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Plant Foods for Human Nutrition 59: 23–27, 2004. C 2004 Springer Science+Business Media, Inc.
23
Fatty Acids and Carotenes in Some Ber (Ziziphus jujuba Mill) Varieties 1,2 ´ J.L. GUIL-GUERRERO,1,∗ A. D´IAZ DELGADO,2 M.C. MATALLANA GONZALEZ, & M.E. TORIJA ISASA1,2 1 Dpto.
de Ingenier´ıa Qu´ımica, Facultad de Ciencias Experimentales, Universidad de Almer´ıa, 04120 Almer´ıa, Spain; 2 Dpto. de Nutrici´on y Bromatolog´ıa II, Facultad de Farmacia de la U.C.M., 28040 Madrid, Spain (∗ author for correspondence; e-mail:
[email protected].)
Abstract. Several jujube varieties from the southeast of Spain were analyzed for fatty acid and carotene contents. Triglycerides having mediumchain fatty acids were most abundant in all samples. The main fatty acids were 12:0 (18.3 ± 9.97), 10:0 (12.5 ± 19.0), 18:2n6 (9.27 ± 7.26), 16:1n7 (8.50 ± 5.77), 16:0 (7.25 ± 4.35), and 18:1n9 (5.34 ± 2.52) on total saponifiable oil. The fruits yield 1.33 ± 0.17 g/100 g saponifiable oil on a dry weight basis. Fatty acid profiles of fruits were found to be influenced by their developmental stage. Multivariable data analyses show that the samples could be grouped on the basis of their fatty acid content. Carotenes were found to be in good agreement with other fruits, varying from 4.12 to 5.98 mg/100 g on a dry weight basis. The contribution to vitamin value reach a mdium of 38 g RE/100 g on a fresh weight basis. Key words: ber, carotenes, fatty acid, medium-chain fatty acids, multivariable data analyses, Ziziphus jujuba
the USDA nutrient database shows that the jujube is rich in carbohydrates, some minerals such as potassium, and vitamin C. To date, analytical data for this species are very scarce. Moreover, the great number of known jujube varieties would make it interesting to differentiate them on the basis of nutritional principle. This work reports on the fatty acid, carotenoids, and βcarotene contents of some ber varieties cultivated in Spain. Data are discussed on the basis of fruit variety, culture type (irrigation or not), location, and developmental stage (mature or raisin). The relationship among the samples is discussed with respect to the fatty acid profile.
Introduction
Materials and Methods
The ber is a hardy fruit tree which grows well, bears heavy crop regularly, and gives handsome economic returns even under constraints of irrigation and fertility and on marginal lands where other fruits, such as some citrus ones and, mango, cannot be grown. In Spain, the fruits ripen during September, but in other countries, such as India, they ripen between February and April, when few other fruits are available [1]. These fruits, besides ingested fresh, can be dried and also preserved in the form of a candy. Some workers have investigated the composition of these fruits and their biological activities. These fruits have a high nutritive value, being a rich source of vitamins C, A, and B complex, and also of Ca, K, Br, Rb, and La [2]. On the other hand, in tests using rats, it was found that systemic irrigation of oral cavities with 10% tincture and cleaning teeth with pastes made from these fruits normalized macroand microelement homeostasis of the solid tissues of the teeth. It was concluded that these fruits could be used to treat disturbed mineral metabolism in teeth [3]. In addition, the extracts of Ziziphus jujuba are used in Japan to treat chronic hepatitis or distress and fullness in the chest and ribs [4]. The volatile constituents of the fruit of Ziziphus jujuba var. inermis shows seventy-eight compounds, among which aliphatic acids and carbonyl compounds accounted for 62.97 and 29.56% of the total oil volatile, respectively. The major components were the decanoic acid (19.98%) and the dodecanoic acid (15.64%) [5]. On the other hand,
Samples Only fruits in a stage fit for consumption (mature or raisin) were used. Fruits were harvested and freeze-dried (Edwards Modulyo-4K freeze drier) and stored at −18 ◦ C until analyses were made. Samples were collected from the locations shown in Table 1. Moisture Determined by drying a representative 2-g sample in an oven with air circulation at 100–105 ◦ C for 40 hours. Fatty Acids Methyl esters were prepared by treatment of the lipidic fraction with acetyl chloride and methanol [6]. The fatty acid methyl esters (FAMEs) of the mixture were analysed by gas chromatography, the FAMEs were identified by comparing their retention times with those of standards (“Rapeseed oil mix” and “PUFAS-1,” from Sigma), in a Hewlett-Packard HP5890 series II chromatograph provided with a flame ionization detector and HP3394 integrator. A capillary column of high polarity fused silica was used (Supelco SP2330; length: 30 m; internal diameter: 0.25 mm; thickness of the film: 0.2 µm). The flow of carrier gas (N2 ) was 0.75 l/min, and the split ratio of the injector was 100:1. The injector temperature was 240 ◦ C and the detector temperature was
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24 Table 1. Sample codes nomenclature Location
Varieties
Development stage Culture type
Ciudad Real (Ci) Ol´las (Ol)
Li (Li) Gorda (Go)
Raisin (Ra) Mature (Ma)
Castell´on (Ca) Antas (An)
Mudeong (Mu) Wild (Wi) Lang (La) Unknown (Un)
Irrigable (Ir) Unirrigated land (Dr)
Note. Samples having the same nomenclature are distinguished by means of a serial letter.
260 ◦ C. The starting temperature of the oven was 205 ◦ C and it was increased at a rate of 6 ◦ C/min until 240 ◦ C (5.83 min). The injection volume was 5 µL and a blank was run after every two analyses. Peaks were identified by using standard FAME and quantified by using methyl heptadecanoate (17:0) as an internal standard. Verification of double bounds was made by gas chromatography–mass spectrometry (GC-MS) in a HewlettPackard HP5890A G.C. provided of a HewlettPackard 5988A M.S. A capillary column of methyl silicorie (HP-1; length: 25 m; internal diameter: 0.2 mm; thickness of the film: 0.33 µm) was used. The flow of the carrier gas (He) was 1 ml/min. Injector temperature was 260 ◦ C, and the pressure at the head of the column was 15 psi. The oven starting temperature was 100 ◦ C, and it was increased at a rate of 10 ◦ C until 280 ◦ C, and then kept at 280 ◦ C for 10 min. The temperature in the interphase was 280 ◦ C, and the temperature of the source in the detector was 180 ◦ C. Carotenoids These were determined spectrophotometrically [7]. β-Carotene The method of extraction and HPLC analysis was that described in [8]. The extraction method involved the blending of produce in acetone, shaking with diethyl ether and water, and collecting the organic layer which was washed with water, dried over anhydrous sodium sulphate, and reduced in volume under vacuum. Saponification was found to be unnecessary. The carotenoid residue was dissolved in a mixture of methanol/methyl-tert-butyl ether and after making up to volume was filtered and immediately analysed by HPLC-mass spectrometry. Analyses were made with a Hewlett-Packard HP11100. The stationary phase was YMC Carotenoid C-30 5 µm, 4.6 × 250 mm. The drying gas flow was 6 l/min, the pebulizer pressure was 40 psig, the drying gas temperature was 325 ◦ C, the vaporizer temperature was 450 ◦ C, the capillary voltage was 2500 V and the corona current was 3 mA. The
mobile phase was a mixture of methanol/methyl-tert-butyl ether. The gradient began with 15% (v/v) of methanol and finished with 100% of methanol at t = 60 min. The eluent flow was 1 ml/min. The interface between the LC and MS was APCI (atmospheric pressure chemical ionization) positive (fragmentor 100 V). Peak identification was based on comparison of HPLC retention times and mass spectra with β-carotene, the chemical standard, which was purchased from IMATRA, S.A. The amount of β-carotene was calculated from chromatographic responses to standard solution. Principal component analyses were performed with the software package Statgraphics for Windows v. 3.3.
Results and Discussion In order to provide a good global comprehension of the influence of the work variables (variety, location, culture type, and developmental stage) on the values obtained for analyzed nutrients, the data were grouped using codes shown in Table 1. Data referring to moisture, total carotenoids, and fatty acid profiles are given in Table 2. Moisture content ranged from 58.9% (AnWiRaIr) to 83.6% (CiMuMaDr b). All mature samples have moisture values of above 70%, while all raisin samples have lower values. Triglycerides having medium-chain-fatty acids were most abundant in all samples. Thus, the 8:0 (3.57 ± 4.45) ranged from 0.12% in CiMuMaDr a to 12.1% in CiGoRaDr; the 10:0 (12.5 ± 18.99) shows also a wide variation, ranging from 1.59% (CiGoRaDr) to 65.5% (AnWiRaIr), and the 12:0 was the prevailing fatty acid in most samples, varying between 1.21% (AnWiRalr) and 45.5% (CiGoRaDr). The fatty acid 14:0 (2.30 ± 1.45) was found in lower amounts, ranging from 0.38% (CiMuMaDr b) to 4.75% (CiLaRaIr). All fatty acids from the C16 series were found in low amounts in the sample An WiRaIr. Thus, 16:0 (7.25 ± 4.35) and 16:1n7 (8.50 ± 5.77) ranged from 0.43 and 0.46% respectively in AnWiRaIr to 13.10% in CaGoRaIr (16:0) and 19.4% (16:1n7) in CiLaRaIr. The two samples from Antas shows the lower amounts for all fatty acids of C18 series, 18:0 being the most scarce. Thus, 18:0 (2.48 ± 1.98) ranged from 0.34% in the two samples from Antas to 6.81% in CiGoMaIr. The 18:1n9 (5.34 ± 2.52) varied between 0.48% (AnWiRaIr) and 8.38% (CiLiMaIr and CiGoRaIr). The 18:1n7 (2.23 ± 1.08) ranged from 0.38% in AnWiRalr to 3.98% in CiLaRaIr. The 18:2n6 (9.27 ± 7.26) was very variable, ranging from 0.23% (AnWiRaIr) to 25.4% (CiMuMaDr b). The 18:3n3 (4.61 ± 4.27) had very disparate amounts, the two samples from Antas showing the lowest percentage (0.35%) and CiWiMaDr b the highest (13.7%).
b On
12.5 19.0
8.33 10.1 5.51 1.59 4.44 3.77 2.20 6.27 6.32 5.70 2.78 5.42 46.8 65.5
10:0
oil obtained by GLC. a dry weight basis.
a Saponifiable
3.57 4.45
10.9 1.79 0.35 12.1 0.12 0.18 0.67 0.27 0.52 0.48 2.28 3.42 6.73 10.1
CiLiMaIr CiLiMaDr CiGoMaIr CiGoMaDr CiMuMaDr a CiMuMaDr b CiMuMaDr c CiMuMaIr CiLaRaIr OlUnRaIr CaGoMaIr CaUnRaIr AnWiMaIr AnWiRaIr
Mean S.D.
8:0
18.3 9.97
15.6 23.2 16.9 45.5 24.7 11.5 14.7 10.4 15.9 25.0 17.4 18.8 15.1 1.21
12:0
2.30 1.45
1.77 3.23 2.97 2.15 0.43 0.38 1.89 0.83 4.75 3.06 4.12 3.78 2.48 0.35
14:0
7.25 4.35
8.90 10.5 11.5 4.42 1.28 2.10 4.34 5.11 11.6 10.1 13.1 12.1 6.22 0.43
16:0
8.50 5.77
7.00 11.1 10.7 8.35 1.34 1.98 3.45 3.24 19.4 13.2 14.4 13.6 10.7 0.46
16:1n7
2.48 1.98
4.19 5.74 6.81 1.74 1.16 1.66 1.35 1.66 1.88 1.77 4.14 1.61 0.34 0.34
18:0
5.34 2.52
8.38 4.44 8.38 2.06 6.55 8.16 6.50 6.18 6.15 5.12 4.84 6.20 1.35 0.48
18:1n9
2.23 1.08
3.19 1.91 3.23 1.10 2.14 2.73 2.15 2.01 3.98 2.46 2.36 3.07 0.46 0.38
18:1n7
9.27 7.26
8.29 7.13 7.56 2.75 17.4 25.4 17.0 17.5 5.15 4.97 6.81 7.97 1.67 0.23
18:2n6
4.61 4.27
3.77 2.23 2.58 0.89 10.4 13.7 9.94 8.44 1.97 2.15 2.76 5.09 0.35 0.35
18:3n3
0.58 0.31
0.54 0.47 0.64 0.31 0.82 1.17 1.13 0.76 0.30 0.52 0.55 0.48 0.13 0.25
20:0
0.91 0.57
0.91 0.47 1.23 0.33 1.53 1.89 2.00 0.95 0.51 0.65 0.89 0.76 0.24 0.35
20:1n9
1.33 1.77
1.25 1.47 1.12 1.45 1.23 1.30 1.27 1.08 1.44 1.57 1.21 1.42 1.14 1.67
Lipids (mg/100 g)a,b
4.95 0.71
4.57 4.78 4.12 5.89 4.22 4.25 3.99 4.54 5.44 5.64 5.40 5.73 4.86 5.98
Carotene (mg/100 g)b
70.4 13.9
74.9 72.3 70.7 74.7 81.4 83.6 83.4 78.4 60.2 68.1 68.9 66.2 79.5 58.9
Moisture (%)
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Samples
Fatty acids (on total saponifiable oil)
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Table 2. Fatty acid and carotenoid contents of some ber (Ziziphus jujuba Mill.) varieties
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26 The C20 series was composed of smaller quantities of 20:0 (0.58 ± 0.31) and 20:1n9 (0.91 ± 0.57). Both ranged from 0.13 and 0.24% in An WiMaIr to 1.17% (CiMuMaDr b) and 2.00% in CiMuMaDr c, respectively. Total saponifiable oil (1.33 ± 1.77 on a dry weight basis) is shown in Table 2. These amounts were in good agreement with those of other fruits, such as apples or peaches. They ranged between 1.08% (CiMuMaIr) and 1.67% (AnWiRaIr). Total carotenoids (4.95 ± 0.71 on a dry weight basis) were also in good agreement with other fruits, varying from 3.99 mg/100 g (CiMuMaDr c) to 5.98 mg/100 g (AnWiRaIr). β-carotene was quantified once in each ber variety by means of HPLC-mass (nine analyses). Analysis reveals that β-carotene comprises 15 ± 6% of total carotenoids. Thus, if we consider that 6 µg β-carotene = 1 µg RE, 100 g of fresh ber would contain a mean of 38 µg RE. Results showed that the main fatty acids were mediumchain-fatty acids, which have been reported recently to improve health. This way, it appears that administration of thermogenetically acting triacylglycerols with medium-chain fatty acids can prevent diet-induced energetic efficiency and can improve the long-term success of dietotherapy of obese patients [9]. Furthermore, results from longest controlled feeding of medium-chain fatty acids studied to date suggest that short-term feeding of diets enriched by medium-chain fatty acids increases total energy expenditure [10]. Thus, a diet enriched with ber can have beneficial effects on human health. This fact has relevance in countries in which jujuba consumption can represent an important part of the diet, such as occurs in some regions of India [1].
Statistical Analyses The correlation coefficients among variables were high and, generally, significant, indicating that the variations could be due to a few related causes. In order to investigate a possible correlation between related variables and sample characteristics, we have conducted a principal components analysis (PCA) by using the fatty acid and carotene profiles. In this analysis, the two first principal components explained 43.7 and 28.6%, respectively, of the total variance. The plot for the two first component weights (Figure 1) shows that there is a grouping of variables. Thus, lipids and carotenes (r = 0.7120; P < 0.01) have a great influence on component 1, and they form a group with the fatty acids 8:0, 10:0, and 12:0, indicating that these are a major constituent of lipids. Other group is composed by the fatty acids 14:0, 16:0, and 16:1n7, located down in the plot. These have positive correlations among them: 14:0 with 16:0 (r = 0.913; P < 0.01), 14:0 with 16:1n7 (r = 0.972; p < 0.01), and 16:0 with 16:1n7 (r = 0.889; p < 0.01). These results suggest that the enzymatic systems that elongated the 14:0 fatty
Figure 1. Plot for first two components weights.
acid and desaturated the 16:0 are coordinated. Next to the previous group, there are variables in a most diffuse group, composed by 18:0, 18:1n9, and 18:1n7. These variables have positive correlations with the variables from the last group: 18:0 with 16:0 (r = 0.613; p < 0.05) and 18:1n9 with 16:0 (r = 0.582; p < 0.05). Simultaneously, 18:1n9 and 18:1n7 are positively correlated (r = 0.854; P < 0.01). Other groups were found up in the plot, next to the previous C18 , which comprises polyunsaturated fatty acids from the C18 series (18:2n6 and 18:3n3) and others from the C20 series (20:0 and 20:1n9). The C18 fatty acids have positive correlation among them: 18:2n6 with 18:1n9 (r = 0.664; p < 0.01), 18:2n6 with 18:3n3 (r = 0.983; p < 0.01), and 18:1n9 with 18:3n3 (r = 0.609; p < 0.05). Fatty acids from the C18 and C20 series also have positive correlations among them: 18:1n9 with 20:1n9 (r = 0.703; p < 0.01), 18:2n6 with 20:0 (r = 0.033; p < 0.01), 18:2n6 with 20:1n9 (r = 0.875; p < 0.01), 18:3n3 with 20:0 (r = 0.924; p < 0.01), and 18:3n3 with 20:1n9 (r = 0.962; p < 0.01). These correlations are a consequence of the metabolic routes in that the fatty acids are biosynthesized. The resulting scatterplot (Figure 2) provides a conceptual overview of the samples by showing 72.3% of the total variance. The component plot and scatterplot can be interpreted together because objects with high scores for a specific PC also have high values for the variables with high loading plots and low values for those with low loadings [11]. The scatterplot showed that similarities between samples were coincident in most cases with varieties. Thus, the two Wild samples from Antas are grouped to the top right of the plot; the two samples of Var. Li are grouped next to the down; the four samples Var. Mudeong form another group located to the left of the plot; the samples Var. Gorda were grouped in the down part of the plot and, finally, only one sample
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27 can be expected by their consumption. It has also been shown that this fruit is a good source of β-carotene like with other fruits of this species. References
Figure 2. Scatterplot for the two first component weights.
each from Var. Lang and Unknown are located to the down in the plot. Influence on groups formation can be assigned to a particular fatty acid, 8:0 and 10:0 on Var. Wild samples; fatty acids from C16 series and 14:0 on Var. Lang and Gorda samples, and the polyunsaturated C18 and C20 in the four Var. Mudeong samples. This situation could be explained by considering that fruits of same varieties have a similar fatty acid profile, although it is not possible to discard environmental factors that affect this situation, such as soil type, insolation, temperature, etc., taking into account that varieties and location are coincident for samples in most cases. As is shown in this work, the ber is a good source of medium-chain fatty acids, thus beneficial effects to health
1. Bakhshi JC, Singh P (1974) The Ber. A good choice form semiarid and marginal soils. Ind Hort 19: 27–30. 2. Tiwari RJ, Banafar RNS (1995) Studies on the nutritive constituents, yield and yield attributing characters in some ber (Zizyphus jujuba) genotypes. Ind J Plant Physiol 38: 88–89. 3. Zhumatov UZ (1996) Elementary compositions of the fruits of Morus nigra and Zizyphus jujuba and their biological activities. Chem Nat Compds 32: 100–101. 4. Yamaoka Y, Kawakita T, Kaneko M, Nomoto K (1996) A polysaccharide fraction of Zizyphi Fructus in augmenting natural killer activity by oral administration. Biol Pharm Bull 19: 936– 939. 5. Wong KC, Chee SG, Tan CH (1996) Volatile constituents of the fruit of Zizyphus jujuba Mill. var. inermis (Bge.) Rehd. J Essen Oil Res 8: 323–326. 6. Lepage G, Roy C (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J Lipid Res. 25: 1391–1396. 7. Whyte JN (1987) Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves. Aquaculture 60: 231–241. 8. Wills RBH, Nurdin H, Wootton M (1988) Separation of carotenes and xanthophylls in fruit and vegetables by HPLC. J Micronutr Anal 4: 87–98. 9. Hainer V, Kunesova M, Stich V, Zak A, Parizkova J (1994) The role of oils containing triacylglycerols and medium-chain fatty acids in the dietary treatment of obesity. The effect on resting energy expenditure and serum lipids. Cas Lek Cesk 133: 373–375. 10. White MD, Papamandjaris AA, Jones PJ (1999) Enhanced postprandial energy expenditure with medium-chain fatty acid feeding is attenuated after 14 d in premenopausal women. Am J Clin Nutr 69: 883–889. 11. Wold S, Eseben K, Geladi P (1987) Principal component analysis. Chemometr Intell Lab Syst 2: 37–52.
