1. Introduction

2. Phytochemistry

3. Pharmacodynamics (in vitro

and in vivo preclinical trials)

4. Clinical efficacy

5. Safety and tolerability

(toxicology)

6. Pharmacokinetics and

metabolism

7. Regulatory affairs

8. Conclusion

9. Expert opinion

Drug Evaluation

Embelin -- a drug of antiquity:shifting the paradigm towardsmodern medicineRadhika PoojariIndian Institute of Technology Bombay, Department of Biosciences and Bioengineering,

Mumbai, India

Introduction: Embelia ribes or Embelia tsjeriam-cottam, more commonly

known as vidanga, is a type of ayurvedic medicine that has been used to treat

various diseases for a number of years. Bright orange embelin-rich fruits have

been well established as ethnomedicinals, for a number of years with their

pharmacological actions attributed to their hydroxybenzoquinone active

constituent. Embelin has become known specifically for its antihelminthic

and contraceptive use.

Areas covered: This drug evaluation provides a historical summary of embelin

along with its therapeutic use, phytochemistry and toxicology. Embelin’s

pharmacotherapeutical properties are also discussed along with its molecular

targets. It is hoped that this article will help to draw the attention of researchers

and biopharmaceutical companies to the untapped potential in bioprospection

for the development of new drugs.

Expert opinion: Embelin is the only known non-peptide small-molecule

X-linked inhibitor of the apoptosis protein (XIAP) -- an anti-apoptotic protein

considered a promising cancer therapeutic target. Embelin acts as an NF-kBblocker and potential suppressor of tumorigenesis. It also exhibits potent

cytotoxic, antioxidant and cancer chemopreventive effects. Given the potential

uses of embelin, it is recommended that further investigations take place to

properly explore its pharmacological and clinical effects.

Keywords: embelin, ethnomedicinal uses, molecular targets, pharmacotherapeutics, vidanga

Expert Opin. Investig. Drugs (2014) 23(3):427-444

1. Introduction

The genus Embelia is a group of shrubs belonging to the Myrsinaceae family, agroup more commonly known as vidanga. For many years, this indigenous drughas been recommended for a number of diseases, with its use recorded in historicaltexts, including the Charaka Samhita in 1949, Sushruta Samhita in 1942 andAshtanga Hridaya in 1950 in great detail. Embelia ribes Burm. f. (Syn. E. gladuliferaWight), Embelia tsjeriam-cottam (Syn. E. robusta, Roxb), is a rambling shrub distrib-uted along the Malabar Coast of Sri Lanka, through Sylhet, Singapore and China, aswell as Assam to Cochin and the hilly parts of Maharashtra and Konkan in India(Figure 1). Embelin (Box 1) gained mention in 1934 in the British PharmaceuticalCodex, followed by the Indian Pharmaceutical Codex in 1953, and was includedas an official drug in 1966 in Pharmacopoeia of India as vidanga, although itsbotanical origin was described as fruits of E. ribes only [1]. The bright orangehydroxybenzoquinone embelin-rich fruits have become well established within eth-nomedicine and their pharmacological action is attributed to this constituent. Thesefruits have been described as a number of things, including acrid, astringent, bitter,pungent, expectorant, depurative, digestive and carminative with antibacterial, sto-machic, diuretic, vermifuge, contraceptive, rejuvenating, alternative, stimulant,

10.1517/13543784.2014.867016 © 2014 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 427All rights reserved: reproduction in whole or in part not permitted

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febrifuge, colic and tonic properties. These fruits are consid-ered to be beneficial in chronic skin diseases as well as respira-tory disorders, helminthiasis and gastrointestinal disorders.They are also thought to promote immune functions, improveblood circulation and possess anti-aging effects [2,3].While there have been a number of papers published on the

therapeutic effects of the fruit extracts of the Embelia species,updated literature on embelin -- the major active constituentof vidanga -- is unavailable. The aim of this review is to sum-marise the history, therapeutic uses, phytochemistry and tox-icology of embelin as well as its molecular aspects in thehope of increasing the awareness of bioprospection for thedevelopment of new drugs. It is also hoped that this reviewwould encourage further research on the pharmacologicaleffects of embelin, both in vitro and in vivo, in order to pro-vide a more in-depth insight into the mechanisms associatedwith the therapeutic effects of embelin. The clinical aspectsof embelin certainly warrant further investigation before itsintegration into medicinal practices as well as for its use inthe formulations for commercialisation.

2. Phytochemistry

Embelin (2,5dihydroxy-3-undecyl-1,4benzoquinone) (Figure2)is the major active constituent (2.3%) in the fruits commonlyknown as vidanga. This naturally occurring hydroxybenzoqui-none consists of a dihydroxyquinone core; its long hydrophobictail is a characteristic feature of the genusEmbelia,whichwas firstchemically investigated by Scott in 1888. Its other constituentsinclude quercitol (1.0%), an alkaloid christembine; tannin;vilangin (methylene-bis-2-5-dihydroxy-4-undicyl 3-6-benzo-quinone) (Figure 3) and fatty ingredients (5.2%), including res-inoid; fixed oil and traces of volatile oil [3,4]. So far, the chemicalexamination of E. ribes has led to its isolation, a better under-standing of its synthesis and its bare constituents. Further,investigations of its analogues and its condensation reactionswith primary amines have also taken place, in addition to inves-tigations in the synthesis of embelin derivatives (diamines andbisquinones) and isolation of vilangin [5-7].

Cherutoi et al. first described the reaction of zinc(II) andcopper(II) ions with embelin in a 1:2 ratio to yield six-coordinate complexes [8]. The embelin molecule, on loss ofprotons, produced a bidentate ligand with five-memberedchelate rings. Further, acidification of this molecule led tothe regeneration of the embelin molecule. A recent synthesisapproach for hydrophilic analogues of embelin was developedto improve the water solubility and biodisponibility ofembelin. Three amines with different carbon chain lengthsbearing a protected benzoquinone were prepared for itshydrophilicity. Such a modification would provide usefulinformation on the influence of polarity of the side chainfor the development of a comprehensive structure--activityrelationship of embelin analogues for anti-cancer activity [9].X-ray crystal structural analysis of plasminogen activatorinhibitor-1 (PAI-1) in complex with antagonist embelin asdrug-designing PAI-1 targets was reported by Lin et al. [10].Nikolovska-Coleska et al. [11] discovered that embelin isthe only known small molecular inhibitor of X-linked inhibi-tor of apoptosis protein (XIAP), identified through a novel,structure-based computational screening of an herbal medi-cine-based compound library. The design, synthesis and eval-uation of new embelin analogues as potent inhibitors of XIAPhave been reported [12]. For example, compound 6 g exhibiteda Ki value of 180 nM binding to XIAP BIR3 in a competitivebinding assay which represented a promising lead compoundfor further optimisation. Very recently, a novel protocol basedon the dihydropyran and dihydropyridin embelin derivativessynthesised through a three-component reaction with embe-lin, with aldehydes and with cyclic enaminones as syntheticimputs for antibacterial activity, has been evaluated [13].

Embelin has been extracted colourimetrically and gravimet-rically [14]. There have been reports on optimisation, quantita-tive estimation of embelin in the fruits of E. ribes and informulations (market tinctures, tablets) spectrophotometri-cally Stability and quality control studies of embelin in ayur-vedic iron formulations (Navayasa Churna used for anaemia,jaundice and piles and Krimimudgara Rasa, an antihelminthicdrug) were carried out using high-performance thin layer

Box 1. Drug summary.

Drug name EmbelinPhase PreclinicalIndication A number of indications including contraceptivePharmacology description A XIAP inhibitor and potent NF--kB blockerRoute of administration OralChemical structure

OH

O

O

CH3HO

Pivotal trial(s) [4,25-27]

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chromatography [15-19]. The developed methods were vali-dated in terms of precision, accuracy, stability, limit of detec-tion and limit of quantification. A simple, sensitive, accurateand reproducible method for embelin marker-based standard-isation and quality assurance of E. ribes and E. tsjeriam-cottamfrom the various extracts of fruits and in polyherbal formula-tions by reversed-phase high-pressure liquid chromatography(RP-HPLC) have been demonstrated [19-23] so far. A sensitiveand accurate HPLC method for the determination of embelinin plasma, urine, faeces and tissue homogenates of brain,heart, liver, kidney, spleen and lungs have been developed.A pharmacokinetic study of embelin 50 mg/kg after oraladministration in rats was successfully quantified up to24 h [24].

3. Pharmacodynamics (in vitro and in vivopreclinical trials)

In recent decades, there has been spurt of reports with respect tothe primary pharmacodynamic activities of embelin, namely,anti-inflammatory, analgesic, anti-fertility effects, lipid metabo-lism, glucose metabolism, hepato-gastroenterological disorders,neurological abnormalities, molecular docking, antioxidanteffects, anti-cancer activity and drug delivery systems. The

mechanism of action has been summarised in this review. Anoverview of the current status of pharmacotherapeutics ofembelin and its molecular targets has been depicted in Figures

4 and 5, respectively.

3.1 Analgesic and anti-inflammatory activityGupta et al. [4], Zutshi et al. [25] and Chitra et al. [26] reporteda promising lead compound, embelin, and its disalt deriva-tives for designing a new class of potent analgesic and anti-inflammatory agents. Kalyan Kumar et al. [27] reported thattopical application of embelin (0.1, 0.3 and 1%) exhibitedanti-inflammatory activities in acute (1 h) and chronic models(10 days) of psoriasis using 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 µg/ml) in induced mouse ear oedema.Embelin inhibited TPA-stimulated tumour necrosis factor-a(TNF-a) in human keratinocytes and showed dose-dependent decrease in bacterial LPS (Escherichia coli sero-type 055:B5)-induced TNF-a levels in mice with an ED50

of 9.28 mg/kg. It inhibited topical oedema in the mouse earwith substantial reductions in skin thickness, tissue weight,inflammatory cytokine production, neutrophil-mediatedmyeloperoxidase activity and ameliorated various histopatho-logical indicators. The inhibitory effect of embelin may bedue to the inhibition of pro-inflammatory cytokines IL-1band TNF-a and the subsequent blockade of leucocyte accu-mulation. Mahendran et al. [28,29] demonstrated potent anal-gesic and anti-inflammatory activities of embelin and itsderivatives. Embelin condensed with various aromatic substi-tuted primary amines yielded 10 new derivatives and one pre-viously reported derivative along with monomethyl embelinand a unique ninhydrin adduct of embelin. Hot plate, tailimmersion, acetic acid-induced writhing methods andcarrageenan-induced paw oedema in mice and rats were testedat 10 and 20 mg/kg intraperitoneal (i.p.) doses of embelinwhich allowed analysis of peripheral and centrally mediatedantinociceptive responses. Potent analgesic activity higherthan the standard pentazocine was observed. Embelin andboth of its derivatives almost completely abolished the aceticacid-induced writhing. The p-sulphonylamine phenylaminoderivative of embelin showed better anti-inflammatory activ-ity than the parent compound embelin with significant reduc-tion in the paw oedema of 19.02% in 30 min in comparisonto 14.74% after 120, 180 and 360 min, respectively. KumaraSwamy et al. [30] studied the wound-healing activity of embe-lin (4 mg/ml of 0.2% sodium alginate gel) which resulted infast epithelialisation of the incision, wound contraction, sig-nificant increase in tensile strength of the incision wound,weight of granulation tissue, collagenation, more fibroblasts,blood vessels and an absence of monocytes. Topical applica-tion of embelin 5% (w/w) ointment on a cutaneous woundin streptozotocin (STZ)-induced diabetic rats using excision,incision and dead space models exhibited a significant increasein wound contraction and better epithelialisation, acceleratingthe healing process. Embelin has been demonstrated tobe active by the oral route (25 and 50 mg/kg) [31].

Figure 2. Illustration of embelin.

Figure 3. Illustration of vilangin.

Figure 1. Embelia tsjeriam-cottam: flowering, fruiting

branch in habitat and dried fruits are shown.

Embelin

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Schaible et al. [32] depicted embelin as a novel chemotype forthe development of dual 5-lipoxygenase (IC50 0.06 µM)/microsomal prostaglandin E2 synthase (IC50 0.2 µM)-1 inhib-itors. A new approach of using embelin and its derivativescould serve as the alternative natural source of crosslinking/stabilising agents [33]. Since their development, crosslinkingagents like formaldehyde, glutaraldehyde and epoxy com-pounds are cytotoxic which limits the clinical applications.Docking of the quinones with type I collagen and type III col-lagen revealed that embelin, 5-O-methyl embelin and potas-sium embelate possessed the best affinities with collagens.Using bioinformatics tools, such as quinone and collagen,molecular interactions could be developed as the potent cross-linking/stabilising agent of collagen preparation in wounddressings for clinical applications.

3.2 Contraceptive activityThere has been wide acceptance of embelin or E. ribes fruitsincorporated in the multi-component ayurvedic formulations(powder/extracts/tablets/capsules) tested in animal models aswell as in clinical studies, but not much has been exploredwith respect to the isolated active form of embelin for its fertil-ity regulation activities. A limited number of survey data is

available to date as discussed here. Powdered berries of E. ribes(100 mg/day) orally administered to male bonnet monkeys for3 months adversely affected the quantity and quality of semen,and testosterone levels were reduced. Ayurvedic formulationscontaining E. ribes, such as ROC-101 and Garbhanivaranaaushadam were reported to impair the fertility of female miceand rats, inducing sterility in male mice. Pippalyadi yoga,AC-4 and Maswin tablets have been used in women for theircontraceptive activities without side effects [34-36].

Embelin possesses anti-implantation activity of 83.33%when administered post-coitally from days 1 to 5 of pregnancyat an oral dose of 60 and 120 mg/kg in rats [37,38]. Similarobservations were made by Rathinam et al. [39] wherein, embe-lin anti-implantation activity of 100% at 10 mg/kg, a muchsmaller oral dose, in rats were reported. It also exhibited highlysignificant anti-ovulatory effects in rabbits. Embelin at an oraldose of 50, 100 and 200 mg/kg was given to male rats for15 days and significant reduction in the sperm count andmotility and weight of the testes were observed [40]. Embelinat subcutaneous (s.c.) dose of 0.3, 0.4 and 0.5 mg/kg was givento male rats for 35 days, resulting in altered testicular histologyand anti-androgenic activity [41]. Embelin at s.c. daily dose of20 mg/kg was given to male rats for 15 or 30 days, which

Anticancer Respiratorydisorders

Contraceptive/antispermatogenic

Anti-infective

Antihyperlipidaemicand antihyperglycemic

Analgesic

Antipyretic

GastrointestinaldisordersNeurodegenerative

disorders

Clinicaldevelopment

Hepatoprotective

Antihelminthic

Nanomedicine

Molecular docking

Antioxidant

Antimitotic

O

O

OH

HO C11H23

Radioprotective

Embelin

XIAP inhibitor

Anti-inflammatory

Figure 4. Pharmacotherapeutics of embelin are shown.

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revealed inhibition of the epididymal motile sperm countand carbohydrate metabolism enzymes. Profound morpholog-ical changes in the spermatozoa such as decapitation of thespermatozoal head, discontinuity of the outer membranoussheath in the mid-piece and the tail region, and alteration inthe shape of the cytoplasmic droplet in the tail were observed.These changes were reversible with a 15- to 30-day recoveryperiod [42]. In male dogs, embelin at an oral dose of 80 mg/kgevery alternate day for 100 days caused significant reductionin the weight of testes and epididymis, the diameter of the sem-iniferous tubule and Leydig cells and biochemical changes intestes and epididymis. Histologically, it showed varying degreesof spermatological alterations which were recouped after a250-day recovery period [43]. Embelin administration to whiteNew Zealand male rabbits (30 mg/kg intramuscular) on alter-nate days for 14 days caused amarked reduction in the testoster-one concentrations within 2 days and up to 90% reduction bythe sixth day. The luteinising hormone level showed a corre-sponding rise with the fall in testosterone levels. Similarly, therewas a rapid increase in the progesterone levels with embelinadministration. The observed changes in the levels of these threehormones suggested that embelin disrupted testosterone pro-duction at the testicular level [44]. Embelin at an oral dose of100 mg/kg given to female rats from days 1 to 5 of pregnancyfailed to increase or decrease the oestrogen-sensitive parameterslike uterine weight, protein, glycogen and alkaline phosphataseactivities of uterine. It had no oestrogenic or anti-oestrogeniceffect [45]. In ovariectomised rats, embelin at an oral dose of25 and 50 mg/kg from day 1 to day 10 with oestradiol 17-b0.4 µg/rat s.c. from day 8 to day 10 inhibited pregnancy andpossessed anti-estrogenic and weak progestational activity [46].Embelin at an oral dose of 10 and 20 mg/kg disrupted the

oestrous cycles with significant depression in the plasma oestra-diol and progesterone. Isolated mixed ovarian cells fromembelin-treated rats produced significantly less progesteroneand estradiol than the controls in vitro. Thus, embelin probablyinterferes with the reproductive functions in female rats bysuppressing the ovarian production of sex steroid hormones [47].Hence, as deciphered from these observations, embelininterfered with the fertility in male and female mammals andconsequently could be developed into a potent contraceptiveagent.

3.3 Gastrointestinal and liver disordersVidanga has been an excellent remedy for diarrhoea, dysen-tery, abdominal pain, constipation, colic and flatulence sinceantiquity. For instance, Vidanga is one of the constituents ofGasex formulation of the Himalaya drug company whichgave excellent results in minimising the gastrointestinalsymptoms in a clinical trial in post-operation where theabdominal discomforts were checked [48]. Daily oral embelin(75 mg/kg/day) administration to male rats for 15 -- 30 dayscaused significant elevation in the uptake of D-glucose,L-alanine, L-leucine and calcium in small intestinal seg-ments. Embelin significantly increased the intestinal brushborder membrane-associated enzymes: sucrase, lactase, malt-ase, alkaline phosphatase and leucine aminopeptidase.A significant increase was also noted for microsomal glu-cose-6-phosphatase and cytosolic lactate dehydrogenase.Increases in the brush border membrane-associated total lip-ids, phospholipids, cholesterol, triacylglycerol, unesterifiedfatty acids and ganglioside sialic acid were seen but not inthe cholesterol:phospholipid molar ratio. All of these changesreturned to control or near control levels with drug

Embelin

Cellproliferation Apoptosis

IκBαp65

Nuclear translocationTRAF 1,2

c-FLIPPPARγ

Caspase 3,7,8,9cIAP 1, cIAP 2

SurvivinPARPNF-κB

p53Bcl-2Bcl-x1Bax

Cyt-cMcl-1RIP

TRAILSTAT3-PTEN

Erb B2Akt/m TOR/S6K1

XIAP

c-FLIPErk1/2

Antioxidant

LPOGSHGSTGPχSODCAT

DNA damage

Immuno-stimulant

Metastasis

MMP-9ICAM-1VE GFCD 31

IL-1β, 6INF αTNF α

XIAP-BIR3PPARγPI3KpAkt

GLUT4PAI-1PCAF

Sex differentiationin ischemic brains

miR-23aCOX-2Cyclin D1

cMycp21Ki67

PCNA

Molecular dockingand bioinformatics

Drug deliverysystems

PEG-derivatized embelinPEG-PLA

PCL microfibersPEG-b-p (CB-co-LA)

PEG-PCD lipopolymerEmbelin-phospholipid

complex

Figure 5. Molecular targets of embelin are shown.

Embelin

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withdrawal [49,50]. In inflammatory bowel disease, a chronicintestinal disorder, a protective effect has been reportedwhen an oral dose of embelin (25 and 50 mg/kg) and sulfa-salazine (100 mg/kg) was given for 5 days before colitisinduction in rats by intrarectal acetic acid (3% v/v) adminis-tration, with continued treatment for 7 days. This treatmentsignificantly reduced the colonic myeloperoxidase activity,lipid peroxides and serum lactate dehydrogenase and signifi-cantly increased glutathione (GSH) which had previouslybeen reduced as an effect of inflammatory bowel disease. Itsignificantly reduced the wet weight of the colon, clinicalactivity, gross lesion score and the percentage of the affectedarea in comparison to colitis control. Histopathologically, theleucocyte infiltration, and oedema and colonic mucosa tissueinjuries were minimal following pre-treatment with embelinindicating its protective effect from ulcerative colitis [51]. Inanother study, colitis was induced in BALB/c mice by 5%dextran sulphate sodium in drinking water for 7 days. Embelin(10, 30 or 50 mg/kg) on oral administration for 7 days attenu-ated the pathological symptoms of colitis. In addition, embelinameliorated colitis by suppressing the pro-inflammatorymediators, such as TNF-a, IL-1b and IL-6 mRNA expressionlevels [52].Although several ayurvedic formulations containing

vidanga (E. ribes) as one of the components for liver disordertreatments are available on the market (such as Livomyn, San-jivani vati, Kumariasava, Liv-52 and Livosin), there are veryfew investigations into the heparoprotective activity of embe-lin alone. Poojari et al. [53] demonstrated significant hepato-protection in rats orally treated with embelin (100 mg/kg)for 5 weeks, including 1 week of pre-treatment prior to car-bon tetrachloride (CCl4, 0.7 ml/kg i.p. once weekly for4 weeks)-induced hepatotoxicity. Embelin treatment signifi-cantly attenuated the elevated enzyme activities of transami-nases, alkaline phosphatase, lipids and enhanced the fall inprotein and albumin levels caused by CCl4, as well asmarkedly reduced the swelling of hepatic cords and inflamma-tory response and minimised mononuclear cell infiltration,thus indicating the effectiveness of the hepatoprotectantdrug. Embelin protected the structural integrity of hepatocytecell membranes or stimulated hepatic regeneration. Previ-ously, the hepatic natural antioxidant potential of embelin(25 mg/kg) taken orally for 15 days against CCl4 (1 ml/kgi.p. once/week for 15 days)-induced liver damage in rats wasevaluated [54]. Peroxidative damage was measured to minimisethe deleterious effects of free radicals including the peroxyradicals and the inhibition of lipid peroxidation (LPO) byembelin reflected the liver recovery from CCl4 toxic effectstowards normal liver cell functioning.

3.4 Antihyperlipidaemic and antihyperglycemic

activityEmbelin possesses lipogenic properties [55]. The lipid levels oftotal cholesterol, phospholipids, triglycerides and free fatty

acids were markedly elevated in methylcholantherene-inducedfibrosarcoma transplanted rats. After 30 days, embelin(50 and 100 mg/kg) treatment orally for 20 days led to signif-icant alterations in these lipid profiles of serum, and liver andkidney tissues [56]. The biochemical effects of embelin admin-istration (50 mg/kg oral dose for 14 weeks) from E. ribesagainst a two-step hepatocarcinogenic regimen of N-nitroso-diethylamine (NDEA, 200 mg/kg single i.p.) and phenobar-bital (PB, 0.05% in drinking water orally for 13 weeks) wasstudied in Wistar rats with respect to the lipid profile, renalfunction tests and blood glucose levels. Rats administeredNDEA/PB showed hypercholesterolaemia, hypertriglyceri-daemia, elevated low-density lipoproteins, free fatty acidsand very-low-density lipoproteins levels and decreased urealevels. Pre- and co-treatment with embelin for 14 weeks sig-nificantly prevented these biochemical alterations inducedby NDEA/PB, suggesting that embelin has protective andhypolipidaemic effects [57]. Chaudhari et al. [58] have shownthe preventive effects of embelin (50 mg/kg) for 21 daysagainst hyperlipidaemia and oxidative stress in rats on ahigh-fat diet with induced obesity. Oral embelin treatmentreduced the gain in body weight, blood pressure, visceral fatpad weight, serum lipid levels, coronary artery risk and ath-erogenic indices, serum glucose (24.77%), insulin (35.03%)and leptin (43.39%) levels. In addition, it significantlydecreased the hepatic thiobarbituric acid-reactive substance(TBARS) levels, while increasing superoxide dismutase(SOD), catalase (CAT) and GSH levels in obese rats. Thus,embelin could be useful as a new drug therapeutic to preventobesity, hyperlipidaemia and oxidative stress.

The anti-diabetic effect of embelin in the management ofdiabetes and severe hyperglycaemia has been established.Wu et al. [59] have shown replication deficient adenoviralvector-encoding human XIAP transduced normal rat insuli-noma cells and human islets on embelin treatment immenselydecreased the XIAP activity by blocking protein synthesis anddecreased the cell viability and insulin production underinflammatory cytokines. It reversed the protective effect ofXIAP in these cells against cytokines, thus suggesting theimportant role of XIAP expression in graft viability and func-tion in a post-transplantation (STZ, 40 mg/kg i.p. for 5 days,induced diabetic NOD-SCID mice, human islets transplanta-tion) model. In another study, embelin (15, 25 and 30 mg/kg)treatment for 21 days caused a significant decrease in bloodglucose, serum nitric oxide, total cholesterol and triglyceridelevels and an increase in low-density lipoprotein cholesterollevels in STZ (50 mg/kg i.p. once weekly)-induced diabeticrats. Further, embelin increased the pancreatic antioxidantdefence enzyme status (SOD, CAT, GSH, glutathione perox-idase [GPx], glutathione S-transferase [GST] and ascorbicacid), and decreased TBARS levels against the reactive oxygenspecies (ROS) produced under hyperglycaemic conditions.The protective action of embelin could be due to the stimula-tion of pancreatic regeneration through improved synthesis ofproteins and/or its accelerated detoxification, reduction of

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deleterious effects of free radicals (including peroxynitrate) andits antioxidant activity. Histologically, degenerated pancreason embelin treatment significantly regenerated islet cells,thereby protecting the pancreatic b-cells against loss and exhib-ited anti-diabetic properties [60-62]. A recent study [63] revealedembelin’s potential to regulate insulin resistance, alter b-celldysfunction and modulate key markers involved in insulin sen-sitivity and glucose transport using high-fat diet fed STZ(40 mg/kg)-induced type 2 diabetic mellitus rats. Embelin(50 mg/kg oral) administration for 30 days exhibited aninsulin-sensitising effect through adipose tissue-specific partialagonism of peroxisome proliferator-activator receptor gamma(PPARg) and activated glucose transport through the translo-cation and activation of glucose transporter protein(GLUT4) expression mediated by the insulin-dependentPI3K/p-Akt signalling pathway in epididymal adipose tissue.Embelin also protected the b-cells from scavenging free radi-cals and alleviated dyslipidaemia in the insulin-resistant animalmodel. This drug can be useful in the prevention and treat-ment of obesity-related type 2 diabetic mellitus. The moleculardocking of embelin into PPARg , PI3K, p-Akt and GLUT4active sites promoting insulin sensitisation and glucose uptakeis a significant finding. Sun et al. [64] demonstrated embelin tobe a potential agent for the prevention and treatment of dia-betic retinopathy, wherein, Muller cell alterations induced byhigh-glucose conditions were counteracted by the XIAPinhibitor embelin.

3.5 Neurodegenerative disordersIn the indigenous systems of medicine, vidanga fruits havebeen used for the treatment of mental disorders, dyspnoea,central nervous system (CNS) disorders and as brain tonic [1,4].The effect of an oral dose of embelin (25 and 50 mg/kg) for4 days on transient global ischaemia/reperfusion-induced neu-ronal damage in rats significantly increased the locomotoractivity and hanging latency time and decreased beam walkinglatency in comparison to ischaemic rats. It also significantlyreduced the LPO and increased the total thiol content andGST activity in brain homogenates. Decreased cerebral infarc-tion area and histopathological alterations, such as normalglial density, decreased oedema, absence of lymphocytes, con-gestion of blood vessels and necrosis in embelin-treatedgroups suggested that embelin as a potent neuroprotectiveagent and would be useful in the adjunct for cerebral stroketreatment [65]. In another study [66], embelin (2.5, 5 and10 mg/kg, i.p.) administration showed significant inhibitionof seizures induced by electroshock and pentylenetetrazole ina dose-dependent manner and the activity was comparableto phenytoin and diazepam. A significant decrease in locomo-tion revealed its CNS-depressant activity. Thus, embelin pos-sesses anticonvulsant activity against both grand mal and petitmal epilepsy. Recently, embelin has been identified byModak et al. [67] as a novel p300/CBP-associated factor(PCAF, KAT 2B)-specific non-competitive inhibitor whichinhibits the lysine acetyltransferase (KAT) activity of PCAF

in vitro (10 µM) and in vivo (5 mg/kg, i.p. for 3 days inmice) which is useful in the muscle differentiation process.This PCAF KAT activity inhibition in turn inhibited the acet-ylation of MyoD, which blocks the differentiation of myo-blasts into myotubes. Microarray analysis of embelin-treateddifferentiated C2C12 cells revealed many altered gene expres-sion levels which are useful to explore the neuro-musculargene regulatory network. In breakthrough research evi-dence [68], embelin treatment (low-dose 10 mg/kg or high-dose 40 mg/kg s.c.) for 3 days significantly exacerbated thestroke-induced injury in female mice but had no effect inmale mice, demonstrating XIAP regulation as a majorcontributor to sex differences after stroke. MicroRNA,miR-23a, directly bound the 3¢ UTR of XIAP and miR-23ainhibition led to an increase in XIAP mRNA. Cerebral ischae-mia induced a significant decrease in the miR-23a expressionlevels in males, whereas it significantly increased after strokein females. Embelin eliminated the stroke-induced decreaseof XIAP and Smac/DIABLO protein expression, whereas thedecrease was still evident in males. Females were more sensi-tive to manipulation of XIAP signalling than males. Anincreased XIAP--caspase-3 interaction was observed in malesafter stroke, but this interaction was decreased in females.This decrease led to enhanced caspase activity after embelintreatment which in turn led to an exacerbation of infarct infemales. Hill et al. [69] reported that inhibition of XIAP priorto hypoxia-ischaemia (HI) caused significant increase in thebehavioural deficits and anatomical abnormalities (ventricularenlargement) in embelin-treated (20 mg/kg s.c.) neonatal HIinjured female rodents in comparison to vehicle-treated HIfemales. XIAP protected female brains from deleterious effectsof early HI injury, while elimination of this protection viaXIAP inhibitor embelin exacerbated both damage and behav-ioural deficits, with no effect on HI males. This reflected thesex-dependent differences in the behavioural and anatomicaloutcome following HI, as well as in the underlying apoptoticmechanisms which is very exciting. Hence, the potential useof embelin as a sex-specific neuroprotectant in clinical practiceremains warranted.

3.6 Anti-cancer activity and the molecular targetsEmbelin is the only known class of non-peptide, cell-perme-able, small-molecule inhibitor that binds to the XIAPBIR3 domain -- a promising cancer therapeutic target. Embe-lin inhibited cell growth (IC50 3.7 µM), induced apoptosis(25 µM with 30% [3-fold] and 50 µM with 75% [9-fold]increase) and activated caspase-9 (20 µM with 33.0%[10-fold] and 40 µM with 62.1% [20-fold] increase) in pros-tate cancer cells with high XIAP levels in comparison to con-trol cells. In stable XIAP-transfected Jurkat cells, embelin(50 µM) effectively overcomes the protective effect of XIAPto apoptosis and enhanced etoposide (10 µM)-induced apo-ptosis with minimal effect in control vector-transfected Jurkatcells [11]. Chen et al. [12] synthesised new XIAP inhibitors withbetter binding affinities than embelin by modifying its

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hydrophobic tail. An embelin derivative [2,5-dihydroxy-3-{2-[4-(2-m-tolyl-ethyl)-phenyl]-ethyl}-[1,4] benzoquinone](embelin 6 g) had a twofold better binding affinity (Ki value0.18 µM) to XIAP BIR3 compared to embelin (Ki value0.40 µM) with potent anti-proliferative effects in humanbreast (IC50 5 µM) and prostate cancer (IC50 5.5 µM) cells.In addition, embelin 6 g (5 µM, p < 0.01) was more potentthan embelin (5 µM, p < 0.05) in repressing XIAP geneexpression at mRNA levels and protein levels by ELISA, byalmost twice that of embelin. However, embelin and embelin6 g exhibited a similar ability to inhibit prostate cancer cellgrowth. Embelin (5 µM) and embelin 6 g (5 µM) alsodecreased androgen receptor (AR) and prostate-specific anti-gen expression by > 50% of the mRNA transcriptional activ-ity levels in comparison to control in LNCaP and C4-2 cells.Embelin prevented AR nuclear translocation from the cyto-plasm to the nucleus in C4-2 cells. A concentration-dependenteffect of AR Ser81 phosphorylation post-embelin treatment(5/10 µM) strongly suggested that embelin suppressedandrogen-mediated AR phosphorylation. Anti-androgen ((S)-N-(4-cyano-3-(trifluoromethyl) phenyl)-3-((4-cyanophenyl)(methyl)amino)-2-hydroxy-2-methylpropanamide)) (CBDIV17,25 µM) and embelin (5 µM) combination was more potent ininhibiting cell growth and inducing apoptosis in advancedprostate cancer in comparison to control or monotherapy.The percentage of cells in the sub-G1 phase for combinationtherapy was 40-fold, 28-fold and 2-fold greater in comparisonto control, embelin and CBDIV17, respectively. This superioreffect signified their supra-additive anti-proliferative and apo-ptotic effects [70]. The protein--protein interactions in theSmac--DIABLO-XIAP and Smac--DIABLO-Survivin com-plexes to design Smac/DIABLO peptidomimetics, which wasinvestigated by Obiol-Pardo et al. [71], suggested the bindingmode of embelin and its two derivatives using molecular dock-ing and molecular dynamics simulations to analyse the ligandand receptor flexibility. This approach could be utilised tofind innovative therapeutic agents for the treatment of malig-nant cancers.Natural products, their derivatives and the synthetic com-

pounds derived from natural products are crucial in the man-agement of pathophysiological conditions of various diseasescaused by ROS. An example of this is quinones which areused as anti-tumour agents [72]. Chitra and ShyamalaDevi [73] reported that potent cytotoxicity and antioxidantproperties were exhibited by embelin (50 and 100 mg/kgorally for 20 days)-treated fibrosarcoma rats where free radicalscavenging properties (i.e., SOD, CAT, GPx, GST and GSHactivities) were significantly enhanced and LPO content wasrestricted sharply in the liver, intestine and kidney tissues incomparison to the control rats. Moreover, an interestingreport [74] showed the cytotoxic effects of embelin (2.5 --20 mM) exhibiting inhibitory effects on the activation ofpolymorphonuclear leucocytes and a dose-dependent decreasein [3H]-thymidine uptake, LPO and GSH levels. The antiox-idant status of this drug on fibrosarcoma-bearing rats with

significant tumour regression and prolonged survival time issignificant, in addition to retardation of cell multiplicationhaving high chemotherapeutic potential. Embelin has beenreported to scavenge 1,1-diphenyl-2-picrylhydrazyl radicalsand inhibit hydroxyl radical-induced deoxyribose degrada-tion. It also inhibited LPO, reduced Fe(III) and restoredimpaired Mn-SOD in rat liver mitochondria. The kineticsand mechanism of reactions of embelin with hydroxyl, one-electron oxidising, organo-haloperoxyl and thiyl radicalshave been studied using a nanosecond pulse radiolysis tech-nique. Its redox potential has been evaluated with cyclic vol-tammetry. Further, its free radical scavenging activity wasbetter than a-tocopherol. These studies suggest that embelinacts as a competitive antioxidant in physiological condi-tions [75,76]. Anti-tumour activity of such quinonic com-pounds is known to be, in part, due to their redox reactionsand formation of semiquinone radicals [77]. Allensworth et al.[78] identified SOD overexpression and decreased induction ofROS-mediated apoptosis in inflammatory breast cancer (IBC)cells with acquired therapeutic resistance in XIAP overexpres-sion models. XIAP inhibition by embelin (12.5 -- 50 µM) incombination with TNF-related apoptosis-inducing ligand(TRAIL,50 ng/ml) caused a synergistic decrease in cell viabil-ity. Embelin treatment resulted in activation of ERK1/2 andROS accumulation, which correlated with downregulationof antioxidant protein SOD and consumption of redox mod-ulator reduced GSH in XIAP-overexpressed cells. Simulta-neous treatment with a SOD mimic, which protects againstROS accumulation, reversed the decrease in cell viabilitycaused by embelin (50 µM) + TRAIL (50 ng/ml) treatment.Embelin primed IBC cells for TRAIL-mediated apoptosis byits direct action on the anti-caspase activity of XIAP and byshifting the cellular redox balance towards oxidative stress-mediated apoptosis. Thus, ROS modulators, such as embelin,represent a novel approach for enhancing the efficacy ofTRAIL-based treatments in IBC.

Studies have shown novel insights into the molecular net-work of apoptosis resulting from embelin treatment on a panelof tumour cancer cells in vitro. Embelin, which prevents thebinding of XIAP to procaspase-9, significantly decreased epi-dermal growth factor receptor-2 (Erb B2) overexpressing IBCcell line SUM190PT cell viability, revealing the inhibitoryeffect of XIAP on caspase-dependent cell death. However, itfailed to affect cell viability in combination with trastuzumabas embelin has no direct effect on XIAP translation or stability.These data have identified a novel functional link between ErbB2 signalling and the anti-apoptotic pathway mediated byXIAP, which established the feasibility of developing a targetedtherapy to potentiate apoptosis in combination with ErbB2-targeted strategies for IBC therapy. Another advancementwas the discovery that XIAP downregulation inrSUM190 and rSUM149 GW583340 (Lapatinib analogue,Erb B1/2 inhibitor)-resistant IBC cells by embelin(20/50 µM) prevented XIAP--procaspase-9 interaction withdecreased cell viability (40 -- 50%) and increased apoptosis

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(p < 0.005) [79-81]. Embelin sensitised XIAP-overexpressingmalignant human pancreatic cancer AsPC-1 cells (20 µM) inTRAIL-mediated apoptosis [82] as well as glioblastoma U251,LN229 and NCH89 cells (25/50 µM) via inhibition ofcellular-FLICE inhibitory protein (c-FLIP) expression or themitochondrial pathway [83,84], as well as non-small-cell lungcancer NSCLC H460 cells (25 µM) [85]. It also induced apo-ptosis in human myeloid HL-60 leukaemia cells (33.97 µM/lor 30 µg/ml) through microtubular disassembly in the G0/G1

phase of the cell cycle, and anti-mitotic activity [86-89] and inhepatocellular carcinoma HepG2 and Huh 7 cells(10 -- 35 µM or 40 -- 120 µg/ml) [90-92] in a dose- and time-dependent manner. It has been shown that p53-dependentlysosomal destabilisation and cathepsin B activation contrib-uted to the increased sensitivity of p21-deficient colon cancercells to embelin with enhanced caspase-9 and -3 activation.A cathepsin B inhibitor reduced cell death and cytochrome crelease in embelin-treated cells, thereby indicating that thelysosomal pathway is upstream of mitochondrial death signal-ling [93]. Embelin has been reported to elicit a rapid increase ofintracellular free Ca2+ leading to activation of endothelial nitricoxide synthase and nitric oxide-induced cyclic GMP accumu-lation in endothelial cells [94]. Embelin (50 µM) also elicitedsuppression of the STAT3 signalling pathway in human mul-tiple myeloma (U266), prostate carcinoma (DU-145) andhead and neck squamous carcinoma (SCC4) cells via proteintyrosine phosphatase PTEN expression. STAT3 suppressionwas mediated through inhibition of the activation ofJAK2 and c-Src. The results indicated that embelin suppressedconstitutive STAT3 activation and downregulated the expres-sion of cell survival, proliferative and angiogenic gene productsleading to suppression of proliferation and induction of apo-ptosis through caspase-3 activation [95]. Embelin-inducedXIAP suppression resulted in an increase of apoptosis via phos-phorylation of p38 in BRAF V600E-mutant thyroid cancercell lines. Thus, regulation of XIAP activity may be potentiallyuseful for thyroid cancer treatment [96]. A new death pathway,demonstrated by Jehan et al. [97] on the treatment of ovariancancer cells with embelin, promoted autophagy as evidencedby the cell death without caspase-3 activation, induced cellularswelling and cytoplasmic vacuolisation with necrotic celldeath. Autophagic hallmarks, increased autophagosomes,punctuated distribution of GFP-LC3, LC3 cleavage andbeclin1 activation depicted the role of embelin as a new che-motherapeutic agent for ovarian cancer treatment. Recent evi-dence [98] revealed that embelin significantly inhibitedmultiple signalling cascades in the Akt/mTOR/S6K1 pathwayalong with downregulation of anti-apoptotic (Bcl-2, Bcl-xL,Survivin, IAP-1 and IAP-2) and proliferative (cyclin D1)proteins, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase in androgen-independent PC-3 prostatecancer cells.

Being a multifaceted drug, embelin not only induces apo-ptosis by inhibiting XIAP but it also potently blocks NF-kBsignalling pathways, thereby leading to downregulation of a

variety of gene products involved in tumour cell survival,proliferation, carcinogenesis, invasion, angiogenesis and infla-mmation, making this agent potentially useful for cancer ther-apy. Embelin (50 µM) inhibited the expression of COX-2,MMP-9, cyclin D1, VEGF and ICAM-1. It sequentiallyinhibited the TNF-a-induced activation of the inhibitorysubunit of NF-kBa (IkBa) kinase, IkBa phosphorylation,IkBa degradation, p65 phosphorylation and nuclear translo-cation. Embelin also suppressed the NF-kB-dependentreporter gene transcription induced by TNF-a, TNFreceptor-1 (TNFR1), TNFR1-associated death domain pro-tein, TNFR-associated factor-2, NF-kB-inducing kinase andIkBa kinase, but not that induced by p65. This downregula-tion was associated with enhanced apoptosis from 2 to 58%by cytokines and chemotherapeutic agents [99,100].

Embelin (25, 50, 100 and 200 mg/kg orally for 20 days)elicited potent anti-proliferative activity and prolonged the life-span of methylcholantherene-induced fibrosarcoma-treated ratswith significant tumour regression [26]. XIAP inhibition withembelin triggered caspase activation, implicating XIAP incaspase blockade in pancreatitis. Embelin (20 mg/kg/day s.c.)for 5 days induced cleavage of receptor-interacting protein(RIP) through activation of caspases-9, -3, -8, stimulatedapoptosis ~ 3-fold and led to a significant decrease in necrosiswith normalisation of pancreatic histoarchitecture in mousecerulein-induced pancreatitis, representing a therapeutic strat-egy for pancreatitis treatment [101]. Moreover, it exhibited che-mopreventive activity against carcinogens NDEA-initiated(200 mg/kg single i.p.) and PB-promoted (0.05% in drinkingwater orally for 13 weeks) two-step hepatocarcinogenesis (pre-neoplastic foci/hyperplastic nodules stage) in Wistar rats.Embelin treatment (50 mg/kg, oral dose) daily for 14 weeksprevented induction of neoplastic nodules, increased levels ofhepatic diagnostic markers and LPO, caused hypoproteinemiaand hyperlipidaemia, decreased hepatic glutathione antioxidantdefence and minimised the histological alterations (dysplasiaand atypical cells with abnormal chromatin pattern) inducedby NDEA/PB [102,103]. In a similar approach, rats receivedNDEA (1 ppm/g body weight in drinking water) for 6 weeksin a liver preneoplasia model and received embelin (50 and100 mg/kg) orally prior, during and after exposure to NDEAfor 20 weeks. Embelin treatment significantly preventedNDEA-induced increase in glutamate pyruvate transaminase,glutamate oxaloacetate transaminase, alkaline phosphatase,g-glutamyl transpeptidase, GST and LPO as well as causinghypoproteinemia, hypoalbuminuria and GSH depletion. Thiswas further substantiated by a marked decrease in the incidenceof preneoplastic foci and inflammatory cells on histopatholog-ical and transmission electron microscopic analysis. Embelinproved remarkable in suppressing and/or arresting the degener-ative changes brought about by NDEA, thus suggesting thatembelin is a promising chemopreventive agent [53]. Dai et al.[104] reported that embelin (30 µM) significantly inhibitedcell proliferation and induced apoptosis in colon cancerHCT116 cells. 1,2-dimethylhydrazine dihydrochloride

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(DMH, 20 mg/kg/week s.c.) administration for 15 weeksinduced colon cancer in PPARg+/+ and PPARg+/- mice. Micefed with embelin daily (100 mg/kg) for 10 days before DMHinjection and continued for 30 more weeks suppressed coloncarcinogenesis in PPARg ligands in mice. Reduced expressionof PPARg significantly sensitised colonic tissues to the carcino-genic effects of DMH. PPARg activation inhibited NF-kBactivity. Embelin suppressed the NF-kB target gene expressionsnamely cyclin D1, survivin, Cox-2 and c-Myc with a moreefficient anti-cancer effect in PPARg+/+ mice.Embelin (20 µM) in combination with ionising radiation

(IR, 4 Gy) potently suppressed prostate cancer PC-3 cell pro-liferation (72%) and triggered caspase-independent apoptosis(14%) when compared with either treatment alone. An oraldose of embelin (60 mg/kg on days 1 to 5/week for 3 weeks)significantly improved tumour response to X-ray radiation(2 Gy fraction on days 1 to 5/week for 2 weeks) in aPC-3 xenograft model. The tumour size in the combinationgroup was 60% of that in the IR alone group and 18% ofthat in the control group. The combination group also pro-longed the time to progression by ~ 5- to 6-folds more thaneither treatment alone with minimal systemic toxicity, sug-gesting a significant combinatorial inhibition by radiationtherapy on tumour suppression and angiogenesis. This find-ing warrants embelin as a novel adjuvant therapeutic candi-date for hormone refractory prostate cancer treatmentresistant to radiation therapy [105,106]. In a co-clinicalapproach, embelin sensitised castration-resistant prostate can-cers (CRPCs) to androgen deprivation therapy [107]. Inhuman prostate cancer cells, dutasteride (10 µM) and/orMDV3100 (10 µM) and/or embelin (5 µM) treatment for2 or 4 days caused a marked increase in apoptotic response.In genetically induced CRPC mouse models, dual combina-tion of bicalutamide (Casodex, 10 mg/kg) and embelin(60 mg/kg) orally for 5 days/week for 4 weeks or triple com-bination treatments with bicalutamide (10 mg/kg), embelin(60 mg/kg) orally for 3 days/week, bicalutamide (10 mg/kg)and dutasteride (2 mg/kg) orally for 2 days/week for 4 weeksled to a significant co-clinical therapeutic approach which wasbeneficial to CRPC patients genetically stratified by XAF1,XIAP and SRD5A1. In another study, pre-treatment of embe-lin (10 and 20 µg/ml) for 60 min inhibited ultraviolet B(290 -- 320 nm for 30 min) radiation-induced oxidative dam-age and DNA damage in peripheral blood human lympho-cytes due to its antioxidant property [108]. The above reportsestablished the potent antioxidant and anti-tumour potentialsof embelin.

3.7 NanomedicinesTransforming traditional drugs such as embelin into designerdrugs using the nanotechnological approach is currently verydemanding. Poor oral bioavailability due to the low aqueoussolubility of embelin leads to poor pharmacokinetic-pharmacodynamic profiles which in turn restricts its use inthe treatment of human diseases with low therapeutic

potency. To counteract this drawback, Lu et al. [109] haverecently developed a formulation based on a hydrophilic poly-mer, polyethylene glycol 5000 (PEG5K)--embelin conjugate,which self-assembled to form stable micelles in aqueous solu-tion and efficiently encapsulated the hydrophobic drug, pacli-taxel (PTX). This novel micelle system (20 -- 30 nm) hadsustained release kinetics over 5 days and exhibits potent cyto-toxicity in DU145 and PC-3 androgen-independent humanprostate cancer and 4T1.2 mouse metastatic breast cancercells. Total body near-infrared fluorescence imaging depictedthat PEG5K--embelin micelles selectively accumulated at atumour site with minimal uptake in major organs, includingthe liver and spleen, and also depicted a safety profile witha maximum tolerated dose of 100 -- 120 mg PTX/kg singleintravenous (i.v.) administration in mice which wassignificantly higher than taxol (15 -- 20 mg PTX/kg). Potentanti-tumour activity at 10 and 20 mg PTX/kg micellar formu-lation i.v. injection in xenograft models of breast (treatmentdays 1, 4, 7, 10 and 13) and prostate cancers (treatmentdays 1, 3, 7, 10, 13, 24 and 28) were demonstrated over taxolformulation. Interestingly, earlier a similar version of thehighly efficient micelle system (20 -- 30 nm) PEG3500--Embelin conjugate exhibited synergism with PTXwith potent anti-tumour activity, representing a dual func-tional delivery system [110]. Poly (ethylene glycol)-b-poly(carbonate-co-lactide) micelles (83 -- 90 nm) prepared by afilm sonication method at 5% drug loading led to encapsula-tion efficiency for CBDIV17 at 91.2% and embelin at 38.4%.A combination of CBDIV17 (25 µM) and embelin (5 µM)micelle formulation was more potent than their monotherapyin prostate cancer (C4-2) treatment with a 20% decrease incell growth, while combination therapy inhibited about70% with superior cell migration inhibition. In a C4-2 xeno-graft tumour model, intratumoral injection of a combinationof 10 mg/kg CBDIV17 and 10 mg/kg embelin-loadedmicelles administered on days 0, 3 and 7 inhibited tumourgrowth more potently and increased tumour-doubling timein comparison to mice treated with void micelles or mono-therapy. On day 7, tumour size in the combination groupwas 50% of that in the CBDIV17 group, 25% of that inthe embelin group and 18% of that in the control groupwith minimal toxicity and therefore appears to be a promisingtherapeutic approach for hormone refractory prostate cancertreatment [70]. In another study, an embelin--phospholipid(Phospholipon 90H, a soya phosphatidylcholine) complex(0.05 -- 0.5 µM) formulated by a mechanical dispersionmethod exhibited embelin 92.44% (w/w) content withimproved water solubility of 3 -- 42 µg/ml in the complex.Free embelin showed only 19% drug release, whereas thecomplex showed 99.80% release at 120 min of dissolutionin distilled water [111]. Li et al. [112] demonstrated thatembelin-loaded poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol) (PEG-PCD) lipopolymeric micelles showed significant inhibition(IC50 10 µM) of C4-2 prostate cancer cell proliferation.

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PEG-PCD lipopolymers with various hydrophobic corelengths showed similar drug release profiles. Poly(ethylene)-b-poly(D,L-lactide) polymeric micelles (30 -- 50 nm) fabri-cated using a film sonication method to solubilise the hydro-phobic drugs, bicalutamide and embelin, led to 60-foldincrease in drug aqueous solubility. Drug-loading capacityincreased up to 20% for embelin and 10% for bicalutamide.In LNCaP xenograft mouse models, sequential exposure tobicalutamide- loaded micelles in intratumoral injection(20 mg/kg) three times a week up to 20 days followed byembelin-loaded micelles from day 28 resulted in regressionof prostate cancer tumours [113]. Thus, micellar delivery ofanti-androgen and XIAP inhibitors has immense potential inthe treatment of advanced prostate cancer.

Natural polymers are promising candidates for functionalmaterials, such as quinones from natural products, and havea number of significant advantages. A biodegradable, biocom-patible polymer matrix of poly("-caprolactone) (PCL) micro-fibre meshes containing embelin were obtained byelectrospinning [114]. Thermal properties revealed that thecrystallinity of embelin was significantly decreased and thedrug almost completely dissolved in the PCL fibres. Animportant aspect to consider in topical drug applicationswas drug-loaded fibrous scaffolds which exhibited an 86%increase in the area-to-volume ratio and provided an effectivearea per unit mass which was 5.8-fold higher than in drug-loaded films. For the meshes, 90% embelin release occurredafter 12 h of PBS exposure, whereas, for the films, a compara-ble level of release occurred only after 72 h. This polymericcarrier would allow a better in situ bioavailability of embelin.The above evidence signifies that nano-embelin is a novel,effective drug delivery system.

4. Clinical efficacy

The drug consists of dried ripe fruits of vidanga which givesprotection against several diseases. Vidanga has gained partic-ular importance due to the proven efficacy in preclinical set-tings and clinical trials investigating its contraceptive andantihelminthic potential. Not many randomised, controlledand double-blind clinical trials are available. In ayurvedic sys-tems of medicine, it forms a chief ingredient found in thecomposition of a number of polyherbal formulations availableon the market such Livomyn, Liv-52, Sanjivani vati, Abana,Koflet, Gasex, Geriforte, Mahamanjisthadi Kwatha, Krumiva-kar Kwath, Livosin, and the like (Table 1) [3,115,116]. Efforts toenhance embelin’s bioavailability in humans are needed inorder to take embelin to the next phase, from the benchto bedside.

5. Safety and tolerability (toxicology)

The LD50 value of embelin from E. ribes was 44 mg/kg byi.p. route. Embelin in doses of 10 mg to 3 g/kg given orallyto rats and mice did not show any toxic effects. Subacute

toxicity on 10 weeks administration of 10 mg/kg of embelinto rats also indicated the drug to be free from toxic effectson heart, liver, kidney and bone marrow, thereby having ahigh margin of safety in acute toxicity studies [39]. The rec-ommended doses in official Ayurvedic Pharmacopoeia andIndian Herbal Pharmacopoeia are 5 -- 10 g of fruit pow-der [117,118] which are considered practically safe withoutside effects. New semi-synthetic derivatives of embelin,such as diamines and bisquinones, were developed of whichembelin and its disalt, 2:5-isobutylamine embelin, exhibitedantipyretic, anti-inflammatory, analgesic, tranquilising andhypotensive activities in varying degrees. These compoundswere effective at 5 -- 10 mg/kg i.p. doses in mice, rats, dogs,cats and rabbits with some side effects [4]. A short-term tox-icity of 6 weeks of embelin 120 mg/kg oral administrationwas observed in female rats including an increase in acidand alkaline phosphatase activities in kidney and adrenalcells, disintegration, necrotic changes, perinuclear vacuola-tion in the liver and kidney, kidney tubular damage andadrenal hypertrophy which were recouped after embelin dis-continuation [119]. Acute toxicity studies in mice treatedwith embelin 50 and 100 mg/kg oral dose showed no signif-icant body weight change, mortality or apparent toxic effects,signifying its safety profile [53].

6. Pharmacokinetics and metabolism

The pharmacokinetic studies of potassium embelate (20 mg/kgoral and i.v.) in rats revealed a bi-exponential kinetic pattern.Absorption was complete (bioavailability 97%) and fast withdrug peak plasma at 0.28 h (9 µg/ml). The disposition half-life was 9.5 h on i.v. and 11 h on oral administration. Highdrug concentrations (8.1 µg/g wet tissue) were found in thebrain between 0.25 h and 2 h in comparison to the liver, heart,lung and spleen and slowly decreased. The kidney played amajor role in drug excretion with 60.9% of the dose found inurine after 24 h [25]. Pharmacokinetics of embelin after oral dos-ing (75 mg/kg/day) for 30 days in rats depicted the highest lev-els in the kidney (249 mg/g), followed by the testis (257 mg/g)and intestine (232mg/g). Significant levels were observed in thebrain (163 mg/g), heart (196 mg/g) and spleen (176 mg/g).Embelin levels in all of the organs were higher than after15 days treatment, with mainly the prostrate, heart, kidney,liver and intestine showing tissue accumulation. However, theselevels slowly declined in all of the organs after 30 days of embe-lin treatment, indicating slow elimination from the body. T1/2

was 21.86 h; embelin on s.c. dosing for 30 days depicted thehighest concentration in the heart (204 mg/g), followed bythe testis (200 mg/g), epididymis (176 mg/g), kidney(159 mg/g) and brain (147 mg/g). Low levels were observedin the spleen (139 mg/g), seminal vesicles (110 mg/g), liver(106 mg/g), intestine (90 mg/g), prostrate (86 mg/g) and lung(82 mg/g). About 30 days after embelin withdrawal,20 -- 75 mg/g levels were observed with T1/2 of 16.5 h [120].

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Table 1. Polyherbal vidanga formulations in market and its uses.

Formulations Chief ingredients Indication/uses Dosage

Vidangadi churna Vidanga, yastimadhu, trikatu,dantamoola, trivrit, chitraka

Antihelminthic, purgative, alternative,flatulence, dyspepsia and tapeworms

3 -- 5 g

Kuberakshadi yoga Putikaranja, vidanga, ajwain, jatiphala,lavangaha, jeeraka

Antihelminthic 200 mg

Vidangadi yoga Vidanga, japapushpa, hingu Anti-fertility 100 mgEladi grita Ela, ajmoda, harad, baheda, amla,

saurastri, shunthi, kalimirch, pippali,arishtak, khadir, sala-sara, bijakasara,bhallataka-suddha, vidanga, ghrta,tavaksiri, sita, madhu, dalchini, ela,chitrak, tejpatra, water

Tuberculosis, anal fistula, dyspepsiaand eye diseases

12 g

Pippalyadi yoga Vidanga, pippali, borax Contraceptive 1 gMadhusnuhi rasayana Shunthi, kalimirch, pippali, harad,

baheda, amla, dalchini, ela, tejpatra,jatiphal, agni, laung, dhaniya, saariva,krsna jirak, vidanga, chavya, kusth,trivtita, pippali, ashwagandha, bhangri,tejovati-bija, kesara, suddha gandha,mahisaksa guggul, ghrita, sita, madhu

Diabetic carbuncle, tumour, goitre,anal fissure, gout, leprosy, piles anditching

12 g

Vidangsaravaleha Vidanga, pippali, triphala Antihelminthic, fistula, sinus, urinarydiseases, ulcers, leprosy and skindiseases

Not available

Sunder vati Vidanga, kutaj, amla, shunti Skin diseases 180 mgKrmimudgar rasa Rasa-suddha, gandhaka-suddha,

ajmoda, vidanga, visamustika-suddha,palaash

Worm infestations and dyspepsia 250 -- 500 mg

Vidangarishta Vidanga, pippali, rasna, kutaj, dalchini,kutaj phala, patha, elavalukamka,amla, ksadura, dhatki, dalchini, ela,tejpatra, priyangu, kanchnaar, lodhra,shunthi, kalimirch, pippali, water

Goitre, anal fissure and cystitis 12 -- 24 ml

Vidangadi lauh Vidanga, clove, borax, jatiphala,lavangaka, shunthi, salt, nutmeg, longpepper, black pepper, lauha bhasma

Vermifuge, dyspepsia, anorexia, piles,anaemia, swelling, fever, asthma andcough

500 mg

Madhukasava Madhuka, vidanga, chitrak, bhallatak,manjishtha, madhu, ela, mrnala,agaru, chandan, water

Bile disorders, leprosy, leucoderma,blood disorders, dyspepsia andemaciation

12 -- 24 ml

Abhyarista Harad, draksha, vidanga, madukabhasma, guda, gokshurtrivrta, dhaniya,dhatki, indravaruni, chavya, danti,madhurika, shunthi, mocarasa, water

Piles, urine retention and dyspepsia 12 -- 24 ml

Kumariasava Kumari rasa, guda, makshika, parvaloha, shunthi, kalimirch, paippali,laung, dalchini, ela, tejpatra,nagkeshar, chitrak, pippalamool,vidanga, chavya, gajpippali, hapusha,dhaniya, poog, katuka, mustak, harad,baheda, amla, rasna, devdaru, haldi,daruharidra, moorva, madhurasa,danti, dhatki, pushkarmool, bala,atibala, kapikachu, gokshur,shatpushpa, hingpatri, akaarkrabh,utingana, svetapunarnava, punarnava,lodhra, dhatumakshika

Dyspepsia, duodenal ulcer,regurgitation, dysuria, stones, epistaxis,seminal disorders, dementia, debility,emaciation, weakness, anorexia andliver disorders

12 -- 24 ml

Vidangasava Vidanga, pippalimula, rasna, kutaja,indrayava, paripatha, amalaki, shunthi,pippali, tamalpatra, bruhatela, tvaktaila

Round worms, thread worms andhook worms

2 -- 4 tsp.

R. Poojari

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7. Regulatory affairs

A number of ayurvedic products are available on the marketcontaining vidanga as a constituent. A critical issue is thatayurvedic medicinal market is poorly regulated by acquiringillegal licenses and over-the-counter sales. Therefore, this leadsto adulteration or substitution of black pepper mixed as spice,allied Embelia sps. or Myrsine africana fruits due to their closeresemblance. Hence, to standardise the content, check the sta-bility and batch-to-batch variation in consistency as well aspreserve its potency, it is necessary to implement stringentquality control standards such as embelin markers andmandatory registration policies for these formulations.

8. Conclusion

Overall, this review provides updated developments of anunder-explored source, embelin, an antique drug known forits vermifuge and contraceptive properties in ayurvedic medi-cine, moving towards an anti-cancer molecule and nanomedi-cine. The current approach tends to focus on the mechanisticmolecular levels of embelin and the issue of poor bioavailabil-ity is challenging. Several approaches, as discussed in thisreview, would resolve the unanswered questions. A plethoraof preclinical trials of embelin provides a rationale for earlyclinical trials for various diseases. Safety and effectivenessneed to be firmly established before such clinical practicescan be endorsed. Embelin acts as a sensitiser and can beused as an adjuvant to current conventional drug treatmentsfor enhanced potency with minimal toxicity. The new waveof multidisciplinary approaches from diverse disciplines,such as chemistry, systems biology, pharmacology, toxicology,pharmaco-genomics, nanoengineering, absorption-drugmetabolism-excretion dynamics, bioinformatics, quality con-trol and patients clinical data would have striking effect onthis investigational, naturally occurring small quinonicmolecule, embelin, and help develop it as powerful chemo-therapeutic for the prevention and treatment of diversemalignancies.

9. Expert opinion

Embelin possesses enormous potential for research and theexploitation of new drugs is impressive. Literature since1934 pertaining to the Embelia species showed investigationsmainly regarding its medicinal properties (crude extracts) andayurvedic formulations. Few attempts had been made toinvestigate the major active constituent, embelin, isolatedfrom the fruits of this plant as a source of drug. Hence, theinterest in the development of newer embelin derivatives,embelin as phytochemical marker for quality control, itsbioprospection as an analgesic, anti-inflammatory, contracep-tive, anti-infective, antioxidant, anti-diabetic, gastro-hepato-protective, neuroprotective, radiation-protective and cancer

chemopreventive therapeutic agents are justified. Moreover,prediction of possible embelin-induced toxic effects could beelucidated by using advanced toxicogenomic analyses ratherthan the conventional techniques. Molecular docking ofembelin for diabetes prevention and management is animportant aspect for designing new targets. Embelin boundto PPARg disclosed stable binding affinities to the active sitesof PI3K, p-Akt and GLUT4. Embelin could improve adiposetissue insulin sensitivity, enhance glycaemic control, protectb-cells from damage and maintain glucose homeostasis. Stud-ies at the miRNA level would immensely improve under-standing of the potential mechanisms of action by whichembelin acts against cancer or neurodegenerative disorders.A crucial finding was that miR-23a inhibition reduces caspaseactivation by enhancing XIAP levels and decreasing cell death.Therefore, development of miRNA-targeted therapies shouldkeep in mind the potential for differential sex regulation.

Embelin, the natural small molecular inhibitor of XIAP, isa lead molecular target for designing new anti-cancer drugsto promote apoptosis in cancer cells. It is an NF-kB blockerand potential suppressor of tumorigenesis, including acuteleukaemia. Embelin-promoted cell death is more effectivein cancer cells with higher levels of XIAP which implicatesembelin therapy in addressing clinical drug-resistant cancers.Embelin inhibits the caspase-inhibitory activity of XIAP bybinding to its BIR3 region and disrupting caspase 9 andSmac binding to XIAP, indicating an anti-apoptotic activityof XIAP in controlling drug-resistant cancer cells. Theembelin + TRAIL-mediated apoptosis pathway is a promis-ing target for anti-cancer therapy. The role of embelin in avariety of tumour cells through the inhibition of IkBakinase, XIAP, c-FLIP expression, activation of PPARg ,microtubular disassembly, induction of lysosomal destabilisa-tion and antioxidant activity have been discussed above. Theeffect of embelin on STAT3 activation, Erb B2 and Akt/mTOR/S6K1 established new mechanistic signalling cas-cades in cancer cells. It also represents a promising adjuvantintervention for enhanced radiotherapy treatment inradiation-resistant hormone refractory prostate cancers. Bear-ing in mind the co-clinical approach of embelin for CRPCs,it is a significant stride for genetically tailored new anti-cancer therapeutics. Although evidence of embelin as aneffective cancer chemopreventive agent against chemicallyinduced cancers in experimental rodents with no side effectshas been established, further research is warranted toelucidate the specific mechanisms involved.

Embelin is water insoluble; PEG, polymeric and lipidmodalities were explored to increase its solubility andenhance its anti-tumour efficacy. Among the new materials,polymers with quinone functionality and biodegradability,such as microfibre meshes containing embelin in PCL matrixwith high bioavailability, are unique. More studies onstructure--activity relationships can be designed using bioin-formatics tools for improved new nano-embelin deliverysystems.

Embelin

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Significant strides have been made in understanding themolecular aspects of embelin, but the clinical progress remainsslow. Few clinical trials of vidanga fruits or multi-componentayurvedic formulations on skin diseases, antihelminthic,gastrointestinal troubles and anti-fertility perspectives areavailable. More in-depth preclinical studies of embelin arerequired. Hence, well-designed clinical trials of embelin forvarious therapeutic applications and its marketability exploita-tion remain warranted. Truly, unravelling the journey of thisancient medicine, embelin, from a potent antihelminthicagent to a novel anti-cancer molecule and nano-embelin inparticular is remarkable.

Acknowledgement

The author is thankful to A Varma from Advanced Centre forTreatment, Research and Education in Cancer (ACTREC),Navi Mumbai, India for critically reviewing the manuscriptand his helpful suggestions.

Declaration of interest

The author states no conflict of interest and has received nopayment in preparation of this manuscript.

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AffiliationRadhika Poojari

Indian Institute of Technology Bombay,

Department of Biosciences and Bioengineering,

Mumbai -- 400076, India

E-mail: drradhikapoojari@gmail.com

R. Poojari

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