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Proximate composition of Xylocarpus moluccensis seeds and their oils
Industrial Crops and Products 41 (2013) 107–112
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Proximate composition of Xylocarpus moluccensis seeds and their oils Setiyo Gunawan a,∗ , Raden Darmawan a , Miranti Nanda a , Akhmad Dhika Setiawan a , Hamzah Fansuri b a b
Department of Chemical Engineering, Faculty of Industrial Technology, Institut Teknologi Sepuluh Nopember, Keputih Sukolilo, Surabaya 60111, Indonesia Center of Excellent in Alternative Energy (COE), Institute of Research and Public Services, Institut Teknologi Sepuluh Nopember, Keputih Sukolilo, Surabaya 60111, Indonesia
a r t i c l e
i n f o
Article history: Received 7 November 2011 Received in revised form 2 April 2012 Accepted 5 April 2012 Keywords: Carbohydrates Fatty acids Lipids Xylocarpus moluccensis seeds X. moluccensis seed oils
a b s t r a c t Mangroves play an important role in protecting shorelines, supporting the food web, and sequestering carbon. In addition, they offer protection against waves, winds, storms, and tsunamis. Indonesian mangrove resources are increasingly being lost due to unsustainable utilization and habitat conversion. Reclamation for aquaculture and agriculture is currently considered the main way to achieve the development of mangrove areas. However, these types of reclamation are costly and have adverse environmental effects. The isolation, identification, and utilization of valuable mangrove products, such as Xylocarpus moluccensis, are other ways to achieve the development of mangrove areas. X. moluccensis is a species of mangrove that has medicinal properties. However, its nutritive and lipids values have not been evaluated. In this study, the proximate composition and mineral content of X. moluccensis seeds, as well as the fatty acid composition of X. moluccensis seed oils, were investigated. The results revealed that X. moluccensis fruit seeds contained crude lipids (10.65–11.09%), crude proteins (4.76–10.14%), ash (10.07–11.59%), crude fibers (7.81–15.85%), and nitrogen free extract (e.g. carbohydrates (52.42–63.32%)). The seeds also contained copper (12.82 ppm), iron (20.25 ppm), manganese (16.22 ppm), zinc (5.89 ppm), potassium (621.98 ppm), and calcium (43.69 ppm). Myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), and docosanoic acid (C22:0) were identified in the hexane extracts of X. moluccensis fruit seeds. It was found that mangrove seeds of moluccensis have potential as biodiesel feedstock due to their lipid content. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Mangroves are highly beneficial, as they yield many valuable products, while also performing, free-of-cost, many important functions that support often dense coastal populations. The largest areas of mangroves in Southeast Asia are found in Indonesia (almost 60 percent of the Southeast Asia total), Malaysia (11.7%), Myanmar (8.8%), Papua New Guinea (8.7%) and Thailand (5.0%) (Giesen et al., 2006). As such, mangroves are widespread along the coast of Indonesia. They are found along the eastern shore of Sumatra, the western and eastern shores of Kalimantan, the western and southern shores of Bird’s Head, the southern shore of Irian Jaya, and the shores of the Aru islands, with limited other areas in southeastern Sulawesi and along the northern shore of Java, totaling 3,493,110 ha (Wilkie et al., 2002). Mangrove forests on the eastern
Abbreviations: TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; GC–MS, gas chromatography–mass spectrometry. ∗ Corresponding author. Tel.: +62 31 5946240; fax: +62 31 5999282. E-mail address: [email protected] (S. Gunawan). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.04.010
coast of Surabaya cover 2182 ha of Keputih, 209 ha of Wonorejo, 848 ha of Medokan Ayu, and 470 ha of Gunung Anyar Tambak. Xylocarpus moluccensis is a mangrove species from the family Meliceae. These beautiful trees used to be seen in mangrove forests on the eastern coast of Surabaya. This species lives solitarily or in a group in sandy areas. They can be recognized by their characteristics, including a tree height of 5–20 m with complex leaves consisting of 2–3 pairs of leaflets (4–12 cm length) (Noor et al., 1999). The complex leaves appear in a spiral form. Their flowers are small and white to reddish in color. The fruit is elliptical (diameter of 8–12 cm) and contains 5–10 seeds. The bark consists of longitudinal fissures. Xylocarpus moluccenssis is harvested twice each year in the middle and at the end of the year. Moreover, the wood is used for firewood and in the construction of houses, boats, and keris handles. The fruit is commonly used during pregnancy and as an appetite enhancer. The bark is used as an astringent and a febrifuge. This plant is also used traditionally in the treatment of dysentry, diarrhea, cholera, and fevers including those caused by malaria (Bandarnayake, 1998). Furthermore, tannin from the bark is used to synthesize a gastrointestinal drug, while the seed is used to cure stomach aches (Noor et al., 1999; Giesen et al., 2006; Lakshmi and Gupta, 2008). Finally, an ointment
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prepared from seed ash, sulfur, and coconut oil is a cure for itch (Ghani, 1998). The most characteristic product of the genus Xylocarpus is xyloccensins, a class of limonoids. Limonoids are active insect antifeedants and anticancer agents. A number of limonoids have been reported previously from the seed of this plant (Connoly et al., 1976; Taylor, 1983; Mulholland and Taylor, 1992; Li et al., 2010; Zhang et al., 2010; Ravangpai et al., 2011). Previous research was limited to the isolation, characterization, and investigation of the biological activities of limonoids from X. moluccensis. The nutritive and lipids values of X. moluccensis seeds have not been evaluated. Recently, there is a growing need to explore an alternative raw material for the production of biodiesel. Therefore, the objective of this work was to determine the proximate composition and mineral content of the X. moluccensis seeds. The fatty acid composition of X. moluccensis seed oils was also investigated. 2. Materials and methods 2.1. Materials X. moluccensis fruits were collected from Wonorejo, Surabaya, East Java. The mangrove seeds were peeled from their husk and put into an 80 ◦ C oven for 12 h to reduce their water content. Afterwards, the seeds were ground to a powdered form and stored at −60 ◦ C until further analysis. Thin-layer chromatograph (TLC) aluminum plates (20 cm × 20 cm × 250 m) were purchased from Merck (Darmstadt, Germany). Standard squalene, fatty acids, monooleylglycerol, diolein, triolein, and tripalmitin were obtained from Sigma Chemicals Company (St. Louis, MO). Myristic, palmitic, stearic, oleic, linoleic, linolenic, arachidic, and docosanoic acid methyl esters were obtained from Supelco (Bellefonte, PA) as standards. Standard -sitosterol (practical grade) was obtained from MP Biomedicals, LLC (Aurora, OH). Various minerals, such as iron, zinc, calcium, manganese, magnesium, potassium, sodium, copper and phosphorus were obtained from Sigma Chemicals Company (St. Louis, MO). All solvents and reagents were obtained from commercial sources and of either high-performance liquid chromatography (HPLC) grade or analytical reagent grade. 2.2. Determination of the moisture content of X. moluccensis seeds Moisture content was analyzed using a Halogen Moisture Analyzer. Briefly, a 1 g sample was weighed accurately in a clean and dried crucible (W1 ). Then, the crucible was placed into the Halogen Moisture Analyzer and held at 105 ◦ C until a constant weight was achieved (W2 ). The percent moisture of X. moluccensis seeds was calculated as: Moisture content (%) =
W1 − W2 × 100 W1
(1)
2.3. Determination of the lipid content of X. moluccensis seeds A Soxhlet extractor, equipped with a condenser system, was employed in this study as described by Shiu et al. (2010). The sample (50 g) was wrapped in filter paper and placed inside the Soxhlet extractor. Neutral lipids, such as fatty acids and acylglycerols, were extracted from the seed with hexane (350 ml) as the solvent. The hexane was then put in a 500 ml round-bottom flask and heated. After a predetermined time, the extraction process was stopped, and the flask that contained the desired extract was removed and replaced immediately by another flask that contained 350 ml of fresh hexane so that the total amount of solvent remained the same
as in the beginning of the run. The first fraction, which was designated as the crude oil, was obtained by extracting with hexane for 4 h. Neutral lipids were not detected in the next fraction, which was obtained after extracting with hexane for another 4 h. The lipid content of X. moluccensis seeds was calculated as: Lipids content (%) =
weight of hexane extract (g) × 100 weight of sample (g)
(2)
2.4. Determination of the crude protein content of X. moluccensis seeds The protein content of X. moluccensis seeds was determined by analyzing its nitrogen content (AOAC, 2003). Total protein was determined by multiplying the amount of nitrogen by the 6.25 correction factor (FAO, 2003). Briefly, a 6 g dried sample (W) was transferred into a Kjeldahl digestion flask. Then, 25 ml of concentrated HCl was added to the digestion flask. The flask was swirled to mix the contents thoroughly, and then placed on a heater to digest until the mixture became clear. Then, distilled water was added to 100 ml and swirled to thoroughly mix the contents. Next, 23 ml of 30% NaOH solution and a few drops of phenolphthalein indicator were added to the Kjeldahl digestion flask. Distillation was finished when there was no flow from the digestion flask. The NH3 produced was collected as NH4 OH in a conical flask containing 50 ml of 4% boric acid solution with a few drops of Conway indicator. The distillate was titrated against 1 N HCl. The crude protein content of X. moluccensis seeds was calculated as: Crude protein content (%)
= 6.25 × (V1 − V2 ) × N × 0.014 ×
f W
× 100
(3)
where V1 , V2 , N, f, W were the sample titration reading, blank titration reading, HCl normality, sample dilution, and sample weight, respectively. The 0.014 constant was the milli equivalent of nitrogen. 2.5. Determination of the ash content of X. moluccensis seeds Ash content was determined by AOAC (2003). Briefly, a clean and empty evaporating dish was heated in a muffle furnace at 600 ◦ C for 1 h, cooled in a desiccator and weighed (W1 ). One gram of sample was then weighed into an evaporating dish. The sample was heated in a muffle furnace at 550 ◦ C for 6 h until it was a grayish white ash. This ash indicated that complete oxidation of all organic matters in the sample had occurred. The evaporating dish was cooled and weighed (W2 ). The percent ash of X. moluccensis seeds was calculated as: Ash content (%) =
W2 − W1 × 100 sample weight
(4)
2.6. Determination of the crude fiber content of X. moluccensis seeds Fiber content was determined by AOAC (2003). A moisture free sample was digested using diluted H2 SO4 followed by diluted KOH solution. The undigested residue was ignited in a muffle furnace. Weight loss after ignition was considered as the crude fiber. A 0.5 g sample (W1 ) was placed in an evaporating dish along with 150 ml of H2 SO4 and a few drops of acetone as foam suppresser. The mixture was heated at 100 ◦ C until it started to boil. Then, the temperature was reduced to 45 ◦ C for 0.5 h. The sediment was filtered and rinsed with distilled water to remove any remaining acid. Afterwards, the same procedure was repeated using KOH instead of sulfuric acid. Filter paper with sediment was dried in an oven at 150 ◦ C for 1 h and then transferred into a desiccator,
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and weighed (W2 ). The sediment and filter paper were placed in an evaporating dish and heated in a furnace for 3–4 h and then transferred into a desiccator and weighed (W3 ). The crude fiber content of the sample was calculated as: Crude fiber content (%) =
W2 − W3 × 100 W1
(5)
2.7. Determination of the nitrogen free extract of X. moluccensis seeds The nitrogen-free extract (NFE) of a material consists of carbohydrates, such as sugars, starches, and hemicelluloses. NFE can be calculated by the following formula (AOAC, 2003): NFE (%) = 100 − (% crude lipids + % crude proteins + % crude fibers + % ash + % moisture)
(6)
Fig. 1. Xylocarpus moluccensis fruit and seeds.
2.8. Determination of the mineral content of X. moluccensis seeds The mineral content was analyzed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-EOS). Briefly, a 2 g sample was mixed with 10 ml of nitric acid and heated at 60 ◦ C for 20 min. Then, 5 ml of HCl was added followed by heating at 60 ◦ C for another 20 min. Then, 100 ml of distilled water was added. The solution was heated at 60 ◦ C until the volume was reduced by half and filtered. The filter paper was rinsed until the minerals dissolved. The obtained solution was diluted to 100 ml and 1 ml of the diluted solution was diluted to 50 ml. Finally, 5–10 ml of the diluted solution was analyzed using ICP-EOS. 2.9. Determination of the total fatty acid content of crude X. moluccensis seed oils Total fatty acids were determined by esterification of X. moluccensis seed oils, as described by Shiu et al. (2010). Crude X. moluccensis seed oils (10 g) were added to a 50 ml tube and treated with boron triflouride (BF3 ) in methanol (10 ml) under nitrogen. The tube was then heated at 60 ◦ C until the reaction was complete (overnight), as verified by analytical TLC (silica gel; eluted with hexane/ethyl–acetate/acetic-acid = 90:10:1, v/v/v). Total fatty acids as fatty acid methyl esters were separated by extraction using nhexane/water. The hexane extract, which contained the total fatty acids, was collected. After the hexane was removed by evaporation, the remaining substance was referred to as the reaction product. The collected product was analyzed by TLC and gas chromatography (GC). 2.10. TLC and GC–MS analyses Individual components in each sample were identified using authentic standards as described by Gunawan et al. (2011). Spots on each plate were visualized by exposing the chromatogram to iodine vapor (Fried, 1996). The identification of fatty acids in the crude oils of X. moluccensis seeds was performed by an HP 6890 GC (Hewlett-Packard, Avondale, PA, USA), equipped with a 5970 mass selective detector, and a 5990A MS Chemstation (HP-UX) (Hewlett-Packard, North Hollywood, CA, USA). Separations were carried out on an HP-5MS (5( -phenyl)-methylsiloxane non-polar column (30 m × 0.25 mm i.d. × 0.25 m film thickness, Hewlett-Packard, Avondale, PA, USA). The temperatures of the injector and detector were both set to 300 ◦ C. The temperature of the column was started at 100 ◦ C, and then was increased to 250 ◦ C at 15 ◦ C/min and held for 10 min. Helium was used as the carrier gas with a linear velocity of 37 cm/s.
Table 1 Proximate composition of Xylocarpus moluccensis seeds.a Constituents
Composition (wt%)
Protein Lipids Fiber Ash Nitrogen free extract a
4.76–10.14 10.65–11.09 7.81–15.85 10.07–11.59 52.42–63.32
Obtained from three independent experiments.
All mass spectra were acquired using the electron impact (EI) mode at 70 eV, an ion current of 50 A, and an ion source temperature of 230 ◦ C. The MS was scanned in the range of m/z 50–450 at 2 scan/s. The identification of fatty acids as fatty acid methyl esters was accomplished by comparison with a spectrum from an authentic standard, by interpretation of the fragmentation pattern from GC–MS data for fatty acid methyl esters from the literature (Qin et al., 2007) and by computerized library matching carried out on the mass spectrum, using the U.S. National Bureau of Standards library. 3. Results and discussion 3.1. X. moluccensis seeds X. moluccensis fruit is broadly ellipsoid, green, and 8–15 cm in diameter, with 5–10 seeds (4–6 cm long) positioned in a tetrahedron (Fig. 1). X. moluccensis is a potential source for insect antifeedant, insect repellant and antiyeast, anti fungal, anti bacterial, anticancer and antiviral drug development (Uddin et al., 2005; Lakshmi and Gupta, 2008; Ravangpai et al., 2011). Mangrove seeds were removed from their husk and put into an 80 ◦ C oven for 12 h to reduce their water content from 42% to 6%. The proximate composition of X. moluccensis seeds is shown in Table 1. It was found that dried X. moluccensis fruit seeds contained significant quantities of crude proteins (4.76–10.14%), crude lipids (10.65–11.09%), crude fibers (7.81–15.85%), ash (10.07–11.59%), and nitrogen-free extract (e.g. available carbohydrates) (52.42–63.32%). These data were obtained from three independent samples. Because no other study has been conducted on the proximate composition of dried X. moluccensis seeds, there are no data to which these values could be compared (Table 1). The proteins content was determined on the basis of total nitrogen content. The nitrogen content was then multiplied by a general factor (6.25) to arrive at the protein content. However, previous
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Fig. 2. GC–MS total ion chromatogram of esterified Xylocarpus moluccensis seed oil.
Table 2 Minerals contents of xylocarpus moluccensis seeds.a Mineral
Content (ppm)
K Ca Fe Mn Cu Zn Mg Na P
600.01–621.98 42.00–43.69 20.10–20.25 15.95–16.22 11.95–12.82 5.23–5.89 ND ND ND
ND, not detected. a Obtained from three independent experiments.
research reported that the use of the general factor introduced errors in protein content ranging from −2 to 9% (FAO, 2003). Carbohydrate content was indirectly calculated rather than analyzed directly (FAO, 2003). This method gives no indication of the composition of the various saccharides comprising the carbohydrate fraction. Available carbohydrates represent that fraction of carbohydrates that can be digested by human enzymes and absorbed into the intermediary metabolism. These carbohydrates do not include dietary fiber, which can be a source of energy only after fermentation. The mineral composition of X. moluccensis seeds is showed in Table 2. It was found that moluccensis contained potassium (600.01–621.98 ppm), calcium (42–43.69 ppm), iron (20.10–20.25 ppm), manganese (15.95–16.22 ppm), copper (11.95–12.82 ppm), and zinc (5.23–5.89 ppm). Among the minerals, zinc was the lowest mineral and potassium was the highest. No other study was found regarding the mineral composition of X. moluccensis seeds. 3.2. Crude X. moluccensis seed oils The lipid content of X. moluccensis seeds (hexane extract) on dry weight basis was 10.65–11.09%. Most of the identified lipids were acylglycerols (triacylglycerols, diacylglycerols, and monoacylglycerols). However, there were also non-acylglycerol components, such as free fatty acids, sterols, wax esters, and phospholipids. It was found that the mangrove seeds of moluccensis were a potential biodiesel feedstock due to their amount of lipids content. This agreed with previous observations that the use of low cost raw materials, such as rice bran (containing 13.5% (Gunawan et al., 2011) and 16–17% lipids (Shiu et al., 2010)), and microalgae (containing 10–17% lipids (Cha et al., 2011)) as substrates for biodiesel production is preferred for economic reasons.
Fatty acids are constituents of all plant cells. They function as membrane components, storage products, metabolites, and as a source of energy (Wada et al., 1994). They are also important nutrient substances and metabolites in living organisms (Chen and Chuang, 2002). In addition, fatty acids play an important role in many functions of the skin. Polyunsaturated fatty acids such as linoleic, linolenic, and arachidonic acids, are necessary for growth and protection of the skin (Elias, 1983). Furthermore, lauric acid is a potential antimicrobial agent, suitable for external application. It is inexpensive and thus useful for infection control in hospitals (Kitahara et al., 2006). Different kinds of fatty acids were characterized by GC-MS as shown in Fig. 2. Eight fatty acids were identified in the crude oils: myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), and docosanoic acid (C22:0). The degree of unsaturation (double bonds) was ascertained from the base peak, with m/z 74, 55, 67, and 79 being the base peaks of saturated, monounsaturated, biunsaturated, and polyunsaturated fatty acids, respectively. The GC–MS fragmentation of peak 1 (retention time (RT) of 7.86 min) showed a molecular ion (M+ ) at m/z 242 (relative intensity: 12%), and other ions at 213 (M+ –C2 H5 ), 199 (M+ –C3 H7 ), 185 (M+ –C4 H9 ), 171 (M+ –C5 H11 ), 157 (M+ –C6 H13 ), 143 (M+ –C7 H15 ), 129 (M+ –C8 H17 ), and 74 (the Mclafferty rearrangement and ␣cleavage), which was the base ion peak as shown in Table 3. Peaks were also observed at m/z 55, 87, 97, and 115. These fragmentation patterns were quite similar to those for tetradecanoic acid methyl ester. Consequently, peak 1 was assigned as tetradecanoic acid methyl ester (myristic acid methyl ester). Peaks 2, 6, 7, and 8 were identified as hexadecanoic, octadecanoic, eicosanoic, and docosanoic acids methyl esters, respectively. Their GC–MS fragmentations were confirmed by M+ , (M+ –C2 H5 ), (M+ –C3 H7 ), (M+ –C4 H9 ), (M+ –C5 H11 ), (M+ –C6 H13 ), (M+ –C7 H15 ), (M+ –C8 H17 ), (M+ –C9 H19 ), (M+ –C10 H21 ), (M+ –C11 H23 ), and m/z 74, which was the base ion peak of the C6 –C26 saturated fatty acid methyl ester. The mass spectrum of 9,12,15-octadecatrienoic acid methyl ester (peak 3, RT = 10.38 min) is shown in Table 3. Its fragmentation mechanism was characterized by the molecular ion (M+ ) at m/z 292, a ␥-hydrogen atom transfer, and i-induced cleavage (M+ –CH3 OH) at m/z 260. Moreover, the peak at m/z 261 (M+ –OCH3 ) was the result of the ␣-cleavage of the carbonyl. The base peak at m/z 79 (C6 H7 + ) was due to the result of double bond transfer and ␣-cleavage. Peaks were also observed at m/z 55, 67, 79, 95, 109, 121, 149, 173, 191, and 236. The GC–MS fragmentation of peak 4 (RT = 10.42 min) showed a molecular ion (M+ ) at 294 (relative intensity: 23%). Its fragmentation mechanism was characterized as follows: after the ␥-hydrogen atom transfer and i-induced cleavage, m/z 262 (M+ –CH3 OH) was produced. Additionally, m/z 263 (M+ –OCH3 ) was the result of the ␣-cleavage of the carbonyl. Ion C5 H7 + , m/z 67, was the result of a double-bond transfer and ␣-cleavage. Other ions were observed at m/z 55, 67, 81, 95, 109, 123, 150, 164, 178, 192, 206, 220, and 234. Qin et al. (2007) reported that the characteristic ions of biunsaturated fatty acid methyl esters were m/z 67, [M-31]+ , and the molecular ion. Therefore, peak 4 was identified as 9,12octadecadienoic acid methyl ester (linoleic acid methyl ester). Peak 5 was identified as 9-octadecenoic acid methyl ester. It had a molecular ion (M+ ) at m/z 296, while the ion at m/z 265 (M+ –OCH3 ) was the result of the ␣-cleavage of the carbonyl. After the ␥-hydrogen atom transfer and i-induced cleavage, an ion at m/z 264 (M+ –CH3 OH) was produced. Other ions were observed at m/z 247 (M+–[OCH3 +CO]), 236 (M+ –[OCH3 +C2 H5 ]), 222 (M+ –[OCH3 +C3 H7 ]), 208 (M+ –[OCH3 +C4 H9 ]), 194 180 (M+ –[OCH3 +C6 H13 ]), 166 (M+ –[OCH3 +C5 H11 ]),
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Table 3 Gas chromatographic retention time and mass spectrometric characteristic ions of fatty acid methyl esters. Peak no. (retention time)
Name (molecular weight)
Characteristic ions m/z (relative intensity)
1. (RT = 7.86 min)
Tetradecanoic acid methyl ester, Myristic acid methyl ester (MW = 242)
M+ 242 (11.67%), 213 (5%), 199 (25%), 185 (6.67%), 171 (1.67%), 157 (6.67%), 143 (30%), 129 (8.33%), 115 (3.33%), 97 (6.67%), 87 (73.33%), 74 (100%), 55 (16.67%)
2. (RT = 9.26 min)
Hexadecanoic acid, methyl ester, Palmitic acid methyl ester (MW = 270)
M+ 270 (19.23%), 241 (5.77%), 227 (23.08%), 213 (3.85%), 199 (9.62%), 185 (9.62%), 171 (7.69%), 157 (3.85%), 143 (26.92%), 129 (9.62%), 115 (3.85%), 97 (7.69%), 87 (76.92%), 74 (100%), 55 (19.23%)
3. (RT = 10.38 min)
9,12,15-Octadecatrienoic acid (Z,Z,Z) methyl ester, Linolenic acid methyl ester (MW = 292)
M+ 292 (7.81%), 261 (4.69%), 236 (6.25%), 191 (3.13%), 173 (6.25%), 149 (15.63%), 121 (25%), 109 (43.75%), 95 (60.94%), 79 (100%), 67 (56.25%), 55 (37.5%)
4. (RT = 10.42 min)
9,12-Octadecadienoic acid (Z,Z) methyl ester, Linoleic acid methyl ester (MW = 294)
M+ 294 (23.19%), 263 (17.39%), 234 (1.45%), 220 (5.8%), 206 (1.45%), 192 (2.9%), 178 (7.25%), 164 (11.59%), 150 (17.39%), 123 (20.29%), 109 (37.68%), 95 (73.91%), 81 (90%), 67 (100%), 55 (8.7%)
5. (RT = 10.52 min)
9-Octadecenoic acid (Z) methyl ester, Oleic acid methyl ester (MW = 296)
M+ 296 (16.67%), 264 (91.67%), 236 (8.33%), 222 (45.83%), 208 (4.17%), 194 (4.17%), 180 (33.33%), 166 (16.67%), 152 (16.67%), 137 (20.83%), 123 (33.33%), 111 (41.67%), 97 (75%), 83 (75%), 69 (83.33%), 55 (100%)
6. (RT = 11.08 min)
Octadecanoic acid methyl ester, Stearic acid methyl ester (MW = 298)
M+ 298 (19.12%), 255 (20.59%), 227 (1.47%), 213 (4.41%), 199 (13.24%), 185 (5.88%), 171 (1.47%), 157 (2.94%), 143 (26.47%), 129 (8.82%), 115 (1.47%), 97 (8.82%), 87 (76.47%), 74 (100%), 55 (19.12%)
7. (RT = 12.23 min)
Eicosanoic acid methyl ester, Arachidic acid methyl ester (MW = 326)
M+ 326 (16.95%), 283 (13.56%), 255 (1.69%), 227 (5.08%), 213 (3.39%), 199 (6.78%), 185 (3.39%), 171 (3.39%), 157 (3.39%), 143 (25.42%), 129 (6.78%), 115 (3.39%), 97 (11.86%), 87 (81.36%), 74 (100%), 55 (25.42%)
8. (RT = 13.48 min)
Docosanoic acid methyl ester (MW = 354)
M+ 354 (17.95%), 311 (11.54%), 255 (5.13%), 227 (1.28%), 213 (2.56%), 199 (6.41%), 185 (5.13%), 171 (1.28%), 157 (2.56%), 143 (25.64%), 129 (7.69%), 115 (3.85%), 97 (15.38%), 87 (87.18%), 74 (100%), 55 (41.03%)
(M+ –[OCH3 +C7 H15 ]), 152 (M+ –[OCH3 +C8 H17 ]), 111 (M+ –[CH3 OH+C11 H21 ]), 97 (M+ –[CH3 OH+C12 H23 ]), 83 (M+ –[CH3 OH+C13 H25 ]), and 69 (M+ –[CH3 OH+C14 H27 ]). The base peak at m/z 55 (M+ –[CH3 OH+C15 H29 ]) was due to ␥-hydrogen atom transfer, i-induced cleavage and the loss of C15 H29 . The ion at m/z 55 was the base ion peak of the C6 –C26 monounsaturated fatty acid methyl esters. 4. Conclusions The proximate and mineral compositions of dried X. moluccensis seeds and their oils were reported. It was found that dried X. moluccensis fruit seeds have a high carbohydrate content. Among the minerals identified, the amount of zinc was the lowest and that of potassium was the highest. Eight major fatty acids were identified in the hexane extracts from X. moluccensis fruit seeds. The present work clearly demonstrated the nutritive value of X. moluccensis seeds for medicinal purposes and the potential use of X. moluccensis seed oils for biodiesel production. Acknowledgment This work was supported by a grant (0750.172/I2.7/PM/2011) provided by the Institute of Research and Public Services, Institut Teknologi Sepuluh Nopember, Indonesia. References AOAC, 2003. Official Methods of Analysis, 17th ed. (2 revision). AOAC International, Gaithersburg, MD, USA. Bandarnayake, W.M., 1998. Traditional and medicinal uses of mangroves. Mangroves and Salt Marshes 2, 133–148. Cha, T.S., Chen, J.W., Goh, E.G., Aziz, A., Loh, S.H., 2011. Differential regulation of fatty acid biosynthesis in two Chlorella species in response to nitrate treatments and the potential of binary blending microalgae oils for biodiesel application. Bioresource Technology 102, 10633–10640. Chen, S.H., Chuang, Y.J., 2002. Analysis of fatty acids by column chromatography. Analytica Chimica Acta 465, 145–155.
Connoly, J.D., Maclellan, M.O.D.A., Taylor, D.A.H., 1976. Limonoids from xylocarpus moluccensis (Lam.) M. Roem. Journal of the Chemical Society–Perkin Transactions 1 (98), 1993–1996. Elias, P.M., 1983. Epidermal lipids, barrier function and desquamation. Journal of Investigative Dermatology 80, 44–49. FAO, 2003. Food energy: methods of analysis and conversion factors. Report of a Technical Nutrition Paper 77. (accessed 1.09.10). Fried, B., 1996. Lipids. In: Sherma, J., Fried, B. (Eds.), Handbook of Thin-layer Chromatography. Marcel Dekker Press, New York, p. 704. Ghani, A., 1998. Medicinal Plants of Bangladesh. Asiatic Society of Bangladesh, Dhaka, pp. 233–239. Giesen, W., Wulffraat, S., Zieren, M., Scholten, L., 2006. Mangrove Guide Book for Southeast Asia. Food and Agriculture Organization (FAO) of the United Nations/Wetlands International Dharmasarn Co., Ltd., Thailand, p. 2. Gunawan, S., Maulana, S., Anwar, K., Widjaja, T., 2011. Rice bran, a potential source of biodiesel production in Indonesia. Industrial Crops and Products 33, 624–628. Kitahara, T., Aoyama, Y., Hirakata, Y., Kamihira, S., Kohno, S., Ichikawa, N., Nakashima, M., Sasaki, H., Higuchi, S., 2006. In vitro activity of lauric acid or myristylamine in combination with six antimicrobial agents against methicillin-resistant Staphylococcus aureus (MRSA). International Journal of Antimicrobial Agents 27, 51–57. Lakshmi, V., Gupta, P., 2008. An overview of the genus Xylocarpus. Natural Product Research 22, 1203–1230. Li, J., Li, M.Y., Feng, G., Xiao, Q., Sinkkonen, J., Satyanandamurty, T., Wu, J., 2010. Limonoids from the seeds of a Godavari mangrove, Xylocarpus moluccensis. Phytochemistry 71, 1917–1924. Mulholland, D.A., Taylor, D.A.H., 1992. Limonoids from Australian members of the Meliaceae. Phytochemistry 31, 4163–4166. Noor, R.Y., Khazali, M., Suryadiputra, N.N., 1999. Mangrove Guide Book for Indonesia. PHKA/WI-IP, Bogor. Qin, W.H., Lan, H.X., Shan, L.X., Fang, H., Xin, Z.Z., Fen, M.Y., 2007. Gas chromatographic retention time rule and mass spectrometric fragmentation rule of fatty acids and its application in food. Chinese Journal of Analytical Chemistry 35, 998–1003. Ravangpai, W., Sommit, D., Teerawatananond, T., Sinpranee, N., Palaga, T., Pengpreecha, S., Muangsin, N., Pudhom, K., 2011. Limonoids from seeds of Thai Xylocarpus moluccensis. Bioorganic and Medicinal Chemistry Letters 21, 4485–4489. Shiu, P., Gunawan, S., Hsieh, W.H., Kasim, N.S., Ju, Y.H., 2010. Biodiesel production from rice bran by a two-step in situ proces. Bioresource Technology 101, 984–989. Taylor, D.A.H., 1983. Limonoid extractives from Xylocarpus moluccensis. Phytochemistry 22, 1297–1299. Uddin, S.J., Shilpi, J.A., Alam, S.M.S., Alamgir, M., Rahman, M.T., Sarker, S.D., 2005. Antidiarrhoeal activity of the methanol extract of the barks of Xylocarpus
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moluccensis in castor oil- and magnesium sulphate-induced diarrhoea models in mice. Journal of Ethnopharmacology 101, 139–143. Wada, H., Gombos, Z., Murata, M., 1994. Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerance temperature stress. Proceedings of the National Academy of Sciences of the United States 91, 4273–4277.
Wilkie, M.L., Fortuna, S., Souksavat, O., 2002. FAO’s database on mangrove area estimates. Forest Resources Assessment Working Paper No. 62. Rome. Zhang, J., Yang, S.X., Yang, X.B., Li, M.Y., Feng, G., Pan, J.Y., Satyanandamurty, T., Wu, J., 2010. Mexicanolides from the Seeds of a Krishna Mangrove, Xylocarpus moluccensis. Chemical and Pharmaceutical Bulletin 58, 552–555.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Proximate composition of Xylocarpus moluccensis seeds and their oils Setiyo Gunawan a,∗ , Raden Darmawan a , Miranti Nanda a , Akhmad Dhika Setiawan a , Hamzah Fansuri b a b
Department of Chemical Engineering, Faculty of Industrial Technology, Institut Teknologi Sepuluh Nopember, Keputih Sukolilo, Surabaya 60111, Indonesia Center of Excellent in Alternative Energy (COE), Institute of Research and Public Services, Institut Teknologi Sepuluh Nopember, Keputih Sukolilo, Surabaya 60111, Indonesia
a r t i c l e
i n f o
Article history: Received 7 November 2011 Received in revised form 2 April 2012 Accepted 5 April 2012 Keywords: Carbohydrates Fatty acids Lipids Xylocarpus moluccensis seeds X. moluccensis seed oils
a b s t r a c t Mangroves play an important role in protecting shorelines, supporting the food web, and sequestering carbon. In addition, they offer protection against waves, winds, storms, and tsunamis. Indonesian mangrove resources are increasingly being lost due to unsustainable utilization and habitat conversion. Reclamation for aquaculture and agriculture is currently considered the main way to achieve the development of mangrove areas. However, these types of reclamation are costly and have adverse environmental effects. The isolation, identification, and utilization of valuable mangrove products, such as Xylocarpus moluccensis, are other ways to achieve the development of mangrove areas. X. moluccensis is a species of mangrove that has medicinal properties. However, its nutritive and lipids values have not been evaluated. In this study, the proximate composition and mineral content of X. moluccensis seeds, as well as the fatty acid composition of X. moluccensis seed oils, were investigated. The results revealed that X. moluccensis fruit seeds contained crude lipids (10.65–11.09%), crude proteins (4.76–10.14%), ash (10.07–11.59%), crude fibers (7.81–15.85%), and nitrogen free extract (e.g. carbohydrates (52.42–63.32%)). The seeds also contained copper (12.82 ppm), iron (20.25 ppm), manganese (16.22 ppm), zinc (5.89 ppm), potassium (621.98 ppm), and calcium (43.69 ppm). Myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), and docosanoic acid (C22:0) were identified in the hexane extracts of X. moluccensis fruit seeds. It was found that mangrove seeds of moluccensis have potential as biodiesel feedstock due to their lipid content. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Mangroves are highly beneficial, as they yield many valuable products, while also performing, free-of-cost, many important functions that support often dense coastal populations. The largest areas of mangroves in Southeast Asia are found in Indonesia (almost 60 percent of the Southeast Asia total), Malaysia (11.7%), Myanmar (8.8%), Papua New Guinea (8.7%) and Thailand (5.0%) (Giesen et al., 2006). As such, mangroves are widespread along the coast of Indonesia. They are found along the eastern shore of Sumatra, the western and eastern shores of Kalimantan, the western and southern shores of Bird’s Head, the southern shore of Irian Jaya, and the shores of the Aru islands, with limited other areas in southeastern Sulawesi and along the northern shore of Java, totaling 3,493,110 ha (Wilkie et al., 2002). Mangrove forests on the eastern
Abbreviations: TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; GC–MS, gas chromatography–mass spectrometry. ∗ Corresponding author. Tel.: +62 31 5946240; fax: +62 31 5999282. E-mail address: [email protected] (S. Gunawan). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.04.010
coast of Surabaya cover 2182 ha of Keputih, 209 ha of Wonorejo, 848 ha of Medokan Ayu, and 470 ha of Gunung Anyar Tambak. Xylocarpus moluccensis is a mangrove species from the family Meliceae. These beautiful trees used to be seen in mangrove forests on the eastern coast of Surabaya. This species lives solitarily or in a group in sandy areas. They can be recognized by their characteristics, including a tree height of 5–20 m with complex leaves consisting of 2–3 pairs of leaflets (4–12 cm length) (Noor et al., 1999). The complex leaves appear in a spiral form. Their flowers are small and white to reddish in color. The fruit is elliptical (diameter of 8–12 cm) and contains 5–10 seeds. The bark consists of longitudinal fissures. Xylocarpus moluccenssis is harvested twice each year in the middle and at the end of the year. Moreover, the wood is used for firewood and in the construction of houses, boats, and keris handles. The fruit is commonly used during pregnancy and as an appetite enhancer. The bark is used as an astringent and a febrifuge. This plant is also used traditionally in the treatment of dysentry, diarrhea, cholera, and fevers including those caused by malaria (Bandarnayake, 1998). Furthermore, tannin from the bark is used to synthesize a gastrointestinal drug, while the seed is used to cure stomach aches (Noor et al., 1999; Giesen et al., 2006; Lakshmi and Gupta, 2008). Finally, an ointment
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prepared from seed ash, sulfur, and coconut oil is a cure for itch (Ghani, 1998). The most characteristic product of the genus Xylocarpus is xyloccensins, a class of limonoids. Limonoids are active insect antifeedants and anticancer agents. A number of limonoids have been reported previously from the seed of this plant (Connoly et al., 1976; Taylor, 1983; Mulholland and Taylor, 1992; Li et al., 2010; Zhang et al., 2010; Ravangpai et al., 2011). Previous research was limited to the isolation, characterization, and investigation of the biological activities of limonoids from X. moluccensis. The nutritive and lipids values of X. moluccensis seeds have not been evaluated. Recently, there is a growing need to explore an alternative raw material for the production of biodiesel. Therefore, the objective of this work was to determine the proximate composition and mineral content of the X. moluccensis seeds. The fatty acid composition of X. moluccensis seed oils was also investigated. 2. Materials and methods 2.1. Materials X. moluccensis fruits were collected from Wonorejo, Surabaya, East Java. The mangrove seeds were peeled from their husk and put into an 80 ◦ C oven for 12 h to reduce their water content. Afterwards, the seeds were ground to a powdered form and stored at −60 ◦ C until further analysis. Thin-layer chromatograph (TLC) aluminum plates (20 cm × 20 cm × 250 m) were purchased from Merck (Darmstadt, Germany). Standard squalene, fatty acids, monooleylglycerol, diolein, triolein, and tripalmitin were obtained from Sigma Chemicals Company (St. Louis, MO). Myristic, palmitic, stearic, oleic, linoleic, linolenic, arachidic, and docosanoic acid methyl esters were obtained from Supelco (Bellefonte, PA) as standards. Standard -sitosterol (practical grade) was obtained from MP Biomedicals, LLC (Aurora, OH). Various minerals, such as iron, zinc, calcium, manganese, magnesium, potassium, sodium, copper and phosphorus were obtained from Sigma Chemicals Company (St. Louis, MO). All solvents and reagents were obtained from commercial sources and of either high-performance liquid chromatography (HPLC) grade or analytical reagent grade. 2.2. Determination of the moisture content of X. moluccensis seeds Moisture content was analyzed using a Halogen Moisture Analyzer. Briefly, a 1 g sample was weighed accurately in a clean and dried crucible (W1 ). Then, the crucible was placed into the Halogen Moisture Analyzer and held at 105 ◦ C until a constant weight was achieved (W2 ). The percent moisture of X. moluccensis seeds was calculated as: Moisture content (%) =
W1 − W2 × 100 W1
(1)
2.3. Determination of the lipid content of X. moluccensis seeds A Soxhlet extractor, equipped with a condenser system, was employed in this study as described by Shiu et al. (2010). The sample (50 g) was wrapped in filter paper and placed inside the Soxhlet extractor. Neutral lipids, such as fatty acids and acylglycerols, were extracted from the seed with hexane (350 ml) as the solvent. The hexane was then put in a 500 ml round-bottom flask and heated. After a predetermined time, the extraction process was stopped, and the flask that contained the desired extract was removed and replaced immediately by another flask that contained 350 ml of fresh hexane so that the total amount of solvent remained the same
as in the beginning of the run. The first fraction, which was designated as the crude oil, was obtained by extracting with hexane for 4 h. Neutral lipids were not detected in the next fraction, which was obtained after extracting with hexane for another 4 h. The lipid content of X. moluccensis seeds was calculated as: Lipids content (%) =
weight of hexane extract (g) × 100 weight of sample (g)
(2)
2.4. Determination of the crude protein content of X. moluccensis seeds The protein content of X. moluccensis seeds was determined by analyzing its nitrogen content (AOAC, 2003). Total protein was determined by multiplying the amount of nitrogen by the 6.25 correction factor (FAO, 2003). Briefly, a 6 g dried sample (W) was transferred into a Kjeldahl digestion flask. Then, 25 ml of concentrated HCl was added to the digestion flask. The flask was swirled to mix the contents thoroughly, and then placed on a heater to digest until the mixture became clear. Then, distilled water was added to 100 ml and swirled to thoroughly mix the contents. Next, 23 ml of 30% NaOH solution and a few drops of phenolphthalein indicator were added to the Kjeldahl digestion flask. Distillation was finished when there was no flow from the digestion flask. The NH3 produced was collected as NH4 OH in a conical flask containing 50 ml of 4% boric acid solution with a few drops of Conway indicator. The distillate was titrated against 1 N HCl. The crude protein content of X. moluccensis seeds was calculated as: Crude protein content (%)
= 6.25 × (V1 − V2 ) × N × 0.014 ×
f W
× 100
(3)
where V1 , V2 , N, f, W were the sample titration reading, blank titration reading, HCl normality, sample dilution, and sample weight, respectively. The 0.014 constant was the milli equivalent of nitrogen. 2.5. Determination of the ash content of X. moluccensis seeds Ash content was determined by AOAC (2003). Briefly, a clean and empty evaporating dish was heated in a muffle furnace at 600 ◦ C for 1 h, cooled in a desiccator and weighed (W1 ). One gram of sample was then weighed into an evaporating dish. The sample was heated in a muffle furnace at 550 ◦ C for 6 h until it was a grayish white ash. This ash indicated that complete oxidation of all organic matters in the sample had occurred. The evaporating dish was cooled and weighed (W2 ). The percent ash of X. moluccensis seeds was calculated as: Ash content (%) =
W2 − W1 × 100 sample weight
(4)
2.6. Determination of the crude fiber content of X. moluccensis seeds Fiber content was determined by AOAC (2003). A moisture free sample was digested using diluted H2 SO4 followed by diluted KOH solution. The undigested residue was ignited in a muffle furnace. Weight loss after ignition was considered as the crude fiber. A 0.5 g sample (W1 ) was placed in an evaporating dish along with 150 ml of H2 SO4 and a few drops of acetone as foam suppresser. The mixture was heated at 100 ◦ C until it started to boil. Then, the temperature was reduced to 45 ◦ C for 0.5 h. The sediment was filtered and rinsed with distilled water to remove any remaining acid. Afterwards, the same procedure was repeated using KOH instead of sulfuric acid. Filter paper with sediment was dried in an oven at 150 ◦ C for 1 h and then transferred into a desiccator,
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and weighed (W2 ). The sediment and filter paper were placed in an evaporating dish and heated in a furnace for 3–4 h and then transferred into a desiccator and weighed (W3 ). The crude fiber content of the sample was calculated as: Crude fiber content (%) =
W2 − W3 × 100 W1
(5)
2.7. Determination of the nitrogen free extract of X. moluccensis seeds The nitrogen-free extract (NFE) of a material consists of carbohydrates, such as sugars, starches, and hemicelluloses. NFE can be calculated by the following formula (AOAC, 2003): NFE (%) = 100 − (% crude lipids + % crude proteins + % crude fibers + % ash + % moisture)
(6)
Fig. 1. Xylocarpus moluccensis fruit and seeds.
2.8. Determination of the mineral content of X. moluccensis seeds The mineral content was analyzed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-EOS). Briefly, a 2 g sample was mixed with 10 ml of nitric acid and heated at 60 ◦ C for 20 min. Then, 5 ml of HCl was added followed by heating at 60 ◦ C for another 20 min. Then, 100 ml of distilled water was added. The solution was heated at 60 ◦ C until the volume was reduced by half and filtered. The filter paper was rinsed until the minerals dissolved. The obtained solution was diluted to 100 ml and 1 ml of the diluted solution was diluted to 50 ml. Finally, 5–10 ml of the diluted solution was analyzed using ICP-EOS. 2.9. Determination of the total fatty acid content of crude X. moluccensis seed oils Total fatty acids were determined by esterification of X. moluccensis seed oils, as described by Shiu et al. (2010). Crude X. moluccensis seed oils (10 g) were added to a 50 ml tube and treated with boron triflouride (BF3 ) in methanol (10 ml) under nitrogen. The tube was then heated at 60 ◦ C until the reaction was complete (overnight), as verified by analytical TLC (silica gel; eluted with hexane/ethyl–acetate/acetic-acid = 90:10:1, v/v/v). Total fatty acids as fatty acid methyl esters were separated by extraction using nhexane/water. The hexane extract, which contained the total fatty acids, was collected. After the hexane was removed by evaporation, the remaining substance was referred to as the reaction product. The collected product was analyzed by TLC and gas chromatography (GC). 2.10. TLC and GC–MS analyses Individual components in each sample were identified using authentic standards as described by Gunawan et al. (2011). Spots on each plate were visualized by exposing the chromatogram to iodine vapor (Fried, 1996). The identification of fatty acids in the crude oils of X. moluccensis seeds was performed by an HP 6890 GC (Hewlett-Packard, Avondale, PA, USA), equipped with a 5970 mass selective detector, and a 5990A MS Chemstation (HP-UX) (Hewlett-Packard, North Hollywood, CA, USA). Separations were carried out on an HP-5MS (5( -phenyl)-methylsiloxane non-polar column (30 m × 0.25 mm i.d. × 0.25 m film thickness, Hewlett-Packard, Avondale, PA, USA). The temperatures of the injector and detector were both set to 300 ◦ C. The temperature of the column was started at 100 ◦ C, and then was increased to 250 ◦ C at 15 ◦ C/min and held for 10 min. Helium was used as the carrier gas with a linear velocity of 37 cm/s.
Table 1 Proximate composition of Xylocarpus moluccensis seeds.a Constituents
Composition (wt%)
Protein Lipids Fiber Ash Nitrogen free extract a
4.76–10.14 10.65–11.09 7.81–15.85 10.07–11.59 52.42–63.32
Obtained from three independent experiments.
All mass spectra were acquired using the electron impact (EI) mode at 70 eV, an ion current of 50 A, and an ion source temperature of 230 ◦ C. The MS was scanned in the range of m/z 50–450 at 2 scan/s. The identification of fatty acids as fatty acid methyl esters was accomplished by comparison with a spectrum from an authentic standard, by interpretation of the fragmentation pattern from GC–MS data for fatty acid methyl esters from the literature (Qin et al., 2007) and by computerized library matching carried out on the mass spectrum, using the U.S. National Bureau of Standards library. 3. Results and discussion 3.1. X. moluccensis seeds X. moluccensis fruit is broadly ellipsoid, green, and 8–15 cm in diameter, with 5–10 seeds (4–6 cm long) positioned in a tetrahedron (Fig. 1). X. moluccensis is a potential source for insect antifeedant, insect repellant and antiyeast, anti fungal, anti bacterial, anticancer and antiviral drug development (Uddin et al., 2005; Lakshmi and Gupta, 2008; Ravangpai et al., 2011). Mangrove seeds were removed from their husk and put into an 80 ◦ C oven for 12 h to reduce their water content from 42% to 6%. The proximate composition of X. moluccensis seeds is shown in Table 1. It was found that dried X. moluccensis fruit seeds contained significant quantities of crude proteins (4.76–10.14%), crude lipids (10.65–11.09%), crude fibers (7.81–15.85%), ash (10.07–11.59%), and nitrogen-free extract (e.g. available carbohydrates) (52.42–63.32%). These data were obtained from three independent samples. Because no other study has been conducted on the proximate composition of dried X. moluccensis seeds, there are no data to which these values could be compared (Table 1). The proteins content was determined on the basis of total nitrogen content. The nitrogen content was then multiplied by a general factor (6.25) to arrive at the protein content. However, previous
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Fig. 2. GC–MS total ion chromatogram of esterified Xylocarpus moluccensis seed oil.
Table 2 Minerals contents of xylocarpus moluccensis seeds.a Mineral
Content (ppm)
K Ca Fe Mn Cu Zn Mg Na P
600.01–621.98 42.00–43.69 20.10–20.25 15.95–16.22 11.95–12.82 5.23–5.89 ND ND ND
ND, not detected. a Obtained from three independent experiments.
research reported that the use of the general factor introduced errors in protein content ranging from −2 to 9% (FAO, 2003). Carbohydrate content was indirectly calculated rather than analyzed directly (FAO, 2003). This method gives no indication of the composition of the various saccharides comprising the carbohydrate fraction. Available carbohydrates represent that fraction of carbohydrates that can be digested by human enzymes and absorbed into the intermediary metabolism. These carbohydrates do not include dietary fiber, which can be a source of energy only after fermentation. The mineral composition of X. moluccensis seeds is showed in Table 2. It was found that moluccensis contained potassium (600.01–621.98 ppm), calcium (42–43.69 ppm), iron (20.10–20.25 ppm), manganese (15.95–16.22 ppm), copper (11.95–12.82 ppm), and zinc (5.23–5.89 ppm). Among the minerals, zinc was the lowest mineral and potassium was the highest. No other study was found regarding the mineral composition of X. moluccensis seeds. 3.2. Crude X. moluccensis seed oils The lipid content of X. moluccensis seeds (hexane extract) on dry weight basis was 10.65–11.09%. Most of the identified lipids were acylglycerols (triacylglycerols, diacylglycerols, and monoacylglycerols). However, there were also non-acylglycerol components, such as free fatty acids, sterols, wax esters, and phospholipids. It was found that the mangrove seeds of moluccensis were a potential biodiesel feedstock due to their amount of lipids content. This agreed with previous observations that the use of low cost raw materials, such as rice bran (containing 13.5% (Gunawan et al., 2011) and 16–17% lipids (Shiu et al., 2010)), and microalgae (containing 10–17% lipids (Cha et al., 2011)) as substrates for biodiesel production is preferred for economic reasons.
Fatty acids are constituents of all plant cells. They function as membrane components, storage products, metabolites, and as a source of energy (Wada et al., 1994). They are also important nutrient substances and metabolites in living organisms (Chen and Chuang, 2002). In addition, fatty acids play an important role in many functions of the skin. Polyunsaturated fatty acids such as linoleic, linolenic, and arachidonic acids, are necessary for growth and protection of the skin (Elias, 1983). Furthermore, lauric acid is a potential antimicrobial agent, suitable for external application. It is inexpensive and thus useful for infection control in hospitals (Kitahara et al., 2006). Different kinds of fatty acids were characterized by GC-MS as shown in Fig. 2. Eight fatty acids were identified in the crude oils: myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidic acid (C20:0), and docosanoic acid (C22:0). The degree of unsaturation (double bonds) was ascertained from the base peak, with m/z 74, 55, 67, and 79 being the base peaks of saturated, monounsaturated, biunsaturated, and polyunsaturated fatty acids, respectively. The GC–MS fragmentation of peak 1 (retention time (RT) of 7.86 min) showed a molecular ion (M+ ) at m/z 242 (relative intensity: 12%), and other ions at 213 (M+ –C2 H5 ), 199 (M+ –C3 H7 ), 185 (M+ –C4 H9 ), 171 (M+ –C5 H11 ), 157 (M+ –C6 H13 ), 143 (M+ –C7 H15 ), 129 (M+ –C8 H17 ), and 74 (the Mclafferty rearrangement and ␣cleavage), which was the base ion peak as shown in Table 3. Peaks were also observed at m/z 55, 87, 97, and 115. These fragmentation patterns were quite similar to those for tetradecanoic acid methyl ester. Consequently, peak 1 was assigned as tetradecanoic acid methyl ester (myristic acid methyl ester). Peaks 2, 6, 7, and 8 were identified as hexadecanoic, octadecanoic, eicosanoic, and docosanoic acids methyl esters, respectively. Their GC–MS fragmentations were confirmed by M+ , (M+ –C2 H5 ), (M+ –C3 H7 ), (M+ –C4 H9 ), (M+ –C5 H11 ), (M+ –C6 H13 ), (M+ –C7 H15 ), (M+ –C8 H17 ), (M+ –C9 H19 ), (M+ –C10 H21 ), (M+ –C11 H23 ), and m/z 74, which was the base ion peak of the C6 –C26 saturated fatty acid methyl ester. The mass spectrum of 9,12,15-octadecatrienoic acid methyl ester (peak 3, RT = 10.38 min) is shown in Table 3. Its fragmentation mechanism was characterized by the molecular ion (M+ ) at m/z 292, a ␥-hydrogen atom transfer, and i-induced cleavage (M+ –CH3 OH) at m/z 260. Moreover, the peak at m/z 261 (M+ –OCH3 ) was the result of the ␣-cleavage of the carbonyl. The base peak at m/z 79 (C6 H7 + ) was due to the result of double bond transfer and ␣-cleavage. Peaks were also observed at m/z 55, 67, 79, 95, 109, 121, 149, 173, 191, and 236. The GC–MS fragmentation of peak 4 (RT = 10.42 min) showed a molecular ion (M+ ) at 294 (relative intensity: 23%). Its fragmentation mechanism was characterized as follows: after the ␥-hydrogen atom transfer and i-induced cleavage, m/z 262 (M+ –CH3 OH) was produced. Additionally, m/z 263 (M+ –OCH3 ) was the result of the ␣-cleavage of the carbonyl. Ion C5 H7 + , m/z 67, was the result of a double-bond transfer and ␣-cleavage. Other ions were observed at m/z 55, 67, 81, 95, 109, 123, 150, 164, 178, 192, 206, 220, and 234. Qin et al. (2007) reported that the characteristic ions of biunsaturated fatty acid methyl esters were m/z 67, [M-31]+ , and the molecular ion. Therefore, peak 4 was identified as 9,12octadecadienoic acid methyl ester (linoleic acid methyl ester). Peak 5 was identified as 9-octadecenoic acid methyl ester. It had a molecular ion (M+ ) at m/z 296, while the ion at m/z 265 (M+ –OCH3 ) was the result of the ␣-cleavage of the carbonyl. After the ␥-hydrogen atom transfer and i-induced cleavage, an ion at m/z 264 (M+ –CH3 OH) was produced. Other ions were observed at m/z 247 (M+–[OCH3 +CO]), 236 (M+ –[OCH3 +C2 H5 ]), 222 (M+ –[OCH3 +C3 H7 ]), 208 (M+ –[OCH3 +C4 H9 ]), 194 180 (M+ –[OCH3 +C6 H13 ]), 166 (M+ –[OCH3 +C5 H11 ]),
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Table 3 Gas chromatographic retention time and mass spectrometric characteristic ions of fatty acid methyl esters. Peak no. (retention time)
Name (molecular weight)
Characteristic ions m/z (relative intensity)
1. (RT = 7.86 min)
Tetradecanoic acid methyl ester, Myristic acid methyl ester (MW = 242)
M+ 242 (11.67%), 213 (5%), 199 (25%), 185 (6.67%), 171 (1.67%), 157 (6.67%), 143 (30%), 129 (8.33%), 115 (3.33%), 97 (6.67%), 87 (73.33%), 74 (100%), 55 (16.67%)
2. (RT = 9.26 min)
Hexadecanoic acid, methyl ester, Palmitic acid methyl ester (MW = 270)
M+ 270 (19.23%), 241 (5.77%), 227 (23.08%), 213 (3.85%), 199 (9.62%), 185 (9.62%), 171 (7.69%), 157 (3.85%), 143 (26.92%), 129 (9.62%), 115 (3.85%), 97 (7.69%), 87 (76.92%), 74 (100%), 55 (19.23%)
3. (RT = 10.38 min)
9,12,15-Octadecatrienoic acid (Z,Z,Z) methyl ester, Linolenic acid methyl ester (MW = 292)
M+ 292 (7.81%), 261 (4.69%), 236 (6.25%), 191 (3.13%), 173 (6.25%), 149 (15.63%), 121 (25%), 109 (43.75%), 95 (60.94%), 79 (100%), 67 (56.25%), 55 (37.5%)
4. (RT = 10.42 min)
9,12-Octadecadienoic acid (Z,Z) methyl ester, Linoleic acid methyl ester (MW = 294)
M+ 294 (23.19%), 263 (17.39%), 234 (1.45%), 220 (5.8%), 206 (1.45%), 192 (2.9%), 178 (7.25%), 164 (11.59%), 150 (17.39%), 123 (20.29%), 109 (37.68%), 95 (73.91%), 81 (90%), 67 (100%), 55 (8.7%)
5. (RT = 10.52 min)
9-Octadecenoic acid (Z) methyl ester, Oleic acid methyl ester (MW = 296)
M+ 296 (16.67%), 264 (91.67%), 236 (8.33%), 222 (45.83%), 208 (4.17%), 194 (4.17%), 180 (33.33%), 166 (16.67%), 152 (16.67%), 137 (20.83%), 123 (33.33%), 111 (41.67%), 97 (75%), 83 (75%), 69 (83.33%), 55 (100%)
6. (RT = 11.08 min)
Octadecanoic acid methyl ester, Stearic acid methyl ester (MW = 298)
M+ 298 (19.12%), 255 (20.59%), 227 (1.47%), 213 (4.41%), 199 (13.24%), 185 (5.88%), 171 (1.47%), 157 (2.94%), 143 (26.47%), 129 (8.82%), 115 (1.47%), 97 (8.82%), 87 (76.47%), 74 (100%), 55 (19.12%)
7. (RT = 12.23 min)
Eicosanoic acid methyl ester, Arachidic acid methyl ester (MW = 326)
M+ 326 (16.95%), 283 (13.56%), 255 (1.69%), 227 (5.08%), 213 (3.39%), 199 (6.78%), 185 (3.39%), 171 (3.39%), 157 (3.39%), 143 (25.42%), 129 (6.78%), 115 (3.39%), 97 (11.86%), 87 (81.36%), 74 (100%), 55 (25.42%)
8. (RT = 13.48 min)
Docosanoic acid methyl ester (MW = 354)
M+ 354 (17.95%), 311 (11.54%), 255 (5.13%), 227 (1.28%), 213 (2.56%), 199 (6.41%), 185 (5.13%), 171 (1.28%), 157 (2.56%), 143 (25.64%), 129 (7.69%), 115 (3.85%), 97 (15.38%), 87 (87.18%), 74 (100%), 55 (41.03%)
(M+ –[OCH3 +C7 H15 ]), 152 (M+ –[OCH3 +C8 H17 ]), 111 (M+ –[CH3 OH+C11 H21 ]), 97 (M+ –[CH3 OH+C12 H23 ]), 83 (M+ –[CH3 OH+C13 H25 ]), and 69 (M+ –[CH3 OH+C14 H27 ]). The base peak at m/z 55 (M+ –[CH3 OH+C15 H29 ]) was due to ␥-hydrogen atom transfer, i-induced cleavage and the loss of C15 H29 . The ion at m/z 55 was the base ion peak of the C6 –C26 monounsaturated fatty acid methyl esters. 4. Conclusions The proximate and mineral compositions of dried X. moluccensis seeds and their oils were reported. It was found that dried X. moluccensis fruit seeds have a high carbohydrate content. Among the minerals identified, the amount of zinc was the lowest and that of potassium was the highest. Eight major fatty acids were identified in the hexane extracts from X. moluccensis fruit seeds. The present work clearly demonstrated the nutritive value of X. moluccensis seeds for medicinal purposes and the potential use of X. moluccensis seed oils for biodiesel production. Acknowledgment This work was supported by a grant (0750.172/I2.7/PM/2011) provided by the Institute of Research and Public Services, Institut Teknologi Sepuluh Nopember, Indonesia. References AOAC, 2003. Official Methods of Analysis, 17th ed. (2 revision). AOAC International, Gaithersburg, MD, USA. Bandarnayake, W.M., 1998. Traditional and medicinal uses of mangroves. Mangroves and Salt Marshes 2, 133–148. Cha, T.S., Chen, J.W., Goh, E.G., Aziz, A., Loh, S.H., 2011. Differential regulation of fatty acid biosynthesis in two Chlorella species in response to nitrate treatments and the potential of binary blending microalgae oils for biodiesel application. Bioresource Technology 102, 10633–10640. Chen, S.H., Chuang, Y.J., 2002. Analysis of fatty acids by column chromatography. Analytica Chimica Acta 465, 145–155.
Connoly, J.D., Maclellan, M.O.D.A., Taylor, D.A.H., 1976. Limonoids from xylocarpus moluccensis (Lam.) M. Roem. Journal of the Chemical Society–Perkin Transactions 1 (98), 1993–1996. Elias, P.M., 1983. Epidermal lipids, barrier function and desquamation. Journal of Investigative Dermatology 80, 44–49. FAO, 2003. Food energy: methods of analysis and conversion factors. Report of a Technical Nutrition Paper 77.
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