The Natural Products Journal, 2012, 2, 000-000
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Bioactive Compounds, Antioxidant Activity and Percent Composition of Jambolão Fruits (Syzygium cumini) Milene T. Barcia*, Paula B. Pertuzatti, Andressa C. Jacques, Helena T. Godoy and Rui Zambiazi Departamento de Ciência e Tecnologia Agroindustrial, Universidade Federal de Pelotas, UFPel, CEP: 96010-900 Capão do Leão, RS, Brasil; Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Unicamp, CEP: 13083- 862, C.P. 6121, Campinas, SP, Brasil Abstract: The study aimed to verify the chemical composition of jambolão fruits collected from three cities in the southern state of Brazil (Rio Grande do Sul). Jambolão fruits collected from Santa Vitória do Palmar, Pelotas and Capão do Leão were found to have about 82% water, 0.4% ash, 0.8% protein, 0.4% fiber, 25% reducing sugars, 51% total sugars, 0.6% pectin, 0.14-0.44 mg.100g-1 total tocopherols, 0.3-0.7 mg/100g total carotenoids, 311-451 mg gallic acid equivalents per 100 g, 17-30% tannins and 7-17 µg/g total anthocyanins. The average antioxidant capacity of the fruits of the jambolão was 505.6 µmol/g TE.
Keywords: Jambolão, phenolics, carotenoids, tannins, anthocyanins. INTRODUCTION The plant Syzygium cumini (L.) Skeels, popularly known as jambolão (family Myrtaceae), is found in several Brazilian states [1-4]. Large parts of the jambolão fruits disappear during harvest season, due to the large production of fruit per tree, the short production period, the short shelf life in natura and the lack of viable technology for its industrialization [5]. Jambolão fruits are small, with 2–3 cm long, ovoid form with a purple-red to black color when ripe, containing a fleshy pink or almost white pulp with astringent taste [5]. Fruit extracts showed antioxidant capacity, which help slow down or prevent the development of several degenerative diseases, due to their abilities to react with free radicals. This protects the tissues in the human body against oxidative stress and pathologies due to cancers, cardiovascular diseases and inflammatory processes [6-8]. These beneficial effects are most probably related to the presence of bioactive compounds, such as carotenoids and phenolic compounds [9]. There are not much literature data about the identification and quantitation of bioactive chemical compounds in jambolão fruits originating in the southern region of the state of Rio Grande do Sul, in Brazil. The study aimed to verify the chemical composition of jambolão fruits collected from three cities in the southern state of Brazil (Rio Grande do Sul). MATERIALS AND METHODS We used jambolão fruits in natura, cultivated in the regions of Pelotas, Capão do Leão e Santa Vitória do Palmar.
*Address correspondence to this author at the Faculdade de Engenharia de Alimentos – Universidade Estadual de Campinas, Unicamp, CEP: 13083862, C.P. 6121, Campinas, SP, Brasil; Tel: (19)35214023; E-mail:
[email protected] 2210-3155/12 $58.00+.00
The plants used were from different regions of these cities (3 plants from each city). The fruits were collected from March to April, 2008. They were selected based on the degree of maturity (determined visually – by their color). They were purple and were collected in the morning. About 1 kg of fruits were collected randomly from the lower parts of each tree. They were frozen at -80ºC until they were analyzed. All the chemicals used were of chromatographic grade. The phenolic standards were purchased from Sigma Chemical Co. (St. Louis, MO) and Fluka (Milwaukee, WI), including the hydroxycinnamic acids: caffeic, ferrulic and pcoumaric; the hydroxybenzoic acids: gallic, ellagic and phydroxybenzoic; the flavonols: quercetin, kaempferol and myricetin; and the flavanols: (+) catechin and (-) epicatechin, all with 96-99% purity; anthocyanins: cyanidin chloride, pelargonidin chloride, malvidin chloride, peonidin chloride, delphinidin chloride, Kuromanin chloride, Keracyanin chloride and malvidin-3-O-galactoside chloride. The standards β-cryptoxantin, lycopene, lutein and zeaxantin, obtained from Chromadex (Irvine, USA) and β-carotene obtained from Fluka (Saint Louis, USA), all 97% pure, were used in the determination of the carotenoids. The chromatographic standard used in the determination of ascorbic acid was L(+)-ascorbic acid, 99% pure, obtained from Synth (Diadema, Brazil). The reagents for the other analyses were all of analytical grade. The physical-chemical determinations (total sugar, reducing sugars, protein, fiber, moisture and pectin) were done in triplicate, according to the AOAC method [10]. The determination of caloric value, expressed in Kcal, was done in accordance to the Brazilian standard method - a Resolução – RDC, nº 360 de 23 de dezembro de 2003 [11]. A Shimadzu HPLC system was used for quantification and identification, equipped with an autosampler SIL 10AF, quaternary gradient pump LC-10AT VP, degasser DGU 14A, column oven CTO-10 AS, UV-visible SPD-10AV and © 2012 Bentham Science Publishers
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fluorescence RF-10A XL detectors and a RP-18 CLC-ODS reverse phase column (5µm, 4.6 mm x 150 mm) with an octadecyl stationary phase, protected by a CLC-GODS guard column and with Class vp computer software. The individual phenolics were extracted by an adapted method [12]. The sample was extracted with methanol and then hydrolyzed by adding hydrochloric acid p.a. (1.2 M HCl, final concentration). The extract was homogenized in a water bath at 35ºC in the absence of light for 24 hours and then filtered. The supernatant was concentrated in a rotary evaporator at 40ºC for 20 minutes. The concentrated residue was re-dissolved in methanol to a final concentration of 5 mL, centrifuged (7000 rpm, 10 minutes) and 30 µL of the resulting supernatant injected into the HPLC. The individual phenolic compounds were quantified based on a calibration curve prepared with external standards of p-coumaric acid, caffeic acid, quercetin, ferrulic acid, epicatechin, phydroxybenzoic acid, gallic acid, ellagic acid, catechin, myricetin and kaempferol. The values for the calibration points were stipulated based on previous studies with the quantification of individual phenolic compounds in small fruits [13]. The HPLC separation was carried out using gradient elution with the mobile phases A (water:acetic acid 99:1) and B (methanol) at a flow rate of 0.8 ml min-1, a column temperature of 25°C and the following gradient: 0 min - 100% A; 25-27 min - 60% A plus 40% B; 37-42 min 95% A plus 5% B; 45 min - 100% A. Anthocyanins were extracted using the method of Zhang et al. [14] (2004). They were extracted with methanol and HCl (99.9:0.1, v/v), followed by 3 hrs of stirring, filtration and concentration on a rotary-evaporator at 30°C. Then, it was dissolved in a solution of 1% HCl in methanol. After the extract was centrifuged (7000 rpm, 10 min), 10 µL of the supernatant was injected on the HPLC. The chromatographic system used was the same as used for total phenols, differing only in the wavelength of the detector (520 nm). The mobile phase consisted of a gradient elution (Supplementary Material), flowing at 0.8 mL/min. Separation was carried out using gradient elution with the mobile phases A (aqueous acid acetic 98:2, v/v), B (methanol) and C (acetonitrile), at a flow rate of 0.8 ml/min. The following gradient: 0 min - 100% A; 10 min- 90%A and 10%B; 15-25 min - 80% A and 10% B and 10% C; 30-35 min - 70% A and 30% B; 40 min - 100% A. The peaks were identified by comparing them to the retention times of the standards and quantified by calibration curves, using external standards of the HCl salts of malvidin, Kuromanin (cyanidin-3-glucoside), Keracyanin (cyanidin3-rutinoside), pelargonidin, peonidin, delfinidin, and the individual anthocyanins, cyanidin and malvidin-3galactosde. The results are expressed in mg/100 g. The carotenoids were analyzed according to the method of Rodrigues-Amaya [15]. That is, samples were extracted with acetone, transferred to petroleum ether and saponified with 1.5 N ethanolic KOH. A 20 µL aliquot of the resulting sample was injected into the HPLC. For all the samples, the carotenoids were separated using a linear gradient of methanol:acetonitrile:EtOAc from 30:70:0 (v/v/v) to 10:80:10 (v/v/v) in 10 min, maintaining this proportion for 5
Barcia et al.
min and then applying another liner gradient to 5:80:15 (v/v/v) in 30 min, returning to the initial conditions in 10 min, giving a total chromatographic time of 40 mins. The flow rate was 1 mL/min and the column temperature was set at 25 °C. The chromatograms were processed at 450 nm. The carotenoids were identified according to the following parameters: chromatographic behavior on the C18 HPLC column and co-chromatography with authentic standards. The carotenoids were quantified by HPLC, using external calibration curves for lutein, β-cryptoxanthin, lycopene and β-carotene, with a minimum of five concentration levels. The carotenoids were quantified by comparison of the peak area of the sample with that of the standard, injected daily. The tocopherols were extracted using the same methodology described for carotenoids [15]. The fluorescence detector was used in the HPLC analysis with excitation and emission wavelengths of 290 and 330 nm, respectively. Separation was carried out by gradient elution using the following mobile phases: methanol:acetonitrile:isopropanol from 40:50:10 (v/v/v) to 30:65:5 (v/v/v) in 10 mins, followed by a linear gradient to 50:40:10 in 2 minutes, maintaining this proportion for a further 15 minutes. The flow rate was 1 ml/min and the column temperature was set at 25 °C. An external standard curve was used to quantify the α-, δ- and γtocopherols, comparing the areas of the sample peaks with those of the samples, injected daily. A 4.5% aqueous metaphosphoric acid solution was used to extract the ascorbic acid, and a 20µL aliquot of sample was injected into the HPLC. The chromatographic separation was carried out by gradient elution using the mobile phases of water:acetic acid (99.9:0.1, v/v/v) (A) and methanol (B), according to the adapted methodology of Vinci et al. and Ayhan et al. [16, 17]. A linear gradient was used from 100% A to 98% A for 5 mins, maintained for 3 mins, and then returned to the starting conditions, giving a total run time of 10 mins. The flow rate was 0.8 ml/min and the column temperature was set at 25 °C. An external standard curve of ascorbic acid was used for quantification, comparing the area of the sample peak with that of the standard, injected daily. Total phenolics were determined according to the method of Badiale-Furlong et al. [18]. It consisted of an extraction with methanol, followed by filtration and clarification with barium hydroxide and zinc sulfate. After obtaining the clarified extract, a solution of sodium carbonate and the Folin-Ciocalteau reagent were added. After a second filtration, the absorbance at 765 nm was read on an Ultrospec 2000 spectrophotometer. A standard curve was constructed using gallic acid. Results are expressed as mg of gallic acid equivalents (mg GAE) per 100 g. Total anthocyanins were determined by the colorimetric method of de Lees e Francis [19], with a few changes. The extraction was done using ethanol, pH 1, and the absorbance at 520 nm was read on an Ultrospec 2.000 UV/Visible spectrophotometer (Pharmacia Biotech). The quantification of anthocyanins was based on the molar extinction coefficient of cyanidin 3-glucoside. Hydrolyzable tannins were determined by the method of de Brune et al. [20]. It consisted of an extraction with
Bioactive Compounds, Antioxidant Activity and Percent Composition
The Natural Products Journal, 2012, Vol. 2, No. 2
methanol and 10 min of shaking, followed by resting 1 hr and subsequent filtration. Then, 2 mL of the filtered methanol extract was mixed with 8 mL of the reagent solution containing ferric ammonium sulfate (FAS). It consisted of 89% urea acetate buffer, 10% solution of gum Arabic, 1% deionized water, and 1% of the solution of ferric ammonium sulfate in 1M HCl. After 20 min reaction, the absorbance at 578 nm was read. The determination of the hydrolyzed tannins was done using a gallic acid calibration curve. The results are expressed as mg GAE/100 g.
using total transmittance, D65 illuminant and observation angle of 10. The hab (hue) and Cab (chroma) values were calculated according to Eqs. (2) and (3), respectively. hab = arctan ( b ) Equation 2 a Cab =
(a ) 2 + (b) 2 Equation 3
The data were evaluated by the analysis of variance (ANOVA). Results, statistically different were evaluated using the Tukey test at levels of 5% and 10% probability, through the Winstat 1.0 software [23].
Condensed tannins were determined by the method of Price et al. [21]. It consisted of a methanol extraction with 1 hr of shaking, followed by filtration. Then, a 1 mL aliquot was added to 5 mL of a solution of 1:1 of vanilla 1% methanol and 4% HCl in methanol. After 15 mins, the absorbance at 500 nm was read. The determination used a standard curve of catechin and the results were expressed as mg per 100 g.
RESULTS AND DISCUSSION The results of the physical-chemical analyses of jambolão are presented in Table 1. The amount of humidity of most fruits is about (74-94%). Others found moisture contents of 85 – 88% [5], similar to the results of this study. Lago, Gomes and Silva [24] working with the same fruits reported 0.34% ash and 0.67% protein. When compared to other species of Myrtaceae such as gabiroba and cherries, one can see that the results are similar, i.e. 75.9% in gabiroba and 90.47% in cherries [25].
The antioxidant activity was determined from the ability of the compounds present in the samples to sequester the stable radical DPPH· (2,2-diphenyl-1-picrylhydrazyl), according to the methodology described by Brand-Williams et al. [22] with slight modifications. The compounds with antioxidant activity present in the fruits were extracted with methanol, and the sample homogenized using an UltraTurrax at maximum speed. After maceration for 24h at a temperature of 3-4ºC, the sample was centrifuged.
The amount of pectin in fruits such as grapes, apples, pomegranates, uvaia, peaches and strawberry are relatively low [26]. This is similar to jambolão which also has only 0.35 to 0.93%. According to Cardoso [27], jambolão juice without the peel has 0.46% pectin, similar to the amount found in this study.
A DPPH stock solution was prepared by dissolving 24 mg of DPPH free radical in 100 mL methanol and storing it under refrigeration. At the moment of analysis, 10 mL of stock solution was removed and diluted in 45 mL methanol to prepare the working solution. The absorbance of this solution was adjusted to 1.1 ± 0.02. To quantify the antioxidant activity, 100 µL of sample extract was added to 3.9 mL of the DPPH working solution in order to complete the volume to 4 mL. The sample was read in the spectrophotometer after 30 minutes and again after 24 hours to certify that the reaction was completed, at a wavelength of 517 nm. A standard curve was prepared with Trolox for quantification of the antioxidant activity.
In the fruits analyzed, fiber content was 0.10 to 0.91%, similar to the 0.28% found by Lago, Gomes and Silva [24]. The jambolão fruits have negligible fat, like the majority of fruits in the region, as reported by Benherlal, Arumughan [5]. Regarding sugars, there is some variation between different plants. Jambolão had an average of 24% reducing sugars and 51% total sugars. The elevated amount of sugars may be caused by the levels of tannins, which can be hydrolyzed to produce sugars. The caloric value was between 61 and 68 Kcal/100g, similar to yellow guava, apples, pears and grapes, which are 54.0, 56.0, 53.0 and 53.0 Kcal/100g [28]. When compared to gabiroba (66.3 Kcal/100g), it is quite similar, since it also belongs to the family Myrtaceae [25]. The pH values (3.05 to 3.35), acidity
The quantitative analyses of total phenolics, anthocyanins, and tannins, as well as antioxidant test, were performed on a spectrophotometer Ultrospec 2.000 UV/Visivel (Pharmacia Biotech). The CIELAB color parameters (L⁄, a⁄, b⁄) of the fruits, were obtained in a spectrocolorimeter (Minolta CR-300) Table 1.
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Physical-chemical Characterization of Jambolão (Syzygium cumini)
Location
Water
Ash
Protein
Fiber
Reducing Sugars
Total Sugars
Pectin
Capão do Leão
82.9±0.6
a
0.35±0.1
a
0.91±0.3
a
0.42±0.3
a
24.5±0.6
b
51.4±1.4
a
0.54±0.5
a
Pelotas
81.7±3.6
a
0.42±0.1
a
0.80±0.1
a
0.40±0.4
a
27.9±1.2
a
53.4±3.8
a
0.65±0.1
a
SVP*
83.9±1.5
a
0.42±0.1
a
0.73±0.0
a
0.57±0.3
a
24.3±0.9b
50.7±1.9
a
0.56±0.0
a
*Santa Vitória do Palmar Results are averages ± standard deviation (n=3). Averages followed by the same letter in the same column indicate that there is no difference between them, according to the Tukey test (p≤0.05);
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Barcia et al.
(0.035 a 0.079% malic acid) and soluble solids (13.8 to 19.0°Brix) are significantly different in different plants. These differences can be associated with variations in soil and climate, which can directly influence the fruit as well as affecting plants in different parts of the same city [29]. Table 1 shows that there are few significant differences between plants in the same city in percent water, ash, protein, fiber, pectin, reducing sugars and total sugars. There is a significant difference in the amount of reducing sugars in fruits from Santa Vitória do Palmar. Fig. (1) shows a chromatogram in which tocopherols were separated in jambolão. In the system used, β- and γtocopherol co-eluted. Table 2 shows the amounts that were found. There was more α-tocopherol than δ or β/γ. The low content of tocopherols can be compared to the similar amounts in the Brazilian blueberry and blackberry, which
have 1.05±0.31 and 3.74±0.94mg tocopherol/100g Chun et al. [30]. The same authors found 0.41±0.07mg tocopherol/ 100g in strawberries, similar to the 0.4 mg/100g found in Jambolão from Pelotas. When compared to the work of Barcia et al. [31], there are similarities in the values found for α-tocopherol (0.437 mg/100g), while the values for δ and β plus were a bit higher (0.0486 mg/100g for both tocopherols) in this study. All three locations produced three carotenoids as shown in Fig. (2) and Table 3. Two of them (lutein and zeaxanthin) were quantified together, since they were not well separated. The fruits from the city of Capão do Leão had lower levels of carotenoids. On the other hand, plants from Pelotas and Santa Vitória do Palmar had an average of 0.666 mg/100g and 0.647mg/100g, respectively. Faria et al. [9] reported that the profile of carotenoids from jambolão is
Fig. (1). Chromatogram showing the separation of delta, gama/beta and alpha tocopherol in jambolão, by HPLC, with a C18 column and fluorescence detection with an excitation and emission wavelengths of 290 and 330 nm, respectively. Table 2.
Levels of Tocopherols (mg/100 g) in Jambolão (Syzygium cumini) α Tocopherol
β/γ Tocopherol
δ Tocopherol
Total Tocopherols
Capão do Leão
0.089 b
0.024 a
0.024 a
0.138 b
Pelotas
0.398 a
0.024 a
0.016 a
0.439 a
SVP*
0.229 a
0.023 a
0.023 a
0.277 b
Location
Average of three replicate analyses. * Santa Vitória do Palmar Averages followed by the same letter in the same column indicate that there is no difference between them, according to the Tukey test (p≤0.05); Coefficient of variation (CV)< 3%
Bioactive Compounds, Antioxidant Activity and Percent Composition
The Natural Products Journal, 2012, Vol. 2, No. 2
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Fig. (2). Chromatogram showing the separation of carotenoids in jambolão, by HPLC, with a C18 column and detection at 450nm. Table 3.
Amounts of Individual Carotenoids (mg/100g) in Jambolão
Location
β-Cryptoxanthin
Lutein+Zeaxanthin
Total Carotenoids
Capão do Leão
0.099 a
0.211 a
0.311 b
Pelotas
0.094a
0.576 b
0.668 a
SVP*
0.133 a
0.513 b
0.647 a
Average of three replicate analyses. * Santa Vitória do Palmar Averages followed by the same letter in the same column indicate that there is no difference between them, according to the Tukey test (p≤0.05); Coefficient of variation (CV)< 6,5%
marked by the presence of lutein, 43.7 % of the total carotenoids, similar to the present study. As far as we know, there are no other studies reporting the composition of carotenoids from jambolão. Marinova and Ribarova [32] reported values of 0.270mg/100g lutein for blackberries and for blueberry 0.230 mg/100g, similar to the amounts found in other plants from Capão do Leão (0.211 mg of lutein and zeaxanthin per 100g, respectively). The same authors found 0.051mg de β-cryptoxanthin per 100 g in blueberry, an amount similar to that found in this study. Fig. (3) shows a chromatogram of L-ascorbic acid in jambolão fruits. The fruit from Capão do Leão had no detectable ascorbic acid, while the fruits from Pelotas and Santa Vitória do Palmar had 16.2 and 4.0 µg/g, respectively. The low levels can be influenced by the season in which they are collected, as well as genetic factors, which are the major determinants of its biosynthesis [33]. Faria et al. [9],
working with the same fruit also found low levels,
