Piper Betel Leaf: a Reservoir of Potential Xenohormetic Nutraceuticals with Cancer-Fighting Properties

Home , Chavibetol, Phenylpropene, Piper (plant)

Published OnlineFirst January 21, 2014; DOI: 10.1158/1940-6207.CAPR-13-0355

Cancer Prevention Review Research

Piper Betel Leaf: A Reservoir of Potential Xenohormetic Nutraceuticals with Cancer-Fighting Properties

Sushma R. Gundala and Ritu Aneja

Abstract Plants contain a much greater diversity of bioactive compounds than any man-made chemical library. Heart-shaped Piper betel leaves are magnificent reservoirs of phenolic compounds with antiproliferative, antimutagenic, antibacterial, and antioxidant properties. Widely consumed in South Asian countries, the glossy leaf contains a multitude of biophenolics such as hydroxychavicol, eugenol, chavibetol, and piperols. Convincing data underscore the remarkable chemotherapeutic and chemopreventive potential of betel leaves against a variety of cancer types. The leaf constituents modulate an extensive array of signaling molecules such as transcription factors as well as reactive oxygen species (ROS) to control multiple nodes of various cellular proliferation and death pathways. Herein, we provide an overall perspective on the cancer- fighting benefits of the phenolic phytochemicals in betel leaves and a comprehensive overview of the mechanisms responsive to dose-driven ROS-mediated signaling cascades conscripted by bioactive phenolics to confer chemotherapeutic and chemopreventive advantages. Intriguingly, these ROS-triggered responses are contextual and may either elicit a protective xenohormetic antioxidant response to premalignant cells to constitute a chemopreventive effect or generate a curative chemotherapeutic response by pro-oxidatively augmenting the constitutively elevated ROS levels in cancer cells to tip the balance in favor of selective apoptosis induction in cancer cells while sparing normal ones. In conclusion, this review provides an update on how distinct ROS levels exist in normal versus cancer cells and how these levels can be strategically modulated and exploited for therapeutic gains. We emphasize the yet untapped potential of the evergreen vine, betel leaf, for chemopreventive and chemotherapeutic management of cancer. Cancer Prev Res; 7(5); 477–86. 2014 AACR.

Introduction noids, isothiocyantes, sulfides, thiols, phenols, and alka- Mankind has relied on plants as a source of medicine for loids in plant extracts have helped cure and/or manage eons. Unsurprisingly, plants contain a much greater diver- several chronic conditions like diabetes, hepatitis, arthritis, sity of bioactive compounds than any known man-made cardiovascular, cerebrovascular diseases, and cancer. Can- chemical library. Even today, as much as 80% of the cer, a malicious disease that gripped mankind since its dawn population in developing countries banks on medicinal in early in fourth century BC (2), has been an alarmingly plants as their only affordable source of medication (1). No increasing cause of death worldwide over the past 20 years. wonder nature has been a matchless source of multicom- Continuing research efforts to identify a cure for cancer have ponent concoctions and single compounds ranging from met with limited success, and the war against this deadly simple phenolics to complex alkaloids that have been neoplasm is growing stronger by the day. Most pharmaceu- developed over the millennia to modulate various cellular tical compositions of various purified compounds and mechanisms for therapeutic gains. The myriad benefits of crude chemopreventive and chemotherapeutic formula- these natural products, which actually emerged because of tions today are from the inexhaustible chemical inventory the interactions among plants and their surrounding envi- of plants. ronment to enhance survival, are prodigious and well appreciated in every aspect of life. Furthermore, the wide Current chemotherapy: one size does not fit all variety of compounds like vitamins, carotenoids, flavo- The greatest impact of plant-derived drugs has been in the anticancer area, wherein the development of plant-derived drugs such as taxol, vinblastine, vincristine, and camptothe-

Authors' Afﬁliation: Department of Biology, Georgia State University, cin has proven to be a boon in the treatment of some of the Atlanta, Georgia deadliest cancers. This perhaps fueled the notion that single Corresponding Author: Ritu Aneja, Department of Biology, Georgia State constituents of medicinal plants dictate their pharmacolog- University, Atlanta, GA 30303. Phone: 404-413-5417; Fax: 404-413-5300; ic activity. Although this paradigm has resulted in several E-mail: [email protected] outstanding synthetic drug molecules and many more doi: 10.1158/1940-6207.CAPR-13-0355 entering the pipeline of clinical trials, it is rather dismaying 2014 American Association for Cancer Research. that about 60% of drug candidates either fail late-stage

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clinical development or shortly after entering the market Asian countries such as Sri Lanka, Indonesia, Nepal, Paki- due to debilitating toxicity, limited efficacy, or emergence of stan, Malaysia, Thailand, and Singapore (12). No wonder drug resistance (3). One obvious explanation for these these leaves are recognized as "Green Gold" due to their failures is that disease pathogenesis involves dysregulation high demand in the South Asian countries (13). These betel of multiple molecular pathways and thus the "silver bullet" leaves are widely cultivated in most parts of South and approach of monotargeting yields limited efficacy. Paradox- Southeast Asia, including India, Bangladesh, Pakistan, Sri ically, the multifactorial nature of cancer requires a pleio- Lanka, Nepal, Burma, Vietnam, Papua New Guinea, Thai- tropic approach wherein a whole plant extract, a multicom- land, Taiwan, and Malaysia. The cultivation of these leaves ponent concoction, or a fractionated mixture of phyto- is mostly limited to areas with moderate climatic conditions chemicals or even a single agent can simultaneously hit accompanied by high humidity. The harvested leaves are multiple targets in consecutive or parallel pathways to also exported to other parts of Asia, Middle East, Europe, achieve optimal clinical efficacy. and the United States. Given that it is a cheap and readily accessible natural Plant extracts: phytochemical potions with miraculous product, various parts of the Piper betel plant especially betel medicinal powers leaves, are used in traditional medicine for treatment of Owing to the unmistakable promise of herbal medica- several conditions such as abscesses, constipation, conjunc- tions, there is an ever-increasing quest for new plants that tivitis, itches, rheumatism, abrasions, and many more (14, could possibly hold the key to prevent or eliminate this 15). Although the roots of Piper betel have served as alter- dreaded disease by conferring superior chemopreventive or native oral contraceptives, the oil obtained from betel leaves chemotherapeutic properties. The secondary metabolites has bactericidal action (13). Consumption of betel leaves produced by plants, referred to, as phytochemicals are often has also been known to stimulate feelings of well-being, plant stress signaling molecules, which have been identified amplified salivation, perspiration, alertness and a sense of to resist stress, fight disease, and improve longevity in increased energy, and warmth (13). A meticulous evalua- animals consuming them, a phenomenon known as xeno- tion of the leaf as a whole and its constituents has yielded hormesis (4–7). Indeed the phytochemicals present in fruits many more medicinal properties such as antifungal, anti- and vegetables (e.g., carotenoids, polyphenolics, anthocya- microbial, wound healing, antioxidant, antimutagenic, and nins, terpenes, alkaloids) are functionally pleiotropic; they chemopreventive activities (refs. 12, 16; Fig. 1). possess multiple intracellular targets, affecting different cell Here below, we attempt to synopsize existing knowledge signaling cascades usually altered in cancer cells with lim- on the cancer-fighting properties of betel leaf and its phy- ited toxicity to normal cells. However, the host of disease- tochemical constituents, and present an overview of cellular combating chemicals is not limited to fruits and vegetables, nuances driven by phenolic phytochemicals that trigger but is widely spread across the major ingredients of our reactive oxygen species (ROS)-mediated signaling circuit- cuisines, spices, and herbs. The nutraceuticals derived from ries to launch a two-pronged offensive; one that underlies spices and herbs have also been shown to modulate mul- chemopreventative benefits produced by a potential xeno- tiple targets, including the transcription factors (NF-kB, hormetic response which fortifies the antioxidant defense Nrf2, hypoxia-inducible factor-1a), kinases, and inflamma- system in premalignant cells, and the other that undergirds tion markers (8). However, there still exist several unex- chemotherapeutic efficacy due to the induction of apoptosis plored exotic condiments with promising disease-fighting by overwhelming the cellular ROS content. potential. Betel leaves: abundant repositories of cancer-fighting Some plants have it all: the versatile pharmacopoeia of phytochemicals Piper betel leaves An enriched source of calcium, vitamin C, niacin, thia- Although known from ancient times, scientific interest in mine, carotene, and riboflavin (17), betel leaves are clearly Piper betel, an evergreen vine, was restricted owing to mis- associated with nutritive benefits. Thus, referring to them as conceptions largely due to the consumption of betel quid, a betel nutraceuticals is supported and well reasoned. Other combination of betel leaf with areca nut, slaked lime, and betel phytochemicals include allylpyrocatechol (APC; 2- tobacco, which is a storehouse of several carcinogens and hydroxychavicol), 4-hydroxycatechol, b-caryophyllene, has been linked to the development of oral cancer (9–11). methyl eugenol, carotenes, starch, diastases, and an essen- Recent years have seen a rekindled interest in pursuing this tial oil containing hydroxychavicol (18, 19). Hydroxycha- member of the natural medicine chest, and several labora- vicol, a phenolic compound quantitatively present at tories, including ours, have begun to examine the medicinal approximately 26% in betel leaves, has been shown to exert powers of this magnificent plant using a rationale-driven antiproliferative activity in prostate cancer (20). Hydroxy- scientific approach. A perennial creeper of the Magnolio- chavicol has also been shown to impede cell-cycle progres- phyta family, the Piper betel plant with glossy and heart- sion of prostate cancer and oral KB carcinoma cells (20–22). shaped leaves flourishes in warm and humid environments Several reports indicate hydroxychavicol as an antimuta- and can grow up to one meter in length (12). Although the genic agent (12, 23) as well as an effective inhibitor of plant originally hails from India, over the past several cyclooxygenase (COX), platelet calcium signaling, and decades, it has gained popularity amongst other South thromboxane B2 production (21). Other studies suggest

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Figure 1. Betel leaf nutraceuticals target multiple cellular events and play a crucial role in inhibiting tumor growth, inflammation by decreasing the levels of cyclooxygenases, COX-1 and COX-2 (21) and stress-inducing events like lipid peroxidation. Furthermore, they activate detoxifying enzymes and upregulate the expression of ABCA1 (ATP-binding cassette transporter) and Liver X receptors, while inhibiting LDL oxidation (62), increase free radical scavenging, and induce apoptosis signified by increased levels of cleaved caspase-3 and PARP levels (20, 63). The betel leaf constituents kill cancer cells by inducing oxidative DNA damage demonstrating their pro-oxidant role (29). PARP, poly (ADP-ribose) polymerase. that hydroxychavicol also known as APC possesses anti- via the urine (26, 27). Chlorogenic acid (ChA), another ulcerogenic activity (24) and has been shown to alleviate active ingredient isolated from betel leaves, has been indomethacin-induced stomach ulceration leading to gas- reported to eliminate cancerous cells without harming tric cancer (25). Hydroxychavicol also inhibits inflamma- normal cells, unlike most conventional chemotherapeutics tory response molecules like inducible nitric oxide synthase (13). In addition to cytotoxic and antimutagenic properties, and COX-2, which are known to enhance tumor growth by the betel bioactive constituents have been studied for their downregulation of the NF-kB) pathway (24). Chavibetol oxidative properties (ref. 28; Fig. 1). For example, high (CHV), along with hydroxychavicol, acts as a radioprotec- hydroxychavicol concentrations provoke its pro-oxidant tant (25), and exhibits substantial immunomodulatory and behavior as demonstrated by induction of oxidative damage free radical scavenging activities (25). We have shown that in liver cancer cells (29). Interestingly, hydroxychavicol was CHV synergizes with hydroxychavicol to exert antiprolifera- found to be a potent antioxidant at lower doses in oral KB tive activity against human prostate cancer PC-3 cells (20). carcinoma, whereas high doses led to apoptosis induction Betel leaf contains large amounts of safrole, which is rapidly by increasing ROS levels (22). degraded in the human body into dihydroxychavicol and Thus, similar to other plant-derived phenolic phyto- eugenol, the antimutagenic agents, which are then excreted chemicals, bioactive betel constituents as single agents exert

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paradoxical anti- and pro-oxidant activities contextually. The phenol groups in plants offer remarkable molecular Given that plant extracts are invariably multicomponent, flexibility resulting in an apparent dichotomy associated their activities can perhaps be more representative of an with the versatile phenolic function. Most betel constituents averaged "profile" of anti- and pro-oxidant behavior, which are composed of an amphiphilic moiety with the hydro- may be an additive summation or an even more complex phobic nature of its planar aromatic nucleus coupled with synergistic interaction among the individual components. the hydrophilic character of its polar hydroxyl group, which There are a lot of conflicting reports on ROS-quenching can act as a hydrogen-bond donor or as an acceptor. The antioxidant mechanisms and ROS-generating pro-oxidant presence of flavonoids, polyphenols, terpenoids, and alka- ones that are believed to majorly drive the chemopreventive loids (30) in betel leaves makes them a remarkable sink as and chemotherapeutic agenda in preneoplastic and neo- well as a source for ROS thus conferring antioxidant as well plastic cells, respectively. This calls into question the unam- as pro-oxidant traits to the leaf extract. As alluded to earlier, biguity in defining a framework for an accurate assessment the paradoxical traits (antioxidant and pro-oxidant) of betel of the chemopreventive and/or chemotherapeutic contri- nutraceuticals empower them to offer both chemothera- butions of these two-faced phytochemicals. Here below, we peutic (cancer cells) and chemopreventive benefits (prema- revisit the paradoxical roles of plant phytochemicals in lignant cells), albeit through different mechanisms (8, 31– general to better comprehend the versatility of the phenolic 34). Once preneoplastic cells undergo malignant transfor- functionality in betel phytochemicals as a double-edged mation into cancer cells, they can perhaps only be elimi- sword. nated through chemotherapeutic strategies, as upon becom- ing cancerous, these cells have surpassed their amenability Chemotherapeutic (toxic pro-oxidant) and to chemopreventive measures. chemopreventive (protective antioxidant) activities Unlike normal cells, cancer cells owing to their enhanced of plant phytochemicals: two healthful sides of the metabolic activity have constitutively higher levels of ROS, same coin which spawn a persistent pro-oxidative environment (35– The most well studied health-promoting phytochemicals 37). Nonetheless, cancer cells despite high ROS concentra- (epigallocatechin gallate, curcumin, resveratrol, quercetin, tions, survive by adapting to the increased oxidative stress caffeic acid, rutin, gingerols, ChA, etc.) belong to the phenol by upregulating prosurvival mechanisms and altering their superfamily. In particular, for betel leaves, the versatile antioxidant systems (Fig. 2). phenolic compounds include hydroxychavicol, eugenol, Normal cells, however, operate within an "optimal win- CHV, piperol, and ChA, and can also serve as examples of dow" to maintain a redox balance between generation and secondary metabolites. These compounds merit a special elimination of free radicals, but also possess a robust capacity mention, as they are the primary defense molecules of to tolerate a sizeable fluctuation in ROS levels. In contrast, plants and have protected them during evolution. In addi- cancer cells with already high basal ROS levels have a much tion, these phenolics have "chemically" contributed to "narrower" window to endure elevations in ROS levels. maintain an ecological equilibrium between plants and This "vulnerability" or susceptibility of cancer cells can be other living organisms feeding on them, including us, exploited by whole betel extract (or its constituent phyto- humans. The magnanimous nature of these betel leaf com- chemicals) to selectively kill cancer cells by augmenting ROS pounds can be attributed to the inherent physicochemical levels beyond the toxic threshold that cancer cells can with- properties packaged within the phenol functional group. stand (Fig. 3). Nevertheless, a "similar-scaled" pro-oxidant

Figure 2. This ﬁgure represents the fate of cancer cells under varying ROS levels. Cancer cells having moderate levels of ROS tend to adapt to the stress conditions and show enhanced expression of prosurvival molecules. When betel nutraceuticals interfere with this situation, causing the moderate ROS levels to increase over the threshold, the cancer cells can no longer withstand the oxidative stress and thus are killed via various possible mechanisms (12, 22, 27, 29, 37, 38, 45, 47, 62, 64–66).

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oxidation processes initiating free radical formation, and (iii) co-oxidation of phenoxyl radicals with NADH, causing improved oxygen uptake and superoxide formation due to the action of peroxidases (39–43). Moreover, the redox- inactive metals like zinc and calcium have also been reported to increase the pro-oxidative nature of certain phenolics (44, 45). In addition, due to the initiation of free-radical chain reactions in the cell membranes, these phenoxyl radicals are stabilized to stay within the cell for a longer time, thus mounting a cytotoxic effect (46). Among eugenol and isoeugenol, the phenylpropene isomers found in betel leaves, the latter has been shown to induce high ROS levels thus proving to be cytotoxic, whereas eugenol has also been implicated in ROS-independent cytotoxic mechanisms (47).

Betel nutraceuticals offer chemopreventive benefits: potential "xenohormetics" A rekindled scientific interest in betel leaves has spurred several studies focused on investigating the chemopreventive Figure 3. "Achilles heel" of cancer cells. The illustration depicts the properties of betel leaves. More evidence to support the levels of ROS in normal, initiated, and cancer cells. The ROS levels potential benefits of betel leaves came from the identification in normal cells are "basal," while they are slightly elevated in an "initiated" of antimutagenic properties of betel leaf constituents, espe- premalignant cell. When these levels elevate beyond a threshold, cially hydroxychavicol, which repressed oral carcinogenesis cell proliferation and survival are promoted (as observed in case of cancer cells). The ROS levels in cancer cells are in the "caution" zone, induced by standard mutagens like benzo[a]pyrene and which, due to exogenous agents like betel nutraceuticals exerting dimethylbenz[a]anthracene in hamsters (48), and tobac- pro-oxidant behavior by possible depletion of cellular glutathione (GSH) co-specific nitrosamines like 4-(methylnitrosamino)-I-(3- levels (22), might increase above the ROS threshold pushing them into the pyridyl)-1-butanone (NNK) and N0-nitrosonornicotine "danger" zone, thus inducing apoptosis. This unique feature, which (NNN) in mice (49, 50), when betel leaf extract was topically makes the cancer cells vulnerable to death, can be exploited to b a selectively kill them, while not harming the surrounding normal cells. applied. In addition, -carotene and -tocopherol of betel leaf extract caused prolonged latency, regression of estab- lished tumors, and decrease in tumor incidence and tumor deviation from redox homeostasis is well tolerated by nor- size in Syrian hamsters exposed to another mutagen named mal cells. This partly explains why plant-based foods, includ- acetoxymethyl nitrosamine, thus indicating a strong chemo- ing betel extract with a concoction of free radical scavenging preventive effect of betel leaves (51). and generating agents, are well tolerated and nontoxic. The Furthermore, studies involving effects of betel leaves width of the "therapeutic window" may be evaluated by the against 7,12-dimethylbenz(a)anthracene (DMBA)-induced redox differences between normal and tumor cells and may breast cancer provided remarkable cues emphasizing their be specific to the nature of the components present in the chemopreventive efficacy. It was observed that when betel plant-based food. leaf extract was fed to rats bearing mammary tumors in the Several reports highlight the pro-oxidant capabilities of initiation phase, the tumor growth was prevented, while rats major betel leaf constituents like hydroxychavicol and with fully developed mammary tumors failed to show any eugenol. Hydroxychavicol has been shown to exploit this reduction/inhibition in tumor growth (52, 53). We have higher ROS level in cancer cells, and increases it even further recently reported that betel leaf extract exhibits significant to preferentially kill them while having no effect on the efficacy in inhibiting growth of prostate cancer xenografts proliferation of normal cells (22, 38). However, these (20). These studies generate compelling foregrounds to phenolic compounds exhibit anti- and/or pro-oxidant warrant future investigation of chemopreventive efficacy of behavior depending on their relative concentrations in vivo betel leaves and/or bioactive betel leaf constituents in along with the redox potential of the surrounding areas. appropriate animal models. Interestingly, phenolics can promote oxidation at elevated Insights into the mechanistic actions of betel leaf consti- concentrations of the quinone form, where they can act as tuents were obtained from several investigations that focused reducing agents causing an increase in ROS species in the on the effect of betel leaf on gastric carcinomas. For instance, form of a phenoxyl radical, which could be detrimental. eugenol, hydroxychavicol, b-carotene, and a-tocopherol This pro-oxidant behavior of phenolic compounds depends were identified to be key players in suppressing ben- on (i) the total number of hydroxyl groups, which result in zo[a]pyrene-induced neoplasms of the gut in mice, when increased production of hydroxyl radicals and in some betel leaf extract was mixed with drinking water (49). Euge- cases, promote production of hydrogen peroxide, (ii) pres- nol was further proven to be inducing intrinsic apoptosis in ence of free transition metal ions like Cu (II), involved in Wistar rats bearing N-methyl-N-nitro-N-nitrosoguanidine–

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Figure 4. Xenohormetic response. The bioactive phenolics of betel leaves may facilitate elimination of initiated cells, thus ensuring that they do not develop into cancer. By "signaling" that there is some "stress" in the environment, ingested betel nutraceuticals are thought to potentiate a systemic stress- combating response in normal cells, which in turn triggers the upregulation of endogenous antioxidant enzymes by modulating the nuclear transcription factor erythroid 2p45 (NF-E2)–related factor 2 (Nrf2) expression or the endogenous thiol glutathione (GSH) levels (7, 67). Because of the systemic activation of this defense system, the initiated cells can be effectively scavenged and eliminated, presumably by the immune system, which is placed on "high- alert." On the other hand, in the cancer cells an opposite effect of further stress induction is observed because of which apoptosis is induced (20) and/or the endogenous Nrf2 and thiol levels are depleted (7, 67).

induced gastric cancer by modulating Bcl-2 family proteins We have alluded to in the above section how polyphe- along with Apaf-1, cytochrome C, and caspases (54). Fur- nolic compounds in whole betel extract with their biophy- thermore, studies involving specific carcinogens like DMBA), sicochemical "flexibility" can tip the redox balance in favor which are well known to cause skin cancer, revealed the of death in cancer cells by taking advantage of their inher- significance of b-cartotene and a-tocopherol in inhibiting ently higher ROS levels, which turns out to be their "Achilles the onset of tumors in mice. Simultaneously, although heel." Nevertheless, it is difficult to reconcile several of these hydroxychavicol exhibited the same response, but to a questions in the chemopreventive context in the absence of greater extent, eugenol, however, could only offer limited a unifying new paradigm called "xenohormesis," which is protection against DMBA-induced skin tumors (55). Other gaining momentum. This hypothesis assumes that poly- studies by Azuine and colleagues suggested that eugenol in phenols were synthesized by common ancestors of plants the betel leaves exhibited specific antiproliferative activity and animals (58). Despite the evolutionary divergence of while partially triggering apoptosis (56) along with eliciting the respective kingdoms, it is intriguing that many crucial superoxide formation, lipid peroxidation, and radical scav- mammalian enzymes and receptors have been conserved enging activity, thus illustrating its antioxidant effects (57). which can be regulated and modulated by plant-produced Eugenol was also shown to hinder the upstream signaling phytochemicals. molecule NF-kB, a key player in regulating the expression of Paramount interest centers on the belief that adverse genes controlling cell proliferation and survival (57). environmental conditions or stress induce the synthesis of Despite the existence of strong evidence to demonstrate phytochemicals, including polyphenols, in plants. Interest- that betel phytochemicals exert a diverse spectrum of ben- ingly, when ingested by humans, these phytochemicals upre- eficial protective and curative effects in various disease gulate the pathways that provide stress resistance in animals models, there are several perplexing questions that await (Fig. 4). Conceivably,this suggests that humans that consume persuasive answers—How can the same polyphenolic phy- phytochemicals via plant-based foods have sensing mechan- tochemical (such as hydroxychavicol from betel leaf) "pre- isms (enzymes/receptors) to perceive these chemical cues and vent" cancer by exercising chemopreventive strategies as elicit beneficial responses that enhance the well-being of well as offer "treatment" by enforcing curative commands humans thus imparting overall health benefits. This phe- for therapeutic gains? How can the long-accepted antioxi- nomenon has been brilliantly termed as "xenohormesis" dant-free radical quenching/scavenging properties of phe- by Howitz and Sinclair fairly recently in 2008, wherein xenos, nols explain their chemopreventive and/or chemothera- is the Greek word for stranger, and hormesis, the term for peutic benefits of betel leaves? How can we correlate efficacy health benefits (58). In simplistic terms, it is health benefits of these phenols with their antioxidant capacity? conferred by a stranger! We envision that ingestion of

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phytochemicals causes the body to be tricked into believing the electrophilic reactive oxygen quenchers like ChA and that it is under some kind of stress, which triggers a system- curcumin contain a Michael acceptor enone system shown wide response to combat stress. While normally, the presence by the a-, b-unsaturated ketone and maintain the effective of a handful of initiated or premalignant cells would not be ROS manipulating system of the dihydroxyl groups. able to trigger a sufficiently strong signal for the body to On the other hand, it is well studied that plant poly- perceive and respond to, phytochemicals may place the phenols containing catechol and/or pyrogallol moieties can system on "high-alert" and stimulate a heightened stress also exert pro-oxidant properties, by reducing iron (III) or response that could effectively eliminate initiated or prema- copper (II) ions that they chelate. Furthermore, the ortho- lignant cells thus emphasizing the workings of plant phyto- hydroxyphenoxy radical produced from the oxidation of a chemicals in the chemopreventive context (Fig. 4). catechol/pyrogallol moiety can also react with a second free- 3 The polyphenolic redox manipulating phytochemicals radical species, including O2, to afford oxidizing ortho- . commonly display an aromatic core with 1,2-hydroxyl quinones and O2 (59). Because of these reactions, the groups, which are found in hydroxychavicol and ChA, or catecholic betel phenolic compounds like hydroxychavicol a hydroxyl group and neighboring methyl ether like in and ChA (Fig. 5) induce DNA breakage in the presence of 3 eugenol and CHV (Fig. 5). These groups have been shown O2 and iron or copper species, thus leading to death. to act as an electron acceptor or hydrogen atom donor and The xenohormetic process thus fills the gaps in knowl- quench the existing ROS and thus prevent the cell from edge on the cancer preventative benefits of plant-based transforming into a cancerous entity. When present in phytochemicals in addition to their roles to kill transformed appropriate concentrations, these powerful phytochemicals cancer cells. The xenohormesis phenomenon aids to com- act as antioxidants. It has been well described throughout prehend the basis of the chemopreventive potential of these the literature that phenolic compounds exist as excellent betel phytochemicals that "prevent" carcinogenesis and electron acceptors due to the resonance stabilization of the intervene early on in the malignant transformation process. radical structure and an excellent hydrogen atom donor for Thus, we believe that it is not unreasonable to include betel similar stabilization of the resulting oxyanion. Furthermore, phytochemicals endowed with "magical" health imparting

Figure 5. A, structural comparison of betel leaf phenolics with known xenohormetic compounds like curcumin and resveratrol suggests that the hydroxyl groups act as electron acceptors or hydrogen atom donors and quench the existing ROS, favoring a xenohormetic response by betel leaf nutraceuticals. Catechol moieties on betel phenolics are highlighted within the red boxes. B, furthermore, free radicals are eliminated because of metal chelating and stabilizing properties of certain phenolic compounds, thus conferring them strong antioxidant activity.

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powers in the armamentarium of the known "xenohor- necessary before conclusions on dose extrapolations, die- metic" molecules. tary incorporation, duration of consumption, etc., can be In the light of these observations, we may rationalize that implemented. in initiated cells, a mild stress induced by betel nutraceuticals, causes the activation of the Nrf2 circuit encouraging the Conclusion expression of endogenous antioxidants and other cytopro- Although the zeal and zest for discovering novel phar- tective genes, which generate a xenohormesis-mediated che- maceutical leads from plant extracts have dwindled in mopreventive response. Although extensive data from sev- recent years, the dominance of plants as superior sources in vivo eral studies demonstrate the chemopreventive efficacy of new drug discovery is unchallengeable. Exploiting the of betel leaf and its constituents, the paucity of supporting bounty of Mother Nature to identify and isolate valuable clinical data demands an urgent need for strategic explora- cancer-fighting agents from fruits, vegetables, and spices is a tion of these effects upon daily consumption in humans. priority considering the increasing incidence of cancer in the world population. In particular, food materials labeled Health in harmony: synergy among betel as "generally recognized as safe" (GRAS) are appealing nutraceuticals sources to identify xenohormetic molecules that offer The complexity of phytochemicals in their natural forms health-promoting effects and that complement the chem- in plant-based foods precludes a definitive conclusion ical space of drugs. No wonder the emergence of Foodinfor- about their mechanisms of action. However, the existence matics, a new variant of ChemInformatics, will uncover of synergistic interactions have been long known and linked potential applications of bioactive food chemicals using to the activity of whole foods. The chemotherapeutic and computational approaches. chemopreventive properties of betel leaf and its constitu- Piper betel, an exotic condiment, reported to be a treasure ents spur the need for examining and identifying the frac- of bioactive phenolics, possesses great potential to fight tion and constituents thereof, which contribute to the against cancers of oral, mammary, prostate, skin, and gastric remarkable efficacy. We have recently reported that the least origin. Much of these powers of betel leaf phytochemicals polar fraction, F2, of betel leaf extract exhibited the highest remain unharnessed and call for extensive research to better antiproliferative activity when tested against human pros- understand their mechanism(s) of action and clearly tate cancer, PC-3 cells (20). F2 was identified to be consist- demarcate their chemopreventive and chemotherapeutic ing of hydroxychavicol and CHV along with other unknown roles. Through this review, we have attempted to summarize phytochemicals, and hydroxychavicol was found to be the the advances in understanding the betel leaf action, its major contributor of betel leaf extract efficacy. Interestingly, constituents, and their anti- and pro-oxidant nature, which this study suggested a possible synergy among F2 constitu- bear chemopreventive and chemotherapeutic ramifica- in vivo ents but additional work, including pharmacokinetic tions. We place into perspective the two seemingly inter- evaluations, is warranted to draw conclusions on the poten- twined preventive and therapeutic activities by discussing tial usefulness of the subfractions or single agents for the physiologic difference in ROS levels in normal versus chemopreventive or therapeutic goals. cancer cells and how this exposes an exploitable vulnera- Considering the potential synergistic interactions among bility of cancer cells, which can be appropriately "tuned" to in vivo betel leaf constituents and the remarkable tumor enhance cancer cell selectivity to enable design of novel growth inhibitory dose of whole betel leaf extract in mice approaches for the management of this insidious disease. bearing human prostate cancer (400 mg/kg body weight; ref. 20), correlations to human health benefits can be Disclosure of Potential Conflicts of Interest obtained. Allometric scaling calculations suggest that upon No potential conflicts of interest were disclosed. extrapolation of mice data to humans (60), the approximate human equivalent dose of whole betel leaf extract was Acknowledgments The authors thank Bazla Shazad and Eric A. Owens for their contributions found to be approximately 32 mg/kg body weight, which to this work. translates to approximately 2.2 g for a 70-kg adult. Further- more, according to the United States Department of Agri- Grant Support culture’s Food Guide pyramid (61), this dose can be This work was supported by grants from the National Cancer Institute at obtained from approximately 41 g, or about one-fourth the NIH (R00CA131489, R01 CA169127; to R. Aneja) and the American cup of fresh betel leaves, which can perhaps easily be Cancer Society (121728-RSG-12-004-01-CNE). incorporated in daily diet. However, detailed pharmacoki- Received October 11, 2013; revised December 19, 2013; accepted January netic investigations and pharmacodynamics analyses are 9, 2014; published OnlineFirst January 21, 2014.

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486 Cancer Prev Res; 7(5) May 2014 Cancer Prevention Research

Piper Betel Leaf: A Reservoir of Potential Xenohormetic Nutraceuticals with Cancer-Fighting Properties

Sushma R. Gundala and Ritu Aneja

Cancer Prev Res 2014;7:477-486. Published OnlineFirst January 21, 2014.

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