Pomegranate Seed Oil (Punica Av

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Research Article *Corresponding author Illana Louise Pereira de Melo, Food and Experimental Nutrition Department - Faculty of Pharmaceutical Science, University of Sao Paulo, Pomegranate Seed Oil (Punica Av. Prof. Lineu Prestes, 580 – Bloco 14, 05508-900 – São Paulo, SP, Brazil, Tel: 55 11 30913688; Fax: 55 11 Granatum L.): A Source of 38154410; E-mail: Submitted: 17 December 2013 Accepted: 14 February 2014 Punicic Acid (Conjugated Published: 17 February 2014 Copyright α-Linolenic Acid) © 2014 de Melo et al. OPEN ACCESS Illana Louise Pereira de Melo*, Eliane Bonifácio Teixeira de Carvalho, Jorge Mancini-Filho Keywords Food and Experimental Nutrition, University of Sao Paulo, Brazil • Pomegranate seed oil • Conjugated fatty acid • Punicic acid Abstract The pomegranate seed oil (PSO) presents a typical fatty acid profile which includes high percentages of the punicic acid (PA) – a conjugated isomer of a-linolenic acid. Conjugated a-linolenic acids (CLnAs) are a collective term for the positional and geometric isomers of octadecatrienoic acid (C18:3) with conjugated double bonds. Recently, conjugated fatty acids have attracted significant attention due to reports of its health benefits in a variety of models of metabolic diseases and chronic inflammatory diseases. However, some work is controversial and there is still no consensus in the literature regarding their effects on animal and human organisms. This article presents a review under the pomegranate seed oil and the potential effects of conjugated a-linolenic acid to health.

Introduction Pomegranate The numerous reported functional properties of pomegranate Pomegranate has been consumed in many parts of the (Punica granatum L.) make it a unique fruit [1]. For instance, world for thousands of years. The use of the fruit dates back the great antioxidant activity of the fruit, as well as its juice, to biblical times and reports of its therapeutic qualities have echoed throughout the millennia. Its seeds were believed to have encouraged further studies into its nutraceutical potential and its resurrecting powers by the Babylonians, to grant invincibility in applicationshas been reported in the foodto have industry beneficial [2]. Its health abundant effects seeds, and normally has also battle by the Persians and to symbolize longevity and immortality waste products from pomegranate processing, are also of great interest, once their oil has a particularly rich composition [3]. by thePomegranate Chinese [8]. (Punica granatum L.), member of the family A high content of phytosterols, tocopherols and a unique Punicaceae, are one of the most ancient edible fruits. They are widely grown in Mediterranean regions (including Iran) and fatty acid composition, mainly consisting of punicic acid (55 %), were observed by Melo [4] when investigating the composition of pomegranate seed oil. Punicic acid, also known as trichosanic India, but sparsely cultivated in the USA, China, Japan and Russia. acid, is an omega-5 long chain polyunsaturated fatty acid and ofIts seeds)edible parts are mostly (corresponding consumed to fresh; 55 - 60 however, % of the they whole are fruit also commerciallyweight, of which available 75 - 85 % as consists processed of juice foods and and 25 drinks,- 15 % consists such as an isomer of conjugated α-linolenic acid (CLnA) with structural juices, wines, liqueurs, jams and canned fruit [3,9]. In addition, (LnA) [5], such as carbon composition, atomic arrangement and the fruit is widely used in therapeutic formulations, cosmetics thesimilarities number toof conjugatedcarbon double linoleic bonds. acid These (CLA) conjugated and α-linolenic fatty acids acid polyphenols and minerals are found in the edible parts of pomegranate;and food seasonings however, [8]. their Sugars, content vitamins, in the polysaccharides, fruit may vary have increasingly attracted scientific interest because of their antitumor, immunomodulatory, anti-atherosclerotic and serum according to several factors, such as cultivars, environmental several potential health benefits [6] including their antioxidant, lipid-lowering activities [7]. This article presents a review under growth conditions, ripening stage, postharvest handling and the pomegranate seed oil and the potential effects of conjugated storage conditions, which, in turn, may affect the quality of the

α-linolenic acid to health. fruit as well as the extent of its beneficial health effects [10,11]. Cite this article: Melo ILP, Carvalho EBT, Mancini-Filho J (2014) Pomegranate Seed Oil (Punica Granatum L.): A Source of Punicic Acid (Conjugated α-Linolenic Acid). J Hum Nutr Food Sci 2(1): 1024. de Melo et al. (2014) Email: Central

Pomegranate, known for its high antioxidant capacity, has been used for medicinal purposes for centuries [12]. Syed et al. cis, 11trans, 13trans) and catalpic [12] investigated their potential antiproliferative, anti-invasive Other isomers9trans,11trans of conjugated,13cis linolenic) occur at acid lower (CLnA), concentrations such as and pro-apoptotic activities against various human cancer cell α-eleostearic acid (C18:3-9 time,acid (C18:3- climatic conditions, among others, have a major effect on showing phytoestrogenic and antioxidant activities have been the[18]. oil However, content of fruit seeds genotypes, as well as cultivationon its fatty acid sites, composition harvesting isolatedlineages from and the in animalseeds, juice models. and Topeel date, of the over fruit 50 and substances from the [19] found that the total lipids in pomegranate seed oil consisted is used in the treatment of stomach-ache and has proved to [3]. Using thin layer chromatography (TLC), Yuan et al. beleaves effective and flowers in preventing of the tree. Dried lipoperoxidation. peel from ripe Fruit pomegranate extracts practicallymainly of triglycerides all in the form (TG). of triglycerides Likewise, Lansky (99%). and The Newman triglyceride [1] well as in suppressing the proliferation of human breast and compositionreported significantly of PSO is variedhigh levels and theof fatty most acids important (over 95%) patterns in PSO, are have succeeded in inhibiting herpes and influenza viruses as severity of depression in ovariectomized rats were observed afterprostate administering cancer cells. pomegranate-juice Reductions in bone concentrate erosion and a in seed the CLnA-CLnA-CLnA and CLnA-CLnA-P [20]. cerebroside: this last one being a key component of the myelin extract [2,13]. Pomegranate products have also been reported sheathCertain in mammals, minor compounds,have also been such found as in sterols, pomegranate steroids seed and oil immunosuppressive activities and protective effects on liver mg to possess, among others, anti-inflammatory, antimicrobial and kg to Al-Muammar and Khan [15], the routine supplementation [1]. Phytosterols are present at high concentrations (4.1 - 6.2 function and lipid and glucose metabolism [10,14]. According of pomegranate juice or extracts may prevent or even correct mg obesity, diabetes, and cardiovascular diseases. As indicated in ) in PSO, mainly as β-sitosterol, campesterol and stigmasterol;) g their review, decreasing energy intake, the intestinal absorption mg tocopherols also occur, mainly as α-tocopherol (161 - 170 of dietary fats by inhibiting pancreatic lipase, and oxidative g 100 and γ-tocopherol (80 - 93 ) [20]. antiobesity effects of pomegranate food as a whole [15]. different pomegranate cultivars100 reported in the literature. Fatty stress and inflammation might be important mechanisms for the Table 1 shows the lipid profile of pomegranate seed oil from The fruit can be divided into three parts: seeds, accounting for acid composition is expressed as a percentage of the total fatty acids. andabout inner 3% totalmembranous weight and walls containing and accounting 20% oil; for juice, approximately accounting for approximately 30% total weight and pericarp, including skin The unique composition of PSO has encouraged specific studies into its health benefits, including weight control, skin repair and Pomegranate seeds are usually waste products from the individuals [2]. According to Koba et al. [23], since pomegranate 67% total weight [1]. aboundspositive alterations in punicic in acid, plasma their lipid seed profiles oil represents in hyperlipidaemic a suitable g

kg isomer. fruit weight processing. and varies Their according content may to cultivars range from [3]. The 40 seeds – 100 have source for the investigation of the physiological roles of this CLnA The chemical formula of punicic acid (PA), an omega-5 long (sugars, polyunsaturated fatty acids, vitamins, polysaccharides, chain polyunsaturated fatty acid and a positional and geometric polyphenolssignificant antioxidant and minerals) capacity [12]. andOn, average,a rich chemical pomegranate composition seeds c,12c,15c grown in Brazil, carbohydrate content was found to account 9c,11t,13c [24,25]. Both chemical structures are represented in isomer of α-linolenic acid (LnA; C18:3-9cis-type double), is C18:3-bonds and 33 % trans Figure 1. Theoretically, PA features 66 % offor pomegranate 43.97 %, followed seeds are by moisture mostly wasted content and 38.30 that %they and can high be -type double bonds [26]. Also, it is structurally processedlipid content to produce14.06 % an[16]. attractive Given the amount fact that of a greatchemically-rich quantities (LnA), which have been reported to have numerous health similar to conjugated linoleic acid (CLA) and α-linolenic acid oil, which may be of great interest to the food industry as food or a nutraceutical ingredient. in vivo andbenefits in vitro [6].. Therefore, conjugated linolenic acids (CLnAs) have Pomegranate seed oil (PSO) also been investigated for their potential beneficial effects Health effects of PSO Thanks to its novelty appeal, good customer acceptance, availability at low prices and rich phytochemical composition, the Pomegranate juice has been widely studied for their oil from pomegranate seeds has attracted increasing attention as a functional ingredient [17]. effectspotential [17,27]. antioxidant For instance, and anti-inflammatory they have been reported activities; to promote in the epidermalsame manner, tissue PSO regeneration has also been[27], reportedboost the toimmune have beneficialsystem in vivo, reduce the accumulation of hepatic triglycerides and display PSO represents between 12% and 20% of the seed total cis,11trans,13cis), an isomer of this chemopreventive activity against hormone-related (prostate and weight and consists chiefly of conjugated octadecatrienoic fatty fatty acid, is found in high amounts in the oil and is synthesized breast) and colon cancers [17]. inacid. situ Punicic from acid linoleic (C18:3-9 acid (a non-conjugated octadecadienoic fatty acid), present in lower amounts (about 7%) in PSO [1]. Given the similarities among CLAs, LnAs and punicic acid, J Hum Nutr Food Sci 2(1): 1024 (2014) 2/11 de Melo et al. (2014) Email: Central

Table 1:

Fatty AcidsFatty acid composition of pomegranate seed oil (PSO). Results are expressed as average (percentage of total fatty acids) ± standard deviation. Reference1 (25) (Number 2of (21) cultivars studied) 3 (15) 4 (6) 5 (1)

14:0 0.7 ± 1.5 0.18 ± 0.21 0.35 ± 0.10 16:0 5.7 ± 4.1 5.07 ± 1.30 2.45 ± 0.19 4.00 ± 0.76 4.04 ± 0.34 18:0 2.1 ± 3.1 4.20 ± 1.56 1.52 ± 0.26 2.92 ± 0.56 2.30 ± 0.21 18:1 (ω-9) 9.0 ± 5.6 7.86 ± 2.25 4.19 ± 0.61 5.68 ± 1.69 5.29 ± 0.25 18:2 (ω-6)c11t13t) 10.8 ± 6.9 8.36 ± 2.36 4.49 ± 0.49 4.08 ± 1.04 6.05 ± 0.53 18:3 (9t11t13t) 10.70 ± 4.44 6.41 ± 0.27 18:3 (9t11t13c) 8.78 ± 5.16 1.03 ± 0.16 18:3 (9c11t13c) 15.24 ± 6.17 3.48 ± 0.34 18:3 (9 71.5 ± 17.9 36.98 ± 10.12 74.11 ± 1.55 81.22 ± 2.15 58.14 ± 2.10 20:0 0.69 ± 0.14 0.39 ± 0.04 0.53 ± 0.18 0.50 ± 0.04 20:1 1.65 ± 1.85 0.61 ± 0.09 0.61 ± 0.05 22:0 0.1 0.18 ± 0.02 24:0 0.97 ± 0.94 1.00 ± 0.24 1. [21] - India; 2. [20] - China and Tunisia; 3.[3] - Turkey; 4. [22] – Georgia, USA; 5. [16] - Petrolina, Brazil - 23.05% unidentified fatty acids). O

OH O

α-Linolenic Acid Punicic acid

Figure 1

Molecular structures of punicic acid (PA) and α-linolenic acid (LnA). amounts in pomegranate seed oil (PSO), weighta CLnA foundand serum in high lipid reducing effects; however, to date, triglyceridedetected, their levels increase in mice showed fed a diet no supplemented statistical significance with PSO [30].than resultsresearch have into been the inconsistent health benefits among of PSO them. has For focused example, on Koba body Yuan et al. [19], in contrast, reported significantly lower liver et al. [23], using different seed oils (pomegranate, bitter gourd, cholesterolthose in rats levels in the were control observed. group and in rats fed CLA-enriched diet; however, no significant changes in phospholipid and total PSO showed a considerable reduction in perirenal adipose tissue Chinese catalpa and pot marigold), reported that only the rats fed To date, there have been few studies researching PSO effects on humans, and the results have also been inconsistent among thatweight punicic compared acid may with have the control played group an important (rats fed role α-linolenic in the dose- acid- enriched linseed oil). In a similar study, Koba et al. [28] suggested dependent reductions observed in perirenal and epididymal levelsthem. Recently, were detected after a four-week in hyperlipidemic treatment patients,with PSO, no whereas changes a in body composition and serum cholesterol and LDL-cholesterol adipose tissue weights in rats fed a basal diet containing 10% [19], however, observed no change in adipose tissue weight in healthydecrease subjectsin triglyceride were levels given took seeds place of Trichosanthes[31]. Yuan et al. kirilowii [24,25], rapeseed oil and 0 %, 0.25 % or 0.50 % punicic acid. Yuan et al. mice fed an experimental diet supplemented with 1 % PSO for anotherobserved natural no significant source ofchanges punicic in acid any (three serum grams lipid levelsper day, when for six weeks.

28 days).Other potential health effects have also been suggested for accumulation in obese, hyperlipidemic rats [29]. Statistically PSO. For instance, reduced body weight gain and leptin and Dietary PSO, rich in punicic acid, attenuates hepatic triglyceride phospholipid levels were observed in rats fed experimental a high-fat diet supplemented with PSO, indicating a potential dietssignificant, containing dose-dependent PSO when comparedincreases in with serum mice triglyceride in the control and risk-reducinginsulin levels wereeffect detected on the development in CD-1 mice of (pathogen-free) type 2 diabetes given [32]. group. Although higher serum total cholesterol levels were also Similarly, diet supplementation with PSO was found to improve

J Hum Nutr Food Sci 2(1): 1024 (2014) 3/11 de Melo et al. (2014) Email: Central glucose tolerance isomer of conjugate [33]. In addition, PSO was reported not to affect liver insulin sensitivity, but to improveand suppress peripheral obesity-related insulin sensitivity inflammation in mice d linolenic acids (CLnAs), which is synthesized from linoleic acid through a specific conjugase enzyme [23,39]. vegetable oils, as a result of isomerization and dehydration of increase carbohydrate oxidative capacity and concluded that it CLnAs may also be produced during the processing of preventsfed a high-fat diet-induced diet [18]. obesity They and also reduces found thatinsulin PSO resistance. was able to [37,43]. Although alkaline isomerization is effective in mass Pomegranate see oil (PSO) was also shown to exhibit in secondary oxidation products of linoleic and α-linolenic acids vivo neutrophil-activation and lipid peroxidation consequences, producing conjugated linolenic acid (CLnAs), the resulting which antioxidant may be useful and in anti-inflammatory the prevention and activities treatment by of limitingseveral acidsmixture have comprises been reported numerous to have isomers some of importantdifferent CLnAs, and singular which physiologicalmakes identification effects, such difficult. as anticarcinogenic However, these and synthetic anti-obesity fatty rheumatoid arthritis and coronary heart disease [34]. It led to improvedinflammatory renal diseases, function suchand reduced as inflammatory protein and bowel lipid disease,damage inhibitory[23]. Coakley effect et on al. cancer [44] investigatedcells observed six may strains be related of intestinal to the nephrotoxicity [35]. The mechanisms involved, however, are yet bifidobacteria for their anticancer activity and suggested that the toin be rats elucidated. undergone hexachloro-1,3-butadiene (HCDB)-induced abilityMetabolism of the bacteria and incorporation to convert LnA to of CLnAs. CLnAs Conjugated α-Linolenic Acid (CLnA) c,11t,15c c,13t,15c) was apparently and, since then, research into their cytotoxic effect on cancer cells Plourde et al. [45] showed that a CLnA equimolarc,11 t mixture) when as wellConjugated as body linoleic fat-reducing acid (CLA) and was lipid first metabolism-normalizing identified in the 1980s (C18:3-9ingested under nutritional and C18:3-9 and physiological conditions. Both absorbed as efficiently as rumenic acid (C18:2-9 They were metabolized by the elongation/desaturation pathway effects has advanced; more recently, since 2000, other fatty acids CLnA isomers were mainly incorporatedc,13t,15c isomer into was neutral converted lipids. containing conjugated double bonds, CLnAs, have also attracted c,11t,15c increasing scientific attention as novel types of functional fatty similarly to LnA. The C18:3-9 acids because of their cytotoxic and antitumor properties [36]. to the 20:5 w-3 conjugated isomer while the C18:3-9 describing a group of octadecatrienoic fatty acid isomers with isomer was converted to the 22:6 w-3 conjugated isomer. Yuan et Conjugated α-linolenic acid (CLnA) is a collective term cis or al. [46] reported that mice fed a six-week diet supplemented with trans 1% punicic acid (PA) showed a significantly higher proportion three conjugated double bonds (C18:3), which may be of of 22:6 w-3 in liver phospholipids than that in mice given a diet Source, configuration biosynthesis at positions and isomers 9,11,13 and of CLnAs8,10,12 [3,27]. consistent with the observations made in a study by Koba et al. supplemented with 1% α-eleostearic acid (α-ESA), which is

[23], who also reported high levels of 22:6 w-3 in the liver of mice CLnAs occur in plant lipids, especially seed oils [37], and seed oil. On the other hand, four-week diet supplementation fed a diet supplemented with 0.5% PA-containing pomegranate represent between 40 and 80 % of total fatty acids [38, 39]. Table 2 shows the five most commonly known isomers found in seed acid (PA), calendic acid, jacaric acid and catalpic acid [24,25]. In with α-ESA-containing bitter gourd seed oil led to increased oils from important plants: α-eleostearic acid (α-ESA), punicic fact, according to Sassano et al. [27], there are other two isomers, proportion of 22:6 w-3 in rat livers [47]. t,11t,13t cells, as well as that of their bioconversion products, 9c,11t- t t,12t), making it seven in total. The distributiont,11t of the CLnA accumulated by the Caco-2 β-eleostearic (β-ESA; C18:3-9 ) and β-calendic (C18:3- 8 ,10 Trichosanthes kirilowii CLA and 9 -CLA, into the different lipid classes was also Maxim. (TK) seeds are edible. The punicic acid content of this determined. It was observed that Caco-2 cells take CLnA (α- and Of all the CLnA-containing seeds, only β-eleostearic, catalpic or punicic acid) up at different rates and then convert them at CLAs, but with varying efficiency depending popular Chinese snack ranges between 32 % and 40 % of total on the structure of the Δ13 double bond. The distribution of CLnA seedThese weight isomers [24,40-42]. are found in select seeds, commonly as the linked to their number of trans double bonds: the higher the most abundant fat number,between the neutral higher lipids the accumu (NL) and phospholipids appeared to be

Table 2: ty acids. Each plant seed accumulates only one lation in the NL fraction [48].

Plant Major isomers of conjugated α-linolenic acid found in plantFattyacid seeds. Isomericconformation % in theoil Tung (Aleutritesfordii) 9cis,11trans,13trans

Bittergourd(Momordicacharantia) α-eleostearic 9cis,11trans,13trans 70 Pomegranate (Punica granatum) α-eleostearicPunicic 9cis,11trans,13cis 6072 Catalpaovata) 9trans,11trans,13cis 31

CatalpaPotmarigold ( (Calendulaofficinalis) Catalpic trans trans,12cis 33 Jacarandasp) Calendic 8cis ,10trans,12cis 32 Jacaranda ( Jacaric 8 ,10 References: [7,23]. J Hum Nutr Food Sci 2(1): 1024 (2014) 4/11 de Melo et al. (2014) Email: Central

In general, in vivo stud with Trichosanthes kirilowii

ies have shown that CLnAs are (TK) seeds, containing 3 g PA, for 28 efficiently absorbed in the organism and metabolized to CLAs days led to 0.47 % and 0.37 % increasesc, 11t in the proportion of PA [4,23,42,49], which has increased interest in CLnAs. These in plasma and red blood cell membranes (RBCM), respectively. In adenineresearchers dinucleotide suggest that phosphate) CLnAs may dependentbe metabolized enzyme, to CLAs which via humansaddition, are the ableproportion to effectively of C18:2-9 incorporate increased PA into from plasma 0.05 and to isa Δ13 either saturation a novel reaction enzyme catalyzed capable by of a recognizing NADPH (nicotinamide conjugated 0.23 % in plasma and from 0.03 to 0.17 % in RBCM. Apparently, trienoic acid or the enzyme active in the leukotriene B4 reductive may be associated with increased 9c,11t pathway. RBCM and the results also suggest that PA supplementation -CLA levels in humans, In a study by Tsuzuki et al. [49], aimed at investigating the probably via a Δ13-saturation reaction. c, 11t The potential ability of organisms to convert CLnAs to 9 lymphabsorption from and the metabolismthoracic duct, of both punicic isomers acid (PA) were and slowly α-eleostearic absorbed consider-CLA hasusing attracted certain seed increasing oils, such interest as Trichosanthes given the reportedkirilowii inacid unaltered (α-ESA) form, in rat and intestine some of using them were a lipid rapidly absorption converted assay into in (TK),beneficial pomegranate biological and effects bitter gourd, of this as CLA alternative isomer. direct One sources should metabolized to 9c,11t CLAs. Likewise, Yuan et al. [42] observed that PA was quickly of CLnAs and indirect sources of CLAs, taking into account their -CLA in rat plasma and different rat tissues. high content of CLnAS, mainly α-ESA e PA, and the moderate Levels of the CLA isomer and PA were undetectable right after Healthsimplicity effects of purification of CLnAs [25,42]. levelstreatment in liver (t=0); tissue both and were plasma detected were in rathigher plasma than and those tissues in the at brain,t=4, 8, heart,12 and kidney 24 h (each and adiposerat fed about tissues. 645mg PA). PA and the CLA Recently, conjugated fatty acids have been the focus of The accumulation of PA in all the tissues examined was scientific research because numerous studies have shown their beneficial effects in a variety of experimental models of metabolic andAlthough chronic inflammatory a consensus aboutdiseases the [18]. health effects of conjugated toobserved 9c,11t to be considerably higher than that of α-ESA [41,46]. fatty acids in animals and humans does not yet exist and They also reported a higher relative rate of conversion of α-ESA knowledge about mechanisms is limited at present, a number -CLA in liver phospholipids and triglycerides. The of studies have reported encouraging results. For instance, in highest conversion rates of α-ESA was found in adipose tissue (91.8 %), spleen (91.4 %) and kidney (90.7 %), whereas the highest conversion rate of PA was observed in the liver (76.2 onstudies human by Grossmann cancer cell et cultures. al. [6], Igarashi They have and also Miyazawa been shown [50] and to %). The conversion rate of both α-ESA and PA was lowest in inhibitYasui et carcinogenesis al. [51], CLnAs [52,53,54]have been shown and alter to have lipid cytotoxic metabolism effect in the heart, 84.6 % and 54.5 %, respectively [46]. As regardscis- chemical structures, PA and α-ESAtrans only-type differ double in terms bond. of Such the animals [23,30,38,55]. Grossmann et al. [6], Tsuzuki et al. [54], structuralconfiguration feature of the either double may bond affect at positionthe degree 13, ofi.e. saturation PA has a of type double bond and α-ESA has a Igarashi and Miyazawa [56] proposed a mechanism of antitumor action of CLnAs by which CLnAs may induce apoptosis through for the conversion enzyme, which may explain the better the conversion enzyme or may make α-ESA a specific substrate lipid peroxidation and the protein kinase C pathway. among isomers [57], however, their mechanisms of action remainInterestingly, to be elucidated. the biological Moreover, activities some in ofvivo CLnAs studies may provided differ conversion rate of α-ESA observed. Yuan et al. [25,42] suggested andthat incorporated CLnA-CLA conversion selectively. enzymes More research, may be however, synthesized is needed in the liver. These results indicate that CLnA isomers are metabolized evidence that CLnAs can be metabolized to CLAs, which may be the actual compounds responsible for the health benefits to fully understand the mechanisms of CLnA conversion to CLA. attributed to CLnAs. Some of the potential beneficial effects of Cao et al. [43] have reported the selective nature of CLnA CLnAsEffects are on discussed body composition, below. lipid profile and glucose used to supplement the diet given to maternal rats, 9c,11t,13c- metabolism incorporationtrans in-type milk double lipids bond) in rats. was Of found the three in highest CLnA levels isomers in milk lipids, followed by 9c,11t,13t trans-type double bonds)CLnA (one and then by 9t,11t,13t trans-type double composition, serum lipids, and glucose metabolism and insulin -CLnA (two resistanceTo date, have studies yielded of the controversial effects of CLnAs results. on weight gain, body -CLnA (three bonds). Interestingly, CLAs were detected in milk from maternal rats fed the diet supplemented with CLnAs, suggesting that they Diet supplementation with 1 % CLnAs (α-ESA and/or PA) for are able to metabolize CLnAs to CLAs. These results also suggest six weeks did not significantly affect food intake neither body and withthat only the oxidoreductaseone double bond catalyzing in the trans the reduction of the Δ13– tissue weights in mice [19,24]. Yamasaki et al. [30] reported no double bond may have exhibited greater affinity for the CLnA seedchanges oil (PSO), in body rich weight in punicic and adipose acid (PA) tissue for three in C57BL/6N weeks. Similarly, mice fed configuration. experimental diets containing 0.12 % and 1.2 % pomegranate investigatedReports on the the metabolism metabolism and andincorporation incorporation of punicic of CLnAs acid in humans are scarce in the literature. Yuan et al. [25] have showedOtsuka Long-Evansno changes Tokushimain abdominal Fatty white (OLETF) adipose rats tissue fed weights a two- week diet supplemented with 9 % safflower oil and 1 % PSO (PA) in healthy young subjects (n=30). Daily supplementation J Hum Nutr Food Sci 2(1): 1024 (2014) 5/11 de Melo et al. (2014) Email: Central

antioxidants naturally found in foods have attracted increasing

[29]. In contrast, when investigating the effects of CLnAs on growth through a mechanism involving lipid peroxidation body fat, Koba et al. [58] observed a reduction in adipose tissue interest [26]. CLnAs have been shown to suppress tumor cell moreweight potently in Sprague-Dawley than linoleic rats acid after (LA), four conjugated weeks of feeding. linoleic Also, acid CLnAs were found to reduce perirenal adipose tissue weight [54,56]. Grossmannµmol et al. [63] found that when breast cancer cells were treated with α-ESA), cell in growth the presence inhibition of the and antioxidant apoptosis L (CLA) and α-linolenic acid (LnA). Koba et al. [28] noted a dose- α-tocotrienol (20 dependent reduction in perirenal adipose tissuet weightt,12c) inled ICR to able to block breast cancer cell proliferation and induce apoptosis decreasedCD-1 mice bodyfed a dietfat content supplemented in mice [59].with PSO for four weeks. Diet througheffects of a mechanism α-ESA were that lost, may suggesting be oxidation that dependent. this CLnA isomerAlthough is supplementation with calendic acid (C18:3-8 ,10 mechanism, many studies have yielded controversial results CLnAs exert their biological activities via an oxidation-related Some studies reported that diet supplementation with CLnAs resulted in significant reductions in serum total cholesterol (TC) [4,7,64]. [58,60], apoB-100 [61] and liver tissue triglyceride (TG) levels [29,61]. Yang et al. [38], in contrast, noted no changes in serum plasmaNoguchi hydroperoxide et al. [47] reported levels and that liver rats lipid treated oxidation, with BGO despite (bitter a total cholesterol (TC) levels neither in serum TG, HDL-C and non- gourd oil), containing α-ESA, showed significant increases in HDL-C levels,g but a significant decrease in liver TC levels when hamsters- 12.7 were fed a diet supplemented with PA or α-ESA (12.2 previous study by Dhar et al. [65], who had reported that rats fed for kg four weeks with the same CLnA, α-ESA from karela (bitter gourd) ) for 42 days. Although Yamasaki et al. [30] reported seeds, at 0.5 % showed lowered lipid peroxidation, assessed supplemented diet, incre when compared to the control group (dietary oil). Mukherjee no significant effects on TC in mice fed a 0.12 % or 1.2 % PSO- by measuring TBARS (thyobarbituricacid reactive substances), ased serum TG and phospholipid levels punicic acid (PA) at different concentrations on lipid peroxidation were found. Koba et al. [28] observed no significant effects on et al. [40] investigated the effects of diet supplementation with serum lipids in mice given PA; however, liver TG levels were however, a pro-oxidant effect was also noted at 1.2 % PA. Under shown to be significantly lower than those in the control group, in rats and observed maximum antioxidant activity at 0.6 % PA; which is consistent with the observations made by Yuan et al. lower hydroperoxide formation by reducing the formation of free the conditions described above, CLnAs may have been able to [19], who reported a significant reduction in liver TG levels, radicals and the peroxidation of PUFAs (polyunsaturated fatty when compared to the control group, but no significant effects on acids) in erythrocyte membranes and other lipids. Alternatively, plasma TG, TC, HDL-C and LDL-C levels when mice were given a biohydrogenation or free radical addition to one of the conjugated six-week diet containing PA or α-ESA. studied to a limited degree. When PSO was administered to formation of conjugated dienes, which, in turn, may have acted hyperlipidaemicThe effect of CLnAs subjects on forthe fourlipid weeks,profile noin humans favorable has effects been double bonds of CLnAs may have taken place, resulting in the

as antioxidants [26,65]. on TC and LDL-C levels, but reductions in TG levels and in the peroxidation in rats with alloxan-induced diabetes mellitus, When investigating the dietary effects of CLnAs on lipid TG:HDL-C ratio were observed [31]. Further studies are required to confirm the reported effects of the intake of CLnAs on body erythrocyte lipid peroxidation in each of the experimental groups their mechanisms of action. Dhar et al. [60] observed significant reductions in LDL and weight gain and blood serum lipid profiles as well as elucidate (0.5 % CLnAs, 0.15 % α-tocopherol and 0.25 % CLnAs + 0.15 % t,11t,13c plasmaHontecillas glucose et and al. [62] insulin found concentrations that diet supplementation and improved with the greaterα-tocopherol) reduction when in livercompared lipid and with membrane the control lipid group. peroxidation The diet catalpicplasma glucose-normalizing acid (C18:3-9 ability) for following 78 days a increasedglucose tolerance fasting containing 0.25 % CLnAs + 0.15 % α-tocopherol also led to a showed that treatment of streptozotocin-induced diabetic albino than the diet containing only α-tocopherol. Saha and Ghosh [66] punicictest in ratsacid (PA). with McFarlin diet-induced et al. obesity. [32] reported Hontecillas that PSO et al. intake [33] lowered oxidative stress, reduced lipid peroxidation and restored have reported similar results in mice fed another CLnA isomer: therats levelswith CLnAs of the (α-ESAantioxidant or PA enzymes at 0.5 % [superoxidasetotal lipids), significantly dismutase developing(average 61.79 type-2 mg diabetesper day) wasmay associated have been with reduced. an improvement In addition, in insulin sensitivity in CD-1 mice, suggesting that the risk of and(SOD), erythrocyte catalase (CAT) lysate. and gluthatione peroxidase (GPx)], reduced gluthatione and nitric oxide (NO) synthase in the pancreas, blood diet-induced obesity and insulin sensitivity in mice. In contrast, These same authors also investigated the antioxidant dietVroegrijk supplementation et al. [18] observed with Trichosanthes that PSO intake kirilowii improved Maxim. high (TK) fat 15 days) against sodium arsenite-induced oxidative stress. fasting serum insulin and glucose levels nor insulin sensitivity, Sodiumactivity of arsenite two isomers altered of the CLnAs activities (0.5 %of totalantioxidant lipids given enzymes for seeds in humans for 28 days had no significant effects on neither in plasma, liver homogenates, the brain and erythrocytes. After resistance) [24]. assessed by HOMA-IR (homeostasis model assessment – insulin Antioxidant activity of CLnAs administration, both CLnA isomers, α-ESA and PA, were capable As oxidative stress-related diseases have advanced, the of increasing the activity of SOD, CAT and GPx and lowering the activity of NO synthase to their normal levels. Although both CLnA isomers were able to successfully lower oxidative stress, J Hum Nutr Food Sci 2(1): 1024 (2014) 6/11 de Melo et al. (2014) Email: Central they performed differently, which can be explained by their distinct distribution of cis-trans trans-type double bonds, protein in Caco-2 cells and in rats [51,53] and tung oil, containing double bonds, α-eleostearic, α-ESA, was shown to activate PPAR-γ in human umbilical vein mixture(α-ESA), of featuring both isomers a greater has shownnumber synergistic of activity and better endothelial cells [52]. Recent studies show that the immune proved to be more effective than punicic acid (PA) [26,67]. But lesionsmodulatory in mice actions with ofdextran α-ESA sodiummay be bothsulfate PPAR-γ induced dependent colitis [5]. and PPAR-γ independent to ameliorate disease activity and intestinal In an study, aimed at evaluating the effectiveness of PA)protection caused thanamelioration individually of renal treated oxidative isomers stress at 0.5 and % the dose isomers [68]. in vitro showedFurthermore, synergistic it has activity.been shown Administration that CLnA isomersof blended (α-ESA product and of both the isomers caused better restoration of renal fatty acids certain CLnA isomers found in pomegranate seed oil as selective estrogen receptor modulators (SERMs), PA was found to inhibit and other altered parameters [69]. estrogen receptors (ER) α and β at 7.2 and 8.8 µM, respectively; F2-isoprostane and a reliable marker of oxidative stress formed α-ESA inhibited ER α and β at 6.5 and 7.8 µM, respectively [13]. Significantly higher levels of 8-iso-PGF2α, the most abundant in vivo as a result of the non-enzymatic, free radical-catalyzed usedThus, as both a potential CLnA isomers breast cancerare effective chemopreventive as SERMs. Theseagent. results peroxidation of arachidonic acid, were found in human urine indicate that PA, abundant in PSO and an effective SERM, could be after diet supplementation with TK seeds, rich in PA, suggesting that punicic acid (PA) is able to increase lipid peroxidation in Saha and Ghosh [66] showed that four week-diet humans [24]. supplementation with CLnAs (α-ESA and PA at 0.5 % total Molecular Actions of CLnAs lipids) led to reduced inflammation in streptozotocin-induced diabetic, albino rats by significantly reducing the expression of The ability of natural dietary components to regulate certain inflammatory cytokines, such as TNF-α and IL-6, in blood and molecular events represents an alternative therapeutic strategy diabetes induction. the expression of hepatic NF-κB (p65), once higher as a result of On the other hand, one study reported that administration disease, rheumatoid arthritis, atherosclerosis and metabolic syndrome.for major health These concerns diseases worldwide, share some such features; as inflammatory for example, bowel the of 400 mg of pomegranate seed oil (rich in punicic acid) twice daily in dyslipidemic patients has no effect on serum TNF-α [71]. overproduction of proinflammatory mediators (TNFα, GM-CSF, found in pomegranate seed oil and other foods may represent a novelThis result therapeutic shows thatalternative although strategy some studies for some indicate diseases, that such CLnAs as cells,IL-1, IL-6, such IL-8, as neutrophils, leukotriene monocytes B4 and PAF) and and macrophages reactive oxygen [34]. species (ROS) and the presence of highly activated inflammatory needed to prove these effects in humans. hyperactivation in vitro inflammatory diseases and cancer [34], additional studies are When investigating PA effects on TNFα-induced neutrophil Final Considerations and on colon inflammation in rats, the same researchers observed that PA inhibited TNFα-induced priming of ROS production and MPO (myeloperoxidase) release abundant presence of punicic acid (PA), an isomer of conjugated In recent years, studies worldwide have confirmed the by neutrophils, suggesting a potent anti-inflammatory effect. in vivo this reason, PSO has been increasingly investigated as a potent the Bassaganya-Rierakeratinocyte growth et factor al. [70] and provided the orphan molecular nuclear evidencereceptor functionalα-linolenic food acid and/or (CLnA), nutraceutical in pomegranate ingredients seed in oil foods. (PSO). But For in that PA intake up regulates colonic PPAR-δ expression, as well as other health effects would have to be considered. RORγ expression and suppresses colonic and M1 macrophage- that case, the impact of the intake of CLnAs on lipid metabolism (i.e.,derived mucosal TNF-α. inductive Also, PA sites). was They shown suggested to increase that PA the modulates levels of in vitro and animal studies have IL-17 and IFN-γ in CD8+T cells in the mesenteric lymph nodes haveEven been though inconclusive numerous and/or contradictory results as well. provided evidence of the bioactive properties of CLnAs, there PAmucosal was reported immune to responseslower fasting and plasma improves glucose gut concentrations, inflammation Furthermore, human studies are scarce and some effects of through PPAR-γ and -δ-dependent mechanisms. Likewise, dietary improve the glucose-normalizing ability, suppress NF-κB healthCLnAs observed remains toin beanimal determined. models differ Further from studies those arereported needed in humans. Therefore, the exact role of CLnAs in improving human Theiractivation results and demonstrate TNF-α expression that PA andcan bind up regulate and robustly PPAR activate α- and to fully understand the mechanisms of action as well as the γ-responsive genes in skeletal muscle and adipose tissue [33]. recommendations regarding its usage. PPAR-γ and increase PPAR-γ-responsive gene expression, which physiological effects of CLnA isomers and to establish safety and will result in improved diabetic and inflammatory status. Studies Acknowledgments of other CLnA isomers have yielded similar results. In a previous study by Hontecillas et al. [62], catalpic acid was shown to up The authors would like to thank São Paulo State Research inregulate mice; suggestingPPAR-α and that its responsivethe metabolic genes effects but notof catalpic to significantly acid on affect PPAR-γ and –δ expression in white adipose tissue (WAT) Foundation (FAPESP), which supports our research (Grants 09/51890–0R eference ands 09/51891–7). rich in 9c,11t,13t- glucose and lipid metabolism may be mediated through a PPAR 1. α-dependent mechanism. Bitter gourd oil, CLnA, was reported to increase the levels of PPAR-γ mRNA and Lansky EP, Newman RA. Punica granatum (pomegranate) and its J Hum Nutr Food Sci 2(1): 1024 (2014) 7/11 de Melo et al. (2014) Email: Central

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Cite this article Melo ILP, Carvalho EBT, Mancini-Filho J (2014) Pomegranate Seed Oil (Punica Granatum L.): A Source of Punicic Acid (Conjugated α-Linolenic Acid). J Hum Nutr Food Sci 2(1): 1024.

J Hum Nutr Food Sci 2(1): 1024 (2014) 11/11