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Variation in protein and oil content and fatty acid composition of Rhus tripartitum fruits collected at different maturity stages in different locations
Industrial Crops and Products 59 (2014) 197–201
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Variation in protein and oil content and fatty acid composition of Rhus tripartitum fruits collected at different maturity stages in different locations Nizar Tlili a,c,∗ , Mariem Tir b , Houda Benlajnef a , Sheyma Khemiri a , Hassen Mejri b , Saloua Rejeb c , Abdelhamid Khaldi c a
Laboratoire de Biochimie, Département de Biologie, Faculté des Sciences de Tunis, Université Tunis El-Manar, Tunis 2092, Tunisie Unité de Recherche de Physiologie et Environnement Aquatiques, Département de Biologie, Faculté des Sciences de Tunis, Université Tunis EL Manar, 2092 Tunis, Tunisie c Institut National de Recherches en Génie Rural, Eaux et Forêts (INRGREF); Université de Carthage, BP: 10, Ariana 2080, Tunisia b
a r t i c l e
i n f o
Article history: Received 20 February 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Keywords: Rhus tripartitum fruits Protein Oil Fatty acid Maturity Location
a b s t r a c t The goal of this study is to evaluate the effects of ripening (immature: yellow; intermediate: mahogany brown; mature: dark brown) on the protein and oil content and fatty acid composition of Rhus tripartitum fruits collected from two Tunisian regions. The high protein and oil content were detected in the intermediate stage (ca. 9.5% and ca. 6%, respectively). Fatty acid profiles did not vary among the growing regions; and palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were the predominant fatty acids. Linoleic acid showed the highest level in the intermediate stage (ca. 21%). In the mature stages palmitic and oleic acids were the major fatty acids (more than 31% and 34%, respectively). Our findings showed that R. tripartitum fruits, especially at the intermediate stage, could therefore represent an attractive source of protein, oil and fatty acid for both food, cosmetic, and pharmaceutical industries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Storage proteins are a source of nutrients for both humans and farm animals. Hence, vegetable proteins have increased in commercial value due to consumer preference for natural food sources (Kaur and Singh, 2007). Vegetable oils are used directly in salad and for cooking, and can also be converted into many products for various uses such as personal care products, soaps, biodiesel, paint formulations, and adhesives (Azmir et al., 2014; Maisonneuve et al., 2013). The importance of oil in industry is determined by its fatty acid composition. Some authors have reported that the quality and flavor of some products can be affected by the fatty acid composition of the lipid (Hinds, 1999). In addition, some researchers have suggested that reducing consumption of saturated fats and increasing that of polyunsaturated fats is effective in lowering serum cholesterol, thus reducing coronary heart disease (Haruenkit et al., 2010). In vegetables, protein and oil content and fatty acid composition are influenced by several factors such as genetic factors,
∗ Corresponding author at: Laboratoire de Biochimie, Département de Biologie, Faculté des Sciences de Tunis, Université Tunis El-Manar, Tunis 2092, Tunisie. E-mail address: [email protected] (N. Tlili). http://dx.doi.org/10.1016/j.indcrop.2014.05.020 0926-6690/© 2014 Elsevier B.V. All rights reserved.
soil, climatic conditions, location, and ripening stages (Breene et al., 2007; Hinds, 1999; Tlili et al., 2011). The effect of ripening stages could be explained by the fact that during maturity, many biochemical, physiological, and structural variations occur to determine the quality of the vegetables (Vendramini and Trugo, 2000). Rhus is a woody genus belonging to the Anacardiaceae family, with about 250 species, which often grow in non-agriculturally viable regions. Species of Rhus are deciduous and multibranched shrubs or trees (Prakash and Van Staden, 2007). Many authors have reported the presence of high levels of bioflavonoids in the aerial parts, thus emphasizing their medicinal importance and supporting their use as spices by previous generations (Rayne and Mazza, 2007). Antimalarial, antimicrobial, and antitumorigenic impacts for Rhus species have also been reported (Ahmed et al., 2001; Choi et al., 2012; Lee et al., 2010). Rhus tripartitum, is a presaharan Tunisian plant (Pottier-Alapetite, 1979), commonly used by Tunisians in herbal treatments of many diseases, such as diarrhea and dysentery (Abbassi and Hani, 2012). The fruit is consumed fresh or stored, or added to water for a refreshing taste (Le Floc’h and Boulous, 2008). To the best of our knowledge, there have been no reports regarding the effect of location and ripening stages on protein and oil content or fatty acid profile of R. tripartitum fruits. The aim of this study was therefore to determine the amount of protein and
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oil and investigate the fatty acid composition of R. tripartitum fruits collected from two regions at three different stages of maturity. This study could be valuable for possible industrial use of R. tripartitum as a raw material source of protein and oil.
acid peaks were integrated and analyzed using HP Chemstation software. Analyses were performed in duplicate.
2. Materials and methods
The Cox value of the oil was calculated based on the percentage of unsaturated C18 fatty acids, applying the formula proposed by Fatemi and Hammond (1980).
2.1. Plant material R. tripartitum fruits were collected in February and March 2013 from two Tunisian regions, Dkhila (36◦ 52 ; 9◦ 42 ) and Ain Jalloula (35◦ 47 ; 9◦ 47 ). Samples were collected during three harvesting periods according to color: immature stage (yellow), intermediate stage (mahogany brown) and mature stage (dark brown). Samples were air-dried at room temperature and then ground for further analysis. Species were identified by Dr. Abdelhamid Khaldi and voucher specimens were deposited in the herbarium of the National Institute for Research in Rural Engineering Water and Forests (INRGREF, Tunisia).
2.5. Calculated oxidizability value (Cox)
Cox =
[1 × (18 : 1%) + 10.3 × (18 : 2%) + 21.6 × (18 : 3%)] 100
2.6. Statistical analyses Each value represented the mean ± SD. The Pearson correlation between mean values was performed using SPSS statistical package (version 17.0). The test was considered significant when the p value was lower than 0.05. 3. Results and discussion
2.2. Protein content Protein content was measured using a Kjeldhal apparatus (AOAC, 1984). Each 100 mg sample was digested for one hour with 100 mg of digestion mixture (8 g K2 SO4 + 20 g CaSO4 + 2 g selenium) and 6 mL of concentrated H2 SO4 . When digestion was completed, the solution became clear. The solution was then placed in a volumetric flask and distilled water was added until the total volume reached 30.0 mL. To determine nitrogen content, 10 mL of 2% boric acid solution was first put in a beaker with a few drops of methyl red as an indicator. Then 10 mL of the digested mixture, 30 mL of 40% NaOH solution, and 10 mL of distilled water were transferred to the distillation chamber. The liberated ammonia combined with NaOH to form NH4 OH, which was then added to the boric acid solution to form ammonium borate (pink to yellow color). The ammonium borate was then titrated with 0.1 N H2 SO4 . The volume of acid that had been added at the point when the color of the distillate changed from yellow to pink was recorded. Protein was calculated according to the following formula: 115%protein = %N × 6.25. Analyses were performed in triplicate. 2.3. Oil extraction Oil content was determined according to ISO method 659:1998 (ISO, 1999). About 30 g of the powder were extracted with petroleum ether in a Soxhlet apparatus for 4 h. The solvent was concentrated using a rotary evaporator under reduced pressure at 50 ◦ C and then a stream of nitrogen was used to dry the oil before storing at −20 ◦ C for later use. Analyses were performed in triplicate.
3.1. Protein content To our knowledge, there is no data about protein content of R. tripartitum fruits. The results in Fig. 1 show the estimated protein content (% dry weight basis) from R. tripartitum fruits collected at different stages of maturity from two locations in Tunisia. Regardless of maturity or location, R. tripartitum fruits contained large quantities of protein, between 4.81% and 10.5%. The highest values were found at the intermediate stage for both locations (9.37% and 10.5% in Ain Jalloula and Dkhila, respectively). These values lend nutritional importance to R. tripartitum fruits due to the fact that protein, as a macronutrient, plays a crucial role in the human diet without any adverse effects. These proteins found here could be important for different population sectors or used as a raw material source in the food industry, especially in Afro-Asian regions where the protein–calorie malnutrition syndrome affects more than 170 million preschool children and nursing mothers (Iqbal et al., 2006). Values clearly showed a difference between samples from the two regions for the same stage of maturity (4.81%, 9.37% and 6.75% in Ain Jalloula and 6.16%, 10.5% and 7.88% in Dkhila, for immature, intermediate and mature stage, respectively). These results were similar to those found by other authors who suggested that protein content of crops can vary with soil, climatic conditions, and cultivars (El Arem et al., 2011; Tlili et al., 2011). When comparing protein levels, in the two locations, based on the stage of maturity, we can conclude firstly that the content
2.4. Fatty acid analysis The fatty acid composition of the oil was determined by gas chromatography as fatty acid methyl esters (FAMEs) using the method described by Cecchi et al. (1985). The FAMEs were separated and identified with a HP 6890 gas chromatograph with a split/splitless injector equipped with a flame ionization detector, and a 30 m HP Innowax capillary column with an internal diameter of 250 m and a film thickness of 0.25 m. The temperatures of the injector and the detector were maintained at 250 ◦ C and 275 ◦ C, respectively; the oven was programmed to rise from 50 to 180 ◦ C at a rate of 4 ◦ C/min, from 180 to 220 ◦ C at 1.33 ◦ C/min and to stabilize at 220 ◦ C for 7 min. Nitrogen was the carrier gas. Fatty acids were identified by comparing retention times of the FAMEs with those of co-injected authentic standards (SUPELCO PUFA-3). Fatty
Fig. 1. Variation in protein content in fruits of Rhus tripartitum collected from two locations at three different maturity stages.
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Table 1 Variation in oil content and fatty acid composition in fruits of Rhus tripartitum collected two locations at three different maturity stages. Ain Jalloula
Samples
Oil (% of dry weight) Fatty acid (% of total fatty acids) 14:0 15:0 15:1 16:0 16:1 17:0 16:3 16:4 18:0 18:1 18:2n-6 18:3n-3 18:4n-3 20:0 20:1 20:2n6 20:3n6 20:4n-6 20:3n-3 20:4n3 20:5n3 22:0 22:1 22:5n-3 22:6n-3
Dkhila
Immature
Intermediate
Mature
Immature
Intermediate
Mature
3.80 ± 0.42
6.41 ± 0.41
3.87 ± 0.89
5.4 ± 0.09
5.85 ± 0.93
3.52 ± 0.09
0.76 ± 0.07 0.17 ± 0.04 0.04 ± 0.00 37.48 ± 1.76 2.59 ± 0.06 0.25 ± 0.11 0.02 ± 0.00 0.03 ± 0.00 0.06 ± 0.00 41.73 ± .024 tr 10.08 ± 0.87 0.20 ± 0.06 1.93 ± 0.33 0.29 ± 0.08 0.14 ± 0.01 0.06 ± 0.02 0.11 ± 0.05 0.04 ± 0.02 0.01 ± 0.00 0.08 ± 0.01 0.03 ± 0.01 1.48 ± 0.33 0.79 ± 0.11 0.68 ± 0.13
0.89 ± 0.56 0.29 ± 0.32 0.05 ± 0.00 37.67 ± 1.84 2.02 ± 2.53 0.25 ± 0.01 0.01 ± 0.00 tr 0.07 ± 0.00 28.89 ± 2.65 21.90 ± 0.74 2.64 ± 0.10 0.14 ± 0.14 1.49 ± 0.73 0.47 ± 0.58 0.15 ± 0.09 0.17 ± 0.10 0.09 ± 0.01 0.29 ± 0.37 0.05 ± 0.00 0.04 ± 0.00 0.28 ± 0.0.34 1.08 ± 0.12 0.47 ± 0.01 0.33 ± 0.13
1.15 ± 0.50 2.84 ± 3.46 1.08 ± 0.93 31.88 ± 1.55 1.47 ± 0.55 0.62 ± 0.48 0.12 ± 0.10 0.19 ± 0.03 2.96 ± 0.22 41.73 ± 2.81 tr 5.09 ± 0.68 0.55 ± 0.38 1.92 ± 0.28 1.35 ± 1.01 0.66 ± 0.01 0.26 ± 0.00 0.36 ± 0.00 0.34 ± 0.39 0.19 ± 0.11 0.18 ± 0.13 0.26 ± 0.19 1.59 ± 0.73 0.69 ± 0.25 1.59 ± 0.62
0.64 ± 0.18 0.02 ± 0.01 0.02 ± 0.02 52.36 ± 13.06 0.20 ± 0.02 0.83 ± 0.06 0.16 ± 0.02 0.01 ± 0.00 0.09 ± 0.02 34.83 ± 15.00 tr 3.25 ± 1.42 0.14 ± 0.10 2.43 ± 0.55 0.07 ± 0.00 0.15 ± 0.00 0.02 ± 0.03 0.15 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.05 ± 0.00 0.19 ± 0.05 1.40 ± 0.58 0.89 ± 0.00 0.98 ± 0.01
1.12 ± 0.06 0.13 ± 0.03 0.33 ± 0.23 39.86 ± 1.64 2.36 ± 0.31 0.32 ± 0.21 0.14 ± 0.09 0.04 ± 0.01 0.48 ± 0.01 16.55 ± 7.28 21.23 ± 13.66 9.27 ± 0.19 0.49 ± 0.48 1.04 ± 0.53 0.12 ± 0.01 0.08 ± 0.02 0.07 ± 0.04 0.14 ± 0.04 0.10 ± 0.10 0.08 ± 0.01 0.03 ± 0.00 0.87 ± 0.17 1.15 ± 0.05 1.69 ± 1.08 1.38 ± 0.52
0.79 ± 0.10 0.09 ± 0.00 0.01 ± 0.00 46.34 ± 1.88 0.09 ± 0.02 1.33 ± 1.03 0.35 ± 0.08 0.04 ± 0.03 0.05 ± 0.03 43.59 ± 3.92 tr 2.29 ± 0.11 0.11 ± 0.02 1.49 ± 0.13 0.04 ± 0.00 0.07 ± 0.00 0.08 ± 0.04 0.16 ± 0.10 0.01 ± 0.00 0.01 ± 0.00 0.06 ± 0.00 0.21 ± 0.17 0.97 ± 0.35 0.74 ± 0.28 0.69 ± 0.30
increased from 4.81% (immature) to 9.37% (intermediate) and from 6.16% (immature) to 10.5% (intermediate) in Ain Jalloula and Dkhila, respectively. The increase in protein levels during ripening might be due to the activation of some enzymes, like cellulase and polygalacturonase, which play significant roles in the softening of the fruit (El Arem et al., 2011). Secondly, the protein content in R. tripartitum decreased to 6.75% and 7.88% in Ain Jalloula and Dkhila, respectively. This decrease was more than 25%. Other authors have reported that protein content can decrease 30% with maturity due to biochemical degradation and also due to the possible secondary metabolite production (Vendramini and Trugo, 2000). This decrease would not affect the nutritional quality but might affect the flavor characteristics of the fruits (Vendramini and Trugo, 2000). 3.2. Oil content The content of oil (% dry weight basis) in all samples of R. tripartitum are presented in Table 1. The results clearly showed a difference in the accumulation of oil between locations. We did not find information about oil accumulation in R. tripartitum fruits, but our results were similar to other authors who reported that oil content in the crops can fluctuate with climatic conditions and cultivars (Tlili et al., 2011). The highest values were detected at the intermediate stage for both locations (6.41% and 5.85% in Ain Jalloula and Dkhila, respectively). These values give R. tripartitum nutritional and industrial importance. Nowadays, research has increased to investigate new plant sources of oil from underexploited seeds. Results showed that the oil content varied between the different stages of maturity (Table 1). First, similar to protein content, the accumulation of oil increased from 3.80% (immature) to 6.41% (intermediate) and from 5.4% (immature) to 5.85% (intermediate) in Ain Jalloula and Dkhila, respectively. Many researchers have also reported an increase in oil content during fruit development (Msaada et al., 2009; Radic, 2006; Rahamatalla et al., 2001). This could be explained by the intervention of some enzymes, such as “Fatty Acid Synthase” which operates differently during fruit
ripening. In the beginning and the middle of maturity, the enzyme system is induced, and lipid accumulation is accelerated, whereas at the end of maturity, these enzymes are inactive, probably by retroinhibition, and the level of oil accumulation can decrease (Msaada et al., 2009). This reduction could also be due to lipase activation in the over-ripened seed (Saidani Tounsi et al., 2011). These findings may explain the decrease of the oil accumulation to 3.87% and 3.52% in Ain Jalloula and Dkhila, respectively. Our results were similar to other reports (Bouali et al., 2013; Saidani Tounsi et al., 2011). 3.3. Fatty acid composition To our knowledge, this was the first study of fatty acid accumulation in R. tripartitum fruits. Twenty five fatty acids were identified (Table 1). Results showed that fatty acid composition in R. tripartitum fruit did not vary between locations, and that palmitic acid (31.7–52.36%), oleic acid (16.55–43.59%), linoleic acid (21.23–21.90%) and linolenic acid (2.29–10.08%) were the most abundant. Previous reports have suggested that the environment is a secondary source of variability for fatty acids (Mukherjee et al., 1984; Uceda and Hermoso, 2001). Based on the ripening stage of R. tripartitum fruits, results did not reveal any variation in the fatty acid profile, but the majority of the fatty acid accumulated varied between stages of maturity (Table 1). Palmitic (16:0) and oleic (18:1) acids were the most abundant fatty acids in the first developmental stage of R. tripartitum fruit. Other authors have suggested the same finding for other crops such as Calophyllum fruits (Hathurusingha et al., 2011) and cuphea seeds (Berti and Johnson, 2008). In samples from Ain Jalloula, palmitic acid level remained stable during the two first stages of maturity (37%) and then fell slightly (31%); while in samples from Dkhila, palmitic acid content decreased from 52.36% (immature) to 39.86% (intermediate), and then increased to reach 46.34% (mature). Other studies have suggested that maturity has an effect on palmitic acid accumulation during ripening (Hathurusingha et al., 2011). Oleic acid content decreased 1.44 fold and 2.10 fold between the immature and intermediate stages and then increased again 1.41
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Table 2 Variation in saturated and unsaturated fatty acid sum and ratio in fruits of Rhus tripartitum collected from two locations at three different maturity stages. Ain Jalloula
Samples
SFAa MUFAa PUFAa UFAa MUFA/SFA PUFA/SFA UFA/SFA 3a 6a 6/3 Cox value
Dkhila
Immature
Intermediate
Mature
Immature
Intermediate
Mature
40.65 46.13 12.19 58.32 1.13 0.30 1.43 11.88 0.31 0.03 2.59
40.94 32.51 26.27 58.78 0.79 0.64 1.44 3.96 22.31 5.63 3.11
41.63 46.32 10.1 56.42 1.11 0.24 1.36 8.63 1.28 0.15 1.51
56.56 36.62 5.66 42.28 0.65 0.10 0.75 5.33 0.32 0.06 1.05
43.82 20.51 34.6 55.11 0.47 0.79 1.26 13.04 21.52 1.65 4.35
50.3 44.7 4.26 48.96 0.89 0.08 0.97 3.91 0.31 0.08 0.93
SFA: saturated fatty acid, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids, UFA: unsaturated fatty acids. a % Of total fatty acids.
fold and 2.63 fold in the mature stage, for samples from Ain Jalloula and Dkhila, respectively. It is clear from Table 1 that the decrease of oleic acid was accompanied by a remarkable increase in the content of linoleic acid (21.9% and 21.23% in Ain Jalloula and Dkhila, respectively). An increase of oleic acid in the last stage was accompanied by a severe decrease of linoleic acid. Our results corresponded to previous studies for other crops such as Calophyllum fruit (Hathurusingha et al., 2011) and Pistacia lentiscus fruit (Trabelsi et al., 2012). The high level of linoleinic acid in Dkhila was detected in the intermediate stage (9.27%); while in samples from Ain Jalloula the highest values were detected in the immature (10.08%) and mature stages (5.09%). Oleic, linoleic and linoleinic acids play an important role in cell components (Berti and Johnson, 2008). Moreover, linoleic acid is used by the personal care products industry due to its beneficial properties for skin (Letawe et al., 1998; Darmstadt et al., 2002). Some fatty acids, such as palmitoleic (16:1), arachidic (20:0) and erucic acid (22:1) were present at an appreciable level. Other fatty acids, such as myristic (14:0), pentadecanoic (15:0), marginic (17:0), stearic (18:0) and arachidonic acids (20:4) were present in trace amounts. It is well known that fatty acid profiles, as an indicator of nutritional value, determine the quality of the comestible oil. The total saturated fatty acid composition in R. tripartitum fruits remained steady during maturity of samples from Ain Jalloula (ca. 40%), while in samples from Dkhila the level decreased from 56.56% (immature) to 43.82% (intermediate), then increased to 50.3% (mature). Similarly, total unsaturated fatty acid content in R. tripartitum fruits also remained stable during maturity of samples from Ain Jalloula (ca. 57%), whereas in samples from Dkhila the values increased from 42.28% (immature) to 55.11 (intermediate), then decreased to 48.96% (mature). The ratios of monounsaturated to saturated (MUFA/SFA), polyunsaturated to saturated (PUFA/SFA) and unsaturated to saturated (UFA/SFA) fatty acids were calculated (Table 2). The ratio of polyunsaturated fatty acids to saturated fatty acids (PUFA/SFA) decreased in the last stage of fruit maturity. A similar result was seen in coriander fruit (Msaada et al., 2009). In most cases, unsaturated fatty acids, especially oleic and linoleic acids, dominate the storage lipid composition, and the ratio of unsaturated/saturated fatty acids exceeds 3 (Connor et al., 2007). The mean of UFA/SFA of R. tripartium fruits was around 1, due to the high level of palmitic acid. The level of PUFA 6 reached the maximum amount in the intermediate stage, due to the highest level of linoleic acid (18:2). The levels of PUFA 3 were more than 3.91% and the maximum values were obtained in the immature and mature stages for Ain Jalloula (11.88% and 8.63%, respectively), while the highest contents in Dkhila were obtained in the immature and intermediate stages (5.33% and 13.04%, respectively). The 6/3 ratio varied
between samples. In fact, nutritionists often recommend a 6/3 ratio of 5:1 for a healthy diet (DACH, 2002). The Cox value decreased in the later stages of maturity. These results were similar to those found in other studies (Trabelsi et al., 2012). A majority of the fatty acid is formed by oleic acid which has a lower weight in the Cox calculation than both linoleic and linolenic acids, since its oxidation time is far less than those of the other C18 unsaturated acids (Gunstone and Hilditch, 1946). 4. Conclusion To our knowledge, this was the first study about phytochemical content (protein and oil) and fatty acid composition of R. tripartitum fruits throughout maturity (immature: yellow; intermediate: mahogany brown; mature: dark brown) at different locations. The highest protein and oil contents were detected in the intermediate stage (ca. 9.5% and ca. 6%, respectively). Palmitic, oleic, linoleic, and linolenic acids were the major fatty acids. Fatty acid profiles did not vary among the growing regions; however, composition varied during maturity. Palmitic and oleic acids were the predominant fatty acids in the mature stage. Linoleic acid was at the highest amount in the intermediate stage (ca. 21%). Our results highlight the possibility of using R. tripartitum fruits as a source of protein and fatty acid for both dietetic and industrial applications. In addition, this work could be used as a reliable guide for estimating the best stage at which to obtain the maximum amount of these compounds. Acknowledgments We are very thankful to INRGREF for help with sample harvesting. The authors also wish to thank Mrs. Aicha Maaroufi and Mrs. Christie Nielsen Chaar for providing language help. The authors are also grateful to the anonymous referees and the editor for their insightful comments on an earlier draft. References Abbassi, F., Hani, K., 2012. In vitro antibacterial and antifungal activities of Rhus tripartitum used as antidiarrhoeal in Tunisian folk medicine. Nat. Prod. Res. 26, 2215–2218. Ahmed, M.S., Galal, A.M., Ross, S.A., Ferreira, D., El Sohly, M.A., Ibrahim, A.R.S., Mossa, J.S., El-Feraly, F.S., 2001. A weakly antimalarial biflavanone from Rhus retinorrhoea. Phytochemistry 58, 599–602. AOAC, 1984. Official Methods of the Association of Official analytical Chemists, 14th ed. AOAC, Arlington, TX (Method 28.110). Azmir, J., Zaidul, I.S.M., Rahman, M.M., Sharif, K.M., Sahena, F., Jahurul, M.H.A., Mohamed, A., 2014. Optimization of oil yield of Phaleria macrocarpa seed using response surface methodology and its fatty acids constituents. Ind. Crops Prod. 52, 405–412. Berti, M.T., Johnson, B.L., 2008. Physiological changes during seed development of cuphea. Field Crops Res. 106, 163–170.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Variation in protein and oil content and fatty acid composition of Rhus tripartitum fruits collected at different maturity stages in different locations Nizar Tlili a,c,∗ , Mariem Tir b , Houda Benlajnef a , Sheyma Khemiri a , Hassen Mejri b , Saloua Rejeb c , Abdelhamid Khaldi c a
Laboratoire de Biochimie, Département de Biologie, Faculté des Sciences de Tunis, Université Tunis El-Manar, Tunis 2092, Tunisie Unité de Recherche de Physiologie et Environnement Aquatiques, Département de Biologie, Faculté des Sciences de Tunis, Université Tunis EL Manar, 2092 Tunis, Tunisie c Institut National de Recherches en Génie Rural, Eaux et Forêts (INRGREF); Université de Carthage, BP: 10, Ariana 2080, Tunisia b
a r t i c l e
i n f o
Article history: Received 20 February 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Keywords: Rhus tripartitum fruits Protein Oil Fatty acid Maturity Location
a b s t r a c t The goal of this study is to evaluate the effects of ripening (immature: yellow; intermediate: mahogany brown; mature: dark brown) on the protein and oil content and fatty acid composition of Rhus tripartitum fruits collected from two Tunisian regions. The high protein and oil content were detected in the intermediate stage (ca. 9.5% and ca. 6%, respectively). Fatty acid profiles did not vary among the growing regions; and palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were the predominant fatty acids. Linoleic acid showed the highest level in the intermediate stage (ca. 21%). In the mature stages palmitic and oleic acids were the major fatty acids (more than 31% and 34%, respectively). Our findings showed that R. tripartitum fruits, especially at the intermediate stage, could therefore represent an attractive source of protein, oil and fatty acid for both food, cosmetic, and pharmaceutical industries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Storage proteins are a source of nutrients for both humans and farm animals. Hence, vegetable proteins have increased in commercial value due to consumer preference for natural food sources (Kaur and Singh, 2007). Vegetable oils are used directly in salad and for cooking, and can also be converted into many products for various uses such as personal care products, soaps, biodiesel, paint formulations, and adhesives (Azmir et al., 2014; Maisonneuve et al., 2013). The importance of oil in industry is determined by its fatty acid composition. Some authors have reported that the quality and flavor of some products can be affected by the fatty acid composition of the lipid (Hinds, 1999). In addition, some researchers have suggested that reducing consumption of saturated fats and increasing that of polyunsaturated fats is effective in lowering serum cholesterol, thus reducing coronary heart disease (Haruenkit et al., 2010). In vegetables, protein and oil content and fatty acid composition are influenced by several factors such as genetic factors,
∗ Corresponding author at: Laboratoire de Biochimie, Département de Biologie, Faculté des Sciences de Tunis, Université Tunis El-Manar, Tunis 2092, Tunisie. E-mail address: [email protected] (N. Tlili). http://dx.doi.org/10.1016/j.indcrop.2014.05.020 0926-6690/© 2014 Elsevier B.V. All rights reserved.
soil, climatic conditions, location, and ripening stages (Breene et al., 2007; Hinds, 1999; Tlili et al., 2011). The effect of ripening stages could be explained by the fact that during maturity, many biochemical, physiological, and structural variations occur to determine the quality of the vegetables (Vendramini and Trugo, 2000). Rhus is a woody genus belonging to the Anacardiaceae family, with about 250 species, which often grow in non-agriculturally viable regions. Species of Rhus are deciduous and multibranched shrubs or trees (Prakash and Van Staden, 2007). Many authors have reported the presence of high levels of bioflavonoids in the aerial parts, thus emphasizing their medicinal importance and supporting their use as spices by previous generations (Rayne and Mazza, 2007). Antimalarial, antimicrobial, and antitumorigenic impacts for Rhus species have also been reported (Ahmed et al., 2001; Choi et al., 2012; Lee et al., 2010). Rhus tripartitum, is a presaharan Tunisian plant (Pottier-Alapetite, 1979), commonly used by Tunisians in herbal treatments of many diseases, such as diarrhea and dysentery (Abbassi and Hani, 2012). The fruit is consumed fresh or stored, or added to water for a refreshing taste (Le Floc’h and Boulous, 2008). To the best of our knowledge, there have been no reports regarding the effect of location and ripening stages on protein and oil content or fatty acid profile of R. tripartitum fruits. The aim of this study was therefore to determine the amount of protein and
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oil and investigate the fatty acid composition of R. tripartitum fruits collected from two regions at three different stages of maturity. This study could be valuable for possible industrial use of R. tripartitum as a raw material source of protein and oil.
acid peaks were integrated and analyzed using HP Chemstation software. Analyses were performed in duplicate.
2. Materials and methods
The Cox value of the oil was calculated based on the percentage of unsaturated C18 fatty acids, applying the formula proposed by Fatemi and Hammond (1980).
2.1. Plant material R. tripartitum fruits were collected in February and March 2013 from two Tunisian regions, Dkhila (36◦ 52 ; 9◦ 42 ) and Ain Jalloula (35◦ 47 ; 9◦ 47 ). Samples were collected during three harvesting periods according to color: immature stage (yellow), intermediate stage (mahogany brown) and mature stage (dark brown). Samples were air-dried at room temperature and then ground for further analysis. Species were identified by Dr. Abdelhamid Khaldi and voucher specimens were deposited in the herbarium of the National Institute for Research in Rural Engineering Water and Forests (INRGREF, Tunisia).
2.5. Calculated oxidizability value (Cox)
Cox =
[1 × (18 : 1%) + 10.3 × (18 : 2%) + 21.6 × (18 : 3%)] 100
2.6. Statistical analyses Each value represented the mean ± SD. The Pearson correlation between mean values was performed using SPSS statistical package (version 17.0). The test was considered significant when the p value was lower than 0.05. 3. Results and discussion
2.2. Protein content Protein content was measured using a Kjeldhal apparatus (AOAC, 1984). Each 100 mg sample was digested for one hour with 100 mg of digestion mixture (8 g K2 SO4 + 20 g CaSO4 + 2 g selenium) and 6 mL of concentrated H2 SO4 . When digestion was completed, the solution became clear. The solution was then placed in a volumetric flask and distilled water was added until the total volume reached 30.0 mL. To determine nitrogen content, 10 mL of 2% boric acid solution was first put in a beaker with a few drops of methyl red as an indicator. Then 10 mL of the digested mixture, 30 mL of 40% NaOH solution, and 10 mL of distilled water were transferred to the distillation chamber. The liberated ammonia combined with NaOH to form NH4 OH, which was then added to the boric acid solution to form ammonium borate (pink to yellow color). The ammonium borate was then titrated with 0.1 N H2 SO4 . The volume of acid that had been added at the point when the color of the distillate changed from yellow to pink was recorded. Protein was calculated according to the following formula: 115%protein = %N × 6.25. Analyses were performed in triplicate. 2.3. Oil extraction Oil content was determined according to ISO method 659:1998 (ISO, 1999). About 30 g of the powder were extracted with petroleum ether in a Soxhlet apparatus for 4 h. The solvent was concentrated using a rotary evaporator under reduced pressure at 50 ◦ C and then a stream of nitrogen was used to dry the oil before storing at −20 ◦ C for later use. Analyses were performed in triplicate.
3.1. Protein content To our knowledge, there is no data about protein content of R. tripartitum fruits. The results in Fig. 1 show the estimated protein content (% dry weight basis) from R. tripartitum fruits collected at different stages of maturity from two locations in Tunisia. Regardless of maturity or location, R. tripartitum fruits contained large quantities of protein, between 4.81% and 10.5%. The highest values were found at the intermediate stage for both locations (9.37% and 10.5% in Ain Jalloula and Dkhila, respectively). These values lend nutritional importance to R. tripartitum fruits due to the fact that protein, as a macronutrient, plays a crucial role in the human diet without any adverse effects. These proteins found here could be important for different population sectors or used as a raw material source in the food industry, especially in Afro-Asian regions where the protein–calorie malnutrition syndrome affects more than 170 million preschool children and nursing mothers (Iqbal et al., 2006). Values clearly showed a difference between samples from the two regions for the same stage of maturity (4.81%, 9.37% and 6.75% in Ain Jalloula and 6.16%, 10.5% and 7.88% in Dkhila, for immature, intermediate and mature stage, respectively). These results were similar to those found by other authors who suggested that protein content of crops can vary with soil, climatic conditions, and cultivars (El Arem et al., 2011; Tlili et al., 2011). When comparing protein levels, in the two locations, based on the stage of maturity, we can conclude firstly that the content
2.4. Fatty acid analysis The fatty acid composition of the oil was determined by gas chromatography as fatty acid methyl esters (FAMEs) using the method described by Cecchi et al. (1985). The FAMEs were separated and identified with a HP 6890 gas chromatograph with a split/splitless injector equipped with a flame ionization detector, and a 30 m HP Innowax capillary column with an internal diameter of 250 m and a film thickness of 0.25 m. The temperatures of the injector and the detector were maintained at 250 ◦ C and 275 ◦ C, respectively; the oven was programmed to rise from 50 to 180 ◦ C at a rate of 4 ◦ C/min, from 180 to 220 ◦ C at 1.33 ◦ C/min and to stabilize at 220 ◦ C for 7 min. Nitrogen was the carrier gas. Fatty acids were identified by comparing retention times of the FAMEs with those of co-injected authentic standards (SUPELCO PUFA-3). Fatty
Fig. 1. Variation in protein content in fruits of Rhus tripartitum collected from two locations at three different maturity stages.
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Table 1 Variation in oil content and fatty acid composition in fruits of Rhus tripartitum collected two locations at three different maturity stages. Ain Jalloula
Samples
Oil (% of dry weight) Fatty acid (% of total fatty acids) 14:0 15:0 15:1 16:0 16:1 17:0 16:3 16:4 18:0 18:1 18:2n-6 18:3n-3 18:4n-3 20:0 20:1 20:2n6 20:3n6 20:4n-6 20:3n-3 20:4n3 20:5n3 22:0 22:1 22:5n-3 22:6n-3
Dkhila
Immature
Intermediate
Mature
Immature
Intermediate
Mature
3.80 ± 0.42
6.41 ± 0.41
3.87 ± 0.89
5.4 ± 0.09
5.85 ± 0.93
3.52 ± 0.09
0.76 ± 0.07 0.17 ± 0.04 0.04 ± 0.00 37.48 ± 1.76 2.59 ± 0.06 0.25 ± 0.11 0.02 ± 0.00 0.03 ± 0.00 0.06 ± 0.00 41.73 ± .024 tr 10.08 ± 0.87 0.20 ± 0.06 1.93 ± 0.33 0.29 ± 0.08 0.14 ± 0.01 0.06 ± 0.02 0.11 ± 0.05 0.04 ± 0.02 0.01 ± 0.00 0.08 ± 0.01 0.03 ± 0.01 1.48 ± 0.33 0.79 ± 0.11 0.68 ± 0.13
0.89 ± 0.56 0.29 ± 0.32 0.05 ± 0.00 37.67 ± 1.84 2.02 ± 2.53 0.25 ± 0.01 0.01 ± 0.00 tr 0.07 ± 0.00 28.89 ± 2.65 21.90 ± 0.74 2.64 ± 0.10 0.14 ± 0.14 1.49 ± 0.73 0.47 ± 0.58 0.15 ± 0.09 0.17 ± 0.10 0.09 ± 0.01 0.29 ± 0.37 0.05 ± 0.00 0.04 ± 0.00 0.28 ± 0.0.34 1.08 ± 0.12 0.47 ± 0.01 0.33 ± 0.13
1.15 ± 0.50 2.84 ± 3.46 1.08 ± 0.93 31.88 ± 1.55 1.47 ± 0.55 0.62 ± 0.48 0.12 ± 0.10 0.19 ± 0.03 2.96 ± 0.22 41.73 ± 2.81 tr 5.09 ± 0.68 0.55 ± 0.38 1.92 ± 0.28 1.35 ± 1.01 0.66 ± 0.01 0.26 ± 0.00 0.36 ± 0.00 0.34 ± 0.39 0.19 ± 0.11 0.18 ± 0.13 0.26 ± 0.19 1.59 ± 0.73 0.69 ± 0.25 1.59 ± 0.62
0.64 ± 0.18 0.02 ± 0.01 0.02 ± 0.02 52.36 ± 13.06 0.20 ± 0.02 0.83 ± 0.06 0.16 ± 0.02 0.01 ± 0.00 0.09 ± 0.02 34.83 ± 15.00 tr 3.25 ± 1.42 0.14 ± 0.10 2.43 ± 0.55 0.07 ± 0.00 0.15 ± 0.00 0.02 ± 0.03 0.15 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.05 ± 0.00 0.19 ± 0.05 1.40 ± 0.58 0.89 ± 0.00 0.98 ± 0.01
1.12 ± 0.06 0.13 ± 0.03 0.33 ± 0.23 39.86 ± 1.64 2.36 ± 0.31 0.32 ± 0.21 0.14 ± 0.09 0.04 ± 0.01 0.48 ± 0.01 16.55 ± 7.28 21.23 ± 13.66 9.27 ± 0.19 0.49 ± 0.48 1.04 ± 0.53 0.12 ± 0.01 0.08 ± 0.02 0.07 ± 0.04 0.14 ± 0.04 0.10 ± 0.10 0.08 ± 0.01 0.03 ± 0.00 0.87 ± 0.17 1.15 ± 0.05 1.69 ± 1.08 1.38 ± 0.52
0.79 ± 0.10 0.09 ± 0.00 0.01 ± 0.00 46.34 ± 1.88 0.09 ± 0.02 1.33 ± 1.03 0.35 ± 0.08 0.04 ± 0.03 0.05 ± 0.03 43.59 ± 3.92 tr 2.29 ± 0.11 0.11 ± 0.02 1.49 ± 0.13 0.04 ± 0.00 0.07 ± 0.00 0.08 ± 0.04 0.16 ± 0.10 0.01 ± 0.00 0.01 ± 0.00 0.06 ± 0.00 0.21 ± 0.17 0.97 ± 0.35 0.74 ± 0.28 0.69 ± 0.30
increased from 4.81% (immature) to 9.37% (intermediate) and from 6.16% (immature) to 10.5% (intermediate) in Ain Jalloula and Dkhila, respectively. The increase in protein levels during ripening might be due to the activation of some enzymes, like cellulase and polygalacturonase, which play significant roles in the softening of the fruit (El Arem et al., 2011). Secondly, the protein content in R. tripartitum decreased to 6.75% and 7.88% in Ain Jalloula and Dkhila, respectively. This decrease was more than 25%. Other authors have reported that protein content can decrease 30% with maturity due to biochemical degradation and also due to the possible secondary metabolite production (Vendramini and Trugo, 2000). This decrease would not affect the nutritional quality but might affect the flavor characteristics of the fruits (Vendramini and Trugo, 2000). 3.2. Oil content The content of oil (% dry weight basis) in all samples of R. tripartitum are presented in Table 1. The results clearly showed a difference in the accumulation of oil between locations. We did not find information about oil accumulation in R. tripartitum fruits, but our results were similar to other authors who reported that oil content in the crops can fluctuate with climatic conditions and cultivars (Tlili et al., 2011). The highest values were detected at the intermediate stage for both locations (6.41% and 5.85% in Ain Jalloula and Dkhila, respectively). These values give R. tripartitum nutritional and industrial importance. Nowadays, research has increased to investigate new plant sources of oil from underexploited seeds. Results showed that the oil content varied between the different stages of maturity (Table 1). First, similar to protein content, the accumulation of oil increased from 3.80% (immature) to 6.41% (intermediate) and from 5.4% (immature) to 5.85% (intermediate) in Ain Jalloula and Dkhila, respectively. Many researchers have also reported an increase in oil content during fruit development (Msaada et al., 2009; Radic, 2006; Rahamatalla et al., 2001). This could be explained by the intervention of some enzymes, such as “Fatty Acid Synthase” which operates differently during fruit
ripening. In the beginning and the middle of maturity, the enzyme system is induced, and lipid accumulation is accelerated, whereas at the end of maturity, these enzymes are inactive, probably by retroinhibition, and the level of oil accumulation can decrease (Msaada et al., 2009). This reduction could also be due to lipase activation in the over-ripened seed (Saidani Tounsi et al., 2011). These findings may explain the decrease of the oil accumulation to 3.87% and 3.52% in Ain Jalloula and Dkhila, respectively. Our results were similar to other reports (Bouali et al., 2013; Saidani Tounsi et al., 2011). 3.3. Fatty acid composition To our knowledge, this was the first study of fatty acid accumulation in R. tripartitum fruits. Twenty five fatty acids were identified (Table 1). Results showed that fatty acid composition in R. tripartitum fruit did not vary between locations, and that palmitic acid (31.7–52.36%), oleic acid (16.55–43.59%), linoleic acid (21.23–21.90%) and linolenic acid (2.29–10.08%) were the most abundant. Previous reports have suggested that the environment is a secondary source of variability for fatty acids (Mukherjee et al., 1984; Uceda and Hermoso, 2001). Based on the ripening stage of R. tripartitum fruits, results did not reveal any variation in the fatty acid profile, but the majority of the fatty acid accumulated varied between stages of maturity (Table 1). Palmitic (16:0) and oleic (18:1) acids were the most abundant fatty acids in the first developmental stage of R. tripartitum fruit. Other authors have suggested the same finding for other crops such as Calophyllum fruits (Hathurusingha et al., 2011) and cuphea seeds (Berti and Johnson, 2008). In samples from Ain Jalloula, palmitic acid level remained stable during the two first stages of maturity (37%) and then fell slightly (31%); while in samples from Dkhila, palmitic acid content decreased from 52.36% (immature) to 39.86% (intermediate), and then increased to reach 46.34% (mature). Other studies have suggested that maturity has an effect on palmitic acid accumulation during ripening (Hathurusingha et al., 2011). Oleic acid content decreased 1.44 fold and 2.10 fold between the immature and intermediate stages and then increased again 1.41
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Table 2 Variation in saturated and unsaturated fatty acid sum and ratio in fruits of Rhus tripartitum collected from two locations at three different maturity stages. Ain Jalloula
Samples
SFAa MUFAa PUFAa UFAa MUFA/SFA PUFA/SFA UFA/SFA 3a 6a 6/3 Cox value
Dkhila
Immature
Intermediate
Mature
Immature
Intermediate
Mature
40.65 46.13 12.19 58.32 1.13 0.30 1.43 11.88 0.31 0.03 2.59
40.94 32.51 26.27 58.78 0.79 0.64 1.44 3.96 22.31 5.63 3.11
41.63 46.32 10.1 56.42 1.11 0.24 1.36 8.63 1.28 0.15 1.51
56.56 36.62 5.66 42.28 0.65 0.10 0.75 5.33 0.32 0.06 1.05
43.82 20.51 34.6 55.11 0.47 0.79 1.26 13.04 21.52 1.65 4.35
50.3 44.7 4.26 48.96 0.89 0.08 0.97 3.91 0.31 0.08 0.93
SFA: saturated fatty acid, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids, UFA: unsaturated fatty acids. a % Of total fatty acids.
fold and 2.63 fold in the mature stage, for samples from Ain Jalloula and Dkhila, respectively. It is clear from Table 1 that the decrease of oleic acid was accompanied by a remarkable increase in the content of linoleic acid (21.9% and 21.23% in Ain Jalloula and Dkhila, respectively). An increase of oleic acid in the last stage was accompanied by a severe decrease of linoleic acid. Our results corresponded to previous studies for other crops such as Calophyllum fruit (Hathurusingha et al., 2011) and Pistacia lentiscus fruit (Trabelsi et al., 2012). The high level of linoleinic acid in Dkhila was detected in the intermediate stage (9.27%); while in samples from Ain Jalloula the highest values were detected in the immature (10.08%) and mature stages (5.09%). Oleic, linoleic and linoleinic acids play an important role in cell components (Berti and Johnson, 2008). Moreover, linoleic acid is used by the personal care products industry due to its beneficial properties for skin (Letawe et al., 1998; Darmstadt et al., 2002). Some fatty acids, such as palmitoleic (16:1), arachidic (20:0) and erucic acid (22:1) were present at an appreciable level. Other fatty acids, such as myristic (14:0), pentadecanoic (15:0), marginic (17:0), stearic (18:0) and arachidonic acids (20:4) were present in trace amounts. It is well known that fatty acid profiles, as an indicator of nutritional value, determine the quality of the comestible oil. The total saturated fatty acid composition in R. tripartitum fruits remained steady during maturity of samples from Ain Jalloula (ca. 40%), while in samples from Dkhila the level decreased from 56.56% (immature) to 43.82% (intermediate), then increased to 50.3% (mature). Similarly, total unsaturated fatty acid content in R. tripartitum fruits also remained stable during maturity of samples from Ain Jalloula (ca. 57%), whereas in samples from Dkhila the values increased from 42.28% (immature) to 55.11 (intermediate), then decreased to 48.96% (mature). The ratios of monounsaturated to saturated (MUFA/SFA), polyunsaturated to saturated (PUFA/SFA) and unsaturated to saturated (UFA/SFA) fatty acids were calculated (Table 2). The ratio of polyunsaturated fatty acids to saturated fatty acids (PUFA/SFA) decreased in the last stage of fruit maturity. A similar result was seen in coriander fruit (Msaada et al., 2009). In most cases, unsaturated fatty acids, especially oleic and linoleic acids, dominate the storage lipid composition, and the ratio of unsaturated/saturated fatty acids exceeds 3 (Connor et al., 2007). The mean of UFA/SFA of R. tripartium fruits was around 1, due to the high level of palmitic acid. The level of PUFA 6 reached the maximum amount in the intermediate stage, due to the highest level of linoleic acid (18:2). The levels of PUFA 3 were more than 3.91% and the maximum values were obtained in the immature and mature stages for Ain Jalloula (11.88% and 8.63%, respectively), while the highest contents in Dkhila were obtained in the immature and intermediate stages (5.33% and 13.04%, respectively). The 6/3 ratio varied
between samples. In fact, nutritionists often recommend a 6/3 ratio of 5:1 for a healthy diet (DACH, 2002). The Cox value decreased in the later stages of maturity. These results were similar to those found in other studies (Trabelsi et al., 2012). A majority of the fatty acid is formed by oleic acid which has a lower weight in the Cox calculation than both linoleic and linolenic acids, since its oxidation time is far less than those of the other C18 unsaturated acids (Gunstone and Hilditch, 1946). 4. Conclusion To our knowledge, this was the first study about phytochemical content (protein and oil) and fatty acid composition of R. tripartitum fruits throughout maturity (immature: yellow; intermediate: mahogany brown; mature: dark brown) at different locations. The highest protein and oil contents were detected in the intermediate stage (ca. 9.5% and ca. 6%, respectively). Palmitic, oleic, linoleic, and linolenic acids were the major fatty acids. Fatty acid profiles did not vary among the growing regions; however, composition varied during maturity. Palmitic and oleic acids were the predominant fatty acids in the mature stage. Linoleic acid was at the highest amount in the intermediate stage (ca. 21%). Our results highlight the possibility of using R. tripartitum fruits as a source of protein and fatty acid for both dietetic and industrial applications. In addition, this work could be used as a reliable guide for estimating the best stage at which to obtain the maximum amount of these compounds. Acknowledgments We are very thankful to INRGREF for help with sample harvesting. The authors also wish to thank Mrs. Aicha Maaroufi and Mrs. Christie Nielsen Chaar for providing language help. The authors are also grateful to the anonymous referees and the editor for their insightful comments on an earlier draft. References Abbassi, F., Hani, K., 2012. In vitro antibacterial and antifungal activities of Rhus tripartitum used as antidiarrhoeal in Tunisian folk medicine. Nat. Prod. Res. 26, 2215–2218. Ahmed, M.S., Galal, A.M., Ross, S.A., Ferreira, D., El Sohly, M.A., Ibrahim, A.R.S., Mossa, J.S., El-Feraly, F.S., 2001. A weakly antimalarial biflavanone from Rhus retinorrhoea. Phytochemistry 58, 599–602. AOAC, 1984. Official Methods of the Association of Official analytical Chemists, 14th ed. AOAC, Arlington, TX (Method 28.110). Azmir, J., Zaidul, I.S.M., Rahman, M.M., Sharif, K.M., Sahena, F., Jahurul, M.H.A., Mohamed, A., 2014. Optimization of oil yield of Phaleria macrocarpa seed using response surface methodology and its fatty acids constituents. Ind. Crops Prod. 52, 405–412. Berti, M.T., Johnson, B.L., 2008. Physiological changes during seed development of cuphea. Field Crops Res. 106, 163–170.
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