Pii: s0378-8741(98)00222-0

Journal of Ethnopharmacology 66 (1999) 11–17 Antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice flavonoids Shay Yehoshua Schubert a, Ephraim Philip Lansky b, Ishak Neeman a,* a Laboratories of Food Engineering and Biotechnology, TechnionIsrael Institute of Technology, Haifa32000, Israel b Rimoni Corporation, Science Park, Nesher, Israel Received 8 April 1998; received in revised form 9 November 1998; accepted 20 November 1998 Abstract
The antioxidant and eicosanoid enzyme inhibition properties of pomegranate (Punica granatum) fermented juice and seed oil flavonoids were studied. The pomegranate fermented juice (pfj) and cold pressed seed oil (pcpso) showedstrong antioxidant activity close to that of butylated hydroxyanisole (BHA) and green tea (Thea sinensis), andsignificantly greater than that of red wine (Vitis 6itifera). Flavonoids extracted from pcpso showed 31–44% inhibitionof sheep cyclooxygenase and 69–81% inhibition of soybean lipoxygenase. Flavonoids extracted from pfj showed21–30% inhibition of soybean lipoxygenase though no significant inhibition of sheep cyclooxygenase. The pcpso wasanalyzed for its polyphenol content and fatty acid composition. Total polyphenols in pcpso showed a concentrationby weight of approximately 0.015%. Pcpso fatty acid composition showed punicic acid (65.3%) along with palmiticacid (4.8%), stearic acid (2.3%), oleic acid (6.3%), linoleic acid (6.6%) and three unidentified peaks from which two(14.2%) are probably isomers of punicic acid (El-Shaarawy, M.I., Nahpetian, A., 1983). Studies on pomegranate seedoil. Fette Seifen Anstrichmittel 83(3), 123–126). 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Pomegranate; Cyclooxygenase; Lipoxygenase; Antioxidant; Eicosanoids; Punica granatum 1. Introduction
main use of pomegranate is as table fruit, butlarge amounts are used in the beverage and liquor Pomegranate (Punica granatum), a small tree industries (Nagy et al., 1990). The pericarp, con- originating in the Orient, belongs to the Puni- taining up to 30% tannins, is used in tanning caceae family (Harde et al., 1970). Pomegranate is grown mainly in Iran, India and the USA, but In folk medicine, pomegranate preparations, also in most Near and Far East countries. The especially of the dried pericarp, but also of theroots, barks of the tree and roots, and the juice of * Corresponding author. Fax: +972-4-832-0742.
the fruit, are employed as per orum medication in 0378-8741/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 9 8 ) 0 0 2 2 2 - 0 S.Y. Schubert et al. / Journal of Ethnopharmacology 66 (1999) 11–17 the treatment of colic, colitis, diarrhea, dysentery, collection from the Neve Yaar Research Station, leucorrhea, menorrhagia, oxyuriasis, paralysis and Volcani Agricultural Research Organization, rectocele, and as external applications to caked Ministry of Agriculture, State of Israel in the breast (Duke and Ayensu, 1985) and to the nape southern Galilee. A sample of mixed cultivars was headache (Ayensu, 1981). Further, a number oftherapeutic actions of these materials have been 2.2. Preparation of fermented plant juice (pfj ) described including vermifugal, taenicidal, astrin-gent, antispasmodic, antihysteric, diuretic, carmi- The seeds of the fruit containing the intact juice native. sudorific, galactogogue and emmenagogue sacs were manually separated from the pericarps, and the sacs ruptured by very light agitation in an Flavonoids, a broad class of polyphenolic com- electric blender for 2–3 s. The mixture of the juice pounds widely distributed among photosynthesiz- and the seeds was then added to a high quality ing cells, possess an impressive array of sterilized plastic jug (ordinarily used for storing pharmacological activity (Hasten, 1983). These spring water). To 16 l of this mixture was added 5 include: free radical scavenging, inhibition of a g of wine yeast, Saccharomycs bayanus (Lalvin vast spectrum of enzymes, and estrogenic activity.
EC-1118) obtained from Lallemand, Montreal, Consequently, a potential role for these com- Canada. A sterile surgical glove was affixed to the pounds in several therapeutic functions is appar- neck of the bottle with a rubber band which ent. As anti-inflammatory agents, flavonoids may served as a pressure release valve, and fermenta- be effective against parodentitis and local pain, tion was allowed to proceed at room temperature without the gastric irritating effects of aspirin and until complete (10 days). A portion of the wine was then decanted and gradually evaporated to (which also act through inhibition of cyclooxyge- one-tenth of its original volume to yield the pfj Flavonoids have also been suggested as cancer-protective agents, if not therapeutic ones (Hasten, 2.3. Preparation of cold pressed pomegranate seed 1983) and the consumption of dietary flavonoids was inversely correlated with coronary heart dis-ease in a population of elderly men (Hertog et al., After the completion of fermentation of the 1993). In the present work we studied pcpso and juice, the seeds were removed by straining and pfj for their antioxidant activity (Hammerschmidt dried in the sun, or alternatively, over an electric and Pratt, 1978) and inhibitory effects on lipoxy- radiator. The dried seeds were then cold pressed genase and cyclooxygenase, key enzymes in the in a Tiby Press Type 55 machine with a 7-mm eicosanoids pathway. Lipoxygenase inhibition was nozzle manufactured by Skeppsta Maskin of Ore- determined using soybean 5-lipoxygenase (Gross- bro, Sweden. A 5.3% yield of oil per dry weight of man and Zakut, 1979) and cyclooxygenase inhibi- tion using sheep cyclooxygenase from sheepvesicular glands (Van der Ouderaa et al., 1977).
2.4. Fla6onoid extraction from pcpso Flavonoid extraction from the pcpso was ac- 2. Materials and methods
complished with the method previously describedfor olive oil (Vazues et al., 1973). A 10-g aliquot of pcpso was moved with 50 ml hexane in aseparation funnel and polyphenols extracted with Plant material was collected by one of the three volumes of 60% methanol. The methanol authors (E. Lansky) through the courtesy of the phase was then moved to a second separation late Professor Dan Palevitch from the cultivar S.Y. Schubert et al. / Journal of Ethnopharmacology 66 (1999) 11–17 methanol phase was then collected and dried taken immediately after addition of the emulsion with anhydrous Na2SO4 and again dried in a to the antioxidant solution against a blank con- vacuum evaporator at 40°C. The resultant taining absolute ethyl alcohol (Carlo Erba, polyphenols were resuspended in methanol and Italy). The tubes were stoppered and placed in a extracted with three portions of chloroform, water bath at 50°C, with readings taken at 15- each half the volume of the methanol phase.
min intervals for 90 min. Controls consisted of The chloroform was removed and the methanol butylated hydroxyanisole (BHA, Sigma), green dried again in the vacuum evaporator at 40°C.
tea (Bi Luo Chun, Hua Sheng Wen Ju Factory, The polyphenols were resuspended in water and Su Zhou, China) and red wine (Cabernet Sauvi- extracted with petrol ether (60–80) until a clear organic phase was obtained. The water phasewas saturated with NaCl and extracted with four portions of ethyl acetate (EA), each a thirdof the water phase volume. The EA fractions Polyphenols were determined using a spec- trophotometric method (AOAC, 1990). Folin Na2SO4. The EA was dried in a vacuum evapo- phomolybdic acid (20 mg, 50 ml) were distilled for 2 h in reflux, chilled and diluted to 1 liter inDDW. Subsequently, 35 g Na 2.5. Fla6onoid extraction from pjf solved in 100 ml DDW, left overnight for crys-tallization and filtered.
The pomegranate fermented juice extract was To obtain a calibration curve, to different combined with two times its volume of EA, concentrations of tannic acid were added 0.5 ml shaken vigorously, and left for 8 h. The EA phase was then dried in the vacuum evaporator lowed by DDW until a total volume of 10 ml was achieved. Readings were taken at 760 nm after 30 min. Polyphenols were determined in asimilar manner, but instead of tannic acid the 2.6. Determination of antioxidant acti6ity Antioxidant activity was determined by mea- suring the coupled oxidation of carotene and linoleic acid (Fluka, Germany), a modificationof a method previously reported (Hammer- schmidt and Pratt, 1978). Approximately 10 mg vesicula seminalis (Yamamoto, 1982). Ten vesi- trans-b-carotene (type 1 synthetic, Sigma, St cles from freshly slaughtered sheep were homog- Louis, MO) was dissolved in 10 ml of chloro- enized in three volumes of potassium phosphate form. The carotene–chloroform solution, 0.2 ml, was pipetted into a boiling flask containing 20 ml linoleic acid and 200 ml Tween-40 (Sigma).
centrifuged at 12 000cg for 15 min and the After removal of the chloroform with N2, 50 ml surfactant centrifuged at 100 000cg for 1 h.
of double distilled water (DDW) was added to The pellet containing the microsomal fraction the flask with vigorous swirling. To tubes con- was dissolved in Tris–HCl buffer (Sigma) con- taining the putative antioxidants in 2 ml ethanol, 5 ml of the aliquots of these emulsions and 20% glycerol, centrifuged at 27 000cg for were each added to final concentrations by 30 min, and the surfactant containing the dis- weight of 0.01%. Spectrophotometric readings at solved enzyme was collected into small contain- 470 nm (Ultraspec II spectrophotometer) were S.Y. Schubert et al. / Journal of Ethnopharmacology 66 (1999) 11–17 2.9. Determination of the acti6ity of flame ionization detector and coupled to a Ku- The activity of cyclooxygenase was determined coated with 10% FFAP. Column temperature was using a polarographic assay employing an O2 programmed from 190 to 210°C. Nitrogen was electrode. Oxygen uptake was measured as the the carrier gas. Mixtures of authentic standard change in dissolved oxygen concentration cata- fatty acids methyl esters were chromatographed lyzed by cyclooxygenase and measured using a under the same conditions for comparison.
Clark (O2) electrode. The substrate was arachi- donic acid 90% purity (Sigma), 0.1 mM in Tris–HCl, pH 8.0 buffer and Hemin (chlorid) (FlukaGermany) 1 M. The enzyme was preincubated for 3. Results and discussion
2 min with the inhibitor, then added to the reac-tion cell containing the substrate at 30°C. Hy- droquinone (Fluka Germany), 0.041 mg/ml, was pomegranate fermented juice (pjf) extract and added immediately prior to the reaction. In- pomegranate cold pressed seed oil extract (pcpso) domethacin (Sigma), a known cyclooxygenase in- are compared with the chemical antioxidant stan- hibitor, was used as a positive control.
dard, BHA, and the most popular botanical an-tioxidants, green tea and red wine. As can be 2.10. Determination of the acti6ity of pomegranate fractions was significantly superiorto that of red wine. Conversely, the antioxidant The activity of soybean lipoxygenase (Sigma) activity of the pomegranate fractions approached, was similarly determined using a polarographic, but did not surpass, the antioxidant activity of oxygen-measuring assay. Oxygen uptake was as- sessed as the change in dissolved oxygen concen- The measurement of antioxidant activity de- tration catalyzed by lipoxygenase and measured picted in the figure is accomplished through a using the aforementioned Clark electrode. The coupled oxidation of linoleic acid to a variety of substrate in this case was linoleic acid, 7.5 mM, future oxidation-provoking oxidation products, dispersed in water with the help of Tween 20, and and b-carotene, whose pigment is readily and

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