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Phenolics, organic acids and minerals in the fruit juice of the indigenous African sourplum (Ximenia caffra, Olacaceae)
South African Journal of Botany 119 (2018) 11–16
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Phenolics, organic acids and minerals in the fruit juice of the indigenous African sourplum (Ximenia caffra, Olacaceae) N.J. Goosen a,⁎, D. Oosthuizen a, M.A. Stander b, A.I. Dabai c, M.-M. Pedavoah d, G.O. Usman e a
Department of Process Engineering, Stellenbosch University, South Africa Central Analytical Facility, Stellenbosch University, South Africa Department of Microbiology, Usmanu Danfodiyo University, Sokoto, Nigeria d Department of Applied Chemistry and Biochemistry, University for Development Studies, Ghana e Department of Food, Nutrition and Home Sciences, Kogi State University, Nigeria b c
a r t i c l e
i n f o
Article history: Received 22 March 2018 Received in revised form 27 April 2018 Accepted 3 August 2018 Available online xxxx Edited by B-E Van Wyk Keywords: Phenolics Flavonoids Phytochemicals Sour plum Indigenous African fruit Underutilized plant Micronutrients Seasonal fruit Rural diet
a b s t r a c t Wild-picked fruits of the indigenous African tree, Ximenia caffra, are widely consumed in Southern Africa when in season, yet compositional data on phytochemicals, organic acids and minerals are lacking. The study therefore aimed to characterize juice obtained from ripe X. caffra fruits, and to identify individual phenolic compounds using liquid chromatography, high-resolution mass spectrometry (LC-HRMS). The juice had high total phenolic (1030 mg 100 ml−1 gallic acid equivalents) and total flavonoid content (852 mg 100 ml−1 catechin equivalents), and LC-HRMS analysis identified procyanidin B1 (12.2 mg 100 ml−1), gallic acid (5.56 mg 100 ml−1) and catechin (2.66 mg 100 ml−1) as the most abundant phenolic compounds, while the dominant organic acid was citric acid (8.05 g 100 ml−1), with lesser levels of tartaric, L-malic and L-lactic acids. LC-HRMS further positively identified and quantified a number of other polyphenolic compounds from the juice. The most prevalent minerals were potassium (525 mg 100 ml−1) and phosphorous (24.6 mg 100 ml−1), while heavy metal content was low. Consumption of X. caffra fruits can significantly contribute toward dietary phytochemical and mineral intake, and consumption of this fruit should therefore be encouraged. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Plant foods gathered from the wild provide essential nutrients and potentially health promoting phytochemicals to especially rural populations, and contribute to global food security (Toledo and Burlingame, 2006; Stadlmayr et al., 2013; Ayessou et al., 2014). Ximenia caffra Sond. is a seasonal wild fruit that forms part of the diet of people throughout its distribution range and it plays an important ethnobotanical role as medicinal plant; the whole fruit including skins, pulp and seeds are edible (Mabogo, 1990; Nair et al., 2013; Maroyi, 2016). The species are widely distributed and indigenous to central, southern and eastern Africa, including Madagascar, and two varieties of X. caffra occur: var. caffra and var. natalensis Sond. (Coates Palgrave, 2003). The Abbreviations: AAE, ascorbic acid equivalents; CE, catechin equivalents; DMPD, N,NDimethyl-p-phenylenediamine dihydrochloride; GAE, gallic acid equivalents; ICP-AES, inductively coupled plasma atomic emission spectroscopy; ICP-MS, inductively coupled plasma mass spectrometry; LC-HRMS, liquid chromatography, high resolution mass spectrometry. ⁎ Corresponding author at: Department of Process Engineering, University of Stellenbosch, Private Bag X1, Stellenbosch, 7602, South Africa. E-mail address: [email protected] (N.J. Goosen).
https://doi.org/10.1016/j.sajb.2018.08.008 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
tree is known under a number of different vernacular names and in English it is mainly known as ‘sour plum’ due to the high acidity of the fruits (Maroyi, 2016). Flowering normally occurs between August and October (Nair et al., 2013), although the exact flowering and fruiting period is heavily influenced by seasonal rainfall (Mr. Dave Rushworth, personal communication). Comprehensive data on nutritionally relevant compounds and phytochemical aspects of both X. caffra varieties are lacking, especially on the fruit pulp and skins. Current nutritional and phytochemical data of the fruit pulp, juice and skin show that the fruits are highly acidic and contain high levels of ascorbic acid, potassium and phenolic compounds, and that methanolic extracts of the fruits and skins exhibit high antioxidant capacity (Wehmeyer, 1966; Ndhlala et al., 2006; 2008; Maroyi, 2016). In contrast to the lack of nutritional data on the fruit, much effort has been spent on analysis of the seeds and the economically valuable seed oil, and data include proximate composition, lipid content and fatty acid profile, protein content and amino acid profile, mineral content, and data on various minor components (Chivandi et al., 2008, 2012; Mitei et al., 2009). These data confirm the high nutritional value of the seeds and the potential contribution that
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consumption of seeds can make to intake of essential and health promoting nutrients. Despite the knowledge that fruits of X. caffra generally contain high levels of organic acids and phenolics, little is known about the nature and levels of the individual compounds. There is further a need to broaden the range and availability of data on compounds of nutritional interest in indigenous fruits in general (Stadlmayr et al., 2013), and for X. caffra in particular, since the plant is known to form a part of rural diets when in season. The aim of the current work was therefore to perform an analysis on water-soluble compounds found in X. caffra fruit collected from the wild through identification and quantification of phenolic compounds, organic acids and macro- and micro-minerals, as these compounds can all be of nutritional relevance in human diets. 2. Materials and methods 2.1. Raw material collection and preparation Ripe fruits of X. caffra were collected in the Hoedspruit area, Limpopo Province, South Africa on January 10, 2015, at the GPS coordinates: S23°54.567′ E029°48.760′. The variety was tentatively identified as var. natalensis by a local botanist, Mr. Dave Rushworth, and fruits were kept on ice until processed. Juice was obtained from the fruits by separating the skin and fleshy portion of the fruit from the seeds through physical means, homogenizing the fruit flesh and skins together in a food processor and filtering the resultant pulp through a cotton cloth. The filtered juice obtained was pooled and mixed to obtain a homogenous sample, and then frozen at −20 °C until analysis. All subsequent analyses were performed on the pooled juice sample. The weight of 42 randomly selected seeds were determined and expressed as the percentage of total fruit weight. Juice pH and Brix were determined using benchtop instruments, while total moisture (AOAC, 2005), total sugars (AOAC, 2003) and titratable acidity (Mora et al., 2009) were measured using standard analytical methods. 2.2. Juice mineral profile Major elements Ca, K, Mg, Na, P and Si were analyzed on a Thermo ICap 6200 inductively coupled plasma atomic emission spectrometer (ICP-AES). The instrument was calibrated using NIST (National Institute of Standards and Technology, Gaithersburg MD, USA) traceable standards purchased from Inorganic Ventures (Christiansburg, Virginia, USA) to quantify selected elements. NIST-traceable quality control standards from De Bruyn Spectroscopic Solutions, Bryanston, South Africa, were analyzed to verify the accuracy of the calibration before sample analysis, as well as throughout the analysis to monitor drift. Trace elements were analyzed on an Agilent 7700 quadrupole inductively coupled plasma mass spectrometer (ICP-MS). Samples are introduced via a 0.4 ml min− 1 micromist nebulizer into a peltier-cooled spray chamber at a temperature of 2 °C, with a carrier gas flow of 1.05 l min−1. The elements V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se were specifically analyzed under He-collision mode to remove polyatomic interferences. The instrument was calibrated using NIST (National Institute of Standards and Technology, Gaithersburg MD, USA) traceable standards to quantify selected elements. NIST-traceable quality control standards of a separate supplier than the main calibration standards were analyzed to verify the accuracy of the calibration before sample analysis. 2.3. Analysis of phenolics and ascorbic acid Spectrophotometric methods were employed to measure total phenolic content and total flavonoid content of the fruit juice in triplicate. The spectrophotometer used for all spectrophotometric analyses was from A&E Lab, United Kingdom, model AE-S60-4U. Total phenolics were determined according to the Folin–Ciocalteu method of Singleton and Rossi (1965), using gallic acid as standard. Values were determined
by measuring absorbance at 765 nm and expressed as gallic acid equivalents (GAE). Total flavonoids were measured using the method described by Amado et al. (2014), with catechin as standard, and absorbance of samples were measured at 410 nm and expressed as catechin equivalents (CE). Total ascorbic acid levels in the juice were determined by way of UV-HPLC according to the method described in Odriozola-Serrano et al. (2007). The column used was a YMC Pack Pro C18 column, and a total runtime and detection wavelength of 35 min and 250 nm were employed, respectively. Mobile phase A was 0.5% trifluoroacetic acid in water, and mobile phase B was 0.5% trifluoroacetic acid in methanol. Column temperature was 25 °C, mobile phase flow rate was constant at 1.0 ml min−1 and sample injection volume was 20 μl. Phenolics were identified and quantified through a liquid chromatography, high resolution mass spectrometry (LC-HRMS) method, as describe previously (Stander et al., 2017), which utilizes a gradient method specifically focussing on phenolic acids and flavonoids. The only difference to the published method was that a Waters BEH C18, 2.1 × 100 mm, 1.7 μm column was used. A Waters Synapt G2 quadrupole time-of-flight mass spectrometer connected to Waters Ultra pressure liquid chromatograph and photo diode array detection was used for the analysis. In short, a 0.1% formic acid (solvent A) to acetonitrile containing 0.1% formic acid (solvent B) gradient was applied up to 28% solvent B, followed by a wash step. The instrument was operated using electrospray ionization in negative MSE mode which consisted of a low collision energy scan (6 V) from m/z 150 to 1500 and a high collision energy scan from m/z 40 to 1500. Positive identification of compounds was based on retention time matching with authentic standards, accurate mass data, UV data as well as MSMS fragmentation data. The juice was diluted 5- and 10-fold in 50% methanol/water, centrifuged and the supernatant injected directly into the system. A cocktail of the standards were injected into the system at (100, 50, 25, 10, 5 and 0.5 mg l−1) and the application manager Targetlynx 4.1 (Waters, MA, US) was used for the quantifications. Preliminary antioxidant capacity was estimated using the spectrophotometric N,N-Dimethyl-p-phenylenediamine dihydrochloride (DMPD) assay of Fogliano et al. (1999), at an absorbance wavelength of 505 nm. Ascorbic acid was used as standard and values were reported as ascorbic acid equivalents (AAE). 2.4. Organic acid determination Individual organic acids and their levels were determined using enzymatic derivatization followed by spectrophotometric detection and quantification. The instrument used was an Arena 20XT Discrete Photometric Analyzer (Thermo Fisher Scientific, Finland). Analysis for each organic acid was performed using a specific analysis kit, and analysis conditions were according to those prescribed by each kit manufacturer. The following organic acids were analyzed for (the specific analytical kit employed is shown in parentheses): acetic acid (Enzytec™ Fluid Acetic acid, catalog number 5226, Thermo Fischer Scientific, Finland), citric acid (R-Biopharm citric acid, Roche catalog number 10139076035, R-Biopharm AG, Darmstadt), D-lactic acid (Enzytec™ Fluid D-Lactic acid catalog number 5240, Thermo Fischer Scientific, Finland), L-lactic acid (Enzytec™ Fluid D-Lactic acid catalog number 5260, Thermo Fischer Scientific, Finland), L-malic acid (Enzytec™ Fluid L-Malic acid catalog number 5280, Thermo Fischer Scientific, Finland), pyruvic acid (Megazyme Pyruvic acid catalog number K-PYRUV 07/12, Megazyme International, Ireland), succinic acid (RBiopharm succinic acid, Roche catalog number 10176281035, RBiopharm AG, Darmstadt), and tartaric acid (Enzytec™ Color Tartaric acid catalog number E3100, Thermo Fischer Scientific, Finland). 3. Results The juice obtained had an attractive red color corresponding to the red color of the fruit, and had a moisture content of 86.4%, a pH of
N.J. Goosen et al. / South African Journal of Botany 119 (2018) 11–16
2.60 and titratable acidity of 7.69% malic acid equivalents. Total sugars were 3.92 g 100 ml−1 and Brix measurements showed a value of 18.5 Brix. The average fruit weighed 11.4 ± 2.47 g (mean ± standard deviation), and seed weight was 18.3 ± 3.47% of total weight. The precision of the analytical methods employed were satisfactory. For spectrophotometric determination of total phenolics, the standard deviation as percentage of the mean was less than 5% in the range of 0–300 mg l−1 GAE, for total flavonoids it was less than 3% in the range 0–100 mg l−1 CE, and for DMPD antioxidant capacity it was less than 2% in the range of 0–100 mg l− 1 AAE. For determination of organic acids, all analyses have a standard deviation as percentage of the mean value of less than 7%, and the error for LC-HRMS quantitation was below 5 ppm for all compounds. 3.1. Analysis and identification of phenolics Table 1 shows values for spectrophotometric determination of total phenolics (1030 mg 100 ml−1 GAE), total flavonoids (852 mg 100 ml−1 CE) and preliminary DMPD antioxidant capacity (6100 mg 100 ml− 1 AAE) of X. caffra juice, and results for HPLC quantification of ascorbic acid (45.0 mg 100 ml−1). LC-HRMS positively identified and quantified 15 phenolics and phenolic glycosides, and tentatively identified a further 7 compounds (Table 4). The phenolics and phenolic glycosides which were positively identified were catechin, epicatechin, gallic acid, hesperetin, hyperoside, isoquercitrin, kaemferol glucoside, luteolin-7-O-glucoside, procyanidin B1, procyanidin B2, quercetin-3O-glucoside, quercetin-3-O-robinobioside, quercetin, rutin and trilobatin. 3.2. Organic acids Table 2 reports the positively identified organic acids and their levels. The most prevalent organic acid was citric acid (8.05 g 100 ml−1), followed by tartaric acid (0.18 g 100 ml−1), L-malic acid (0.16 g 100 ml−1) and L-lactic acid (0.13 g 100 ml−1). D-lactic acid was present in the sample, but could not be accurately quantified. Concentrations of acetic, pyruvic and succinic acids were below detection levels.
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Table 2 Identity and concentrations of specific organic acids as found in the juice of Ximenia caffra. Organic acid
Concentration (g 100 ml−1)
Acetic acid Citric acid D-Lactic acid L-Lactic acid L-Malic acid Pyruvic acid Succinic acid Tartaric acid
b0.003 8.05 Presenta 0.13 0.16 b0.001 b0.001 0.18
a D-Lactic acid was present in low concentration, but could not be quantified accurately.
of seed weight (18.3 ± 3.47%) found in this study agree well with a prior investigation where average fruit weight was 11.8 g, and seed weight was 17.8% of total fruit weight (Ligthelm et al., 1954). Due to the relative contribution of the fruit flesh and skins to overall fruit weight, much of the potentially beneficial nutritional and phytochemical components found in X. caffra fruit could be located in the fruit flesh and skins. The seeds have been well-characterized and it is known that seeds have high oil and protein contents, as well as other potentially beneficial minor components including phytosterols, vitamins and phospholipids (Ligthelm et al., 1954; Chivandi et al., 2008, 2012; Mitei et al., 2009). Total content of phenolics and flavonoids was found to be relatively high in the current study, which is in line with previous investigations. Based on spectrophotometric analyses, a large proportion (82.7%) of the total phenolics consisted of flavonoids, which is also in agreement with previous work where methanol extracts of fruit pulp and skins both showed high phenolic and flavonoid content and high antioxidant capacity (Ndhlala et al., 2006, 2008). LC-HRMS analysis supports this finding, where 14 of the 16 positively identified compounds were flavonoids or flavonoid glycosides. The values obtained in the current study for total phenolics and flavonoids might be more representative of actual phenolic and flavonoid ingestion during fruit consumption, as skins are many times discarded when the fruits are consumed (Mabogo, 1990).
3.3. Mineral profile The major and trace mineral profile of the X. caffra juice is reported in Table 3. The most abundant major minerals were potassium (525 mg 100 ml−1), phosphorous (24.6 mg 100 ml−1), magnesium (15.9 mg 100 ml−1) and calcium (3.04 mg 100 ml−1). The most abundant trace minerals were manganese (320 μg 100 ml−1), zinc (180 μg 100 ml− 1), iron (170 μg 100 ml− 1) and aluminum (100 μg 100 ml−1). Both selenium and mercury levels were below detection limits, while concentrations of arsenic, cadmium, cobalt, chromium, molybdenum, lead, antimony and vanadium were all below 6 μg 100 ml−1. 4. Discussion The largest proportion of the fruits consisted of the pulp and skins, while the seeds contributed 18.3 ± 3.47% of the total fruit weight on wet weight basis. The fruit weights (11.4 ± 2.47 g) and contribution Table 1 Mean values of spectrophotometric assays of total phenolics, total flavonoids and DMPD antioxidant capacity, and HPLC identification and quantification of ascorbic acid. Assay/compound
Concentration −1
a
Total phenolics (mg 100 ml GAE ) Total flavonoids (mg 100 ml−1 CEa) DMPD (mg 100 ml−1 AAEa) Ascorbic acid (mg 100 ml−1)
1030 852 6100 45.0
a Abbreviations: GAE – gallic acid equivalents; CE - catechin equivalents; AAE – ascorbic acid equivalents.
Table 3 Mineral profile of Ximenia caffra juice, showing both macro- and micro-minerals. Element
Concentration
Macro minerals (mg 100 ml−1) Ca K Mg Na P Si
3.04 525 15.9 0.82 24.6 0.52
Micro-minerals (μg 100 ml−1) Al As Ba Cd Co Cr Cu Fe Hg Mn Mo Ni Pb Sb Se Sr V Zn
100 0.1 8.8 0.1 3.5 3.6 72 170 b 0.1 320 2.1 7.1 1.5 0.4 b 3.0 58 0.3 180
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Table 4 LC-HRMS data obtained for the juice from Ximenia caffra, detailing compound identity and the concentration for the identified compounds.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Compound name
m/za
Retention time (min)
[M-H]−
MSE fragmentsb
Concentration (mg 100 ml−1)
Positively identified compounds Catechin Citric acid Epicatechin Gallic acid Hesperetin Hyperoside Isoquercitrin Kaemferol glucoside Luteolin-7-O-glucoside Procyanidin B1 Procyanidin B2 Quercetin-3-O-glucoside Quercetin-3-O-robinobioside Quercitin Rutin Trilobatin
289.0713 191.0187 289.0698 169.0129 301.1643 463.0878 463.0876 447.0938 447.0935 577.1317 577.1345 463.0886 609.1432 301.0353 609.1458 435.1284
11.48 3.12 13.57 5.8 24.49 17.51 17.51 18.06 18.06 10.68 12.72 17.81 17.06 23.99 17.27 18.75
C15H13O6 C6H7O7 C15H13O6 C7H5O5 C15H25O6 C21H19O12 C21H19O12 C21H19O11 C21H19O11 C30H25O12 C30H25O12 C21H19O12 C27H29O16 C15H9O7 C27H29O16 C21H23O10
289,125,203,245,151 191,111,87,173 weak 125,169,111 weak 300,463,271,255 300,463,271,301,255, 285,169,447 285,284,169,125,447 289,407,425,577 289,407,425,577 300,271,463,255,125 300,609,271,125 125,169 300,609,271,255 315,345
2.66 295.2 0.03 5.56 0.01 0.96 1.09 0.60 0.02 12.2 0.25 0.11 0.12 0.02 0.37 0.07
9.14 24.5 11.18 10.68 16.63 16.94 19.63 18.89 18.83 14.37 11.37 5.49 5.68 5.83 5.93 21.403 9.37
C6H5O6 C16H31O4 C16H17O8 C30H25O12 C28H23O16 C28H23O16 C21H19O11 C21H19O11 C20H17O11 C15H18NO8 C21H31O10 C18H27O17 C18H27O17 C18H27O17 C18H27O17 C24H19O8 C8H11O7
111 287 163,119,191,337 289,407,425,577 300,615,463,255,169 300,463,615 300,271,255,447,243 300,271,255,447, 300,271,255,433,315 161,101,85 443,289,303 515,111,173 515,111,173 515,111,173 515,111,173 341,189,125,435 111,219,87
Tentatively identified and unknown compoundsc Aconitic acid 173.0089 Dihydroxy hexadecanoic acid 287.2236 p-Coumaorylquinic acid 337.0916 Procyanidin 577.1344 Quercetin galloyl glucoside 615.0979 Quercetin galloyl glucoside 615.0977 Quercetin rhamnoside 447.0927 Quercetin rhamnoside 447.0927 Quercetin-3-O-pentoside 433.0764 Unknown 340.1035 Unknown 443.1913 Unknown 515.1246 Unknown 515.1245 Unknown 515.1255 Unknown 515.1253 Unknown 435.107 Unknown 219.0506 a b c
The mass accuracy for all compounds was better than 5 ppm. Most abundant fragment mentioned first. Concentrations of epicatechin, procyanidin B2 and all tentatively identified and unknown compounds could not be determined.
Various phenolic compounds were positively identified in the juice, which, to the best of our knowledge, is the first instance that many of these phenolic compounds have been identified from X. caffra fruits. The major phenolic compounds were procyanidin B1 (12.2 mg 100 ml− 1), gallic acid (5.56 mg 100 ml− 1) and catechin (2.66 mg 100 ml−1), while LC-HRMS analysis also confirmed the high levels of citric acid. Procyanidin B1 is a potent antioxidant known to exert high radical scavenging ability (Villaño et al., 2007), and forms part of the group of compounds known as condensed catechins, which are responsible for a range of potentially beneficial health effects (Matsui, 2015). Gallic acid is a phenolic compound with known antioxidant, antimicrobial and health promoting activity, and could potentially be employed for a range of medicinal and industrial purposes (Borges et al., 2013; Badhani et al., 2015). Catechin and catechin derivatives are associated with direct beneficial effects upon ingestion, including increased blood plasma antioxidant activity and decreased oxidation of LDL cholesterol (Williamson and Manach, 2005). The presence of the other minor polyphenols, despite their relatively low concentrations, may also be nutritionally significant due to apparent synergistic health benefits when multiple polyphenolics are ingested (Pandey and Rizvi, 2009; Shahidi and Ambigaipalan, 2015). It should be noted that the composition and total amounts of the phenolic compounds found in the fruits will be different during different stages of ripening of the fruits (Amira et al., 2012); however, the present work is in agreement with previous reports of high polyphenolic content in X. caffra fruits (Ndhlala et al., 2008). The ascorbic acid content of the juice (45.0 mg 100 ml−1) is considered to be nutritionally significant, and is comparable to ascorbic acid concentrations found in other fruits like papaya, kiwifruit, strawberries
and citrus, which are generally considered to contain high levels of ascorbic acid (Nishiyama et al., 2004; Wall, 2006; Pantelidis et al., 2007; Rekha et al., 2012). The value is higher than previously found for X. caffra fruit, where an ascorbic acid content of 22.5 mg ascorbic acid per 100 g edible fruit (presumably referring to the fruit pulp) was reported (Wehmeyer, 1966). There are two prior reports which state an ascorbic acid content of 27% in X. caffra fruit (Ndhlala et al., 2008; Maroyi, 2016), although neither of these investigations measured the ascorbic acid content, and this value is likely an overestimation of true ascorbic acid content of the fruits. The DMPD antioxidant capacity assay of the juice indicated a high value of 6100 mg 100 ml−1AAE, and is likely due to the combined effect of the polyphenolic compounds, ascorbic acid and citric acid. The value obtained, however, can only be viewed as preliminary and needs to be investigated further through more detailed analyses to determine the actual antioxidant potential when the fruits are ingested as part of a normal diet. The antioxidant capacity as measured using in-vitro methods such as the DMPD assay does not necessarily correlate with the actual bioavailability and potentially health-promoting effects in humans, as phenolic compounds differ in respect to the total uptake and rate of uptake in the digestive system (Holst and Williamson, 2008). Organic acid quantification revealed that citric acid is the dominant organic acid in X. caffra juice, but contrary to a previous investigation which reported that citric acid alone accounts for the high fruit acidity (Ligthelm et al., 1954), the current study has also established the presence of D- and L-lactic acids, L-malic acid and tartaric acid. It is probable that the much higher citric acid concentration compared to those of the other organic acids prohibited detection of any acidifying effect by any of the other acids in the previous study, thereby leading to the
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premature conclusion that citric acid is the sole organic acid present in the fruit. Despite the differences between the current and previous study, there is good agreement in total acidity levels: 8.52 g 100 ml−1 total acidity (as the sum of all enzymatically quantified acids in the juice, or approximately 8.5% of the juice) in the current study, vs. 8.7% expressed as citric acid in the work of Ligthelm et al. (1954). The high total acid content is confirmed by the total titratable acidity (7.69% malic acid equivalent), and is responsible for the low pH of 2.60 measured in the fruit juice, and the characteristic sour taste of the fruit that gives this species its English vernacular name. Quantitative values for total sugar content of X. caffra juice is reported for the first time. The only data to which this could be compared relate to those calculated as total carbohydrates, but which also include complex carbohydrates, fiber and potential non-carbohydrate components. Total carbohydrates of which sugars are a component, were determined by difference and reported as 26.3% (Wehmeyer, 1966) and 78.8% (Saka and Msonthi, 1994) in two previous studies, however, neither of these values can be considered as accurate estimates of carbohydrate or sugar content of the fruits. The work done by Wehmeyer (1966) discounts the significant organic acid and polyphenolic contents in the calculation of total carbohydrates, while the value reported by Saka and Msonthi (1994) was incorrectly calculated and should have been 4.4% when calculated according to the protocol of the FAO (2003), instead of the reported 78.8%. The recalculated value of 4.4% is reasonably close to the HPLC-determined value of 3.92 g 100 ml−1 for total sugars found the current investigation, but these values are not directly comparable as the methods by which they were obtained are very different. The fruit of X. caffra is a good source of beneficial minerals, especially the macro-minerals potassium, phosphorous and magnesium. It is generally accepted that X. caffra has a high potassium content (Wehmeyer, 1966; Saka and Msonthi, 1994); however, literature data show less agreement in the level of magnesium and phosphorous. The magnesium contents of the current study is 15.6 mg 100 ml− 1 juice, while prior values range between 2.0 and 45.9 mg 100 g−1; the phosphorous content of 24.6 mg 100 ml−1 juice also falls between the two prior values of 14.5 and 167.4 mg 100 g− 1 that are available in literature (Wehmeyer, 1966; Saka and Msonthi, 1994). Most of the data regarding mineral composition of the fruit of X. caffra rely on analysis done by the authors of the two above-mentioned reports, and values are only reported for calcium, copper, iron, magnesium, phosphorous, potassium and sodium. The current study adds to prior work through reporting of the contents of additional minerals. The mineral analysis further shows that the fruit contains low levels of heavy metals. As there is no specific reference standard for fruit juice for maximum allowable levels, the data obtained for heavy metals were compared to the Codex Standard 193–1995 (CCCF, 1995), which provides guidance for contaminants in food products, but only advises on levels of arsenic, cadmium, mercury, lead and tin. Levels of all of these minerals were below guidance levels, except for tin which was not measured. The results of the current study indicates that consumption of X. caffra fruits can make a significant contribution toward obtaining important micronutrients and phytochemicals which are a requirement in a balanced, healthy diet. Despite the high fruit acidity, X. caffra fruits are collected from the wild and consumed fresh or as part of traditional dishes when in season (Mabogo, 1990; Chivandi et al., 2008; Maroyi, 2016). The importance of obtaining sufficient amounts of secondary plant metabolites (e.g., phenolic compounds) and micronutrients for prevention of especially age-related and chronic diseases has been repeatedly highlighted (Visioli et al., 2000; Holst and Williamson, 2008; Haminiuk et al., 2012), and the role that indigenous and underutilized plants and crops can play in this regard is increasingly being recognized (Ebert, 2014; FOA, 2014). Within this context, X. caffra is an important species, as the high concentrations of total phenolics and flavonoids, significant amounts of ascorbic acid and certain macro-minerals in X. caffra fruit are a good indication that consumption of the fruits provides
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important dietary components required in a balanced diet. Further, even though micro-minerals are present in low concentrations, they may still play an important nutritional role (Maroyi, 2016). 5. Conclusion In conclusion, the current study has contributed toward a better understanding of the phytochemical and mineral contents, and the makeup of the organic acids within the fruit of X. caffra. The juice contains a number of potentially beneficial phytochemicals and minerals, and preliminary evaluation point toward a high antioxidant capacity of the fruit. As an indigenous African species that has a wide distributions range, the consumption of X. caffra fruits can therefore make an important contribution toward ensuring sufficient intake of phytochemicals and minerals that are required in a balanced diet, and consumption of this indigenous fruit should therefore be encouraged. Declaration of interest The authors have no real or potential conflict of interest to declare. Acknowledgements This research was supported by the International Foundation for Science, Stockholm, Sweden, through a grant to Dr. Neill Goosen (Grant number J/5503-1). Mr. Dave Rushworth in his personal capacity and Ms. Moloko Mojapelo from the South African Department of Agriculture, Forestry and Fisheries are gratefully acknowledged for assistance with sourcing and identification of the fruit used in this study. References Amado, I.R., Franco, D., Sanches, M., Zapata, C., Vazquez, J.A., 2014. Optimisation of antioxidant extraction from Solanum tuberosum potato peel waste by surface response methodology. Food Chemistry 165, 290–299. Amira, E.A., Behija, S.E., Beligh, M., Lamia, L., Manel, I., Mohamed, H., Lotfi, A., 2012. Effects of the ripening stage on phenolic profile, phytochemical composition and antioxidant activity of date palm fruit. Journal of Agricultural and Food Chemistry 60, 10896–10902. AOAC (Ed.), 2003. AOAC 982.14 - glucose, fructose, sucrose and maltose in presweetened cereals, Official methods of analysis of AOAC International, 17th ed. AOAC International, Gaithersburg, Maryland. AOAC, 2005. AOAC Official Method 934.01 - Moisture in animal feed, Official methods of analysis of AOAC International. 18th ed. American Organisation of Analytical Chemists, Gaithersburg, Maryland. Ayessou, N.C., Ndiaye, C., Cissé, M., Gueye, M., Sakho, M., Dornier, M., 2014. Nutrient composition and nutritional potential of wild fruit Dialium guineense. Journal of Food Composition and Analysis 34, 186–191. Badhani, B., Sharma, N., Kakkar, R., 2015. Gallic acid: a versatile antioxidant with promising therapeutic and industrial applications. RCS Advances 5, 27540–27557. Borges, A., Ferreira, C., Saavedra, M.J., Simões, M., 2013. Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microbial Drug Resistance 19, 256–265. CCCF, 1995. Codex committee on contaminants in foods: codex general standard for contaminants and toxins in food and feed. Codex Stan 193-1995, 1–44. Chivandi, E., Davidson, B.C., Erlwanger, K.H., 2008. A comparison of the lipid and fatty acid profiles from the kernels of the fruits (nuts) of Ximenia caffra and Ricinodendron rautenenii from Zimbabwe. Industrial Crops and Products 27, 29–32. Chivandi, E., Davidson, B.C., Erlwanger, K.H., 2012. The red sour plum (Ximenia caffra) seed: a potential non-conventional energy and protein source for livestock feeds. International Journal of Agriculture and Biology 14, 540–544. Coates Palgrave, K., 2003. Trees of Southern Africa. 3rd Edition ed. Struik, Cape Town. Ebert, A.W., 2014. Potential of underutilized traditional vegetables and legume crops to contribute to food and nutritional security, income and more sustainable production systems. Sustainability 6, 319–335. FAO, 2003. Food energy – Methods of analysis and conversion factors. FAO food and nutrition paper. Food and Agricultural Organization of the United Nations, Rome, Italy. FOA, 2014. In: Durst, P., Bayasgalanbat, N. (Eds.), Promotion of underutilized indigenous food resources for food security and nutrition in Asia and the Pacific. Food and Agricultural Organisation of the United Nations, Rome, Italy. Fogliano, V., Verde, V., Randazzo, G., Ritieni, A., 1999. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. Journal of Agricultural and Food Chemistry 47, 1035–1040. Haminiuk, C.W.I., Maciel, G.M., Plata-Oviedo, M.S.V., Peralta, R.M., 2012. Phenolic compounds in fruits - an overview. International Journal of Food Science and Technology 47, 2023–2044.
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Pantelidis, G.E., Vasilakakis, M., Manganaris, G.A., Diamantidis, G., 2007. Antioxidant capacity, phenol, anthocyanin and ascorbic acid contents in raspberries, blackberries, red currants, gooseberries and cornelian cherries. Food Chemistry 102, 777–783. Rekha, C., Poornima, G., Manasa, M., Abhipsa, V., Devi, J.P., Kumar, H.T.V., Kekuda, T.R.P., 2012. Ascorbic acid, total phenol content and antioxidant activity of fresh juices of four ripe and unripe citrus fruits. Chemical Science Transactions 1, 303–310. Saka, J.D.K., Msonthi, J.D., 1994. Nutritional value of edible fruits of indigenous wild trees in Malawi. Forest Ecology and Management 64, 245–248. Shahidi, F., Ambigaipalan, P., 2015. Phenolics and polyphenolics in foods, beverages and spices: antioxidant activity and health effects – a review. Journal of Functional Foods 18, 820–897. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. American Journal of Enology and Viticulture 16, 144–158. Stadlmayr, B., Charrondiére, U.R., Eisenwagen, S., Jamnadass, R., Kehlenbeck, K., 2013. Nutrient composition of selected indigenous fruits from sub-Saharan Africa. Journal of the Science of Food and Agriculture 93, 2627–2636. Stander, M.A., Van Wyk, B.-E., Taylor, M.J.C., Long, H.S., 2017. Analysis of phenolic compounds in rooibos tea (Aspalathus linearis) with a comparison of flavonoid-based compounds in natural populations of plants from different regions. Journal of Agricultural and Food Chemistry 65, 10270–10281. Toledo, Á., Burlingame, B., 2006. Biodiversity and nutrition: a common path toward global food security and sustainable development. Journal of Food Composition and Analysis 19, 477–483. Villaño, D., Fernández-Pachón, M.S., Moyá, M.L., Troncoso, A.M., García-Parrilla, M.C., 2007. Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta 71, 230–235. Visioli, F., Borsani, L., Galli, C., 2000. Diet and prevention of coronary heart disease: the potential role of phytochemicals. Cardiovascular Research 47, 419–425. Wall, M.M., 2006. Ascorbic acid, vitamin a, and mineral composition of banana (Musa sp.) and papaya (Carica papaya) cultivars grown in Hawaii. Journal of Food Composition and Analysis 19, 434–445. Wehmeyer, A.S., 1966. The nutrient composition of some edible wild fruits found in the Transvaal. S.A. Medical Journal 40, 1102–1104. Williamson, G., Manach, C., 2005. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. American Journal of Clinical Nutrition 81, 243S–255S.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Phenolics, organic acids and minerals in the fruit juice of the indigenous African sourplum (Ximenia caffra, Olacaceae) N.J. Goosen a,⁎, D. Oosthuizen a, M.A. Stander b, A.I. Dabai c, M.-M. Pedavoah d, G.O. Usman e a
Department of Process Engineering, Stellenbosch University, South Africa Central Analytical Facility, Stellenbosch University, South Africa Department of Microbiology, Usmanu Danfodiyo University, Sokoto, Nigeria d Department of Applied Chemistry and Biochemistry, University for Development Studies, Ghana e Department of Food, Nutrition and Home Sciences, Kogi State University, Nigeria b c
a r t i c l e
i n f o
Article history: Received 22 March 2018 Received in revised form 27 April 2018 Accepted 3 August 2018 Available online xxxx Edited by B-E Van Wyk Keywords: Phenolics Flavonoids Phytochemicals Sour plum Indigenous African fruit Underutilized plant Micronutrients Seasonal fruit Rural diet
a b s t r a c t Wild-picked fruits of the indigenous African tree, Ximenia caffra, are widely consumed in Southern Africa when in season, yet compositional data on phytochemicals, organic acids and minerals are lacking. The study therefore aimed to characterize juice obtained from ripe X. caffra fruits, and to identify individual phenolic compounds using liquid chromatography, high-resolution mass spectrometry (LC-HRMS). The juice had high total phenolic (1030 mg 100 ml−1 gallic acid equivalents) and total flavonoid content (852 mg 100 ml−1 catechin equivalents), and LC-HRMS analysis identified procyanidin B1 (12.2 mg 100 ml−1), gallic acid (5.56 mg 100 ml−1) and catechin (2.66 mg 100 ml−1) as the most abundant phenolic compounds, while the dominant organic acid was citric acid (8.05 g 100 ml−1), with lesser levels of tartaric, L-malic and L-lactic acids. LC-HRMS further positively identified and quantified a number of other polyphenolic compounds from the juice. The most prevalent minerals were potassium (525 mg 100 ml−1) and phosphorous (24.6 mg 100 ml−1), while heavy metal content was low. Consumption of X. caffra fruits can significantly contribute toward dietary phytochemical and mineral intake, and consumption of this fruit should therefore be encouraged. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Plant foods gathered from the wild provide essential nutrients and potentially health promoting phytochemicals to especially rural populations, and contribute to global food security (Toledo and Burlingame, 2006; Stadlmayr et al., 2013; Ayessou et al., 2014). Ximenia caffra Sond. is a seasonal wild fruit that forms part of the diet of people throughout its distribution range and it plays an important ethnobotanical role as medicinal plant; the whole fruit including skins, pulp and seeds are edible (Mabogo, 1990; Nair et al., 2013; Maroyi, 2016). The species are widely distributed and indigenous to central, southern and eastern Africa, including Madagascar, and two varieties of X. caffra occur: var. caffra and var. natalensis Sond. (Coates Palgrave, 2003). The Abbreviations: AAE, ascorbic acid equivalents; CE, catechin equivalents; DMPD, N,NDimethyl-p-phenylenediamine dihydrochloride; GAE, gallic acid equivalents; ICP-AES, inductively coupled plasma atomic emission spectroscopy; ICP-MS, inductively coupled plasma mass spectrometry; LC-HRMS, liquid chromatography, high resolution mass spectrometry. ⁎ Corresponding author at: Department of Process Engineering, University of Stellenbosch, Private Bag X1, Stellenbosch, 7602, South Africa. E-mail address: [email protected] (N.J. Goosen).
https://doi.org/10.1016/j.sajb.2018.08.008 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
tree is known under a number of different vernacular names and in English it is mainly known as ‘sour plum’ due to the high acidity of the fruits (Maroyi, 2016). Flowering normally occurs between August and October (Nair et al., 2013), although the exact flowering and fruiting period is heavily influenced by seasonal rainfall (Mr. Dave Rushworth, personal communication). Comprehensive data on nutritionally relevant compounds and phytochemical aspects of both X. caffra varieties are lacking, especially on the fruit pulp and skins. Current nutritional and phytochemical data of the fruit pulp, juice and skin show that the fruits are highly acidic and contain high levels of ascorbic acid, potassium and phenolic compounds, and that methanolic extracts of the fruits and skins exhibit high antioxidant capacity (Wehmeyer, 1966; Ndhlala et al., 2006; 2008; Maroyi, 2016). In contrast to the lack of nutritional data on the fruit, much effort has been spent on analysis of the seeds and the economically valuable seed oil, and data include proximate composition, lipid content and fatty acid profile, protein content and amino acid profile, mineral content, and data on various minor components (Chivandi et al., 2008, 2012; Mitei et al., 2009). These data confirm the high nutritional value of the seeds and the potential contribution that
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consumption of seeds can make to intake of essential and health promoting nutrients. Despite the knowledge that fruits of X. caffra generally contain high levels of organic acids and phenolics, little is known about the nature and levels of the individual compounds. There is further a need to broaden the range and availability of data on compounds of nutritional interest in indigenous fruits in general (Stadlmayr et al., 2013), and for X. caffra in particular, since the plant is known to form a part of rural diets when in season. The aim of the current work was therefore to perform an analysis on water-soluble compounds found in X. caffra fruit collected from the wild through identification and quantification of phenolic compounds, organic acids and macro- and micro-minerals, as these compounds can all be of nutritional relevance in human diets. 2. Materials and methods 2.1. Raw material collection and preparation Ripe fruits of X. caffra were collected in the Hoedspruit area, Limpopo Province, South Africa on January 10, 2015, at the GPS coordinates: S23°54.567′ E029°48.760′. The variety was tentatively identified as var. natalensis by a local botanist, Mr. Dave Rushworth, and fruits were kept on ice until processed. Juice was obtained from the fruits by separating the skin and fleshy portion of the fruit from the seeds through physical means, homogenizing the fruit flesh and skins together in a food processor and filtering the resultant pulp through a cotton cloth. The filtered juice obtained was pooled and mixed to obtain a homogenous sample, and then frozen at −20 °C until analysis. All subsequent analyses were performed on the pooled juice sample. The weight of 42 randomly selected seeds were determined and expressed as the percentage of total fruit weight. Juice pH and Brix were determined using benchtop instruments, while total moisture (AOAC, 2005), total sugars (AOAC, 2003) and titratable acidity (Mora et al., 2009) were measured using standard analytical methods. 2.2. Juice mineral profile Major elements Ca, K, Mg, Na, P and Si were analyzed on a Thermo ICap 6200 inductively coupled plasma atomic emission spectrometer (ICP-AES). The instrument was calibrated using NIST (National Institute of Standards and Technology, Gaithersburg MD, USA) traceable standards purchased from Inorganic Ventures (Christiansburg, Virginia, USA) to quantify selected elements. NIST-traceable quality control standards from De Bruyn Spectroscopic Solutions, Bryanston, South Africa, were analyzed to verify the accuracy of the calibration before sample analysis, as well as throughout the analysis to monitor drift. Trace elements were analyzed on an Agilent 7700 quadrupole inductively coupled plasma mass spectrometer (ICP-MS). Samples are introduced via a 0.4 ml min− 1 micromist nebulizer into a peltier-cooled spray chamber at a temperature of 2 °C, with a carrier gas flow of 1.05 l min−1. The elements V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se were specifically analyzed under He-collision mode to remove polyatomic interferences. The instrument was calibrated using NIST (National Institute of Standards and Technology, Gaithersburg MD, USA) traceable standards to quantify selected elements. NIST-traceable quality control standards of a separate supplier than the main calibration standards were analyzed to verify the accuracy of the calibration before sample analysis. 2.3. Analysis of phenolics and ascorbic acid Spectrophotometric methods were employed to measure total phenolic content and total flavonoid content of the fruit juice in triplicate. The spectrophotometer used for all spectrophotometric analyses was from A&E Lab, United Kingdom, model AE-S60-4U. Total phenolics were determined according to the Folin–Ciocalteu method of Singleton and Rossi (1965), using gallic acid as standard. Values were determined
by measuring absorbance at 765 nm and expressed as gallic acid equivalents (GAE). Total flavonoids were measured using the method described by Amado et al. (2014), with catechin as standard, and absorbance of samples were measured at 410 nm and expressed as catechin equivalents (CE). Total ascorbic acid levels in the juice were determined by way of UV-HPLC according to the method described in Odriozola-Serrano et al. (2007). The column used was a YMC Pack Pro C18 column, and a total runtime and detection wavelength of 35 min and 250 nm were employed, respectively. Mobile phase A was 0.5% trifluoroacetic acid in water, and mobile phase B was 0.5% trifluoroacetic acid in methanol. Column temperature was 25 °C, mobile phase flow rate was constant at 1.0 ml min−1 and sample injection volume was 20 μl. Phenolics were identified and quantified through a liquid chromatography, high resolution mass spectrometry (LC-HRMS) method, as describe previously (Stander et al., 2017), which utilizes a gradient method specifically focussing on phenolic acids and flavonoids. The only difference to the published method was that a Waters BEH C18, 2.1 × 100 mm, 1.7 μm column was used. A Waters Synapt G2 quadrupole time-of-flight mass spectrometer connected to Waters Ultra pressure liquid chromatograph and photo diode array detection was used for the analysis. In short, a 0.1% formic acid (solvent A) to acetonitrile containing 0.1% formic acid (solvent B) gradient was applied up to 28% solvent B, followed by a wash step. The instrument was operated using electrospray ionization in negative MSE mode which consisted of a low collision energy scan (6 V) from m/z 150 to 1500 and a high collision energy scan from m/z 40 to 1500. Positive identification of compounds was based on retention time matching with authentic standards, accurate mass data, UV data as well as MSMS fragmentation data. The juice was diluted 5- and 10-fold in 50% methanol/water, centrifuged and the supernatant injected directly into the system. A cocktail of the standards were injected into the system at (100, 50, 25, 10, 5 and 0.5 mg l−1) and the application manager Targetlynx 4.1 (Waters, MA, US) was used for the quantifications. Preliminary antioxidant capacity was estimated using the spectrophotometric N,N-Dimethyl-p-phenylenediamine dihydrochloride (DMPD) assay of Fogliano et al. (1999), at an absorbance wavelength of 505 nm. Ascorbic acid was used as standard and values were reported as ascorbic acid equivalents (AAE). 2.4. Organic acid determination Individual organic acids and their levels were determined using enzymatic derivatization followed by spectrophotometric detection and quantification. The instrument used was an Arena 20XT Discrete Photometric Analyzer (Thermo Fisher Scientific, Finland). Analysis for each organic acid was performed using a specific analysis kit, and analysis conditions were according to those prescribed by each kit manufacturer. The following organic acids were analyzed for (the specific analytical kit employed is shown in parentheses): acetic acid (Enzytec™ Fluid Acetic acid, catalog number 5226, Thermo Fischer Scientific, Finland), citric acid (R-Biopharm citric acid, Roche catalog number 10139076035, R-Biopharm AG, Darmstadt), D-lactic acid (Enzytec™ Fluid D-Lactic acid catalog number 5240, Thermo Fischer Scientific, Finland), L-lactic acid (Enzytec™ Fluid D-Lactic acid catalog number 5260, Thermo Fischer Scientific, Finland), L-malic acid (Enzytec™ Fluid L-Malic acid catalog number 5280, Thermo Fischer Scientific, Finland), pyruvic acid (Megazyme Pyruvic acid catalog number K-PYRUV 07/12, Megazyme International, Ireland), succinic acid (RBiopharm succinic acid, Roche catalog number 10176281035, RBiopharm AG, Darmstadt), and tartaric acid (Enzytec™ Color Tartaric acid catalog number E3100, Thermo Fischer Scientific, Finland). 3. Results The juice obtained had an attractive red color corresponding to the red color of the fruit, and had a moisture content of 86.4%, a pH of
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2.60 and titratable acidity of 7.69% malic acid equivalents. Total sugars were 3.92 g 100 ml−1 and Brix measurements showed a value of 18.5 Brix. The average fruit weighed 11.4 ± 2.47 g (mean ± standard deviation), and seed weight was 18.3 ± 3.47% of total weight. The precision of the analytical methods employed were satisfactory. For spectrophotometric determination of total phenolics, the standard deviation as percentage of the mean was less than 5% in the range of 0–300 mg l−1 GAE, for total flavonoids it was less than 3% in the range 0–100 mg l−1 CE, and for DMPD antioxidant capacity it was less than 2% in the range of 0–100 mg l− 1 AAE. For determination of organic acids, all analyses have a standard deviation as percentage of the mean value of less than 7%, and the error for LC-HRMS quantitation was below 5 ppm for all compounds. 3.1. Analysis and identification of phenolics Table 1 shows values for spectrophotometric determination of total phenolics (1030 mg 100 ml−1 GAE), total flavonoids (852 mg 100 ml−1 CE) and preliminary DMPD antioxidant capacity (6100 mg 100 ml− 1 AAE) of X. caffra juice, and results for HPLC quantification of ascorbic acid (45.0 mg 100 ml−1). LC-HRMS positively identified and quantified 15 phenolics and phenolic glycosides, and tentatively identified a further 7 compounds (Table 4). The phenolics and phenolic glycosides which were positively identified were catechin, epicatechin, gallic acid, hesperetin, hyperoside, isoquercitrin, kaemferol glucoside, luteolin-7-O-glucoside, procyanidin B1, procyanidin B2, quercetin-3O-glucoside, quercetin-3-O-robinobioside, quercetin, rutin and trilobatin. 3.2. Organic acids Table 2 reports the positively identified organic acids and their levels. The most prevalent organic acid was citric acid (8.05 g 100 ml−1), followed by tartaric acid (0.18 g 100 ml−1), L-malic acid (0.16 g 100 ml−1) and L-lactic acid (0.13 g 100 ml−1). D-lactic acid was present in the sample, but could not be accurately quantified. Concentrations of acetic, pyruvic and succinic acids were below detection levels.
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Table 2 Identity and concentrations of specific organic acids as found in the juice of Ximenia caffra. Organic acid
Concentration (g 100 ml−1)
Acetic acid Citric acid D-Lactic acid L-Lactic acid L-Malic acid Pyruvic acid Succinic acid Tartaric acid
b0.003 8.05 Presenta 0.13 0.16 b0.001 b0.001 0.18
a D-Lactic acid was present in low concentration, but could not be quantified accurately.
of seed weight (18.3 ± 3.47%) found in this study agree well with a prior investigation where average fruit weight was 11.8 g, and seed weight was 17.8% of total fruit weight (Ligthelm et al., 1954). Due to the relative contribution of the fruit flesh and skins to overall fruit weight, much of the potentially beneficial nutritional and phytochemical components found in X. caffra fruit could be located in the fruit flesh and skins. The seeds have been well-characterized and it is known that seeds have high oil and protein contents, as well as other potentially beneficial minor components including phytosterols, vitamins and phospholipids (Ligthelm et al., 1954; Chivandi et al., 2008, 2012; Mitei et al., 2009). Total content of phenolics and flavonoids was found to be relatively high in the current study, which is in line with previous investigations. Based on spectrophotometric analyses, a large proportion (82.7%) of the total phenolics consisted of flavonoids, which is also in agreement with previous work where methanol extracts of fruit pulp and skins both showed high phenolic and flavonoid content and high antioxidant capacity (Ndhlala et al., 2006, 2008). LC-HRMS analysis supports this finding, where 14 of the 16 positively identified compounds were flavonoids or flavonoid glycosides. The values obtained in the current study for total phenolics and flavonoids might be more representative of actual phenolic and flavonoid ingestion during fruit consumption, as skins are many times discarded when the fruits are consumed (Mabogo, 1990).
3.3. Mineral profile The major and trace mineral profile of the X. caffra juice is reported in Table 3. The most abundant major minerals were potassium (525 mg 100 ml−1), phosphorous (24.6 mg 100 ml−1), magnesium (15.9 mg 100 ml−1) and calcium (3.04 mg 100 ml−1). The most abundant trace minerals were manganese (320 μg 100 ml−1), zinc (180 μg 100 ml− 1), iron (170 μg 100 ml− 1) and aluminum (100 μg 100 ml−1). Both selenium and mercury levels were below detection limits, while concentrations of arsenic, cadmium, cobalt, chromium, molybdenum, lead, antimony and vanadium were all below 6 μg 100 ml−1. 4. Discussion The largest proportion of the fruits consisted of the pulp and skins, while the seeds contributed 18.3 ± 3.47% of the total fruit weight on wet weight basis. The fruit weights (11.4 ± 2.47 g) and contribution Table 1 Mean values of spectrophotometric assays of total phenolics, total flavonoids and DMPD antioxidant capacity, and HPLC identification and quantification of ascorbic acid. Assay/compound
Concentration −1
a
Total phenolics (mg 100 ml GAE ) Total flavonoids (mg 100 ml−1 CEa) DMPD (mg 100 ml−1 AAEa) Ascorbic acid (mg 100 ml−1)
1030 852 6100 45.0
a Abbreviations: GAE – gallic acid equivalents; CE - catechin equivalents; AAE – ascorbic acid equivalents.
Table 3 Mineral profile of Ximenia caffra juice, showing both macro- and micro-minerals. Element
Concentration
Macro minerals (mg 100 ml−1) Ca K Mg Na P Si
3.04 525 15.9 0.82 24.6 0.52
Micro-minerals (μg 100 ml−1) Al As Ba Cd Co Cr Cu Fe Hg Mn Mo Ni Pb Sb Se Sr V Zn
100 0.1 8.8 0.1 3.5 3.6 72 170 b 0.1 320 2.1 7.1 1.5 0.4 b 3.0 58 0.3 180
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N.J. Goosen et al. / South African Journal of Botany 119 (2018) 11–16
Table 4 LC-HRMS data obtained for the juice from Ximenia caffra, detailing compound identity and the concentration for the identified compounds.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Compound name
m/za
Retention time (min)
[M-H]−
MSE fragmentsb
Concentration (mg 100 ml−1)
Positively identified compounds Catechin Citric acid Epicatechin Gallic acid Hesperetin Hyperoside Isoquercitrin Kaemferol glucoside Luteolin-7-O-glucoside Procyanidin B1 Procyanidin B2 Quercetin-3-O-glucoside Quercetin-3-O-robinobioside Quercitin Rutin Trilobatin
289.0713 191.0187 289.0698 169.0129 301.1643 463.0878 463.0876 447.0938 447.0935 577.1317 577.1345 463.0886 609.1432 301.0353 609.1458 435.1284
11.48 3.12 13.57 5.8 24.49 17.51 17.51 18.06 18.06 10.68 12.72 17.81 17.06 23.99 17.27 18.75
C15H13O6 C6H7O7 C15H13O6 C7H5O5 C15H25O6 C21H19O12 C21H19O12 C21H19O11 C21H19O11 C30H25O12 C30H25O12 C21H19O12 C27H29O16 C15H9O7 C27H29O16 C21H23O10
289,125,203,245,151 191,111,87,173 weak 125,169,111 weak 300,463,271,255 300,463,271,301,255, 285,169,447 285,284,169,125,447 289,407,425,577 289,407,425,577 300,271,463,255,125 300,609,271,125 125,169 300,609,271,255 315,345
2.66 295.2 0.03 5.56 0.01 0.96 1.09 0.60 0.02 12.2 0.25 0.11 0.12 0.02 0.37 0.07
9.14 24.5 11.18 10.68 16.63 16.94 19.63 18.89 18.83 14.37 11.37 5.49 5.68 5.83 5.93 21.403 9.37
C6H5O6 C16H31O4 C16H17O8 C30H25O12 C28H23O16 C28H23O16 C21H19O11 C21H19O11 C20H17O11 C15H18NO8 C21H31O10 C18H27O17 C18H27O17 C18H27O17 C18H27O17 C24H19O8 C8H11O7
111 287 163,119,191,337 289,407,425,577 300,615,463,255,169 300,463,615 300,271,255,447,243 300,271,255,447, 300,271,255,433,315 161,101,85 443,289,303 515,111,173 515,111,173 515,111,173 515,111,173 341,189,125,435 111,219,87
Tentatively identified and unknown compoundsc Aconitic acid 173.0089 Dihydroxy hexadecanoic acid 287.2236 p-Coumaorylquinic acid 337.0916 Procyanidin 577.1344 Quercetin galloyl glucoside 615.0979 Quercetin galloyl glucoside 615.0977 Quercetin rhamnoside 447.0927 Quercetin rhamnoside 447.0927 Quercetin-3-O-pentoside 433.0764 Unknown 340.1035 Unknown 443.1913 Unknown 515.1246 Unknown 515.1245 Unknown 515.1255 Unknown 515.1253 Unknown 435.107 Unknown 219.0506 a b c
The mass accuracy for all compounds was better than 5 ppm. Most abundant fragment mentioned first. Concentrations of epicatechin, procyanidin B2 and all tentatively identified and unknown compounds could not be determined.
Various phenolic compounds were positively identified in the juice, which, to the best of our knowledge, is the first instance that many of these phenolic compounds have been identified from X. caffra fruits. The major phenolic compounds were procyanidin B1 (12.2 mg 100 ml− 1), gallic acid (5.56 mg 100 ml− 1) and catechin (2.66 mg 100 ml−1), while LC-HRMS analysis also confirmed the high levels of citric acid. Procyanidin B1 is a potent antioxidant known to exert high radical scavenging ability (Villaño et al., 2007), and forms part of the group of compounds known as condensed catechins, which are responsible for a range of potentially beneficial health effects (Matsui, 2015). Gallic acid is a phenolic compound with known antioxidant, antimicrobial and health promoting activity, and could potentially be employed for a range of medicinal and industrial purposes (Borges et al., 2013; Badhani et al., 2015). Catechin and catechin derivatives are associated with direct beneficial effects upon ingestion, including increased blood plasma antioxidant activity and decreased oxidation of LDL cholesterol (Williamson and Manach, 2005). The presence of the other minor polyphenols, despite their relatively low concentrations, may also be nutritionally significant due to apparent synergistic health benefits when multiple polyphenolics are ingested (Pandey and Rizvi, 2009; Shahidi and Ambigaipalan, 2015). It should be noted that the composition and total amounts of the phenolic compounds found in the fruits will be different during different stages of ripening of the fruits (Amira et al., 2012); however, the present work is in agreement with previous reports of high polyphenolic content in X. caffra fruits (Ndhlala et al., 2008). The ascorbic acid content of the juice (45.0 mg 100 ml−1) is considered to be nutritionally significant, and is comparable to ascorbic acid concentrations found in other fruits like papaya, kiwifruit, strawberries
and citrus, which are generally considered to contain high levels of ascorbic acid (Nishiyama et al., 2004; Wall, 2006; Pantelidis et al., 2007; Rekha et al., 2012). The value is higher than previously found for X. caffra fruit, where an ascorbic acid content of 22.5 mg ascorbic acid per 100 g edible fruit (presumably referring to the fruit pulp) was reported (Wehmeyer, 1966). There are two prior reports which state an ascorbic acid content of 27% in X. caffra fruit (Ndhlala et al., 2008; Maroyi, 2016), although neither of these investigations measured the ascorbic acid content, and this value is likely an overestimation of true ascorbic acid content of the fruits. The DMPD antioxidant capacity assay of the juice indicated a high value of 6100 mg 100 ml−1AAE, and is likely due to the combined effect of the polyphenolic compounds, ascorbic acid and citric acid. The value obtained, however, can only be viewed as preliminary and needs to be investigated further through more detailed analyses to determine the actual antioxidant potential when the fruits are ingested as part of a normal diet. The antioxidant capacity as measured using in-vitro methods such as the DMPD assay does not necessarily correlate with the actual bioavailability and potentially health-promoting effects in humans, as phenolic compounds differ in respect to the total uptake and rate of uptake in the digestive system (Holst and Williamson, 2008). Organic acid quantification revealed that citric acid is the dominant organic acid in X. caffra juice, but contrary to a previous investigation which reported that citric acid alone accounts for the high fruit acidity (Ligthelm et al., 1954), the current study has also established the presence of D- and L-lactic acids, L-malic acid and tartaric acid. It is probable that the much higher citric acid concentration compared to those of the other organic acids prohibited detection of any acidifying effect by any of the other acids in the previous study, thereby leading to the
N.J. Goosen et al. / South African Journal of Botany 119 (2018) 11–16
premature conclusion that citric acid is the sole organic acid present in the fruit. Despite the differences between the current and previous study, there is good agreement in total acidity levels: 8.52 g 100 ml−1 total acidity (as the sum of all enzymatically quantified acids in the juice, or approximately 8.5% of the juice) in the current study, vs. 8.7% expressed as citric acid in the work of Ligthelm et al. (1954). The high total acid content is confirmed by the total titratable acidity (7.69% malic acid equivalent), and is responsible for the low pH of 2.60 measured in the fruit juice, and the characteristic sour taste of the fruit that gives this species its English vernacular name. Quantitative values for total sugar content of X. caffra juice is reported for the first time. The only data to which this could be compared relate to those calculated as total carbohydrates, but which also include complex carbohydrates, fiber and potential non-carbohydrate components. Total carbohydrates of which sugars are a component, were determined by difference and reported as 26.3% (Wehmeyer, 1966) and 78.8% (Saka and Msonthi, 1994) in two previous studies, however, neither of these values can be considered as accurate estimates of carbohydrate or sugar content of the fruits. The work done by Wehmeyer (1966) discounts the significant organic acid and polyphenolic contents in the calculation of total carbohydrates, while the value reported by Saka and Msonthi (1994) was incorrectly calculated and should have been 4.4% when calculated according to the protocol of the FAO (2003), instead of the reported 78.8%. The recalculated value of 4.4% is reasonably close to the HPLC-determined value of 3.92 g 100 ml−1 for total sugars found the current investigation, but these values are not directly comparable as the methods by which they were obtained are very different. The fruit of X. caffra is a good source of beneficial minerals, especially the macro-minerals potassium, phosphorous and magnesium. It is generally accepted that X. caffra has a high potassium content (Wehmeyer, 1966; Saka and Msonthi, 1994); however, literature data show less agreement in the level of magnesium and phosphorous. The magnesium contents of the current study is 15.6 mg 100 ml− 1 juice, while prior values range between 2.0 and 45.9 mg 100 g−1; the phosphorous content of 24.6 mg 100 ml−1 juice also falls between the two prior values of 14.5 and 167.4 mg 100 g− 1 that are available in literature (Wehmeyer, 1966; Saka and Msonthi, 1994). Most of the data regarding mineral composition of the fruit of X. caffra rely on analysis done by the authors of the two above-mentioned reports, and values are only reported for calcium, copper, iron, magnesium, phosphorous, potassium and sodium. The current study adds to prior work through reporting of the contents of additional minerals. The mineral analysis further shows that the fruit contains low levels of heavy metals. As there is no specific reference standard for fruit juice for maximum allowable levels, the data obtained for heavy metals were compared to the Codex Standard 193–1995 (CCCF, 1995), which provides guidance for contaminants in food products, but only advises on levels of arsenic, cadmium, mercury, lead and tin. Levels of all of these minerals were below guidance levels, except for tin which was not measured. The results of the current study indicates that consumption of X. caffra fruits can make a significant contribution toward obtaining important micronutrients and phytochemicals which are a requirement in a balanced, healthy diet. Despite the high fruit acidity, X. caffra fruits are collected from the wild and consumed fresh or as part of traditional dishes when in season (Mabogo, 1990; Chivandi et al., 2008; Maroyi, 2016). The importance of obtaining sufficient amounts of secondary plant metabolites (e.g., phenolic compounds) and micronutrients for prevention of especially age-related and chronic diseases has been repeatedly highlighted (Visioli et al., 2000; Holst and Williamson, 2008; Haminiuk et al., 2012), and the role that indigenous and underutilized plants and crops can play in this regard is increasingly being recognized (Ebert, 2014; FOA, 2014). Within this context, X. caffra is an important species, as the high concentrations of total phenolics and flavonoids, significant amounts of ascorbic acid and certain macro-minerals in X. caffra fruit are a good indication that consumption of the fruits provides
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important dietary components required in a balanced diet. Further, even though micro-minerals are present in low concentrations, they may still play an important nutritional role (Maroyi, 2016). 5. Conclusion In conclusion, the current study has contributed toward a better understanding of the phytochemical and mineral contents, and the makeup of the organic acids within the fruit of X. caffra. The juice contains a number of potentially beneficial phytochemicals and minerals, and preliminary evaluation point toward a high antioxidant capacity of the fruit. As an indigenous African species that has a wide distributions range, the consumption of X. caffra fruits can therefore make an important contribution toward ensuring sufficient intake of phytochemicals and minerals that are required in a balanced diet, and consumption of this indigenous fruit should therefore be encouraged. Declaration of interest The authors have no real or potential conflict of interest to declare. Acknowledgements This research was supported by the International Foundation for Science, Stockholm, Sweden, through a grant to Dr. Neill Goosen (Grant number J/5503-1). Mr. Dave Rushworth in his personal capacity and Ms. Moloko Mojapelo from the South African Department of Agriculture, Forestry and Fisheries are gratefully acknowledged for assistance with sourcing and identification of the fruit used in this study. References Amado, I.R., Franco, D., Sanches, M., Zapata, C., Vazquez, J.A., 2014. Optimisation of antioxidant extraction from Solanum tuberosum potato peel waste by surface response methodology. Food Chemistry 165, 290–299. Amira, E.A., Behija, S.E., Beligh, M., Lamia, L., Manel, I., Mohamed, H., Lotfi, A., 2012. Effects of the ripening stage on phenolic profile, phytochemical composition and antioxidant activity of date palm fruit. Journal of Agricultural and Food Chemistry 60, 10896–10902. AOAC (Ed.), 2003. AOAC 982.14 - glucose, fructose, sucrose and maltose in presweetened cereals, Official methods of analysis of AOAC International, 17th ed. AOAC International, Gaithersburg, Maryland. AOAC, 2005. AOAC Official Method 934.01 - Moisture in animal feed, Official methods of analysis of AOAC International. 18th ed. American Organisation of Analytical Chemists, Gaithersburg, Maryland. Ayessou, N.C., Ndiaye, C., Cissé, M., Gueye, M., Sakho, M., Dornier, M., 2014. Nutrient composition and nutritional potential of wild fruit Dialium guineense. Journal of Food Composition and Analysis 34, 186–191. Badhani, B., Sharma, N., Kakkar, R., 2015. Gallic acid: a versatile antioxidant with promising therapeutic and industrial applications. RCS Advances 5, 27540–27557. Borges, A., Ferreira, C., Saavedra, M.J., Simões, M., 2013. Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microbial Drug Resistance 19, 256–265. CCCF, 1995. Codex committee on contaminants in foods: codex general standard for contaminants and toxins in food and feed. Codex Stan 193-1995, 1–44. Chivandi, E., Davidson, B.C., Erlwanger, K.H., 2008. A comparison of the lipid and fatty acid profiles from the kernels of the fruits (nuts) of Ximenia caffra and Ricinodendron rautenenii from Zimbabwe. Industrial Crops and Products 27, 29–32. Chivandi, E., Davidson, B.C., Erlwanger, K.H., 2012. The red sour plum (Ximenia caffra) seed: a potential non-conventional energy and protein source for livestock feeds. International Journal of Agriculture and Biology 14, 540–544. Coates Palgrave, K., 2003. Trees of Southern Africa. 3rd Edition ed. Struik, Cape Town. Ebert, A.W., 2014. Potential of underutilized traditional vegetables and legume crops to contribute to food and nutritional security, income and more sustainable production systems. Sustainability 6, 319–335. FAO, 2003. Food energy – Methods of analysis and conversion factors. FAO food and nutrition paper. Food and Agricultural Organization of the United Nations, Rome, Italy. FOA, 2014. In: Durst, P., Bayasgalanbat, N. (Eds.), Promotion of underutilized indigenous food resources for food security and nutrition in Asia and the Pacific. Food and Agricultural Organisation of the United Nations, Rome, Italy. Fogliano, V., Verde, V., Randazzo, G., Ritieni, A., 1999. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. Journal of Agricultural and Food Chemistry 47, 1035–1040. Haminiuk, C.W.I., Maciel, G.M., Plata-Oviedo, M.S.V., Peralta, R.M., 2012. Phenolic compounds in fruits - an overview. International Journal of Food Science and Technology 47, 2023–2044.
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