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Solvents extraction efficiency for lycopene and β-carotene of GAC fruit ( cochinchinensis, Spreng) cultivated in Iraq

Article in Bioscience Research · October 2017

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Shurook Saadedin Salwa J Abdullah Al-Awadi University of Baghdad Al-Nahrain University , college of biotechnology ,Iraq,Baghdad

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The user has requested enhancement of the downloaded file. Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2017 14(4): 788-800. OPEN ACCESS

Solvents extraction efficiency for lycopene and β- carotene of GAC fruit (Momordica cochinchinensis, Spreng) cultivated in Iraq

Shurook Mohammad Kadhim Saadedin1, Iqbal Harbi Mohammed Al-Zaidi1, 2 and Salwa Jaber Abdullah AL-Awadi3

1Institute of Genetic Engineering and Biotechnology, University of Baghdad, Baghdad, Iraq 2Ministry of Agriculture office –Horticulture, Baghdad, Iraq 3College of Applied Biotechnology – Al Nahrain University, Baghdad, Iraq .. *Correspondence: [email protected] Accepted: 20 Sep. 2017 Published online: 07 Nov. 2017 Momordica cochinchinensis Spreng or Gac fruits is a tropical vine originating from South and South-East Asia . The has the potential to be a high-value as anticancer supplements, particularly fruit aril for its lycopene contain. The plant introduced and cultivated in Iraqi 2016. The usefulness of the fruit products is put emphasis on processing methods. In the present study, three different organic solvents mixture, Ethanol\ethyl acetate (6:4), Hexane\ ethyl acetate\ Ethanol (2:1:1), Hexane\acetone\ Ethanol (2:1:1) and 100% Hexane were investigated to find the solvents that extracts the highest lycopene and β-carotene from Gac aril fruit powders. The extracts analyzed by high-performance liquid chromatography (HPLC) to measure the concentration of these bioactive compounds. Result showed that the combination of ethanol / ethyl acetate (6:4) had the best extraction efficiency for lycopene (40640 μg/g) while Hexane\acetone\ Ethanol (2:1:1) had the best extraction efficiency for β-carotene (2912 μg/g) Keywords: lycopene and β-carotene, Gac aril powders, Organic solvents, HPLC, Extraction efficiency

INTRODUCTION of (Tran 2007). The fruits contain Gac (Momordica cochinchinensis, (Lour.) lycopene and β-carotene a natural source of Spreng.) is considered as an indigenous tropical antioxidants (Kim et al. 2007; Tuyen et al. 2013). plant to grown in many countries In the meantime, β-carotene transforms to vitamin including Vietnam, Laos, Thailand, China, India, A in the body. This vitamin is necessary for the Cambodia, Philippines, Malaysia and Bangladesh health and cell membranes growth (Kubola and (Vuong et al. 2006; Kubola and siriamornpun Siriamornpun 2011). 2011; Wimalasiri 2015) Gac, is called a fruit from Few containing lycopene are known. heaven belongs to the family. A Tomato and its products are the main source of synonym is Momordica macrophylla Gage, lycopene (George et al. 2004). Tomato and other common name in English: Chinese Bitter fruits and vegetables consist lycopene are shown Cucumber. Vernacular name in China: Da Ye Mu in table 1. Though, many researches point out that Bie Zi, Vietnam: Dia Ta Piea, Thailand: Bai- Khai- Gac fruits are the most important source of Du, Malaysia: Teruah (Lim 2012). The red seed (β-carotene and lycopene), mainly the membrane (seeds aril) and aril oils are used as a red seed membrane that comprise of 70 times constituent of medicine in different ethnic societies more lycopene than tomato (Rao and Rao 2007). Saadedin et al. Solvents extraction efficiency for lycopene of Gac fruit

Table 1: Lycopene content of lycopene-rich fruit and vegetables μg/g modified from different references:

Sources of Lycopene μg/g References

Bitter melon DW 411 Tran and Raymundo1999 Bitter melon FW 142-170 Interiano 2009 Carrot ( Red) 61 Surles et al. 2004 Guava 54 Mangels et al.1993 Momordica subangulata Singh, et al, 2014 aril 57 pulp 52 (turning yellow) Momordica subangulata Singh et al. 2014 aril 142 pulp 453 (ripe) Papaya 20–53 Rao and Rao 2007 Papaya 16-34 De Souza et al. 2008 Papaya 11 Malviya 2014 Persimmon 0.2 -0.4 Olives- Barba et al. 2006

Pink grapefruit 34 Mangels et al. 1993 Tomato 27 Malviya 2014 Tomato 43- 60 Shahzad et al. 2014 Tomato 100-150 Dumas et al. 2003 Tomato (GM) 300 Mehta et al. 2002 Tomato ( Cherry) 89 Lilwani and Nair 2015 Tomato ( Pakistani) 56 Lilwani and Nair 2015 Tomato juice 100 Rao and Rao 2007 Tomato ketchup 124 Rao and Rao 2007 Tomato paste 365 Rao and Rao, 2007 Tomato peel DW 63 -107 Choksi and Joshi 2007 Tomato peel FW 5-11 Choksi and Joshi 2007 Tomato pulp DW 51 - 125 Choksi and Joshi 2007 Tomato pulp FW 2- 5 Choksi and Joshi 2007 Tomato Raw 31 Holden et al.1999 Tomato Raw 8.8–42 Rao and Rao 2007 Tomato sauce 131 Rao and Rao 2007 Water melon 41 Mangels et al.1993

Water melon 23-72 Rao and Rao 2007

Water melon 49 Holden et al. 1999

Water melon 65-73 Olives- Barba, et al. 2006

Water melon 75 Lilwani and Nair 2015 Water melon 32 Malviya 2014

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. The highest content of lycopene in fresh aril in fully ripe fruit was reported as 3.728 Outcomes indicate the potential use of Gac mg/g whereas β –carotene levels were 0.379 aril extracts as promising source of antimicrobial mg/g (Nhung et al. 2010). The levels of β- against several bacteria (Sajjan et al. 2010; carotene and Lycopene in dried aril powders were Innuna 2012 and Tinrat 2014).The plant aril has 0.9-1.8 g/100g and 3-5 g/100g respectively (parks been shown to help prevent coronary artery et al. 2013). Extraction methods and an evaluation disease (Akkarachaneeyakorn et al. 2015), to be of the levels of lycopene and β –carotene from active as an anticancer agent against colon different references is shown in Table 2. cancer (Tien et al. 2005) and associated with Lycopene is C40H56 , thermal reduce risk of several types of cancers as prostate resistant determined with different spectral and cancer, lung cancer and digestive (Goula and chromatographic methods (Choudhary et al. Adamopoulos, 2005; Tuyen et al. 2013). Develop 2009) such as High Performance Liquid cell to cell gap joint communication, defeat of Chromatographic (HPLC), Ultraviolet-Visible, insulin-like growth factor and stimulation of Fourier Transformer Infrared spectroscopy, and apoptosis (Mein et al. 2008). others. The choice of extraction methods for lycopene Human health is an important issue basically and β-carotene analysis from Gac aril is very affected by food consumption in both qualified and important; there is no exclusively established quantified directions to provide energy and method for carotenoid extraction. The common maintain health and prevent or minimize diseases accepted method is extraction with organic especially cancers. The rate of cancer cases solvents, polar or non – polar solvent dependent shows increasing trend in Iraq over the last 15 on the polarity of carotenoids. Mainly used years (Mousa 2012). The experiments were solvents for carotenoid extraction are, acetone- performed to introduce and cultivate Gac plant to chloroform (1:2) and hexane-acetone-ethanol Iraqi as a source of lycopene, then quantify (2:1:1) (Olives- Barba et al. 2006), Hexane- lycopene and β- carotene from Gac seeds aril Acetone (1:1), Hexane- Ethyl acetate (1:1), powder, using high-pressure liquid hexane- ethanol (1:1) ( Strati and Oreopoulou chromatography (HPLC), as influenced by 2011). different non-toxic environmental friendly In a review on carotenoid extractions De extraction solvents as compared with most used Quirós, and Costa ( 2006) stated, Acetone is one (hexane/acetone/ethanol, 2:1:1 v/v/v) and widely used for carotenoid extraction and listed determine their efficiency. solvents as tetrahydrofuran (THF), n-hexane, pentane and mixtures as: dichloromethane: MATERIALS AND METHODS methanol (6:1) acetone: petroleum-ether (1:1), THF: methanol (1:1), n-hexane: toluene (1:1), n- Plant materials hexane: acetone: ethanol (2:1:1). THF: Diethyl M.cochinchinensis (Lour.)Spreng seeds aril oil, ether: methanol used by (Ishida, et al. 2004). lycopene 0.4% and Gac seeds were imported Acetone: Diethyl ether: Acetonitrile (Aoki et al. from Sabiwe Vietnam Co., Ltd- Vietnam. 2002) and Hexane and THF (Vuong et al. 2006). Seeds were introduced from Vietnam for THF- Methanol (1:1) and Methanol- Ethyl acetate cultivation. The hard outer covering of the seeds (6:4) were the most effective in extracting removed before germination, the germinated carotenoids from Corn (Rivera and Canela 2012). seedlings were transplanted into 150 mm pots The references point to results of lycopene (sand: peat moss 1:1) growing media, Individual and β-carotene analyses in Gac fruit were very plants were transplanted in April 2016 to broad, extraction methods and contents of University of Baghdad Research Station (44° lycopene and β –carotene are shown in Table 2. 24'E, 30° 20'N and 34.1 m altitude). Sun-shade For instance, Lycopene was found to be mainly Green plastic net in summer and plastic existing in concentration of up to 380 µg\g of Gac greenhouse cover in winter were used to protect seed aril and β-carotene 101 µg\g (Aoki et al. the plants. Irrigation used as the plants need to 2002), (408.4 μg/g) lycopene and β-carotene avoid drought and fertilizer applied in two weeks (83.3 μg/g) (Vuong et al. 2006). While (Tran et al. interval. In September the female flowers were 2016) reported that the peak content of lycopene 0.45 mg/g and β –carotene 0.33 mg/g in fresh weight was in fruit harvested after 14 weeks from

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Table 2. Extraction methods and contents μg/g or ml of Lycopene and β-carotene in Gac fruit modified from different references Lycopene µg\g or ml β –carotene µg\g Extraction solvents References or ml aril oil 50 192 Screw Press Akkarachaneeyakorn et al. 2015 380 FW aril 101 FW acetone Aoki et al. 2002 Peel 2000 DWTotal Ethyl acetate Chuyen, et al. 2017 Carotenoids aril 1342 -2972 FW 636-836 FW Ice cold methanol Ishida et al. 2004 Aril 847 DW 29 DW Ethanol 80% : Hexane Jang, and Kim 2014 (1:1)+ 2% pyrogallol: BHT (100 mg/L) Aril oil 4140 1400 Microwave -drying, Kha et al. 2013 steaming and hydraulic pressure Aril oil 5110 1740 Microwave-drying Kha et al. 2014 prior to hydraulic pressing Aril 7020 300–540 FW hexane/acetone/ Kubola and Siriamornpun Pulp 180–620 FW Ethanol: 2:1:1 v/v/v). 2011 Pulp (green)1690 FW Peel 160–340 mg/g FW 160–590 FW Aril oil 490 1180 chloroform:methanol Kubola et al. 2013 Aril 450 90 (2:1 v/v) Aril 2378 – 3728 FW 257 – 379 FW Nhung et al. 2010 Aril 30000-50000 DW Parks et al. 2013 Aril 2000–2300 FW Rao and Rao 2007 Aril 270190 DW 197750 DW Acetone. Tanongkankit et al. 2014 Aril 450 FW 330 FW ethanol: hexane (4:3 v/v) Tran et al. 2016 Aril 408 FW 83 FW Vuong et al. 2006 aril oil 2990 2710 Vuong, and King. 2003 Aril 95 - 3798 FW Ethanol Wihong et al. 2017 (determined by (n-hexane/DW/ethanol, spectrophotometer) 56:10:34 v/v/v) (n- hexane/acetone/ethanol, 2:1:1 v/v/v) Aril 300- 4015 FW hexane/acetone/ethanol, Wihong et al. 2017 (determined by HPLC) 2:1:1 v/v/v Aril 3050 FW 100-770 FW Wimalasiri 2015 Himedja (India). Methanol and tetrahydrofuran HPLC-grade were purchased from Fisher hand pollinated. At maturity, the ripe fruits with red Scientific. All chemicals and reagents used in the skin were harvested. The seeds aril was experiments were of analytical grade. separated from the fruits and freeze- dried for 15 hours (Tran 2007). The freeze-dried seeds aril Extraction solvents were ground in a mill and passed through a 500 Four solvents or solvents mixtures were used for mesh sieve, then stored at -20ºC until use. extraction: Hexane 100% (Kubola et al. 2013), Hexane\acetone\ Ethanol (2:1:1v/v/v) (Olives- Reagents and standards: Barba 2006 and Kubola and siriamornpun 2011), Standards trans-lycopene and β-carotene were ethanol\ethyl acetate (6:4v/v) and Hexane\ ethyl purchased from Sigma (St. Louis, MO, USA). The acetate\ Ethanol (2:1:1v/v/v) (first time used for grade solvents used in liquid–liquid extraction Gac carotenoids extraction). were supplied by Baker (Germany) in case of Acetone and Hexane, by Baker (Netherlands) in Lycopene and β-carotene extraction case of Ethanol absolute, and Ethyl acetate by In order to reduce oxidation and isomerization, Lycopene and β-carotene extraction performed

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Saadedin et al. Solvents extraction efficiency for lycopene of Gac fruit quickly, with minimum light, oxygen and heat (De A = 4 (factor comes from 4 mL of re-disbanded Quirós and Costa 2006). Extractions were solvents) performed using modified method of Tran, 2007 B = 7.02(factor to change the 150mg (with 5.06% and Tanongkankit, et al. 2014. A total of 150 mg moisture) to 1g dry weight) of the dried seeds aril powder of Gac fruit was Moisture content calculates flow the method placed in a test tube. Five mL of the extraction 984.25 (AOAC 2000). solvents were added. The tube was vortexed for 30 s and centrifuged at 3000 rpm for 10 minutes. Fourier Transformer Infrared Spectroscopy The pellet was re-extracted twice following the (FTIR): previous steps then combined with the first FTIR spectra with infrared instrumentation ABB TM extract, then filtered through Whatman No. 1 filter Spectro Lab Horizon MB , UK were used. Drop paper, dried in a rotary evaporator under vacuum of lycopene standard placed between two KBr (70 mbar), in water bath at 50 Co. The dried plates and turn gently to obtain a nice even film. extracts were re-suspended to volume of 4 mL. All The plates were sited into the FTIR Spectroscopy experiments were conducted using glassware holder and a spectrum was obtained. 2 mg of wrapped with aluminum foil. Ethanol\ethyl acetate (6:4) extract dissolve in Dichloromethane to prepare concentrated solution High Performance Liquid Chromatography to obtain a spectrum of it using the same method (HPLC): Gac seeds aril powder extracts were placed in RESULTS ultrasonic bath for 20 min and passed through a The cultivated plants start flowering in filter (Whatman No. 1). After filtration, 20 μl of September, flowerings period were for two months each of standard and diluted sample were injected for the male plants and for shorter period for into the HPLC for the quantification of lycopene female plants. On average, a plant produces 10 and β- Carotene using RP-Kromasil C18 column fruits and it takes about 70 days for a fruit to (250 x 4.6 mm), 5 µm particles size. Mobile mature, the ripe fruits were harvested in phase: methanol: THF tetrahydrofuran (90:10; November. The fruits are oblong, 20-22 cm in v/v), Isocratic elution, flow rate: 1.2 ml/min., length, 24-26 diameters and weigh 1.5-1.8 kg. detector: ultraviolet with wavelength 472 nm as Moisture content of Gac seeds aril powder described by Roh, et al. (2013) with the minor was 5.1%. The calculations of Lycopene and β- modifications, for evaluation of extraction Carotene content per gm adjusted to 0% moisture efficiency: content.

Calculation: Concentration of sample µg / mL =

× conc.of standard × dilution factor × A × B where:

Figure 1: M. cochinchinensis A: Germinated seed B: Seedling C: Vine leaves D: Flower bracts E: Male flower F: Female flower G: Immature fruit different stages

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The results of retention time from six HPLC to the solvent front in comparison with standard). runs are shown in figure. 1 and table.3 the From the retention times it can be stated, that all Lycopene and β- Carotene retention time of the extracts contain Lycopene and β- Carotene standards and Hexane, Hexane\acetone\ Ethanol constituents. All solvents that were examined had (2:1:1v/v/v), Hexane\ ethyl acetate\ Ethanol a sharp peak for lycopeneand β- Carotene in the (2:1:1v/v/v) extracts, Gac crude Oil were run HPLC chromatogram indicating no contaminants consecutively. Ethanol\ethyl acetate (6:4v/v) were present. extract was run a few seconds later (this applied

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Figure. 1. Chromatogram s of lycopene and β- Carotene of standard solution, four different extracts of Gac seeds aril powder, of Gac grown in Iraq and crude Gac Oil from Vietnam. Chromatographic conditions: RP- Kromasil C18 column (250 x 4.6 mm),5 µm particles size. Mobile phase: methanol: THF tetrahydrofuran (90:10; v/v), Isocratic elution, flow rate: 1.2 mL/min., detector: ultraviolet with wavelength 472 nm.

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The effect of different solvents or solvent mixture lowest yield of β-carotene and lycopene (887, showed variation in extracts content of Lycopene 24365 µg/gm respectively). Among the studied and β- Carotene of Gac seeds aril powder as solvents, those containing ethyl acetate exhibited seen in table 3, figure 3 and 4. Ethanol\ethyl the highest lycopene extraction efficiency, and acetate (6:4) resulted in the highest content of similar extracted β-carotene content. When ethyl lycopene 40640µg/gm and second highest β- acetate replaced with acetone in Hexane\acetone\ carotene 1033 µg/gm compared to the other Ethanol mixture, it gave the highest content of β- solvents. Whereas hexane extract showed the carotene 2912 µg/gm. Table 3: β-carotene and lycopene retention time/min, area, Concentration in samples and contents µg\g or ml Carotene of standard solution, four different extracts of Gac seeds aril powder of Gac grown in Iraq and crude Gac Oil from Vietnam Extraction method Constituents Retention Area Concentration Content time/min In samples µg\g or ml Standard β- carotene 2.93 94904 20µg/ml 20µg/ml Lycopene 5.65 31239 20µg/ml 20µg/ml exane β- carotene 2.925 15005 31.6 887 Lycopene 5.678 135540 867.7 24365 Ethanol\ethylacetate β- carotene 3.248 17497 36.8 1033 :4 Lycopene 6.037 226071 1.447,3 40640 Hexane\ ethylacetate\ β- carotene 2.925 17176 36.2 1016 Ethanol 2:1:1 Lycopene 5.697 200444 1.283,3 36035 Hexane\acetone\ β- carotene 2.897 49208 103.7 2912 Ethanol 2:1:1 Lycopene 5.697 158839 1.017 28557 Crude il β- carotene 2.942 54858 578 578 Lycopene 5.688 1315774 4.346,3 4346 Dilution factor 10 dilution factor 50 The lycopene contents of lycopene-rich fruit and degree of ripeness. In this study β- carotene and vegetables were range 0.2-453 µg\g table 1. lycopene of Gac seeds aril powder were range While β- carotene and lycopene of fresh or dry 887-2912 and 24365-40640 µg\g or ml weight of Gac aril were range 29- 197750 and 50- respectively as seen in table 3. These perhaps 270190 µg\g respectively cited from different depend on modifications of extractions solvents references as shown in table 2. These may be and environmental conditions determined by the variety, altitude, latitude, environmental conditions, cultivation methods and

Crude Oil 0.43 2.85 Hexane\ ethylacetate\ Ethanol 3.60 4.06 Hexane 2.43

Figure. 3. Lycopene content g\100g of Gac fruit powder by different extraction solvents

Crude Oil 0.05 0.29 Hexane\ ethylacetate\ Ethanol 0.10 0.10 Hexane 0.08

Figure. 4. β –carotene content g\100g of Gac fruit powder by different extraction solvents

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To verify the experimental results, crude oil of to carotene and cis-lycopene isomerase; these seeds aril with lycopene 0.4%, from Sabiwe results are in agreement with a previous study of Vietnam Co., Ltd- Vietnam were examined. Ishida et al., 2004 when Isocratic separations Lycopene content resolute by HPLC method for were accomplished using a reversed phase, C30 quantitative estimation of Lycopene in the crude column (250 - 4.6-mm I. D.), 3- μm particle oil was 0.43% as shown in fig 3. It is similar to diameter polymeric. The mobile phase was Sabiwe company percentage. MTBE: MeOH: EtOAc (40:50:10 v/v/v) and the Chromatograms in fig.1. Show that the flow rate was 1 ml/min . At a column temperature number of pecks between lycopene peck and β- of 28°C and detection wavelength was set Carotene ranges from 3-5. These peaks are due between 300 and 700 nm as shown in fig.5.

Figure.5. Isocratic RF-HPLC C30 column (250 _ 4.6-mm I. D.), 3- μm particle diameter polymeric.Mobile phase: MTBE: MeOH :EtOAc 4:5:1 (v/v/v) , flow rate: 1 ml/min . column temperature of 28°C detection wavelength 300 - 700 nm Ishida. et al., 2004

Figure. 6. FTIR spectra of GAC fruit aril powder Ethanol\ethylacetate(6:4,v/v) extract. The spectra show typical bands arising from stretching C-C, C=C (1168- 2928 [cm-1] as well as asymmetrical and symmetrical stretching C-H and 3440 cm-1 [cm-1]) (stretch OH). Other bands -1 -1 -1 occur at 1033 cm (stretching CH) 1024 cm (CH3 stretching of poly (C=C) system), 1056 cm (C=O, glucose), and 1264 cm-1 (stretching of HC-OH in carbohydrate

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FTIR analysis: Administration FDA ( Strati and Oreopoulou Infrared spectroscopy is qualitative and 2011). That makes them the best solvents for quantitative technique used for analysis of carotene extraction. Another important parameter organic, inorganic, and metallo-organic is the price of industrial ethanol which makes it compounds depending on functional group cost effective comparing to the commonly used vibration character. Lycopene (Fig. 6) as organic solvents, and have a relatively high flash point. compound contains several functional groups that Acetone has been chosen because it is widely showed their vibration modes when exposed to used for carotenoid extraction alone ( Aoki et al. infrared radiation. Lycopene-FTIR spectrum in gac 2002, De Quirós and Costa 2006 and fruit showed (1168, 1640, 2856, and 2928) cm-1 Tanongkankit et al. 2014) or as a mixture with belonged to stretching C-C, C=C as well as hexane, mostly hexane/acetone/ethanol, (2:1:1 asymmetrical and symmetrical stretching C-H. v/v/v) see Table 2. Also, 3440 cm-1 (stretch OH), 1033 cm-1 As shown in Figure 1, the total area of Hexane -1 (stretching CH) 1024 cm (CH3 stretching of poly peaks was equal to 299167, which is the lowest (C=C) system), 1056 cm-1 (C=O, glucose), and area compared to different solvents mixture. 1264 cm-1 (stretching of HC-OH in carbohydrate) Ethanol: Ethylene acetate mixture of 6: 4 v/v has a confirmed the presence of lycopene in Gac aril total peaks area of 502877, which is the highest extract beside other molecules such as area when compared with the other solvents carbohydrate. These experimental data of used, because ethanol can dissolve polar and identical spectral bands are essential in chemical non-polar materials, thereby helping to extract study of lycopene in the present work and can be more lycopene and β- Carotene. compared with previous publications (Rubio-Diaz. These results correspond to what Ishida, et al., et al. 2010; De Nardo. et al. 2009 and Rubio-Diaz 2004, obtained when extracting carotenoids from 2010). maize endosperm; Ishida et al., used Methanol/ethyl acetate 6:4 v/v and found that they DISCUSSION were the most effective mixture. Extraction with organic solvents is a familiar In HPLC reverse phase, when the solvent used in technique in food production and pharmaceutical the mobile phase is polar, the more polar industry. The method is reliable, but it requires the constituents, β- Carotene in this case, elute first use and disposal of organic solvents (Colle, et al., from the column. While lower polarity constituents 2010). If the organic solvent used to extract the such as lycopene here will remain absorbed on lycopene is toxic, then residual traces will make the column and elute later. Acetone has higher the extract unsuitable for human consumption polarity compared with ethyl acetate so it can (Sapkale, et al., 2010 and Zuknik et al., 2012). extract more beta-carotene, from the same weight When extraction using solvents such a hexane, of sample, according to the expression "like methanol and chloroform, they are (Class 2 dissolves like". solvents), mildly toxic, there will always be some The results showed that FTIR spectroscopy is remaining solvent left in the extract, causes valuable techniques to fingerprint and to analyze difficulties with proper disposal after use and it biomolecules. All FTIR data will be correlated in had some level of environmental contamination future with the detailed HPLC analysis of the from their use. While solvents include ethanol and same extracts, in order to validate FTIR method ethyl acetate (Class 3 solvents) are suitable for its as a good tool to investigate fingerprint and to minor remaining, and are used in the food and predict composition. pharmaceutical productions (Ishida and Chapman 2009), although acetone is included in this CONCLUSION category, but according to Occupational Health Seeds aril Lycopene content of Gac fruit grown in and Safety Administration (OHSA ,2006) Hexane Iraq was higher than other lycopene-rich fruit and and Acetone have many hazards , as they are vegetables including Gac of other regions, if the flammable, harmful to the human health and acute plant grown in Iraq environment without difficulty, hazards to the aquatic environment (GHS single it would-be developed as a functional food. The word: Danger). use of environmentally friendly solvents ethanol Ethyl acetate and ethanol, were chosen because and ethyl lactate (6:4 v/v) for extraction of they are non-toxic, biodegradable permitted as lycopene was found to be the best extraction additives by the U.S. Food and Drug solvent tested due to its high efficiency.

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. McGlynn W, 2009. Rapid estimation of CONFLICT OF INTEREST lycopene concentration in watermelon and The authors declared that present study was tomato puree by fiber optic visible reflectance performed in absence of any conflict of interest”. spectroscopy, Postharvest Biol Technol 52: 103-109 ACKNOWLEGEMENT Chuyen HV, Tran XT, Nguyen MH, Roach PD, The paper is a part of PhD Dissertation. Authors Parks SE, Golding JB, 2017. Yield of would like to thanks Institute of Genetic carotenoids, phenolic compounds and Engineering and Biotechnology, University of antioxidant capacity of extracts from Gac Baghdad, Ministry of Agriculture, Office – peel as affected by different solvents and Horticulture and College of Applied Biotechnology extraction conditions JOAAT 4: 87-91 – Al Nahrain University for the support Colle IJP, Lemmens L, Tolesa GN, Van Buggenhout S, De Vleeschouwer K, Van AUTHOR CONTRIBUTIONS Loey AM, Hendrickx ME, 2010. Lycopene SMK Saadedin designed the experiment, degradation and isomerization kinetics during cultivated the plant and wrote and reviewed the thermal processing of an olive oil/tomato manuscript, IHM Al-Zaidi performed the emulsion, J Agric Food Chem, 58:12784- experimental part, pre-wrote and reviewed the 12789 manuscript, SJ AL-Awadi prepared the extract De Quirós AR, Costa HS, 2006. Analysis of and reviewed the manuscript. All authors read and carotenoids in vegetable and plasma approved the final version. samples: A review. J Food Compos Anal, 19: 97–111 De Nardo T, Shiroma-Kian C, Halim Y, Francis D, Copyrights: © 2017 @ author (s). Rodriguez-Saona, L, 2009. Rapid and This is an open access article distributed under the Simultaneous Determination of Lycopene terms of the Creative Commons Attribution License and Beta-Carotene Contents in Tomato Juice (CC BY 4.0), which permits unrestricted use, by Infrared Spectroscopy, J of Agric and distribution, and reproduction in any medium, Food Chem, 57: 1105-1112 provided the original author(s) and source are De Souza LM, Ferreira KS, Chaves JBP, Teixeira credited and that the original publication in this SL, 2008. L-ascorbic acid, β-carotene and journal is cited, in accordance with accepted lycopene content in papaya fruits (Carica academic practice. No use, distribution or papaya) with or without physiological skin reproduction is permitted which does not comply freckles, Sci Agric (Piracicaba, Braz.), 65: with these terms. 246-250 Dumas Y, Dadomo M, Di Lucca G, Grolier P, REFERENCES 2003. Effects of environmental factors and Akkarachaneeyakorn S, Boonrattanakom A, agricultural techniques on antioxidant content Pukpin P, Rattanawaraha S, Khantaphant S, of tomatoes, J Sci Food Agric 83: 369-382 2015. Optimization of Oil Extraction from Gac George B, Kaur D, Kapoor H, 2004. Antioxidants (Momordica cochinchinensis Spreng) Aril in tomato (Lycopersium esculentum) as a Using a Screw Press. Chiang Mai Uni J of function of genotype, Food Chem 84: 45-51 Nat Sci, 14: 257-269 Goula MA, Adamopoulos KG, 2005. Stability of AOAC, 2000. Official methods of analysis,17th ed. lycopene during spray drying of tomato pulp, The Association of Official Analytical LWT - Food Sci Technol 38: 479-487. Chemists, Gaithersburg, M.D. Holden JM, Eldridge AL, Beecher GR, Buzzard Aoki H, Kieu NT, Kuze N, Tomisaka K, Chuyen IM, Bhagwat SA, Davis CS, Douglass LW, VN, 2002. Carotenoid pigments in gac fruit Gebhardt SE, Hayowitz DB, Shakel S, 1999. (Momordica cochinchinensis Spreng), Biosci Carotenoid content of U.S. foods: an update Biotechnol Biochem 66: 2479–2482 of the database, J Food Compost Anal Choksi PM, Joshi VY, 2007. 'A Review on 12:169–196 Lycopene - Extraction, Purification, Stability Innuna A, 2012. Antimicrobial activity of Gac fruit and Applications', Int J of Food Prop 10: 289 (Momordica cochinchinensis) Proceeding - Science and Engineering Symposium, – 298 th Choudhary R, Bowser T, Weckler P, Maness N, 4 International Science, Social Science, Engineering and Energy Conference 2012

Bioscience Research, 2017 volume 14(4): 788-800 798

Saadedin et al. Solvents extraction efficiency for lycopene of Gac fruit

:1–6 MR, Lanza E, 1993. Carotenoid content of Interiano LRJ, 2009. Proximate Composition and fruits and vegetables: an evaluation of Determination of Lycopene in Bitter Melon analytic data, J Am Diet Assoc 93: 284–296 Seed Aril, Thesis, Department of Chemical Mehta RA, Cassol N, Li N, Ali N, Handa AK, Engineering, University of Arkansas. Scholar Mattoo AK, 2002. Engineered polyamine works.uark.edu/cgi/view content. accumulation in tomato enhances cgi?article=1017&context. Accessed 14 June phytonutrient content, juice quality, and vine 2017. life, Nat Biotechnol 20: 613-618 Ishida BK, Chapman MH, 2009. Carotenoid Mein JR, Lian F, Wang XD, 2008. Biological extraction from plants using a novel, activity of lycopene metabolites: implications environmentally friendly solvent, J Agric for cancer prevention, Nutr Rev 66: 667-683. Food Chem 57:1051–1059 Mousa AA, 2012. Cancer in Maysan Compared to Ishida BK, Turner C, Chapman MH, McKeon T, Cancer in 3 Other Provinces, TQMJ 6:132- 2004. and carotenoid composition 142 of Gac (Momordica cochinchinensis Spreng) Nhung DTT, Bung PN, Ha NT, Phong TK, 2010. fruit, J Agric Food Chem 52: 274–279 Changes in lycopene and beta carotene Jang M, Kim G, 2014. Antioxidant Activity and contents in aril and oil of gac fruit during HPLC Analysis of Lycopene, β-carotene and storage, Food Chem 121: 326–331 Α-Tocopherol from GEUK (Momordica OHSA, 2006. A Guide to the Globally Harmonized cochinchinensis Spreng) Fruit, J Int Sci Publ System of Classification and Labeling of : Agric. Food 2: 430-438 Chemicals (GHS), Occupational Health and Kha TC, Nguyen, MH, Roach PD, Stathopoulos Safety Administration, 2006, retrieved July CE, 2013. Effects of Gac aril microwave 16, 2017 processing conditions on oil extraction Olives- Barba AI, Hurtado MC, Mata MCS, Ruiz efficiency, and β-carotene and lycopene VF, de Tejada MLS, 2006. Application of a contents, J Food Eng 117: 486-491 UV-vis detection-HPLC method for a rapid Kha TC, Nguyen MH, Roach PD, Stathopoulos determination of lycopene and beta-carotene CE, 2014 . Effect of Drying Pre-treatments in vegetables, Food Chem 95: 328-336. on the Yield and Bioactive Content of Oil Parks S, Nguyen M, Gale D, Murray C, 2013. Extracted from Gac Aril, IJFE 10: 103–112 Assessing the Potential for a Gac Kim YN, Giraud DW, Driskell JA, 2007. (Cochinchin ) Industry in Australia, Tocopherol and carotenoid contents of Rural Industries Research and Development selected Korean fruits and vegetables, J Corporation (RIRDC), Publication No. Food Compos Anal 20:458–465 13/060, Project No. PRJ-00581 Kubola J, Siriamornpun S, 2011. Phytochemicals Rao A, Rao LG, 2007. Carotenoids and human and antioxidant activity of different fruit health, Pharmacol Res 55: 207-216. fractions (peel, pulp, aril and seed) of Thai Roh M, Jeon M, Moon J, Moon W, Park S, Choi J, gac (Momordica cochinchinensis Spreng), 2013. A simple mrthod for the isolation of Food Chem, 127:1138-1145 Lycopene from Lycopersicon esculentum, Kubola J, Meeso N, Siriamornpun S, 2013. Bot Sci 91 : 187-192 Lycopene and beta carotene concentration in Rivera S, Canela R, 2012. Influence of Sample aril oil of gac (Momordica cochinchinensis Processing on the Analysis of Carotenoids in Spreng) as influenced by aril-drying process Maize, Molecules 17: 11255-11268 and solvents extraction, Food Res Int 50: Rubio-Diaz DE, 2010. Effect of Plant Genotype 664-669. and Processing Techniques on Stability and Lilwani S, Nair V, 2015. Extraction and Isolation of Content of Tomato Carotenoids by Infrared Lycopene Form Various Natural Sources. Spectroscopy, Ph.D. Dissertation, the IOSR JBB 1:49-51 Graduate School of The Ohio State Lim TK, 2012. Edible Medicinal And Non- University. Medicinal Plants Volume 2, Fruits, Springer Rubio-Diaz DE, De Nardo T, Santos A, de Jesus Science and Business Media, pp 369 S, Francis, D, Rodriguez Saona LEV, 2010. Malviya N, 2014. Isolation and Quantification of Profiling of nutritionally important carotenoids Lycopene from Watermelon, Tomato and from genetically-diverse tomatoes by infrared Papaya, Res J Recent Sci 3:68-70 spectroscopy, Food Chem 120: 282-289 Mangels AR, Holden JM, Beecher GR, Forman

Bioscience Research, 2017 volume 14(4): 788-800 799

Saadedin et al. Solvents extraction efficiency for lycopene of Gac fruit

Sajjan S, Chetana SH, Paarakh PM, Vedamurthy (Momordica cochinchinensis Spreng.), Food AB, 2010. Antimicrobial activity Momordica Sci Nutr 4: 305–314 cymbalaria Fenzl aerial parts extracts, Indian Tuyen CK, Minh HN, Paul DR, Sophie EP, J Nat Prod Resour. 1: 296-300 Constantinos S, 2013. Gac fruit: Nutrient and Sapkale GN, Payil SM, Surwase US, Bhatbhage Phytochemical Composition, and Options for PK, 2010. Supercritical Fluid Extraction A Processing, Food Rev Int 29: 92-106. review, Int. J. Chem. Sci, 8: 729-743 Vuong LT, Franke AA, Custer LJ, Murphy SP, Shahzad T, Ahmad I ,Choudhry S, Saeed MK, 2006. Momordica Cochinchinnensis Spreng Khan MN, 2014. Dpph free radical (Gac) fruit carotenoids reevaluated. J Food scavenging activity of tomato, cherry tomato Compost Anal 19: 664-668. and watermelon: lycopene extraction, Vuong LT, King JC, 2003. A method of preserving purification and quantification, Int J Pharm and testing the acceptability of gac fruit oil, Pharm Sci, 6: 223-228 agood source of β-carotene and essential Singh S, Singh DR, Bharati LK, Dam Roy S, 2014. fatty acids, Food and Nutr Bull 24: 224-230 Antioxidant rich fruit fractions from Wihong P, Songsri P, Suriharn B, Lomthaisong K, Momordica spp. and their commercial Lertrat A, 2017. Rapid Assessment potential in functional food industry, Lycopene and β-carotene in Spiny Bitter JCBPS,10: 3456-3463 Ground (Momordica cochinchinensis ( Lour.) Strati IF, Oreopoulou V, 2011. Effect of extraction Spreng), Pak J Bot 49: 493-497 parameters on the carotenoid recovery from Wimalasiri DC, 2015. Genetic diversity, nutritional tomato waste, Int J Food Sci Tech 46: 23-29. and biological activity of Momordica Surles RL, Weng N, Simon PW, Tanumihardjo cochinchinensis (Cucurbitaceae). A thesis- SA, 2004. Carotenoid profiles and consumer School of Applied Sciences - College of sensory evaluation of specialty carrots Science Engineering and Health, RMIT (Daucus carota, L.) of various colors, J Agri University- Sri Lanka, Food Chem 52: 3417-3421. https://researchbank.rmit.edu.au/eserv/rmit:1 Tanongkankit T, SutthaphanT, Kaewmanas J, 61591/Wimalasiri.pdf . Accessed 25 June Poonnoy P, Narkprasom K, 2014. Evolutions 2017 of β-carotene and Lycopene in a Natural Zuknik MH, Norulini, NAN Omar, AKM 2012. Food Colorant from Gac (Momordica Supercritical carbon dioxide extraction of cochinchinensis Spreng) Arils during Drying lycopene: A review, J Food Eng 112: 253– 3rd International Conference on Nutrition and 262. Food Sciences IPCBEE vol. 71 . Tien PG, Kayama F, Konishi F, Tamemoto H, Kasono K, Hung NT, Kuroki M, Ishikawa SE, Van CN, Kawakami M, 2005. Inhibition of tumor growth and angiogenesis by water extract of Gac fruit (Momordica cochinchinensis Spreng), Int J Oncol, 26: 881-889 Tinrat S, Akkarachaneeyakorn S, Singhapol C, 2014. Evaluation of antioxidant and antimicrobial activities of Momordica Cochinchinensis Spreng (Gac fruit) ethanolic extract, Int J Pharm Sci Res 5: 3163-3169 Tran TLH,Raymundo LC, 1999. Biosynthesis of carotenoids in bitter melon at high temperature, Phytochemistry 52:275-280. Tran TH, 2007. Producing carotenoid-rich powder from Gac fruit. University of Western Sydney, College of Health and Science: Centre for Plant and Food Science. Tran XT, Parks SE, Roach PD, Golding JB, Nguyen MH, 2016. Effects of maturity on physicochemical properties of Gac fruit

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