychopathologies such as drug addiction. Indeed, memory processes in honeybees strongly depend on HAT activity and histone H3 acetylation by treatment with garcinol or C646. [89]

Miscellaneous Activities and Applications
The establishment of garcinol as a proper drug is in progress. The Indian company Sami Labs Ltd. isolated garcinol by extraction of dried kokum plum rinds with hexane followed by column chromatography and crystallization from hexane and this company has standardized garcinol (40 % garcinol in microcrystalline cellulose) and evaluated the toxicity of 40 % garcinol in Wistar rats. In fact, 40 % garcinol exhibited a low toxicity and no signs of any side-effects at doses of up to 100 mg/kg/day (orally) after weeks and months. [90] There are also successful efforts to produce fruit wines from kokum and the fermentation of a mixture of kokum juice with banana juice generated an excellent wine which has the potential to conquer the exotic wine market in the future. [91] The preparation of effective sunscreens with kokum extract is also possible. The ethyl acetate extract of kokum, which is rich in garcinol, when washed with water in order to remove hydroxycitric acid, showed significant UV-radiation protective effects. A cosmetic cream containing 5 % of the extract revealed a sun protection factor of 3.43 with boot star rating 5. [92] Garcinol isolated from the fruits of the Thai plant Garcinia dulcis also showed vasorelaxant activity. It might be a suitable drug for the treatment of hypertension due to its antioxidant activity. Injection of hypertensive 2 K1 C (2-kidneys-1-clip) rats with low doses of garcinol (0.1 mg/kg, i. v.) led to hypotensive effects and reduced arterial blood pressure, heart rate, plasma malondialdehyde values, and eNOS expression. [93] Eryptosis is a programmed cell death process to eliminate defective erythrocytes (prior to nephrotoxic hemolysis) analogously to apoptosis of damaged cells with nuclei. [94] In particular, eryptosis has been identified as a mechanism to fight infection with Plasmodium (malaria), for example, in sickle-cell trait, in order to eliminate infected erythrocytes and the parasites therein. Human erythrocytes treated with garcinol (5 μM) underwent eryptosis according to increased annexin-V binding, increased ROS formation and reduced ATP level of the cytosol. [95] Thus, garcinol has the potential to fight Plasmodium-caused diseases via this peculiar mode of action.

Conclusions
The chemistry and biology of garcinol and isogarcinol are active and prospering fields of research. The total synthesis of garcinol by Socolsky and Plietker represents a chemical highlight of the past years and enables the fine-tuning of the garcinol molecule concerning improved biological activities by preparation of synthetic garcinol derivatives. The identification of new HATs as targets of garcinol or isogarcinol broadens the scope of application of these natural products including activities against viral and parasitic models. In addition, their distinct activities against cancer stem-like cells warrant studies against further tumor models. One report has appeared that described a promoting effect on LPS-induced inflammation processes. This is in stark contrast to many other publications which describe garcinol or isogarcinol as potent agents against various inflammation processes. Future studies will show if this is just a solitary case. It is noteworthy that garcinol was also found active against models of various neurological diseases such as EAE, Parkinson disease, epilepsy or drug addiction. These discoveries underline once more the potential of garcinol and isogarcinol as valuable drug candidates.