The Effect of Maturity Status on Biochemical Composition,Antioxidant Activity,and Anthocyanin Biosynthesis Gene Expression in a Pomegranate (Punica granatum L.) Cultivar with Red Flowers,Yellow Peel,and Pinkish Arils

Pomegranate (Punica granatum L.) has widely been used as a fruit and in folk medicine since ancient civilizations in the world. It is now known that bioactive compounds present in pomegranates attribute to its therapeutic potential. Harvesting at the correct maturity stage is one of the key factors deciding the quality of harvest for consumption in fresh or value-added forms. Identification of the correct maturity stage (harvesting index) is particularly difficult for cultivars having yellowish peel and pinkish arils (sarcotesta). We studied the changes in total phenolic content (TPC), antioxidant activity (AOX), punicalagin α and β contents, color indices, total soluble solids (TSS), and expression of anthocyanin biosynthetic pathway genes from flowering to harvesting in the pomegranate cultivar Nimali, having red flowers, yellow peels, and pinkish arils at maturity over two growing seasons. Interestingly, there was no seasonal variation observed in any of the parameters over two cultivation seasons. Although the β punicalagin content did not change, the TPC, AOX, punicalagin α contents in both peel and arils gradually decreased from flowering to maturity. Though the TPC of both peels and arils, AOX and total punicalagin content of peel did not change significantly 140 days after flower initiation, the TPC and the total punicalagin of arils reached a stable level at 160 days. The TSS in both peels and arils increased significantly with the maturity having the highest values at 180 days. The peel color changed from green to yellow with maturity with a significant increase in l* and b* values and significant decrease in a* value. Nevertheless, the aril color changed from pale white to pink with the maturity with significant reduction of l* and b* values and significant increase of a* value. Changes in pomegranate dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), and leucoanthocyanidin dioxygenase (anthocyanidin synthase-ANS) gene expression in Nimali arils correlated with its color changes during maturity. These findings support to identify the harvesting index of Nimali ensuring the maximum nutritional and health benefits of pomegranate flower, arils, and peels for different downstream uses.

     

In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice

Pomegranate (Punica granatum L.) fruits are widely consumed as juice (PJ). The potent antioxidant and anti-atherosclerotic activities of PJ are attributed to its polyphenols including punicalagin, the major fruit ellagitannin, and ellagic acid (EA). Punicalagin is the major antioxidant polyphenol ingredient in PJ. Punicalagin, EA, a standardized total pomegranate tannin (TPT) extract and PJ were evaluated for in vitro antiproliferative, apoptotic and antioxidant activities. Punicalagin, EA and TPT were evaluated for antiproliferative activity at 12.5-100 microg/ml on human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620) and prostate (RWPE-1, 22Rv1) tumor cells. Punicalagin, EA and TPT were evaluated at 100 microg/ml concentrations for apoptotic effects and at 10 microg/ml concentrations for antioxidant properties. However, to evaluate the synergistic and/or additive contributions from other PJ phytochemicals, PJ was tested at concentrations normalized to deliver equivalent amounts of punicalagin (w/w). Apoptotic effects were evaluated against the HT-29 and HCT116 colon cancer cell lines. Antioxidant effects were evaluated using inhibition of lipid peroxidation and Trolox equivalent antioxidant capacity (TEAC) assays. Pomegranate juice showed greatest antiproliferative activity against all cell lines by inhibiting proliferation from 30% to 100%. At 100 microg/ml, PJ, EA, punicalagin and TPT induced apoptosis in HT-29 colon cells. However, in the HCT116 colon cells, EA, punicalagin and TPT but not PJ induced apoptosis. The trend in antioxidant activity was PJ>TPT>punicalagin>EA. The superior bioactivity of PJ compared to its purified polyphenols illustrated the multifactorial effects and chemical synergy of the action of multiple compounds compared to single purified active ingredients.     

八种水果中的多酚含量及其抗氧化性

测定了苹果、石榴、橄榄、芒果、香蕉、菠萝、葡萄和龙眼的壳、肉及核中的多酚含量及其抗氧化性。以70%丙酮(v/v)为提取溶剂,室温下超声波辅助浸提样品后得到提取液。采用普鲁士兰(Prussian blue)法测定了提取液中多酚和单宁的含量,利用FRAP法测定其抗氧化性。结果表明:提取液中多酚和单宁的含量均与其抗氧化性成正相关关系;石榴、橄榄、芒果、葡萄和龙眼等水果的壳与核有望成为天然抗氧化剂的新来源。     

Pomegranate juice and punicalagin attenuate oxidative stress and apoptosis in human placenta and in human placental trophoblasts

The human placenta is key to pregnancy outcome, and the elevated oxidative stress present in many complicated pregnancies contributes to placental dysfunction and suboptimal pregnancy outcomes. We tested the hypothesis that pomegranate juice, which is rich in polyphenolic antioxidants, limits placental trophoblast injury in vivo and in vitro. Pregnant women with singleton pregnancies were randomized at 35～38 wk gestation to 8 oz/day of pomegranate juice or apple juice (placebo) until the time of delivery. Placental tissues from 12 patients (4 in the pomegranate group and 8 in the control group) were collected for analysis of oxidative stress. The preliminary in vivo results were extended to oxidative stress and cell death assays in vitro. Placental explants and cultured primary human trophoblasts were exposed to pomegranate juice or glucose (control) under defined oxygen tensions and chemical stimuli. We found decreased oxidative stress in term human placentas from women who labored after prenatal ingestion of pomegranate juice compared with apple juice as control. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in term villous explants and primary trophoblast cultures exposed to hypoxia, the hypoxia mimetic cobalt chloride, and the kinase inhibitor staurosporine. Punicalagin, but not ellagic acid, both prominent polyphenols in pomegranate juice, reduced oxidative stress and stimulus-induced apoptosis in cultured syncytiotrophoblasts. We conclude that pomegranate juice reduces placental oxidative stress in vivo and in vitro while limiting stimulus-induced death of human trophoblasts in culture. The polyphenol punicalagin mimics this protective effect. We speculate that antenatal intake of pomegranate may limit placental injury and thereby may confer protection to the exposed fetus.     

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 microM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein-diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 microM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 microM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).     

Polyphenolic extracts from pomegranate and watermelon wastes as substrate to fabricate sustainable silver nanoparticles with larvicidal effect against Spodoptera littoralis

The agricultural wastes adversely affect the environment; however, they are rich in polyphenols; therefore, this study aimed to employ polyphenol-enriched waste extracts for silver nanoparticles synthesis, and study the larvicidal activity of silver nanoparticles fabricated by pomegranate and watermelon peels extracts (PPAgNPs and WPAgNPs) against all larval instars of Spodoptera littoralis. The polyphenol profile of pomegranate and watermelon peel extracts (PP and WP) and silver nanoparticles was detected by HPLC. The antioxidant activity was estimated by DPPH, and FARP assays and the antimicrobial activity was evaluated by disc assay. The Larvicidal activity of AgNPs against Egyptian leaf worm was performed by dipping technique. The obtained AgNPs were spherical with size ranged 15–85 nm and capped with proteins and polyphenols. The phenolic compounds in silver nanoparticles increased about extracts; therefore, they have the best performance in antioxidant/reducing activity, and inhibit the growth of tested bacteria and yeast. The PPAgNPs were the most effective against the first instar larvae instar (LC₅₀ = 68.32 µg/ml), followed by pomegranate extract with (LC₅₀ = 2852 µg/ml). The results indicated that obvious increase in polyphenols content in silver nanoparticles enhance their larvicidal effect and increasing mortality of 1^st larval of S. littoralis Egyptian leafworms causing additive effect and synergism. We recommend recycling phenolic enriched agricultural wastes in producing green silver nanoprticles to control cotton leafworm that causes economic loses to crops.     

Punicalagin Targets Atherosclerosis: Gene Expression Profiling of THP-1 Macrophages Treated with Punicalagin and Molecular Docking

Atherosclerosis is an important cause of cardiovascular disorders worldwide. Natural botanical drugs have attracted attention due to their antioxidant, anti-inflammatory, and antiatherogenic properties in the treatment of atherosclerosis. Punicalagin is the major bioactive component of pomegranate peel, and has been shown to have antioxidant, anti-inflammatory, antiviral, anti proliferation, and anticancer properties. To explore its antiatherogenic properties at a molecular level, we investigated the genome-wide expression changes that occur in differentiated THP1 cells following treatment with a non-toxic dose of punicalagin. We also conducted a molecular docking simulation study to identify the molecular targets of punicalagin.     

Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir

Influenza epidemics cause numerous deaths and millions of hospitalizations each year. Because of the alarming emergence of resistance to anti-influenza drugs, there is a need to identify new naturally occurring antiviral molecules. We tested the hypothesis that pomegranate polyphenol extract (PPE) has anti-influenza properties. Using real time PCR, plaque assay, and TCID 50% hemagglutination assay, we have shown that PPE suppresses replication of influenza A virus in MDCK cells. PPE inhibits agglutination of chicken red blood cells (cRBC) by influenza virus and is virucidal. The single-cycle growth conditions indicated that independent of the virucidal effect PPE also inhibits viral RNA replication. PPE did not alter virus ribonucleoprotein (RNP) entry into nucleus or translocation of virus RNP from nucleus to cytoplasm in MDCK cells. We evaluated four major Polyphenols in PPE (ellagic acid, caffeic acid, luteolin, and punicalagin) and demonstrated that punicalagin is the effective, anti-influenza component of PPE. Punicalagin blocked replication of the virus RNA, inhibited agglutination of chicken RBC's by the virus and had virucidal effects. Furthermore, the combination of PPE and oseltamivir synergistically increased the anti-influenza effect of oseltamivir. In conclusion, PPE inhibited the replication of human influenza A/Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.     

Antioxidant activity of polyphenolic extracts of Ichnocarpus frutescens

In the present study antioxidant activities by (1,1-diphenyl-2-picrylhydrazyl radical (DPPH), hydrogen peroxide, hydroxyl radical inhibition, hemolysis by hydrogen peroxide assay, reducing power and total antioxidant activities of polyphenolic extract of Ichnocarpus frutescens leaves were investigated. The flavonoids and total polyphenolic contents of the extract were also determined using standard methods. Phytochemical analyses revealed the presence of flavonoids, polyphenols, anthocyanins and simple phenolic acids. The results of antioxidant activities of polyphenol extract obtained by different in vitro methods were varied depending on the method used. Nevertheless, polyphenol extract showed significant inhibitory activities in all in vitro reactive oxygen species scavenging, might be attributed due to the high level of polyphenolic compound. Also, these various antioxidant activities were compared to α-tocopherol and l-ascorbic acid as reference antioxidant compounds. These findings provide evidence that the polyphenolic extract of I. frutescens is a natural source of antioxidant against oxidative damage.     

Phenolic production and antioxidant properties of some Macedonian medicinal plants

Investigations have been made to study the production of phenolic compounds (total phenolics, flavonoids and phenylpropanoids) and total antioxidant capacity in 27 Macedonian traditional medicinal plants to improve its potential as a source of natural antioxidants. Antioxidant potential of plant extracts was analyzed by five different assays: cupric reducing antioxidant capacity (CUPRAC), phosphomolybdenum method (PM), reducing power (RP), 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS^·+) radical scavenging activity. Origanum vulgare extract consistently exhibited the highest content of phenolic compounds and the strongest antioxidant capacity based on the tests performed, and can be proposed as a promising source of natural antioxidants. Melissa officinalis and Salvia ringens were also identified as valuable sources of antioxidant compounds. A positive linear correlation between antioxidant activity and total phenolics, flavonoids and phenylpropanoids indicates that these compounds are likely to be the main antioxidants contributing to the observed activities of evaluated plants. These findings suggest that the medicinal plants studied in this paper are good sources of bioactive compounds for the food and pharmaceutical industries.     

Enhancing beneficial antioxidants in fruits: a plant physiological perspective

The aim of this review is to present an outline of the physiological perspectives of beneficial antioxidant production in fruit. The drive to enhance the consumption of fruit and vegetables in the human diet is linked with positive effects of beneficial antioxidants impacting on health promotion. We briefly outline our physiological understanding of environmental processes which induce the production of reactive oxygen species and how antioxidants prevent plant cellular damage. More specifically, we describe the impact that environmental stresses, such as drought and radiation, have on the production of endogenous antioxidants and how these stresses can be incorporated into novel experimental crop growing systems to achieve high antioxidant concentrations in fruits. This includes in particular the use of irrigation application techniques and enhanced light reflectance to increase the concentrations of bioactive compounds such as ellagic acid and ascorbic acid.     

Phenolics as potential antioxidant therapeutic agents: mechanism and actions

Accumulating chemical, biochemical, clinical and epidemiological evidence supports the chemoprotective effects of phenolic antioxidants against oxidative stress-mediated disorders. The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression. The antioxidant capacities of phenolic compounds that are widely distributed in plant-based diets were assessed by the Trolox equivalent antioxidant capacity (TEAC), the ferric reducing antioxidant power (FRAP), the hypochlorite scavenging capacity, the deoxyribose method and the copper-phenanthroline-dependent DNA oxidation assays. Based on the TEAC, FRAP and hypochlorite scavenging data, the observed activity order was: procyanidin dimer > flavanol > flavonol > hydroxycinnamic acids > simple phenolic acids. Among the flavonol aglycones, the antioxidant propensities decrease in the order quercetin, myricetin and kaempferol. Gallic acid and rosmarinic acid were the most potent antioxidants among the simple phenolic and hydroxycinnamic acids, respectively. Ferulic acid displayed the highest inhibitory activity against deoxyribose degradation but no structure–activity relationship could be established for the activities of the phenolic compounds in the deoxyribose assay. The efficacies of the phenolic compounds differ depending on the mechanism of antioxidant action in the respective assay used, with procyanidin dimers and flavan-3-ols showing very potent activities in most of the systems tested. Compared to the physiologically active (glutathione, -tocopherol, ergothioneine) and synthetic (Trolox, BHA, BHT) antioxidants, these compounds exhibited much higher efficacy. Plant-derived phenolics represents good sources of natural antioxidants, however, further investigation on the molecular mechanism of action of these phytochemicals is crucial to the evaluation of their potential as prophylactic agents.     

Phenolic compounds and antioxidant activity from red grape marc extracts

The aims of this work were to determine the amounts of the different classes of phenolic compounds in an ethanolic extract from red grape marc and its components, peels and seeds, and to evaluate their antioxidant activities by the beta-carotene bleaching test for their utilization as natural antioxidants. The results showed that red grape marc was rich in polyphenol compounds with a clear antioxidant activity. The extracts, in fact, at very low concentration (20 ppm) in total phenols showed an antioxidant activity (AA) higher than 43% on average, while at higher concentration (80-160 ppm) all the fractions had an AA comparable to that of butlylated-hydroxytoluene. Grape seeds seemed to give the highest contribution to such AA, as they contained high quantities of proanthocyanidines, a type of flavonoid known for its high antioxidant properties.     

Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage

Potato peels are waste by-product of the potato processing industry. They are reportedly rich in polyphenols. Our earlier studies have shown that extracts derived from potato peel (PPE) possess strong antioxidant activity in chemical and biological model systems in vitro, attributable to its polyphenolic content. The main objective of this study was to investigate the ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protection rendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage, morphological alterations as well as membrane structural alterations. The total polyphenolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids present in PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid. We chose the experimental prooxidant system: FeSO₄ and ascorbic acid to induce lipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipid peroxidation with similar effectiveness in both the systems (about 80–85% inhibition by PPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the erythrocytes, under the experimental conditions, PPE significantly inhibited the H₂O₂-induced morphological alterations in rat RBCs as revealed by scanning electron microscopy. Further, PPE was found to offer significant protection to human erythrocyte membrane proteins from oxidative damage induced by ferrous–ascorbate. In conclusion, our results indicate that PPE is capable of protecting erythrocytes against oxidative damage probably by acting as a strong antioxidant.     

Evaluating the Potential Role of Pomegranate Peel in Aluminum-Induced Oxidative Stress and Histopathological Alterations in Brain of Female Rats

Studies have shown that pomegranate, Punica granatum Linn. (Lythraceae), has remarkable biological and medicinal properties. However, the effects of pomegranate peel methanolic extract (PPME) on the aluminum-induced oxidative stress and histopathological change have not been reported yet. To determine the effect of PPME (200?mg/kg bwt) on the aluminum chloride (AlCl₃; 34?mg/kg bwt)-induced neurotoxicity, aluminum accumulation in brain and oxidant/antioxidant status were determined. The change of brain structure was investigated with hematoxylin and eosin, and anti-apoptosis effects of PPME were analyzed by immunohistochemistry. The present study showed an indication of carcinogenicity in the AlCl₃-treated group representing an increase in tissue tumor markers such as tumor necrosis factor-?? and angiogenin and inflammation by inducing an increase in prostaglandin E2 and prostaglandin F2??. PPME protected brain through decreasing the aluminum accumulation and stimulating antioxidant activities and anti-apoptotic proteins namely Bcl-2. Therefore, these results indicated that pomegranate peel methanolic extract could inhibit aluminum-induced oxidative stress and histopathological alternations in brain of female rats, and these effects may be related to anti-apoptotic and antioxidants activities.     

Preparative separation of punicalagin from pomegranate husk by high-speed countercurrent chromatography

Punicalagin, the main ingredient of pomegranate (Punica granatum L.) husk, is a high molecular weight polyphenolic compound. It has shown remarkable pharmacological activities attributed in the presence of dissociable OH groups. To isolate punicalagin, previous methods included labor intensive and expensive solid phase extractions by column chromatography (C-18, polyamides, dellulose, Sephadex Lipophilic LH-20, Diaion HP20). High-speed countercurrent chromatography (HSCCC) was used for isolation and purification of punicalagin from pomegranate husk. Using preparative HSCCC about a 350 mg amount of the crude extract was separated, yielding 105 mg of punicalagin at a high-purity of over 92%. Eighty milligrams of gallic acid was simultaneously separated as another product, at a purity of 75%.     

Free radical scavenging and antioxidant potential of mangrove plants: a review

Free radicals derived from reactive oxygen species and reactive nitrogen species are generated in our body by normal cellular metabolism which is enhanced under stress conditions. The most vulnerable biological targets of free radicals are cell structures including proteins, lipids and nucleic acids. Since antioxidants synthesized in the body are not sufficient under oxidative stress, their exogenous supply is important to prevent the body from free radical-induced injury. Recent researches have shown that antioxidants of plant origin with free radical scavenging property could have great importance as therapeutic agents in management of oxidative stress. Mangrove plants growing in inhospitable environment of the intertidal regions of land and sea in tropics and sub-tropics are equipped with very efficient free radical scavenging system to withstand the variety of stress conditions. These mangrove plants possess variety of phytochemical and are rich in phenolic compounds such as flavonoids, isoflavones, flavones, anthocyanins, coumarins, lignans, catechins, isocatechins, etc., which served as source of antioxidants. Isolation and identification of these antioxidant compounds offer great potential for their pharmaceutical exploitations. However, no comprehensive literature is available on antioxidants’ studies in mangrove plants in particular. Hence, the present review discusses the antioxidant potential of mangrove plants with its specific role under salt stress as well as the progress made so far in evaluation of antioxidant activities of different mangrove species.     

Identification of bioactive phytochemical from two Punica species using GC–MS and estimation of antioxidant activity of seed extracts

Punica species are medicinally important plants belonging to the family Lythraceae. The pomegranate is widely reported to exhibit antiviral, antioxidant, anticancer, anti-proliferative activities. In the present study the ethanolic extract of the peel seeds of two species of Punica (Punica granatum and Punica protopunica) were subjected to GC–MS analysis. Twenty-one and 14 compounds were identified in P. granatum and P. protopunica peel seeds, respectively. The main chemical constituents in P. granatum-peel seeds were propanoic acid, benzenedicarboxylic acid, methoxypropionic acid and methyl amine. The corresponding constituents of P. protopunica peel seeds were benzenedicarboxylic acid, benzoic acid and propanoic acid. Moreover, the antioxidant effects of the aqueous ethanolic extracts were estimated in vitro. The two tested extracts contained significantly different phenolic and total flavonoid contents in P. granatum than in P. protopunica. Different in vitro methods of antioxidant activity determination produced varying results. In malondialdehyde (MDA), hydrogen peroxide (H₂O₂) scavenging and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays, the two peel seed extracts exhibited very high antioxidant activities, with higher activity observed for the P. granatum extract.     

Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer

Cancer prevention and treatment using traditional Chinese medicines have attracted increasing interest. This study characterizes antioxidant activity and phenolic compounds of traditional Chinese medicinal plants associated with anticancer, comprising 112 species from 50 plant families. The improved ABTS(*+) method was used to systematically assess the total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) of the medicinal extracts. The TEAC values and total phenolic content for methanolic extracts of herbs ranged from 46.7 to 17,323 micromol Trolox equivalent/100 g dry weight (DW), and from 0.22 to 50.3 g of gallic acid equivalent/100 g DW, respectively. A positive, significant linear relationship between antioxidant activity and total phenolic content (all R(2) values>/=0.95) showed that phenolic compounds were the dominant antioxidant components in the tested medicinal herbs. Major types of phenolic compounds from most of the tested herbs were preliminarily identified and analyzed, and mainly included phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. These medicinal herbs exhibited far stronger antioxidant activity and contained significantly higher levels of phenolics than common vegetables and fruits. Traditional Chinese medicinal plants associated with anticancer might be potential sources of potent natural antioxidants and beneficial chemopreventive agents.