Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013 Extraction, Separation and Phytochemical Analysis of Lignan from Nutmeg ( Myristica Fragrans) Seeds aEssam F. Al-Jumaily,aShymaa I. Al Barzanchi aBiotechnology Department, Genetic Engineering and Biotechnology Institute for postgraduate students; University of Baghdad, Iraq Abstract In this study natural lignan dimer was isolated from nutmeg seeds using organic solvent, partially purified using liquid/liquid partiation and purified using anion exchanger and chemically characterized using Molish’s, Benedict’s, Fehing’s and Ferric chloride tests screening, then by the aid of UFLC-PDA-IT-TOF-MS System the molecular weight (657.221 ) and the molecular formula(C 40 H48 O8 ) of this dimer were determined after that the free radical scavenging activities was studied using stable free radical compound DPPH, results showed that this purified dimer had 75% scavenging activity while the partial purified dimer had 45% compared with vitamin C. KEYWORDS :Lignan; ( Myristica fragrance ) nutmeg; phytochemical analysis Introduction Myristica fragrance belongs to Myristicaceae family in order Magnoliales which contain about 150 genera and more than 3000 species. (Asgarponah and Kazemivash, 2012). Nutmeg produces two separate spiciousos, namely nutmeg and mace. Nutmeg is the dried Kernel of the seed and mace is the dried aril surrounding it. Myristice species are indigenous to India and Sri Lanka and now cultivated in many tropical countries (Barceloux, 2008). Nutmeg contains about 2% of lignan, which are nonvolatile dimmers. Lignan refers to a group of natural compounds comprising n-phenyl propane bound to B-position of the n- propyl side chain and is widely distributed in nature. There have been studies on the various physiological activities of lignan. Such as blood glucose- lowering action, anticancer action, anti- asthmatic action and whitening action. for example. It was reported that lignans isolated from sesame, such as sesamin, episesamin, sesaminol, sesamolin and episesaminol, have anti-inflammatory effects (Korean Patent Publication No. 1997-7001043). Lignan compounds isolated from Magnoliae floss can be used antiasthma tic agents (Korean patent Registration No. 0263439). Moreover macelignan is atypical lignan compound found in Myristica fragrans (Tuchinda. et al .,2002 ). Park, ( 2004) was reported to have various activities, such as the activation of caspase-3 during apoptosis . The aims of this study are to extraction and separation of ligana from nutmeg (Myristica fragrans) Seeds. Materials and Methods The dried seeds of Myristica fragrans were collected from local market in Baghdad during November /2011 and identified by the botanist professor Dr. Ali Almosawi in the College of Sciences/ Baghdad University. Firstly, nutmeg seed was cleaned from derbies which include other plants seeds, some parts of vegetarian of nutmeg and dust, Secondly www.oiirj.org ISSN 2249-9598 Page 1 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013 nutmeg seed was grinded by a grinder by a coffee grinder to a fine powder and stored in a closely tight container until used. Defatting Layer Stage This stage involved defatting of nutmeg oils by using Soxhelt apparatus according to (AACC, 1984). So (50) g. of powder was put in a thumble and (300) ml of petroleum ether was added within (40-60) 0C for (6) hours when time was over, petroleum ether was substituted by Chloroform with the same volume and the same time, continuously on the same program. Extraction of Crud Lignan The method which was described by Rickard et al, (1996) was used, (25)g of defatted powder was treated with a mixture of Dioxan and Ethanol alcohol (1:1)(v:v),respectively, with a ratio (1:8),(w:v),(powder: solvent),sample was placed on magnetic stir for (4)hrs., then filtrated by whatmann filter paper No.1. The solvent was evaporated by rotary evaporator at (40) 0C to obtain crud lignan. Separation of Lignan The process of separation Alkaline hydrolysis of SDG oligomers was done by dissolving a certain amount of crud dry lignanin an alkaline hydrolysis solution (a methanolicNaOH , 20 mM,pH=8) at 50 ºC for hydrolyzing SDG oligomers.(Li et al . ,2008).The mixture was filtered by filter paper then the supernatant was concentrated with a rotary evaporator within (45) 0C. Eventually, a thick sticky texture material was obtained ,pH was corrected into 3.0 through adding drops of sulfuric acid 2 molar then the sample was stored in (4) 0C. Liquid/Liquid Partition This method involves (liquid/ liquid) separation according to Westcott and Muir,(1996).There were two separating solvent systems which were differed in their polarity these systems include: Ethyl acetate: distilled water with ratio(1:7). Two layers were formed and take aqueous layer. This process repeated twice and the aqueous layer was concentrated with the rotary evaporator at (45) 0C to produce dry crude. Preparative Thin Layer Chromatography There was a method mentioned byHarborne (1973) was used. Aluminum plated (20x20cm) coated with silica gel thickness 1mm type (60F254). The separation solution ( Benzene:ethanol) was used with ratio (1:9)(v:v).When the solvent system moves about (15) cm from the spots plate directly pulled out and dried by air and illuminated under UV light source. The plate was examined at (280) nm wave length and the Rf value was calculated according to this equation: Rf = distance of sample (cm) /distance of solvent (cm) www.oiirj.org ISSN 2249-9598 Page 2 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013 Purification of Lignan: The anion exchanger Sephadex Q25 QAE (Pharmacia Co.) was used for lignan purification and was prepared according to Westcott and Muri (1998) then packaged as acetate form in (2.5×15) cm and the flow rate is 1 ml/ min. 2ml fractions were collected , then the D.W. was replaced by using 50% glacial acetic acid in 15% ethanol to remove substances that attached to the exchanger.All fractions were detected for lignan using Benedict reagent. Chemical tests for the Lignan compound By using the Molish’s, Benedict’s, Fehing’s and Ferric chloride test there were done according to (AOAC ,1984). Lignan Characterization By HPLC-TOF-MS System Shimadzu UFLC-PDA-IT-TOF-MS system , it is combination of HPLC and Mass spectrum that connected with fractionation system, so we able to purify the compounds by fractionation the samples according to the retention time and in the same time high sensitive TOF- mass identify molecular weight of compounds by measuring the time of fly (TOF) for each single atom and suggest possibility of compound identification using software data base.Shimadzu system (advance analytical HPLC) consist of dual pump solvent delivery system. Programmable fluorescence detector and water XBidge 2.2×50 mm column. The sample injection volume of auto injector was set to 100 µL, the solvent system consisting of water: acetonitrile:methanol (30:35:35 vol/vol%) as a mobile phase, the flow rate was 1 ml/min. Antioxidant Activity Antioxidant activity of partial purified lignan and purified lignan was detected using DPPH Radical Scavenging Assay according to Romeilah et al , (2010). 130 µl of methanol was added to each well of microtiter plate.20 µl of each sample (partial purified lignan, purified lignan and vitamin C) were added separately .Serial 10 fold dilution were done for each sample.50 µl of DPPH (Freshly prepared DPPH solution (0.004% w/v) was added for each well.Microtiter plate was incubated at 37 C for one hour.Radical scavenging activity of samples against the staple DPPH radical was determined spectrophotometrically using ELISA , the colorimetric changes ( from deep- violet to light- yellow) when DPPH is reduced were measured at 517 nm . The Scavenging activity of DPPH (%) was calculated by using the following equation – % DPPH radical scavenging = [(Absorbance of control - Absorbance of test Sample) / (Absorbance of control)] x100.Were the absorbance of negative control which was (1:9) (vol:vol) of ( DMSO : Methanol ) mixture and Abs. sample was the absorbance of samples( partial purified lignan, purified lignan and vitamin C which was the positive control). Results and Discussion Chemical Detection When purification of lignin was done by colum chromatography, chemical examination was carried out by using chemical reagents. These were specified for sugar www.oiirj.org ISSN 2249-9598 Page 3 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013 groups that are bound to lignan structurally, lignan contained two sugar moieties, therefore these reagents were used to detect these groups ,including firstly Molish’sreagent and the violet color appearance is an indication of lignan presence, While in Benedict’s test, the orange color which then changed into red sediment in the bottom of test tube is an indication for lignan presence. This result was supported by Fehling’s reagent where red sediment appeared , while in Ferric Chloride test the appearance of Intense Green colure was another evidence for sugar moieties presence in lignan compound and this agrees with AL-Awaad (2001) and AL-Al-Juamily(2012) whose used these reagents for lignan chemical detection . Table (1) shows chemical reagents which had been used in detection of the sugar moieties in lignan compound.