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Evaluation of valuable nutrients in selected genotypes of marula (Sclerocarya birrea ssp. caffra)
Scientia Horticulturae 117 (2008) 321–328
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Evaluation of valuable nutrients in selected genotypes of marula (Sclerocarya birrea ssp. caffra) Zipi Hillman a, Yosef Mizrahi a, Elie Beit-Yannai b,* a b
Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Department of Clinical Pharmacology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 March 2008 Received in revised form 7 May 2008 Accepted 11 May 2008
Different marula clones domesticated in the arid Negev desert were evaluated for their horticultural behavior physiological and antioxidant characteristics of their fruits. Two types of abscission pattern were detected, early and late. Fruit yields fluctuated from year to year (6–45 kg/tree) with large differences between clones (few fruits to 45 kg/tree). The clones also differed in fruit weight (34.41 0.91 to 56.74 1.65 g), shape, and juice content. Ascorbic acid content was found to be high in all the clones with significant differences among clones and time post-abscission. Clone no 12 at 1 week post-abscission had the higher ascorbic acid content, 21.177 mg/g dry weight and clone no 4 at the abscission day had the lower ascorbic acid content, 7.142 mg ascorbic acid/g dry weight. The total antioxidant and polyphenol content of the juice varied among clones and with time post-abscission, as did the activity of superoxide dismutase (about 1.40 IU/g dry weight of SOD at week 0 for all clones, to 2.67 IU/g dry weight at the third week postabscission). Fresh marula juice had, on average, four times more antioxidant than the juice of either orange or pomegranate (marula ascorbic acid equivalent: 8.52 0.76 mM to 23.26 2.20 mM versus 2.72 0.39 mM to 7.38 0.18 mM and 2.56 0.41 mM to 3.92 0.18 mM, respectively), considered to be rich in antioxidants. Marula ice cream and jam manufactured according to industrial protocols were rich in ascorbic acid 45 days post-production. In conclusion, selected genotypes of marula exhibited superior horticultural traits and high antioxidant contents. These data should be of value in selecting marula clones for commercial planting. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Antioxidants Cyclic voltammetry Clones Breeding Industrial crop Nutraceuticals Fruit Ripening
1. Introduction Marula (Sclerocarya birrea ssp. caffra) is a large decidous savannah tree belonging to the Anacardiaceae that is indigenous to southern Africa. It bears pale yellow plum-sized fruits 3–4 cm in diameter with a plain tough peel and a fibrous juicy sweet-sour mucilaginous flesh. The flesh surrounds a hard nut consisting of 1– 4 soft white edible kernels rich in oil and protein (Fig. 1). The seeds enclosed in locules embedded in the lignified tissue of the drupe. The seeds are about 10% of the drupe dry weight. The fruits abscise when green and firm, turning yellow as they ripen (Fig. 2). They are highly aromatic with a sweet-sour taste and can be eaten fresh or used for the preparation of juices, jams, conserves, dry fruit rolls, and alcoholic beverages (Weinert et al., 1990). Fresh marula fruit can be consumed by biting or cutting through the thick leathery
* Corresponding author. Tel.: +972 8 6477374; fax: +972 8 6477629. E-mail addresses: [email protected] (Z. Hillman), [email protected] (Y. Mizrahi), [email protected] (E. Beit-Yannai). Abbreviations: SOD, superoxide dismutase; CV, cyclic voltammetry; PBS, phosphate buffer saline. 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.05.008
skin and sucking the juice or chewing the mucilaginous flesh after removal of the peel. The seeds have a high content of oil (56%) and proteins (28%) are edible, eaten fresh or roasted and utilized for oil production (Moganedi et al., 2007). Moreover the nuts are highly nutritious owing to their elevated protein and oil content and profile (Mariod et al., 2004). The peel can be used for essential oil production for cosmetic purposes. Several paper reported medicinal effects of the marula bark and leaves (Eloff, 2001; Ojewole, 2003). During the last two decades several marula domestication processes were taken place (Holtzhausen et al., 1990; Mizrahi et al., 1996; Taylor et al., 1996). More recently, Leakey and Simons were involved in a programme launched by the International Centre for Research in Agroforestry, in which subsistence farmers are the planned beneficiaries of the marula domestication activities (Leakey et al., 2005). The genetic stability of grafted marula trees was described (Moganedi et al., 2007). Over the last two decades, we have successfully domesticated wild marula clones in the arid Negev region of Israel in a program that has included clonal propagation of genotypes selected for horticultural parameters and nutritive constituents (Nerd, 1993; Gutman et al., 1999). However, in order to proceed from smallscale cultivation to commercial planting of marula orchards for
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Fig. 1. Internal cross-section of the marula fruit. (A) Marula fruit internal cross-section representing the fruit peel, flash and drupe. (B) Horizontal (upper) and vertical sections of the marula nut representing the seed and the drupe.
fresh fruit consumption and industrial processing, it is necessary to further characterize the different genotypes. An important factor to be considered is nutritive value. The protection against disease provided by fruits and vegetables has been attributed to the various antioxidants contained in these foods (Rice-Evans and Miller, 1985; Ames et al., 1993). There is now overwhelming evidence indicating that free radicals cause oxidative damage to lipids, proteins, and nucleic acids (Halliwell, 2006). Free radicals are thought to be implicated in the etiology or natural history of a number of diseases, including cancer and heart, vascular, and neurodegenerative diseases (Halliwell, 1994; Valko et al., 2007). Antioxidants, which can neutralize free radicals, may thus be of central importance in the prevention of these pathological states. Wild marula fruits are known to be rich in ascorbic acid (Weinert et al., 1990) and polyphenols (Braca et al., 2003). Both classes of antioxidants have recently gained much attention owing, among others, to their antioxidant functions and possible impact on human health. Many polyphenols have redox properties (Sang et al., 2005) that enable them to act as reducing agents and singlet oxygen quenchers (Devasagayam et al., 1995), thereby contributing to the antioxidant capacity of fruits and vegetables. Recently the polyphenol oxidase and peroxidase from marula fruit, enzymes involved in enzymatic browning were evaluated (Mdluli, 2005). In the study described below, seven marula genotypes were examined for their horticultural characters and antioxidant capacity. The objectives were: To characterize the horticultural characters of the different marula clones.
To characterize and evaluate the antioxidant content of the fruit juice from the different clones at different intervals postabscission. To compare the antioxidant capacity of marula juice with that of other fruits known to be rich in antioxidant capacity. To evaluate the antioxidant capacity of marula food products. 2. Materials and methods 2.1. Plant material The subjects of the study were 6-year-old marula clones growing in an orchard in Beer-Sheva, which is situated in the Negev, a region of loess soil (pH 7.8) characterized by 205 mm of rainfall annually and temperatures ranging from 1 to 20 8C in winter and 20 to 40 8C in summer. These finding are in support of former results (Nerd, 1993; Nerd and Mizrahi, 2000). Marula fruits were collected from an experimental orchard located in BeerSheva that had been planted with several selected clones grafted on marula seedlings (Gutman et al., 1999). The fruits were collected from the ground every morning, the day of abscission being recorded as time zero. The fruits were washed in tap water and stored at 20 8C for 3 weeks. Every week 10 fruits were removed from each clone for analysis. To extract the juice, a small hole was made in the fruit peel and the juice was squeezed out by hand (together with some pulp). The juice was then frozen and lyophilized, and the lyophilized samples were stored at 20 8C until analysis. Orange (Valencia, Shamouti and an unknown variety purchased at the local market) and three clones of
Fig. 2. Skin color changes in marula fruits at different time intervals post-abscission. One representative marula fruit (clone no. 6) photographed at 1-week intervals. As ripening proceeded, fruit color changed from green to yellow.
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pomegranate (Hershkovitz, Rosh-Pered and an unknown variety purchased at the local market) were squeezed and centrifuged. The supernatants were diluted 10 in PBS pH 7.4 and evaluated immediately by cyclic voltammetry for their total antioxidant content. 2.2. Reagents Sodium phosphate dibasic, potassium phosphate, L-methionine, ( )-riboflavin, gallic acid, superoxide dismutase (SOD), nitrotetrazolium blue chloride, and ascorbic acid were purchased from Sigma [Rehovot, Israel]. Folin-Ciocalteu reagent was purchased from Merck [Rosh Ha A´yin, Israel]. 2.3. Cyclic voltammetry 0.5 g of lyophilized marula was homogenized in 5 ml of 100 mM Phosphate buffer saline (PBS), pH 7.4. Solution of marula extract was placed in a cyclic voltammeter (CV) cell equipped with a working electrode (3.2 mm in diameter, glassy carbon), a reference electrode (Ag/AgCl), and an auxiliary electrode (platinum wire). The potential was applied linearly to the working electrode at a constant rate (100 mV/s) toward the positive potential (evaluation of reducing equivalents). An electrochemical working station (CH Instruments Inc., 610B, Austin, TX, USA) was used. During operation of the CV, a potential current curve was recorded (Kohen et al., 1999).
Fig. 3. Fresh fruit yields of different marula genotypes growing in a Beer-Sheva orchard. Fruits were collected every day or two from each tree from first to last fruit abscission (one tree per clone). Both fruit weight and number of fruits were recorded.
2.7. Statistical analysis All experiments were conducted in triplicate. The data were subjected to one-way ANOVA. Significant differences among treatment means were evaluated by applying an LSD post hoc test (p < 0.05) using SPSS software. 3. Results
2.4. Determination of total polyphenols
3.1. Fruit yield per clone
0.25 g of lyophilized marula powder was homogenized in acetone/distilled water (7:3, v:v). The homogenate was centrifuged and the supernatant collected and diluted to 1:10 with distilled water. Samples were heated to 90 8C for 3 h in a warm bath. Total polyphenols were determined by the Folin-Ciocalteu method (George et al., 2005). Absorption was measured at 760 nm (Ultrospec 2100 pro UV–vis spectrophotometer). The total phenolic content was expressed as gallic acid equivalents in milligrams per gram of dry matter.
Marula fruits were collected over two consecutive years in order to evaluate fresh fruit mass produced by clones belonging to different genotypes (Fig. 3). Yields fluctuated from year to year, revealing large differences between clones.
2.5. Superoxide dismutase (SOD) assay Protein extract from the lyophilized marula was prepared according to Mdluli (Mdluli, 2003). The SOD activity of the extract was evaluated indirectly using the nitro blue tetrazolium (NBT) assay (Beauchamp and Fridovich, 1971) with slight modifications. The reaction medium contained 50 mM K-phosphate buffer pH 7.8, 0.1 mM EDTA, 0.025% Triton X-100, 14 mM methionine, 75 mM NBT, 4 mM riboflavin, and the required amount of enzyme extract from lyophilized marula samples. The tubes were illuminated for 10 min at 25 8C, and subsequently absorbance at 560 nm was measured with an UV–vis spectrophotometer (Ultrospec 2100 pro). One unit of SOD activity was defined by standard SOD activity (sigma S-2515). Linearity was obtained between 0–6 units of enzyme (R2 = 0.96).
3.2. Distribution of fruit abscission Two major patterns of fruit abscission emerged: in the first, the majority of the fruits dropped in a typical Gaussian distribution within the first 10 days after the start of abscission; in the second, abscission also described a Gaussian curve but the majority of the fruits dropped later, from the end of week 1 to the end of week 3 (Fig. 4). 3.3. Fruit weight and appearance Although grown under the same conditions, the marula genotypes exhibited significant differences in average fruit weight (Fig. 5A), as well as differences in fruit shape, hue and size (Fig. 5B). These differences persisted throughout the abscission stage.
2.6. Food products Marula sorbet was prepared by an industrial process. A parallel preparation of sorbet free of marula served as control. Marula jam was made by an industrial process. Briefly, marula juice (210 g) and pieces of marula peel (40 g) were cooked with sugar (1:1). Next, potassium sorbate (0.4 g) and pectin (2.5 g) were added and the jam was pasteurized at 93 8C for 14 min. After cooling the jam was stored in a jar.
Fig. 4. The two typical abscission patterns displayed by marula genotypes. Clone no. 17 represents an early-abscission type and clone no. 2 a type with relatively prolonged abscission. Fruits were collected every day or two from each tree from first to last fruit abscission. Data from 2005.
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representative genotypes are shown in Fig. 6B and C one with relatively low flesh content (40% of fresh weight) and the other with relatively high flesh content (55%). With the lapse of time post-abscission the color of the peel changed from green to yellow (Fig. 2), corresponding to the progress of fruit ripening as well as to changes in the antioxidant capacity of the fruits. 3.5. Antioxidant capacity of marula fruits In this part of the study we investigated the major compounds associated with antioxidant function in marula juice. This was done by evaluating ascorbic acid and polyphenol contents, as well as the total antioxidant capacity of the unfractionated juice. The results were then compared with corresponding data for orange and pomegranate juice, both known to have relatively high contents of antioxidants. 3.6. Cyclic voltammetry analysis of marula juice
Fig. 5. Fruit weight, shape, hue and size displayed by different marula genotypes. (Panel A) Differences in fruit weight between the marula clones. Each bar represent an average for at least 32 fruits from each tree S.E. Different letters are significantly different from each other at p < 0.05 as established by ANOVA. (Panel B) Photograph showing representative fruit shape, hue, and relative size 1-week post-abscission.
3.4. Fresh fruit components The fruits produced by the different marula genotypes were assayed for percentage of fruit components (Fig. 6A). Two
The cyclic voltammetry approach was used to analyze the total reducing capacity of the marula juice content (Kohen et al., 1999; Butera et al., 2002). We were able to identify two major reducing groups of low molecular weight antioxidants representing the total hydrophilic antioxidants (Fig. 7). The first anodic wave (A) was mainly composed of ascorbic acid, polyphenols, and small amounts of uric acid (Beit-Yannai et al., 2005). The definite composition of the first anodic wave was confirmed as mainly ascorbic acid using an HPLC equipped with an electrochemical detector, as well as by addition of ascorbate oxidase to the juice and CV tracing (data not shown). We also examined the antioxidant capacity of the various marula clones at different time intervals post-abscission as expressed by the currents of the first anodic wave. The current is proportional to the low molecular
Fig. 6. Fruit fresh weight components for different marula genotypes. One-week post-abscission 10 fruits from each genotype were separated into flesh, stone and peel and weighed. (A) Summarizes the weight differences between fruit components. Data represent the means for ten fruits S.E. (B) and (C) show distribution of components for typical low (clone no. 2) and high (clone no. 6) fruit flesh content, respectively.
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Fig. 8. Effect of time post-abscission and clone on ascorbic acid equivalents in marula fruit juice. Ascorbic acid content evaluated by cyclic voltammetry (glassy carbon working electrode 3.2 mm, Ag/AgCl reference electrode, Pt wire as counter electrode; scan rate 100 mV/s). Values were calculated vs. ascorbic acid standard (R2 = 0.99). The values are means S.E. for six fruits from each tree in each week postabscission. Significant differences were found between clones in the same week as established by ANOVA (*p < 0.05 vs. clone no. 4, #p < 0.05 vs. clone no. 6).
Fig. 7. Typical cyclic voltammogram of marula fruit juice. Cyclic voltammetry measurements were carried out as described in Section 2. Cyclic voltammograms of marula juice from abscission day (––) and 3 weeks post-abscission day (+++). Lyophilized marula, 0.5 g was homogenized in 5 ml of 100 mM PBS, pH 7.4. Cyclic voltammetry measurements were carried out as described in Section 2. Two groups of reducing equivalents can be detected (A and B).
(Fig. 9A–C). Differences in polyphenol content were found between the different marula genotypes. Polyphenol content peaked at 3 weeks post-abscission in all the clones tested. Clone no. 6 exhibited the highest concentration of polyphenols and clone no. 12 the lowest.
weight antioxidant content at potential 450 50 mV (Table 1) and 1000 50 mV (Table 2). 3.7. Ascorbic acid content in the different marula genotypes Marula fruits are known to be relatively rich in ascorbic acid content. In our study we found that ascorbic acid content varied between 7 and 21 mg/g dry weight depending on clone and fruit ripening stage (Fig. 8).
3.9. Superoxide dismutase The superoxide dismutase activity of marula extract from different clones was measured by the NBT method (Beauchamp and Fridovich, 1971) (Fig. 10A–C). Significant differences in SOD activity were found between clones. All the clones displayed a significant increase in SOD activity with time post-abscission.
3.8. Polyphenol content Polyphenol content was evaluated using Folin-Ciocalteau reagent with gallic acid as reference value (George et al., 2005)
Table 1 Antioxidant capacity of seven marula clones at different time intervals post-abscission as expressed by the currents at potential 450 50 mV Clone number
2 4 6 8 12 17 20
Abscission day
One-week post-abscission
Two weeks post-abscission
Three weeks post-abscission
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
125 82 144 138 198 148 152
c d b bc a b b
135 119 164 164 244 113 144
cd cd b b a d bc
186 124 150 161 239 232 146
b c bc bc a a c
158 144 150 166 219 116 136
b bc b b a c bc
Values are means for six fruits from each clone at each time point. Samples were scanned three times and results subjected to ANOVA (p < 0.05). Different letters indicate significant differences among columns. Table 2 Antioxidant capacity of seven marula clones at different time intervals post-abscission as expressed by the currents at potential at 1000 50 mV Clone number
2 4 6 8 12 17 20
Abscission day
One-week post-abscission
Two weeks post-abscission
Three weeks post-abscission
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
71 60 91 89 113 99 91
c d b b a b b
82 81 99 100 130 78 92
c c b b a c bc
101 81 86 88 121 121 91
b c bc bc a a bc
96 77 86 87 116 75 84
b c bc bc a c bc
Values are means for six fruits from each clone at each time point. Samples were scanned three times and results subjected to ANOVA (p < 0.05). Different letters indicate significant differences among columns.
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Fig. 9. Effect of time post-abscission on polyphenol content in lyophilized marula fruit juice from three different clones. (A) Clone no. 4, (B) clone no. 6, (C) clone no.12. Polyphenol content was evaluated by the Folin-Ciocalteu method using gallic acid as a standard. Values are means S.E. for six fruits from each tree in each week postabscission. Different letters are significantly different from each other at p < 0.05, as established by ANOVA.
3.10. Comparison of ascorbic acid contents of pomegranate, orange and marula fresh juice The ascorbic acid contents of fresh juices of marula (Beer-Sheva and Besor orchard), different orange clones (Valencia, Shamouti and an unknown clone purchased at the local market), and three clones of pomegranate (Hershkovitz, Rosh-Pered and an unknown clone purchased at the local market) were compared. The juices were analyzed by cyclic voltammetry and the current values converted into ascorbic acid equivalent. The ascorbic acid content of the best marula clone tested was found to be approximately 10 time higher than orange and pomegranate from the local market. At least 30% increase of ascorbic acid content was found in the marula (clone no 13) versus the best pomegranate clone tested (Hershkovitz). The lower ascorbic acid content marula clone no. 13, show a 100% increase versus the best orange clone (Valencia) (Fig. 11). 3.11. Antioxidant capacity of marula food products The ascorbic acid content of marula sorbet and marula jam prepared according to industrial protocols was evaluated by cyclic
Fig. 10. Effect of time post-abscission on SOD activity of lyophilized fruit juice from three marula clones. (A) Clone no. 4, (B) clone no. 6, (C) clone no. 12. Superoxide dismutase analysis was performed by the NBT method using an SOD activity curve with known enzyme activity. Values are means S.E. for five fruits from each clone in each week. All the reactions were performed in triplicate. Different letters are significantly different from each other at p < 0.05 as established by ANOVA.
voltammetry. Non-marula ingredients in the sorbet and jam were found to be free of ascorbic acid. The ascorbic acid concentrations were evaluated on the day of production and again after 45 days (Fig. 12). After 45 days the jams prepared with marula juice from clones 8, 12 and 17 retained only about 50% of the ascorbic acid content measured on the production date, while the jam prepared from clone no. 6 retained 84% of the original content. The ascorbic acid content remained high despite the pasteurization to which the jam had been subjected. The sorbet prepared from clone no. 6 showed no loss of ascorbic acid at 45 days as compared with the production date; the original reduction is due mainly to the preparation procedure. 4. Discussion For domestication of newly introduced fruit clones to be successful, the crop must possess favorable horticultural traits, such as high yield per clone, large edible parts, and a long yielding season, as well as a high nutrient content. Several years ago a number of marula clones were selected for high yields, good taste and seasonality and subsequently tested for horticultural char-
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Fig. 11. Antioxidant content as ascorbic acid equivalents in marula, orange and pomegranate fresh juice. Fresh fruits were squeezed and centrifuged. The supernatants were diluted 10 in PBS pH 7.4 and evaluated by cyclic voltammetry for their total antioxidant content. Cyclic voltammetry measurements were carried out as described in Section 2. Values are means S.E. of at least three fruits of each species and clone.
acteristics in the arid Israeli Negev. The aim of the present study was to examine the same marula clones more closely as potential new candidates for commercial cultivation. In particular, we looked at their horticultural characteristics and the content of antioxidants in the fruit flesh (especially ascorbic acid and total polyphenols). The marula genotypes were found to exhibit significant differences in annual fruit yield, from about 45 kg per clone for
Fig. 12. Ascorbic acid content of marula jam and marula sorbet as evaluated by cyclic voltammetry. Ascorbic acid content of marula jam (A) and marula sorbet (B) prepared by industrial protocols and evaluated on day of production and 45 days later.
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the highest yielding individuals (clones 2, 17 and 20) to 1–3 kg per clone for the low yielders (14 and 18). The data presented in Fig. 3 seem to suggest that marula is an alternate bearing species. Differential yield from year to year is an important trait from the perspective of commercial planting, especially in the case of a new species intended for the horticultural industry. As marula is considered to be a dioecious species, male clones were also planted in the grove. It is reasonable to speculate that unsynchronized flowering of the female and male clones prevented optimal pollination and hence affected yields; however, the flowering patterns of female and male individuals have not been consistently followed in this study. Moreover, male and female flowers have occasionally been observed blooming together on the same clone in the Beer-Sheva orchard as well as at other sites; such a phenomenon could interfere with optimal pollination. We note that the present study was limited to a 2-year-period, which is too brief time to settle the question of alternate bearing in marula. Besides yield per clone, fruit weight and fruit size are also important criteria for selecting clones for fresh fruit production (Frary et al., 2000). The seven clones tested displayed a relatively wide range of fruit weights, namely from 19 to 63 g. Consumption of fresh marula fruits is inconvenient owing to their thick peel, so industrial processing may prove to be the preferred option, in which case the ratio between the fruit components would be an important characteristic. Flesh content, which is not correlated with fruit fresh weight (r2 = 0.3, data not shown), might then be a key point in clone selection. We have identified two typical groups of marula clones in relation to fruit flesh, namely with relatively high (58%) and low (39%) contents; both values are higher than the average for wild populations, where it is around 25%. The stone may be consumed as a nut or used for oil extraction. In principle, different clones might be recommended for oil extraction versus juice production; however, as marula stones should routinely be available as a by-product of the juice industry, it is unlikely that there would be a demand for clones developed exclusively for the stone. The marula clones exhibited two abscission profiles, one in which the majority of the fruits abscised early and the other in which the majority dropped late, the time differential being about 10 days. This trait could be relevant for mechanical harvesting. The orchard chosen for this study included diverse clones which together provided a long harvesting season, an important consideration for marketing and export. In future orchards, clones should be planted in rows to allow efficient mechanical harvesting. Many studies in recent decades have focused on the occurrence of antioxidant molecules and the link between diets rich in fruits and vegetables and the onset and/or prevention of a number of oxidative stress related diseases (Sardesai, 1995; Halliwell, 2002; Ignarro et al., 2007). Accordingly, we examined the antioxidant capacity of the fruits produced by our selected marula clones. The total reducing capacity measured by the cyclic voltammetry method (Silva et al., 2001; Butera et al., 2002; Roginsky et al., 2003) revealed significant differences in antioxidant content between the different clones. Two main groups of low molecular weight antioxidants were found (1st and 2nd anodic waves: 450 50 and 1000 50 mV, respectively). The first anodic wave consisted mainly of ascorbic acid (diminished by the addition of ascorbate oxidase, data not shown) and small amounts of uric acid (Beit-Yannai et al., 2005). The peak shifted by 50 mV compared to ascorbic acid standard corresponds to other low molecular weight antioxidants. The second peak is probably composed of several polyphenols containing 3-5 hydroxyl groups (such as quercetin) (Jorgensen et al., 1999; Aaby et al., 2004). We were able to show a rise in the hydrophilic antioxidant content with time post-abscission. This increase in
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reducing capacity is mainly due to elevated amounts of several polyphenols, as well as to ascorbic acid. In parallel with the increase in low molecular weight antioxidants during ripening, a significant increase in SOD activity was found in all clones. The significant differences in SOD activity between the different clones suggests differences in the ability of the marula genotypes to cope with oxidative stress generated by abscission of superoxide radicals. The two large anodic waves revealed by the cyclic voltammetry suggest that marula fruit owes its high defense capability in the face of different oxidative stresses to the presence of low molecular weight antioxidants and the key antioxidant enzyme. All the marula clones exhibited the same pattern of increase in antioxidant capability with ripening, even though the clones differed significantly in total antioxidant capacity. Clone no. 6 exhibited the highest polyphenol content and SOD activity during ripening. Clone no. 12 had the highest ascorbic acid content, which reached 22 mM at 2 weeks postabscission. In the present study we compared the ascorbic acid content in fresh juice of several marula clones to different orange and pomegranate. Kiwi is another well known source for vitamin C. Szeto et al. (2002) analyzed the ascorbic acid of different fruits, finding that the kiwi content is about 520 mg/kg versus 330 mg/kg for orange. According to these values Kiwi has about 1.6 times more ascorbic acid then orange. Yet the marula content is almost two times higher then the kiwi content. The finding that the juice of marula has a much higher antioxidant capacity than that of species commonly thought of as particularly rich in antioxidants could facilitate its acceptance as a new fruit product. The first semi-commercial orchard of selected clones was planted 4 years ago in the Arava valley of Israel, where high temperatures combine with saline soil and water to create highly stressful conditions. The fact that marula proved capable of tolerating these conditions may be attributable to its high content of antioxidants. 5. Conclusion In our study we showed that marula has a good potential to become a fruit crop on a commercial scale since the fruits are tasty, rich in antioxidants and particularly suitable for industrial uses. The current study contributes to validation of indigenous knowledge on marula and more so to the knowledge about the quality of our selected clones. Many aspects of fruit quality and many clones, other than those investigated here, remain to be studied, hence, it is necessary to continue analyze additional selected clones to satisfy the needs for establishing new emerging fruit crop. Acknowledgement We thank Ms. Aliza Sen for editing the manuscript. References Aaby, K., Hvattum, E., Skrede, G., 2004. Analysis of flavonoids and other phenolic compounds using high-performance liquid chromatography with coulometric array detection: relationship to antioxidant activity. J. Agric. Food Chem. 52 (15), 4595–4603. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 90 (17), 7915–7922. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44 (1), 276–287. Beit-Yannai, E., Jaglitz, S., Trembovler, V., Mizrahi, Y., 2005. Reducing Properties of Marula Fruit Evaluated by Cyclic Voltammetry. 2nd International meeting of the Society for Free Radical Research-Asia, Shanghai, PRC. Braca, A., Politi, M., Sanogo, R., Sanou, H., Morelli, I., Pizza, C., Tommasi, N.D., 2003. Chemical composition and antioxidant activity of phenolic compounds from wild and cultivated Sclerocarya birrea (Anacardiaceae) Leaves. J. Agric. Food Chem. 51 (23), 6689–6695. Butera, D., Tesoriere, L., Di Gaudio, F., Bongiorno, A., Allegra, M., Pintaudi, A.M., Kohen, R., Livrea, M.A., 2002. Antioxidant activities of sicilian prickly pear
(Opuntia ficus indica) fruit extracts and reducing properties of its betalains: betanin and indicaxanthin. J. Agric. Food Chem. 50 (23), 6895–6901. Devasagayam, T.P., Subramanian, M., Singh, B.B., Ramanathan, R., Das, N.P., 1995. Protection of plasmid pBR322 DNA by flavonoids against single-stranded breaks induced by singlet molecular oxygen. J. Photochem. Photobiol. B 30 (2/3), 97–103. Eloff, J.N., 2001. Antibacterial activity of Marula (Sclerocarya birrea (A. rich.) Hochst. subsp. caffra (Sond.) Kokwaro) (Anacardiaceae) bark and leaves. J. Ethnopharmacol. 76 (3), 305–308. Frary, A., Nesbitt, T.C., Grandillo, S., Knaap, E., Cong, B., Liu, J., Meller, J., Elber, R., Alpert, K.B., Tanksley, S.D., 2000. fw2.2: a quantitative trait locus key to the evolution of tomato fruit size. Science 289 (5476), 85–88. George, S., Brat, P., Alter, P., Amiot, M.J., 2005. Rapid determination of polyphenols and vitamin C in plant-derived products. J. Agric. Food Chem. 53 (5), 1370– 1373. Gutman, F., Nerd, A., Mizrahi, Y., Bar-Zvi, D., Raveh, D., 1999. Amplification of random amplified polymorphic DNA markers for identification of marula genotypes. HortScience 34 (7), 1256–1258. Halliwell, B., 1994. Free radicals and antioxidants: a personal view. Nutr. Rev. 52 (8 Pt 1), 253–265. Halliwell, B., 2002. Effect of diet on cancer development: is oxidative DNA damage a biomarker? Free Radic. Biol. Med. 32 (10), 968–974. Halliwell, B., 2006. Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 97 (6), 1634–1658. Holtzhausen, L.C., E., S. v. R. R., 1990. Propagation of the marula Sclerocarya birrea subsp. caffra. Acta Hortic. 275, 323–334. Ignarro, L.J., Balestrieri, M.L., Napoli, C., 2007. Nutrition, physical activity, and cardiovascular disease: an update. Cardiovasc. Res. 73 (2), 326–340. Jorgensen, L.V., Madsen, H.L., Thomsen, M.K., Dragsted, L.O., Skibsted, L.H., 1999. Regeneration of phenolic antioxidants from phenoxyl radicals: an ESR and electrochemical study of antioxidant hierarchy. Free Radic. Res. 30 (3), 207– 220. Kohen, R., Beit-Yannai, E., Berry, E.M., Tirosh, O., 1999. Overall low molecular weight antioxidant activity of biological fluids and tissues by cyclic voltammetry. Methods Enzymol. 300, 285–296. Leakey, R., S., S., P., P. d., 2005. Domestication potential of Marula (Sclerocarya birrea subsp caffra) in South Africa and Namibia. 1. Phenotypic variation in fruit traits. Agroforestry Syst. 64, 25–35. Mariod, A., Matthaus, B., Eichner, K., 2004. Fatty acid, tocopherol and sterol composition as well as oxidative stability of three unusual Sudanese oils. J. Food Lipids 11, 179–189. Mdluli, K.M., 2005. Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit (Sclerocarya birrea subsp. caffra). Food Chem. 92 (2), 311. Mdluli, K. M., O.-A., R., 2003. Enzymatic browning in Marula fruit. 1. Effect of endogenous antioxidants on Marula fruit polyphenol oxidase. J. Food Biochem. 27 (Part 1), 67–82. Mizrahi, Y., A., N., 1996. New crops as a possible solution for the troubled Israeli export market. American Society for Horticultural Science. Proceedings of the Third National New Crops Symposium. Moganedi, K.L.M., Colpaert, N., Breyne, P., Sibara, M.M., Goyvaerts, E.M.A., 2007. Determination of genetic stability of grafted marula trees using AFLP markers. Sci. Hortic. 111 (3), 293. Nerd, A., Mizrahi, Y., 2000. Introduction of Marula, an unexploited fruit tree from southern Africa, to the Israeli Negev. Israel J. Plant Sci. 48 (3), 217–222. Nerd, A.M., Y., 1993. Domestication and Introduction of Marula (Sclerocarya birrea sbsp caffra) as a New Crop for the Negev Desert of Israel, Wiley Press. Ojewole, J.A., 2003. Hypoglycemic effect of Sclerocarya birrea [(A. Rich.) Hochst.] [Anacardiaceae] stem-bark aqueous extract in rats. Phytomedicine 10 (8), 675– 681. Rice-Evans, C.A., Miller, N.J., 1985. Antioxidants: the case of fruit and vegetables in the diet. Br. Food J. 97, 35–40. Roginsky, V., Barsukova, T., Hsu, C.F., Kilmartin, P.A., 2003. Chain-breaking antioxidant activity and cyclic voltammetry characterization of polyphenols in a range of green, oolong, and black teas. J. Agric. Food Chem. 51 (19), 5798– 5802. Sang, S., Hou, Z., Lambert, J.D., Yang, C.S., 2005. Redox properties of tea polyphenols and related biological activities. Antioxid Redox Signal 7 (11/12), 1704– 1714. Sardesai, V.M., 1995. Role of antioxidants in health maintenance. Nutr. Clin. Pract. 10 (1), 19–25. Silva, D.H., Pereira, F.C., Zanoni, M.V., Yoshida, M., 2001. Lipophyllic antioxidants from Iryanthera juruensis fruits. Phytochemistry 57 (3), 437–442. Szeto, Y.T., Tomlinson, B., Benzie, I.F., 2002. Total antioxidant and ascorbic acid content of fresh fruits and vegetables: implications for dietary planning and food preservation. Br. J. Nutr. 87 (1), 55–59. Taylor, F.W., S.M., M., K.J., B., 1996. A holistic approach to the domestication and commercialisation of non-timber forest products. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M., Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39 (1), 44–84. Weinert, I., van Wyk, P.J., Hoitzhausen, L.C., 1990. Marula. In: Nagy, S., Show, S.E., Nardowsky, W.F. (Eds.), Fruit of Tropical and Subtropical Origin. Florida Science Source, FL, pp. 88–115.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Evaluation of valuable nutrients in selected genotypes of marula (Sclerocarya birrea ssp. caffra) Zipi Hillman a, Yosef Mizrahi a, Elie Beit-Yannai b,* a b
Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Department of Clinical Pharmacology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 March 2008 Received in revised form 7 May 2008 Accepted 11 May 2008
Different marula clones domesticated in the arid Negev desert were evaluated for their horticultural behavior physiological and antioxidant characteristics of their fruits. Two types of abscission pattern were detected, early and late. Fruit yields fluctuated from year to year (6–45 kg/tree) with large differences between clones (few fruits to 45 kg/tree). The clones also differed in fruit weight (34.41 0.91 to 56.74 1.65 g), shape, and juice content. Ascorbic acid content was found to be high in all the clones with significant differences among clones and time post-abscission. Clone no 12 at 1 week post-abscission had the higher ascorbic acid content, 21.177 mg/g dry weight and clone no 4 at the abscission day had the lower ascorbic acid content, 7.142 mg ascorbic acid/g dry weight. The total antioxidant and polyphenol content of the juice varied among clones and with time post-abscission, as did the activity of superoxide dismutase (about 1.40 IU/g dry weight of SOD at week 0 for all clones, to 2.67 IU/g dry weight at the third week postabscission). Fresh marula juice had, on average, four times more antioxidant than the juice of either orange or pomegranate (marula ascorbic acid equivalent: 8.52 0.76 mM to 23.26 2.20 mM versus 2.72 0.39 mM to 7.38 0.18 mM and 2.56 0.41 mM to 3.92 0.18 mM, respectively), considered to be rich in antioxidants. Marula ice cream and jam manufactured according to industrial protocols were rich in ascorbic acid 45 days post-production. In conclusion, selected genotypes of marula exhibited superior horticultural traits and high antioxidant contents. These data should be of value in selecting marula clones for commercial planting. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Antioxidants Cyclic voltammetry Clones Breeding Industrial crop Nutraceuticals Fruit Ripening
1. Introduction Marula (Sclerocarya birrea ssp. caffra) is a large decidous savannah tree belonging to the Anacardiaceae that is indigenous to southern Africa. It bears pale yellow plum-sized fruits 3–4 cm in diameter with a plain tough peel and a fibrous juicy sweet-sour mucilaginous flesh. The flesh surrounds a hard nut consisting of 1– 4 soft white edible kernels rich in oil and protein (Fig. 1). The seeds enclosed in locules embedded in the lignified tissue of the drupe. The seeds are about 10% of the drupe dry weight. The fruits abscise when green and firm, turning yellow as they ripen (Fig. 2). They are highly aromatic with a sweet-sour taste and can be eaten fresh or used for the preparation of juices, jams, conserves, dry fruit rolls, and alcoholic beverages (Weinert et al., 1990). Fresh marula fruit can be consumed by biting or cutting through the thick leathery
* Corresponding author. Tel.: +972 8 6477374; fax: +972 8 6477629. E-mail addresses: [email protected] (Z. Hillman), [email protected] (Y. Mizrahi), [email protected] (E. Beit-Yannai). Abbreviations: SOD, superoxide dismutase; CV, cyclic voltammetry; PBS, phosphate buffer saline. 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.05.008
skin and sucking the juice or chewing the mucilaginous flesh after removal of the peel. The seeds have a high content of oil (56%) and proteins (28%) are edible, eaten fresh or roasted and utilized for oil production (Moganedi et al., 2007). Moreover the nuts are highly nutritious owing to their elevated protein and oil content and profile (Mariod et al., 2004). The peel can be used for essential oil production for cosmetic purposes. Several paper reported medicinal effects of the marula bark and leaves (Eloff, 2001; Ojewole, 2003). During the last two decades several marula domestication processes were taken place (Holtzhausen et al., 1990; Mizrahi et al., 1996; Taylor et al., 1996). More recently, Leakey and Simons were involved in a programme launched by the International Centre for Research in Agroforestry, in which subsistence farmers are the planned beneficiaries of the marula domestication activities (Leakey et al., 2005). The genetic stability of grafted marula trees was described (Moganedi et al., 2007). Over the last two decades, we have successfully domesticated wild marula clones in the arid Negev region of Israel in a program that has included clonal propagation of genotypes selected for horticultural parameters and nutritive constituents (Nerd, 1993; Gutman et al., 1999). However, in order to proceed from smallscale cultivation to commercial planting of marula orchards for
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Fig. 1. Internal cross-section of the marula fruit. (A) Marula fruit internal cross-section representing the fruit peel, flash and drupe. (B) Horizontal (upper) and vertical sections of the marula nut representing the seed and the drupe.
fresh fruit consumption and industrial processing, it is necessary to further characterize the different genotypes. An important factor to be considered is nutritive value. The protection against disease provided by fruits and vegetables has been attributed to the various antioxidants contained in these foods (Rice-Evans and Miller, 1985; Ames et al., 1993). There is now overwhelming evidence indicating that free radicals cause oxidative damage to lipids, proteins, and nucleic acids (Halliwell, 2006). Free radicals are thought to be implicated in the etiology or natural history of a number of diseases, including cancer and heart, vascular, and neurodegenerative diseases (Halliwell, 1994; Valko et al., 2007). Antioxidants, which can neutralize free radicals, may thus be of central importance in the prevention of these pathological states. Wild marula fruits are known to be rich in ascorbic acid (Weinert et al., 1990) and polyphenols (Braca et al., 2003). Both classes of antioxidants have recently gained much attention owing, among others, to their antioxidant functions and possible impact on human health. Many polyphenols have redox properties (Sang et al., 2005) that enable them to act as reducing agents and singlet oxygen quenchers (Devasagayam et al., 1995), thereby contributing to the antioxidant capacity of fruits and vegetables. Recently the polyphenol oxidase and peroxidase from marula fruit, enzymes involved in enzymatic browning were evaluated (Mdluli, 2005). In the study described below, seven marula genotypes were examined for their horticultural characters and antioxidant capacity. The objectives were: To characterize the horticultural characters of the different marula clones.
To characterize and evaluate the antioxidant content of the fruit juice from the different clones at different intervals postabscission. To compare the antioxidant capacity of marula juice with that of other fruits known to be rich in antioxidant capacity. To evaluate the antioxidant capacity of marula food products. 2. Materials and methods 2.1. Plant material The subjects of the study were 6-year-old marula clones growing in an orchard in Beer-Sheva, which is situated in the Negev, a region of loess soil (pH 7.8) characterized by 205 mm of rainfall annually and temperatures ranging from 1 to 20 8C in winter and 20 to 40 8C in summer. These finding are in support of former results (Nerd, 1993; Nerd and Mizrahi, 2000). Marula fruits were collected from an experimental orchard located in BeerSheva that had been planted with several selected clones grafted on marula seedlings (Gutman et al., 1999). The fruits were collected from the ground every morning, the day of abscission being recorded as time zero. The fruits were washed in tap water and stored at 20 8C for 3 weeks. Every week 10 fruits were removed from each clone for analysis. To extract the juice, a small hole was made in the fruit peel and the juice was squeezed out by hand (together with some pulp). The juice was then frozen and lyophilized, and the lyophilized samples were stored at 20 8C until analysis. Orange (Valencia, Shamouti and an unknown variety purchased at the local market) and three clones of
Fig. 2. Skin color changes in marula fruits at different time intervals post-abscission. One representative marula fruit (clone no. 6) photographed at 1-week intervals. As ripening proceeded, fruit color changed from green to yellow.
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pomegranate (Hershkovitz, Rosh-Pered and an unknown variety purchased at the local market) were squeezed and centrifuged. The supernatants were diluted 10 in PBS pH 7.4 and evaluated immediately by cyclic voltammetry for their total antioxidant content. 2.2. Reagents Sodium phosphate dibasic, potassium phosphate, L-methionine, ( )-riboflavin, gallic acid, superoxide dismutase (SOD), nitrotetrazolium blue chloride, and ascorbic acid were purchased from Sigma [Rehovot, Israel]. Folin-Ciocalteu reagent was purchased from Merck [Rosh Ha A´yin, Israel]. 2.3. Cyclic voltammetry 0.5 g of lyophilized marula was homogenized in 5 ml of 100 mM Phosphate buffer saline (PBS), pH 7.4. Solution of marula extract was placed in a cyclic voltammeter (CV) cell equipped with a working electrode (3.2 mm in diameter, glassy carbon), a reference electrode (Ag/AgCl), and an auxiliary electrode (platinum wire). The potential was applied linearly to the working electrode at a constant rate (100 mV/s) toward the positive potential (evaluation of reducing equivalents). An electrochemical working station (CH Instruments Inc., 610B, Austin, TX, USA) was used. During operation of the CV, a potential current curve was recorded (Kohen et al., 1999).
Fig. 3. Fresh fruit yields of different marula genotypes growing in a Beer-Sheva orchard. Fruits were collected every day or two from each tree from first to last fruit abscission (one tree per clone). Both fruit weight and number of fruits were recorded.
2.7. Statistical analysis All experiments were conducted in triplicate. The data were subjected to one-way ANOVA. Significant differences among treatment means were evaluated by applying an LSD post hoc test (p < 0.05) using SPSS software. 3. Results
2.4. Determination of total polyphenols
3.1. Fruit yield per clone
0.25 g of lyophilized marula powder was homogenized in acetone/distilled water (7:3, v:v). The homogenate was centrifuged and the supernatant collected and diluted to 1:10 with distilled water. Samples were heated to 90 8C for 3 h in a warm bath. Total polyphenols were determined by the Folin-Ciocalteu method (George et al., 2005). Absorption was measured at 760 nm (Ultrospec 2100 pro UV–vis spectrophotometer). The total phenolic content was expressed as gallic acid equivalents in milligrams per gram of dry matter.
Marula fruits were collected over two consecutive years in order to evaluate fresh fruit mass produced by clones belonging to different genotypes (Fig. 3). Yields fluctuated from year to year, revealing large differences between clones.
2.5. Superoxide dismutase (SOD) assay Protein extract from the lyophilized marula was prepared according to Mdluli (Mdluli, 2003). The SOD activity of the extract was evaluated indirectly using the nitro blue tetrazolium (NBT) assay (Beauchamp and Fridovich, 1971) with slight modifications. The reaction medium contained 50 mM K-phosphate buffer pH 7.8, 0.1 mM EDTA, 0.025% Triton X-100, 14 mM methionine, 75 mM NBT, 4 mM riboflavin, and the required amount of enzyme extract from lyophilized marula samples. The tubes were illuminated for 10 min at 25 8C, and subsequently absorbance at 560 nm was measured with an UV–vis spectrophotometer (Ultrospec 2100 pro). One unit of SOD activity was defined by standard SOD activity (sigma S-2515). Linearity was obtained between 0–6 units of enzyme (R2 = 0.96).
3.2. Distribution of fruit abscission Two major patterns of fruit abscission emerged: in the first, the majority of the fruits dropped in a typical Gaussian distribution within the first 10 days after the start of abscission; in the second, abscission also described a Gaussian curve but the majority of the fruits dropped later, from the end of week 1 to the end of week 3 (Fig. 4). 3.3. Fruit weight and appearance Although grown under the same conditions, the marula genotypes exhibited significant differences in average fruit weight (Fig. 5A), as well as differences in fruit shape, hue and size (Fig. 5B). These differences persisted throughout the abscission stage.
2.6. Food products Marula sorbet was prepared by an industrial process. A parallel preparation of sorbet free of marula served as control. Marula jam was made by an industrial process. Briefly, marula juice (210 g) and pieces of marula peel (40 g) were cooked with sugar (1:1). Next, potassium sorbate (0.4 g) and pectin (2.5 g) were added and the jam was pasteurized at 93 8C for 14 min. After cooling the jam was stored in a jar.
Fig. 4. The two typical abscission patterns displayed by marula genotypes. Clone no. 17 represents an early-abscission type and clone no. 2 a type with relatively prolonged abscission. Fruits were collected every day or two from each tree from first to last fruit abscission. Data from 2005.
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representative genotypes are shown in Fig. 6B and C one with relatively low flesh content (40% of fresh weight) and the other with relatively high flesh content (55%). With the lapse of time post-abscission the color of the peel changed from green to yellow (Fig. 2), corresponding to the progress of fruit ripening as well as to changes in the antioxidant capacity of the fruits. 3.5. Antioxidant capacity of marula fruits In this part of the study we investigated the major compounds associated with antioxidant function in marula juice. This was done by evaluating ascorbic acid and polyphenol contents, as well as the total antioxidant capacity of the unfractionated juice. The results were then compared with corresponding data for orange and pomegranate juice, both known to have relatively high contents of antioxidants. 3.6. Cyclic voltammetry analysis of marula juice
Fig. 5. Fruit weight, shape, hue and size displayed by different marula genotypes. (Panel A) Differences in fruit weight between the marula clones. Each bar represent an average for at least 32 fruits from each tree S.E. Different letters are significantly different from each other at p < 0.05 as established by ANOVA. (Panel B) Photograph showing representative fruit shape, hue, and relative size 1-week post-abscission.
3.4. Fresh fruit components The fruits produced by the different marula genotypes were assayed for percentage of fruit components (Fig. 6A). Two
The cyclic voltammetry approach was used to analyze the total reducing capacity of the marula juice content (Kohen et al., 1999; Butera et al., 2002). We were able to identify two major reducing groups of low molecular weight antioxidants representing the total hydrophilic antioxidants (Fig. 7). The first anodic wave (A) was mainly composed of ascorbic acid, polyphenols, and small amounts of uric acid (Beit-Yannai et al., 2005). The definite composition of the first anodic wave was confirmed as mainly ascorbic acid using an HPLC equipped with an electrochemical detector, as well as by addition of ascorbate oxidase to the juice and CV tracing (data not shown). We also examined the antioxidant capacity of the various marula clones at different time intervals post-abscission as expressed by the currents of the first anodic wave. The current is proportional to the low molecular
Fig. 6. Fruit fresh weight components for different marula genotypes. One-week post-abscission 10 fruits from each genotype were separated into flesh, stone and peel and weighed. (A) Summarizes the weight differences between fruit components. Data represent the means for ten fruits S.E. (B) and (C) show distribution of components for typical low (clone no. 2) and high (clone no. 6) fruit flesh content, respectively.
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Fig. 8. Effect of time post-abscission and clone on ascorbic acid equivalents in marula fruit juice. Ascorbic acid content evaluated by cyclic voltammetry (glassy carbon working electrode 3.2 mm, Ag/AgCl reference electrode, Pt wire as counter electrode; scan rate 100 mV/s). Values were calculated vs. ascorbic acid standard (R2 = 0.99). The values are means S.E. for six fruits from each tree in each week postabscission. Significant differences were found between clones in the same week as established by ANOVA (*p < 0.05 vs. clone no. 4, #p < 0.05 vs. clone no. 6).
Fig. 7. Typical cyclic voltammogram of marula fruit juice. Cyclic voltammetry measurements were carried out as described in Section 2. Cyclic voltammograms of marula juice from abscission day (––) and 3 weeks post-abscission day (+++). Lyophilized marula, 0.5 g was homogenized in 5 ml of 100 mM PBS, pH 7.4. Cyclic voltammetry measurements were carried out as described in Section 2. Two groups of reducing equivalents can be detected (A and B).
(Fig. 9A–C). Differences in polyphenol content were found between the different marula genotypes. Polyphenol content peaked at 3 weeks post-abscission in all the clones tested. Clone no. 6 exhibited the highest concentration of polyphenols and clone no. 12 the lowest.
weight antioxidant content at potential 450 50 mV (Table 1) and 1000 50 mV (Table 2). 3.7. Ascorbic acid content in the different marula genotypes Marula fruits are known to be relatively rich in ascorbic acid content. In our study we found that ascorbic acid content varied between 7 and 21 mg/g dry weight depending on clone and fruit ripening stage (Fig. 8).
3.9. Superoxide dismutase The superoxide dismutase activity of marula extract from different clones was measured by the NBT method (Beauchamp and Fridovich, 1971) (Fig. 10A–C). Significant differences in SOD activity were found between clones. All the clones displayed a significant increase in SOD activity with time post-abscission.
3.8. Polyphenol content Polyphenol content was evaluated using Folin-Ciocalteau reagent with gallic acid as reference value (George et al., 2005)
Table 1 Antioxidant capacity of seven marula clones at different time intervals post-abscission as expressed by the currents at potential 450 50 mV Clone number
2 4 6 8 12 17 20
Abscission day
One-week post-abscission
Two weeks post-abscission
Three weeks post-abscission
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
125 82 144 138 198 148 152
c d b bc a b b
135 119 164 164 244 113 144
cd cd b b a d bc
186 124 150 161 239 232 146
b c bc bc a a c
158 144 150 166 219 116 136
b bc b b a c bc
Values are means for six fruits from each clone at each time point. Samples were scanned three times and results subjected to ANOVA (p < 0.05). Different letters indicate significant differences among columns. Table 2 Antioxidant capacity of seven marula clones at different time intervals post-abscission as expressed by the currents at potential at 1000 50 mV Clone number
2 4 6 8 12 17 20
Abscission day
One-week post-abscission
Two weeks post-abscission
Three weeks post-abscission
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
Current (mA)
Statistic
71 60 91 89 113 99 91
c d b b a b b
82 81 99 100 130 78 92
c c b b a c bc
101 81 86 88 121 121 91
b c bc bc a a bc
96 77 86 87 116 75 84
b c bc bc a c bc
Values are means for six fruits from each clone at each time point. Samples were scanned three times and results subjected to ANOVA (p < 0.05). Different letters indicate significant differences among columns.
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Fig. 9. Effect of time post-abscission on polyphenol content in lyophilized marula fruit juice from three different clones. (A) Clone no. 4, (B) clone no. 6, (C) clone no.12. Polyphenol content was evaluated by the Folin-Ciocalteu method using gallic acid as a standard. Values are means S.E. for six fruits from each tree in each week postabscission. Different letters are significantly different from each other at p < 0.05, as established by ANOVA.
3.10. Comparison of ascorbic acid contents of pomegranate, orange and marula fresh juice The ascorbic acid contents of fresh juices of marula (Beer-Sheva and Besor orchard), different orange clones (Valencia, Shamouti and an unknown clone purchased at the local market), and three clones of pomegranate (Hershkovitz, Rosh-Pered and an unknown clone purchased at the local market) were compared. The juices were analyzed by cyclic voltammetry and the current values converted into ascorbic acid equivalent. The ascorbic acid content of the best marula clone tested was found to be approximately 10 time higher than orange and pomegranate from the local market. At least 30% increase of ascorbic acid content was found in the marula (clone no 13) versus the best pomegranate clone tested (Hershkovitz). The lower ascorbic acid content marula clone no. 13, show a 100% increase versus the best orange clone (Valencia) (Fig. 11). 3.11. Antioxidant capacity of marula food products The ascorbic acid content of marula sorbet and marula jam prepared according to industrial protocols was evaluated by cyclic
Fig. 10. Effect of time post-abscission on SOD activity of lyophilized fruit juice from three marula clones. (A) Clone no. 4, (B) clone no. 6, (C) clone no. 12. Superoxide dismutase analysis was performed by the NBT method using an SOD activity curve with known enzyme activity. Values are means S.E. for five fruits from each clone in each week. All the reactions were performed in triplicate. Different letters are significantly different from each other at p < 0.05 as established by ANOVA.
voltammetry. Non-marula ingredients in the sorbet and jam were found to be free of ascorbic acid. The ascorbic acid concentrations were evaluated on the day of production and again after 45 days (Fig. 12). After 45 days the jams prepared with marula juice from clones 8, 12 and 17 retained only about 50% of the ascorbic acid content measured on the production date, while the jam prepared from clone no. 6 retained 84% of the original content. The ascorbic acid content remained high despite the pasteurization to which the jam had been subjected. The sorbet prepared from clone no. 6 showed no loss of ascorbic acid at 45 days as compared with the production date; the original reduction is due mainly to the preparation procedure. 4. Discussion For domestication of newly introduced fruit clones to be successful, the crop must possess favorable horticultural traits, such as high yield per clone, large edible parts, and a long yielding season, as well as a high nutrient content. Several years ago a number of marula clones were selected for high yields, good taste and seasonality and subsequently tested for horticultural char-
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Fig. 11. Antioxidant content as ascorbic acid equivalents in marula, orange and pomegranate fresh juice. Fresh fruits were squeezed and centrifuged. The supernatants were diluted 10 in PBS pH 7.4 and evaluated by cyclic voltammetry for their total antioxidant content. Cyclic voltammetry measurements were carried out as described in Section 2. Values are means S.E. of at least three fruits of each species and clone.
acteristics in the arid Israeli Negev. The aim of the present study was to examine the same marula clones more closely as potential new candidates for commercial cultivation. In particular, we looked at their horticultural characteristics and the content of antioxidants in the fruit flesh (especially ascorbic acid and total polyphenols). The marula genotypes were found to exhibit significant differences in annual fruit yield, from about 45 kg per clone for
Fig. 12. Ascorbic acid content of marula jam and marula sorbet as evaluated by cyclic voltammetry. Ascorbic acid content of marula jam (A) and marula sorbet (B) prepared by industrial protocols and evaluated on day of production and 45 days later.
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the highest yielding individuals (clones 2, 17 and 20) to 1–3 kg per clone for the low yielders (14 and 18). The data presented in Fig. 3 seem to suggest that marula is an alternate bearing species. Differential yield from year to year is an important trait from the perspective of commercial planting, especially in the case of a new species intended for the horticultural industry. As marula is considered to be a dioecious species, male clones were also planted in the grove. It is reasonable to speculate that unsynchronized flowering of the female and male clones prevented optimal pollination and hence affected yields; however, the flowering patterns of female and male individuals have not been consistently followed in this study. Moreover, male and female flowers have occasionally been observed blooming together on the same clone in the Beer-Sheva orchard as well as at other sites; such a phenomenon could interfere with optimal pollination. We note that the present study was limited to a 2-year-period, which is too brief time to settle the question of alternate bearing in marula. Besides yield per clone, fruit weight and fruit size are also important criteria for selecting clones for fresh fruit production (Frary et al., 2000). The seven clones tested displayed a relatively wide range of fruit weights, namely from 19 to 63 g. Consumption of fresh marula fruits is inconvenient owing to their thick peel, so industrial processing may prove to be the preferred option, in which case the ratio between the fruit components would be an important characteristic. Flesh content, which is not correlated with fruit fresh weight (r2 = 0.3, data not shown), might then be a key point in clone selection. We have identified two typical groups of marula clones in relation to fruit flesh, namely with relatively high (58%) and low (39%) contents; both values are higher than the average for wild populations, where it is around 25%. The stone may be consumed as a nut or used for oil extraction. In principle, different clones might be recommended for oil extraction versus juice production; however, as marula stones should routinely be available as a by-product of the juice industry, it is unlikely that there would be a demand for clones developed exclusively for the stone. The marula clones exhibited two abscission profiles, one in which the majority of the fruits abscised early and the other in which the majority dropped late, the time differential being about 10 days. This trait could be relevant for mechanical harvesting. The orchard chosen for this study included diverse clones which together provided a long harvesting season, an important consideration for marketing and export. In future orchards, clones should be planted in rows to allow efficient mechanical harvesting. Many studies in recent decades have focused on the occurrence of antioxidant molecules and the link between diets rich in fruits and vegetables and the onset and/or prevention of a number of oxidative stress related diseases (Sardesai, 1995; Halliwell, 2002; Ignarro et al., 2007). Accordingly, we examined the antioxidant capacity of the fruits produced by our selected marula clones. The total reducing capacity measured by the cyclic voltammetry method (Silva et al., 2001; Butera et al., 2002; Roginsky et al., 2003) revealed significant differences in antioxidant content between the different clones. Two main groups of low molecular weight antioxidants were found (1st and 2nd anodic waves: 450 50 and 1000 50 mV, respectively). The first anodic wave consisted mainly of ascorbic acid (diminished by the addition of ascorbate oxidase, data not shown) and small amounts of uric acid (Beit-Yannai et al., 2005). The peak shifted by 50 mV compared to ascorbic acid standard corresponds to other low molecular weight antioxidants. The second peak is probably composed of several polyphenols containing 3-5 hydroxyl groups (such as quercetin) (Jorgensen et al., 1999; Aaby et al., 2004). We were able to show a rise in the hydrophilic antioxidant content with time post-abscission. This increase in
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reducing capacity is mainly due to elevated amounts of several polyphenols, as well as to ascorbic acid. In parallel with the increase in low molecular weight antioxidants during ripening, a significant increase in SOD activity was found in all clones. The significant differences in SOD activity between the different clones suggests differences in the ability of the marula genotypes to cope with oxidative stress generated by abscission of superoxide radicals. The two large anodic waves revealed by the cyclic voltammetry suggest that marula fruit owes its high defense capability in the face of different oxidative stresses to the presence of low molecular weight antioxidants and the key antioxidant enzyme. All the marula clones exhibited the same pattern of increase in antioxidant capability with ripening, even though the clones differed significantly in total antioxidant capacity. Clone no. 6 exhibited the highest polyphenol content and SOD activity during ripening. Clone no. 12 had the highest ascorbic acid content, which reached 22 mM at 2 weeks postabscission. In the present study we compared the ascorbic acid content in fresh juice of several marula clones to different orange and pomegranate. Kiwi is another well known source for vitamin C. Szeto et al. (2002) analyzed the ascorbic acid of different fruits, finding that the kiwi content is about 520 mg/kg versus 330 mg/kg for orange. According to these values Kiwi has about 1.6 times more ascorbic acid then orange. Yet the marula content is almost two times higher then the kiwi content. The finding that the juice of marula has a much higher antioxidant capacity than that of species commonly thought of as particularly rich in antioxidants could facilitate its acceptance as a new fruit product. The first semi-commercial orchard of selected clones was planted 4 years ago in the Arava valley of Israel, where high temperatures combine with saline soil and water to create highly stressful conditions. The fact that marula proved capable of tolerating these conditions may be attributable to its high content of antioxidants. 5. Conclusion In our study we showed that marula has a good potential to become a fruit crop on a commercial scale since the fruits are tasty, rich in antioxidants and particularly suitable for industrial uses. The current study contributes to validation of indigenous knowledge on marula and more so to the knowledge about the quality of our selected clones. Many aspects of fruit quality and many clones, other than those investigated here, remain to be studied, hence, it is necessary to continue analyze additional selected clones to satisfy the needs for establishing new emerging fruit crop. Acknowledgement We thank Ms. Aliza Sen for editing the manuscript. References Aaby, K., Hvattum, E., Skrede, G., 2004. Analysis of flavonoids and other phenolic compounds using high-performance liquid chromatography with coulometric array detection: relationship to antioxidant activity. J. Agric. Food Chem. 52 (15), 4595–4603. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 90 (17), 7915–7922. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44 (1), 276–287. Beit-Yannai, E., Jaglitz, S., Trembovler, V., Mizrahi, Y., 2005. Reducing Properties of Marula Fruit Evaluated by Cyclic Voltammetry. 2nd International meeting of the Society for Free Radical Research-Asia, Shanghai, PRC. Braca, A., Politi, M., Sanogo, R., Sanou, H., Morelli, I., Pizza, C., Tommasi, N.D., 2003. Chemical composition and antioxidant activity of phenolic compounds from wild and cultivated Sclerocarya birrea (Anacardiaceae) Leaves. J. Agric. Food Chem. 51 (23), 6689–6695. Butera, D., Tesoriere, L., Di Gaudio, F., Bongiorno, A., Allegra, M., Pintaudi, A.M., Kohen, R., Livrea, M.A., 2002. Antioxidant activities of sicilian prickly pear
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