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5003-5012, 2013 ISSN 1819-544X This Is a Refereed Journal and All Articles Are Professionally Screened and Reviewed

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5003-5012, 2013 ISSN 1819-544X This Is a Refereed Journal and All Articles Are Professionally Screened and Reviewed 5003 Journal of Applied Sciences Research, 9(8): 5003-5012, 2013 ISSN 1819-544X This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLES Efficient Extraction and in-vitro Control of Trichomonas vaginalis of Peelsʹ Total Phenols and Flavonoids for Selected Egyptian Pomegranate Cultivars. Abdel-Hady, N.M. Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. ABSTRACT Pomegranate peels contain significant quantities of phenol compounds which possess significant versatile biological activities; different methods, solvents and conditions were used for efficient extraction of the total phenols and flavonoids from two selected Egyptian pomegranate cultivars (Wardy and Assuity), the efficiency of extraction was expressed quantitatively using reported spectrophotometric methods, the gained results revealed that the Wardy cultivar exhibited higher total phenol and flavonoid contents compared to the Assuity cultivar, the highest calculated values of total phenols were (252.09±3.10 & 329.57± 2.85 mg GAE g-1) and (237.70±3.61 324.30±3.06 mg GAE g-1) for Wardy and Assuity cultivars using soxhlet and microwave-assisted extraction methods, the most efficient solvents for extraction were 50%, 25% methanol and water respectively while the highest calculated values of total flavonoids were (166.38±2.15 & 191.90±1.85 mg QE g-1) and (154.56±1.81 & 188.40±1.79 mg QE g-1) in Wardy and Assuity cultivars using soxhlet and microwave-assisted extraction methods, the most efficient solvents for extraction were 75%, 100% and 50% methanol respectively. In-vitro screening of anti-trichomoniasis vaginalis effect of the prepared extracts revealed that all exhibited promising results compared to the reference standard Metronidazole where the effect can be attributed to the total phenol content rather than the total flavonoid content. Kew words: Pomegranate, Phenols, Flavonoids, Soxhlet, Microwave, in-vitro, Trichomoniasis vaginalis, Metronidazole. Introduction Pomegranate (Punica granatum L. , family; Punicaceae) is a phytochemical rich nutrient source which is widely consumed as fresh and in commercial products as juices, jams, and wines (Seeram et al., 2006; Miguel et al., 2010; Nuamsetti et al., 2012). The world pomegranate production reaches approximately one billion and half tons where peels constitute approximately 50-60% of fruits weight (Shabtay et al., 2008; Mirzaei- Aghsaghali et al., 2011; FAOSTAT-FAO, 2012). Pomegranate peel is characterized by the existence of numerous polyphenol compounds which are of hydroxybenzoic acid derivatives as gallic and ellagic acids, hydroxycinnamic acid derivatives as caffeic, chlorogenic and p-coumaric acids or of cyclitol carboxylic acid derivatives as quinic acid (Hassanpour et al., 2011, Al Hazzani et al., 2013), in addition to flavonoids as catechin, epicatechin, epigallocatechin-3-gallate, quercetin, kaempferol, luteolin, kaempferol-3-O-glycoside, kaempferol-3-O-rhamnoglycoside and naringin, moreover, peel contains also anthocyanins as cyanidin, pelarginidin and delphinidin, ellagitannins as punicallin, punicalagin, corilagin, casuarinin, gallagyldilacton, pedunculagin, tellimagrandin, granatin A and granatin B(Wang et al., 2010; Prakash and Prakash , 2011, Maddah et al., 2012), such content of phenol compounds have been implicated in most pharmacological actions referred to pomegranate peel (Althunibat et al., 2010; Viuda-Martos et al., 2010) Pomegranate peel exerted diverse pharmacological actions as antioxidant (Thring et al., 2009; Ahmed and Ali 2010), antifertility (Umadevi et al., 2013), cytotoxic (Kulkarni et al., 2007; Abdel motaal and Shaker 2011), hepatoprotective (Murthy et al., 2002), hypoglycemic (Hontecillas et al., 2009), anti-atherosclerotic (Parmar and Kar 2007), antibacterial (Naz et al., 2007), anti-candidal (Tayel and El-Tras 2010) and antiviral (Zhang et al., 1995) activities. Solvent extraction is the traditional procedure to extract phenol compounds from plant sources to liberate them from the vacuolar structures (Wang et al., 2011), cold and hot extraction are associated with large amounts of solvents and extended periods of time used while hot extraction has the same disadvantages in addition to the possibility of degradation of labile chemicals (Dai et al., 2001; Singh et al., 2011;Wissam et al., 2012) while modern techniques as microwave-assisted extraction (MAE) and ultrasonic extraction (USE) targeted avoidance of such disadvantages. MAE provides a modern technique that use microwave energy to heat the Corresponding Author: Abdel-Hady, N.M., Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. E-mail: [email protected] 5004 J. Appl. Sci. Res., 9(8): 5003-5012, 2013 solvent and the sample aiming extraction of specific compounds from the sample into the solvent have been used for extraction of bioactive compounds from wide variety of plants and natural residues (Wang et al., 2007; Nemes and Orsat, 2010; Singh et al., 2011). Trichomoniasis vaginalis is an important worldwide health problem where Metronidazole has so far been used in treatment in spite of development of metronidazole-resistant strains and its unpleasant adverse effects, hence screening of medicinal plants as alternative regimens for treatment becomes an urgent demand to provide effective, safe and inexpensive drug (El-Sherbini et al., 2010; El-Sherbini and Shoukry, 2012). The present study was conducted to access the most efficient methods for extraction of total phenols and flavonoids from pomegranate peel monitored with in-vitro screening of the anti- Trichomoniasis vaginalis effect of different prepared extracts. Material and Methods Plant Material: Samples of pomegranate cultivars used for this study were collected from Assuit and El-Badary, Upper Egypt, Egypt on September 2011 and were kindly identified by Prof. Dr. Moneer Abdel-Ghany, Prof. of Plant Taxonomy, Faculty of Science, Cairo, University. Fruits for each cultivar were manually peeled; the peels were separately air-dried, finely powdered and kept in tightly closed amber coloured glass containers, protected from light at low temperature as possible. Voucher specimens are kept in herbarium, Pharmacognosy Department, Faculty of Pharmacy Al -Azhar University, Cairo, Egypt. Chemicals: Folin-Ciocalteu's reagent was purchased from Sigma Chemical Co., Saint Louis, MO, USA; gallic acid, quercetin, aluminum chloride were purchased from E. Merck, Darmstadt, Germany; Metronidazole (Amrizole® tablets 500 mg), were purchased from Al Amrya co. for pharmaceuticals while all the solvents used were of analytical grade. Apparatus: Soxhlet; rotatory evaporator (BUCHI Rotavapor ® R-210/R-215, Germany); CEM-MARS-5 Microwave (CEM, Matthews, NC); Thermo Fisher Scientific USA, Genesys Spectrophotometer (Milton Roy, INC., Rochester, NY); 96 Micro-well plates and haemo-cytometer (Neubauer cell-counter chamber). Preparation of peel extracts: - Soxhlet extracts; 15 of g powdered peel of each sample were extracted separately in a soxhlet with 100 ml of distilled water, 25%, 50%, 75 % v/v aqueous methanol, pure methanol and ethyl acetate at three temperature levels 25, 50 and 75 0C for two time periods 1 and 2h, filtered under vacuum through Whatmann No.1 filter paper and dried under vacuum at 400 C to yield different extracts. - Microwave-assisted extracts: 15 g of powdered peel of each sample were extracted separately with 100 ml of distilled water, 25%, 50%, 75 % v/v aqueous methanol, pure methanol and ethyl acetate separately in pyrex vessels and placed inside the microwave at 800 w, medium stirring and holding conditions for 10, 20 and 30 min; extracts were filtered under vacuum through Whatmann No.1 filter paper and dried under vacuum at 400 C to yield different extracts. Estimation of total phenol content: The total phenol content of individual extracts of each cultivar was determined spectrophotometrically using Folin-Ciocalteu's reagent where standard curve was done using different concentrations of gallic acid in methanol. The concentrated extracts were dissolved each in least methanol volume then completed to 10ml, 100µl of these extracts were separately diluted with 8 ml distilled water, to each sample 0.5 ml of 50% Folin- Ciocalteu's reagent was added and left 8 min, and then 1.5 ml of 5% sodium carbonate was added, mixed and allowed to stand for 60 min. protected from light. Their absorbance was measured at 725 nm using methanol as 5005 J. Appl. Sci. Res., 9(8): 5003-5012, 2013 blank and the concentration of the total phenolic content was calculated as mg gallic acid equivalents per g dry weight (mg GAE g-1) (Zhou and Yu 2006). Estimation of total flavonoid content: The total flavonoid content of individual extracts of each cultivar was determined colourimetrically using aluminum chloride solution where standard curve was done using different concentrations of quercetin in methanol, 100µl were added to a 96 Micro-well plate and then 100µl of 2% aluminum chloride solution in methanol were added, after 10 min, their absorbance was measured at 415 nm using methanol as blank and the concentration of total flavonoid content was calculated as mg quercetin equivalent per g dry weight (mg QE g-1) (Djeridane et al., 2006). In-vitro screening of anti- Trichomoniasis vaginalis effect: - Parasites and culture: T. vaginalis was isolated from female patients attending the Obstetrics and Gynecology Clinic at Al- Zah’raa University hospitals, Cairo, the trophozoites were
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