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Carotenoids and antioxidant capacities from Canarium odontophyllum Miq. fruit
Food Chemistry 124 (2011) 1549–1555
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Carotenoids and antioxidant capacities from Canarium odontophyllum Miq. fruit K. Nagendra Prasad a, Lye Yee Chew a, H.E. Khoo a, Bao Yang b, Azrina Azlan a, Amin Ismail a,c,⇑ a
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China c Laboratory of Analysis and Authentication, Halal Products Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b
a r t i c l e
i n f o
Article history: Received 14 May 2010 Received in revised form 12 July 2010 Accepted 3 August 2010
Keywords: Antioxidant activity Canarium odontophylluim Carotenoids Haemoglobin oxidation
a b s t r a c t Carotenoids were isolated and identified from peel, pulp and seed fractions of Canarium odontophyllum Miq., and their antioxidant capacities were evaluated. all-trans-b-carotene was present in a large amount in peel (69.5 ± 1.0 mg/kg), followed by pulp (31.1 ± 0.76 mg/kg) and seed (15.1 ± 3.0 mg/kg). Additionally, 15-cis-b-carotene, 9-cis-b-carotene and 13-cis-b-carotenes were also major contributors to carotenoid contents in peel, pulp and seed fractions. Pulp exhibited excellent b-carotene bleaching activity, significantly higher than peel and seed; high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, whereas peel exhibited significantly higher scavenging activity of 2,2’-azino-bis(3-ethylbenzthiazoline6-sulphonic acid) (ABTS) radicals. All the extracts exhibited good inhibitory effect against hydrogen peroxide-induced haemoglobin oxidation, ranging from 45.3 to 59.7%. This is the first report about carotenoids and antioxidant capacities from C. odontophyllum fruit, and indicates that this fruit can be explored and promoted as a potential source of natural antioxidants. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Carotenoids are lipophilic compounds, responsible for red, orange and yellow hues of fruits and vegetables (Rao & Rao, 2007). Many studies have proven certain cancers, coronary heart disease and other degenerative diseases could be reduced through eating carotenoid-rich fruits and vegetables (Ziegler, 1989). Recently, carotenoids from underutilised fruits have gained much attention in the Malaysian diet, especially among rural communities (Khoo, Ismail, Mohd-Esa, & Idris, 2008). Canarium odontophyllum Miq. belongs to Burseraceae family and is classified as an underutilised fruit, found in the tropical rainforest of Sarawak, Malaysia (Lau, 2009). The fruit is called ‘dabai’ and eaten by the native people. The fruits are oblong in shape, measuring 3–4 cm in length and weighing 10–13 g. The peel of the fruit is dark purple in colour with yellow pulp and a single seed in the centre. Peel constitutes 10% of the fruit weight, while pulp and seed contribute 46 and 44%, respectively. The fruit is considered rich in minerals, proteins, carbohydrates and fat (Voon & Kueh, 1999). The objective of the present study was to make a systematic comparison, and to investigate the carotenoid composition of peel, pulp and seed fractions from C. odontophyllum fruit fractions, and to demonstrate their antioxidant capacities.
2.1. Plant material
⇑ Corresponding author at: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +603 89472435; fax: +603 8942679. E-mail address: [email protected] (A. Ismail). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.08.010
Fresh fruits of C. odontophyllum Miq. (20 kg) at the commercially mature stage were provided by Agriculture Research Centre, Department of Agriculture, Kuching, Sarawak, Malaysia. The fruits were packed in an ice-box and transported by air on the same day to Universiti Putra Malaysia, Serdang, Selangor, Malaysia. Fruits free from pest and physical damage were chosen, and washed with tap water and air dried. The fruit peel, pulp and seeds were manually separated, freeze-dried (Virtis, New York, NY) and were ground into powder using a blender then sieved. 2.2. Chemicals and standards Carotenoid standards (all-trans-b-carotene and all-trans-lutein, all HPLC grade), 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2’(azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), Tween 20, and linoleic acid, were obtained from Sigma–Aldrich Co. (St. Louis, MO). Ethanol, acetonitrile, butanol (HPLC grade), and n-hexane were purchased from Fisher Scientific (Pittsburgh, PA). Hydrogen peroxide (H2O2) was obtained from Merck (Darmstadt, Germany). All other chemicals and solvents used were of analytical grade. 2.3. Extraction of sample Extraction of carotenoids was carried out according to the method developed by Chen, Tai, and Chen (2004). Lyophilised peel,
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pulp and seed samples (10 g) were mixed with 50 ml of hexane:acetone:ethanol (70:15:15, v/v/v) containing 0.1% magnesium carbonate and 0.05% BHT. The mixture was stirred for 1 h using an orbital shaker (Heidolph, Schwabach, Germany). Later, 5 ml of 40% methanolic KOH were added and the solution was saponified at 25 °C in the dark for 2 h. Then, 30 ml of hexane were added, the mixture shaken vigorously and the upper layer was collected. The lower layer was extracted twice and the supernatant was also collected and filtered through sodium sulphate powder to remove traces of water. The supernatant obtained was pooled, concentrated and evaporated using a rotary evaporator (Büchi, Flawil, Switzerland). The resultant extract obtained was dissolved in methanol:methylene chloride (50:50, v/v) and filtered through a 0.2-lm membrane filter for HPLC analysis. The extraction yield obtained for peel, pulp and seeds were determined to be 0.213, 0.113 and 0.015 g/100 g of sample, respectively.
of Tween 20. The chloroform in the mixture was evaporated to dryness under vacuum and 100 ml of deionised water was added. The mixture was shaken vigorously to form an emulsion. Emulsion (1 ml) and 100 ll of the samples at different concentrations were taken in different test tubes and incubated at 45 °C for 2 h. Control samples received only the emulsion without any sample, while blank consisted of emulsion without b-carotene and the sample. The absorbance of the solutions was monitored at 470 nm. The rate of bleaching of b-carotene was calculated as antioxidant activity coefficient (AAC) and calculated using the equation:
AAC ¼ ½Asð120Þ Acð120Þ =Acð0Þ Acð120Þ 1000 where As (120) is the absorbance of the sample at time 120 min, Ac (120) is the absorbance of the control at 120 min, Ac (0) is the absorbance of control at 0 min, Ac (120) is the absorbance of control at 120 min. The higher the AAC values are, the higher the antioxidant activity.
2.4. HPLC determination of carotenoids Identification and quantification of carotenoids in C. odontophyllum peel, pulp and seed fractions were carried out using an HPLC method as described by Lin and Chen (2003) with some modifications. HPLC analysis was carried out using a Hewlett Packard 1100 system (Agilent Technologies, Santa Clara, CA) coupled with diode array detector. A 150 4.6 mm, 3 lm C30 analytical column (Waters Corporation, Milford, MA) was used in this analysis. The mobile phase consisted of acetonitrile:1-butanol (7:3, v/v, A) and methylene chloride (B). Separation was carried out with a flow rate of 1 ml/min by gradient elution, with 99% A and 1% B initially, increasing to 4% B in 20 min and then returned to 1% B in 22 min. The injection volume was 20 ll and the column temperature was maintained at 25 °C and detection was carried out at 476 nm. 2.5. Identification of carotenoids The carotenoids from peel, pulp and seeds of C. odontophyllum were identified using the HPLC system software by comparing retention time (RT) and absorption spectra of unknown peaks with reference standards. Furthermore, the cis-isomers of carotenoids were assigned based on the spectral characteristics and Q-ratio values reported in the literature. Quantification was done by external standard method based on peak area. 2.6. Preparation of standard curve and recovery Different concentrations of all-trans forms of b-carotene (10– 50 mg/l) and lutein, were injected into the HPLC, and the linear regression equation for each standard curve was acquired by plotting the quantity of standard compound injected against the peak area. The correlation coefficient r2 for all standards was greater than 0.99. The limit of detection and quantification were measured based on the method described by Wang, Chang, Inbaraj, and Chen (2010). Three concentrations of lutein, and b-carotene were prepared for recovery determination. 2.7. Antioxidant activity determination 2.7.1. Beta-carotene bleaching assay Beta-carotene bleaching assay was performed according to the method of Velioglu, Mazza, Gao, and Oomah (1998) with some modifications. For a typical assay, 1 ml of b-carotene solution, (2 g/l dissolved in chloroform), was added into an amber-coloured, round-bottomed flask containing 0.02 ml of linoleic acid and 0.2 ml
2.7.2. ABTS radical-scavenging activity The radical-scavenging activity of the samples against ABTS was carried out according to the method described by Re et al. (1999). ABTS radical cation was produced by reacting ABTS stock solution (7 mM) with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 16 h. The ABTS solution was diluted to get an absorbance of 0.70 at 734 nm and equilibrated at 30 °C. ABTS solution (1 ml) was mixed with the sample solution (100 ll) at different concentrations and the decrease in absorbance after 6 min of incubation was monitored using a spectrophotometer. Control samples received only the ABTS solution, while distiled water was used as blank. The inhibition of ABTS radicals by the test samples was calculated as, scavenging activity (%) = ((control ODsample OD)/control OD) 100where OD is optical density. 2.7.3. DPPH radical-scavenging assay DPPH radical-scavenging activities of peel, pulp and seed extracts of C. odontophyllum were determined according to Blois (1958) with some modifications. Different concentrations (10, 20, 30 and 40 mg/l) of the sample extracts and b-carotene were placed in different test tubes and were mixed with 1 ml of 0.2 mM DPPH (dissolved in methanol). The reaction mixture was shaken vigorously and incubated at 28 °C in a dark room for 40 min. The control was prepared as above without any extract, and methanol was used as blank. The changes in the absorbance of the samples were measured at 517 nm using a spectrophotometer. The inhibition of DPPH radicals by the test samples was calculated as, scavenging activity (%) = ((control ODsample OD)/control OD) 100. 2.7.4. Haemoglobin oxidation assay Fasting venous blood (10 ml) from healthy volunteers (aged 20– 25 years) was collected in EDTA tubes (0.4 g/l) and centrifuged at 1600g for 20 min at 4 °C, to separate plasma and red blood cells (RBC). RBC were washed with phosphate buffered saline (PBS) three times. Haemoglobin oxidation was performed on the same day of blood withdrawal as described by Rodríguez et al. (2006), with some modification. The RBC were suspended with PBS to obtain 5% of RBC and pre-incubated at 37 °C for 10 min in the presence of 1 mM sodium azide. Subsequently, 1.6 ml of RBC was transferred to test tubes for experimental analysis. All test tubes except control were added with 10 mM of H2O2 and with or without the sample extracts (5, 10, 15, 20 mg/l, 0.2 ml). Following 60min incubation at 37 °C, the mixture was kept for 60 s in an ice bath and then centrifuged at 1853g for 10 min at 4 °C. Malondialdehyde (MDA) levels of haemoglobin oxidation were measured
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using TBA assay as described by Buege and Aust (1978). The percentage inhibition of the sample extracts against haemoglobin oxidation was also calculated using the following equation
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percentage inhibition ¼ ððabsorbance of H2 O2 -induced haemoglobin sample absorbanceÞ=absorbance of H2 O2 induced haemoglobinÞ 100
Fig. 1. HPLC chromatogram of carotenoids of Canarium odontophyullum peel, pulp and seed extracts. The peak identification of carotenoids are (1) all-trans-lutein; (2) 9-cislutein; (3) 13-cis-lutein; (4) di-cis-b-carotene; (5) 15-cis-b-carotene; (6) 9-cis-b-carotene; (7) all-trans-b-carotene and (8) 13-cis-b-carotene.
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2.8. Statistical analysis Data were expressed as means ± standard deviations (SD) of three replicate determinations and then analysed by SPSS V.13 (SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) and Duncan’s new multiple-range test were used to determine the differences among the means. Values with p less than 0.05 were considered to be significant. 3. Results and discussion 3.1. HPLC analysis of carotenoids in C. odontophyllum peel, pulp and seed extracts HPLC analysis was performed to determine the carotenoids in C. odontophyllum peel, pulp and seed extracts. The percent recoveries for lutein and b-carotene were in the range of 90–105%. The detection limits for all-trans-lutein, and all-trans-b-carotene were 0.22 and 5.46 mg/l, while quantification limits were 0.66 and 16.38 mg/l, respectively. A total of 8 carotenoids were effectively resolved from peel, pulp and seed (Fig. 1) with peak purities higher than 90%. The separation factor (a) values were greater than 1 for all the peaks, showing that a good selectivity of mobile phase to carotenoid composition was attained (Table 1). Moreover, the retention factor (k0 ) values for all peaks ranged from 1.47 to 6.13, which also signifies that a proper solvent strength was maintained. Carotenoids were mainly identified based on comparison of retention time, absorption spectra from the standards and Q-ratio. The UV absorption spectra and Q-ratios obtained in the present investigation are in good agreement with various authors (Lin & Chen, 2003; Rodriguez-Amaya, Bobbio, & Bobbio, 1983). Peel exhibited the highest concentration of all the carotenoids identified (Table 2). All-trans-b-carotene content (69.5 ± 1.0 mg/ kg) was the highest in peel, followed by pulp (31.1 ± 0.76 mg/kg) and seed (15.1 ± 3.0 mg/kg). In addition b-carotene isomers, namely 15-cis-b-carotene, 9-cis-b-carotene and 13-cis-b-carotene isomers were the other main contributors to carotenoid composi-
tion. Carotenoid composition in pulp was 31.1 ± 0.76 mg/kg being the highest for all-trans-b-carotene and 0.10 ± 0.02 mg/kg being the lowest for 9-cis-lutein. Seed contained lower levels of all the carotenoids, compared with peel and pulp. The relative standard deviation (RSD) between 4 and 10% is acceptable, showing a good precision (AOAC, 2002). Most of the results obtained in the present investigation are within this range and this shows the method we have followed is a valid one. Additionally, our results are in good agreement with other reports where the carotenoid composition of peel is higher than pulp and seeds. Kreck, Kurbel, Ludwig, and Paschold (2006) have reported higher content of carotenoids in pumpkin peel, compared to pulp and juice. Gross, Gabai, and Lifshitz (1973) have reported higher concentration of b-carotene (10.8%), and lutein (55.8%) in avocado peel, compared to pulp (4.0 and 25%, respectively). A similar observation was also noticed in Cyphomandra betacea fruits where the peel contained high concentrations of b-carotene and zeaxanthin than the pulp (Rodriguez-Amaya et al., 1983). Normally, fruits are exposed to sunlight during their growth. Sunlight favours the breakdown of chlorophyll and induction of carotenogenesis. Specific changes in carotenoids patterns could be mediated by redox signals, probably from reactive oxygen species generated under excessive sunlight and by ethylene, the formation of which is intensified under photooxidative conditions (Gross et al., 1973). Solovchenko, Avertcheva, and Merzlyak (2006) have also reported that detachment of fruit from the tree after ripening accelerates breakdown of chlorophyll and synthesis of carotenoids, due to a shift in the hormonal balance in favour of ethylene, resulting in an increase in the carotenoid content during the postharvest storage period. 3.2. Antioxidant activity of chemical assay 3.2.1. Beta-carotene bleaching assay Beta-carotene bleaching method is widely used to measure antioxidant activity of plant extracts. It is an in vitro assay that measures the inhibition of coupled auto-oxidation of linoleic acid
Table 1 Retention time (min), factor (k0 ), and separation factor (a) and other tentative identification data for all-trans and cis forms of carotenoids from Canarium odontophyllum.
a
Peak no.
Compound
Retention time (min)
k0
a
kmax (nm)
1 2 3 4 5 6 7 8
all-trans-lutein 9-cis-lutein 13-cis-lutein di-cis-b-carotene 15-cis -b-carotene 9-cis-b-carotene all-trans-b-carotene 13-cis-b-carotene
1.27 1.87 2.17 2.54 3.14 3.67 4.70 5.29
1.47 2.16 2.51 2.94 3.64 4.25 5.45 6.13
1.47 1.16 1.17 1.23 1.09 1.28 1.12 1.12
418 424 422 426 426 426 430 428
442 448 444 450 452 450 456 452
470 476 470 478 478 478 482 478
Q-ratio
Q-ratio reported
0.16 0.34 0.64 0.34 0.16 0.19
0.19 0.34 0.68 0.37 0.10 0.20
a
Ref: Lin and Chen (2003).
Table 2 Carotenoids from Canarium odontophyllum peel, pulp and seed extracts. Carotenoids (mg/kg fresh weight)
Peel
Pulp
Mean all-trans-lutein 9-cis-lutein 13-cis-lutein di-cis-b-carotene 15- cis - b-carotene 9-cis-b-carotene all -trans-b-carotene 13-cis-b-carotene Total a–c
RSD (%) a
1.62 ± 0.03 0.32 ± 0.01a 0.62 ± 0.01a 0.69 ± 0.01a 18.3 ± 2.4a 39.6 ± 0.28a 69.52 ± 1.0a 19.4 ± 1.2a 149
1.85 3.12 1.61 1.45 1.12 0.71 1.44 6.18
Seeds
Mean
RSD (%) b
0.36 ± 0.01 0.10 ± 0.02b 0.16 ± 0.009b 0.35 ± 0.0005b 11.9 ± 0.3b 5.8 ± 0.69b 31.1 ± 0.76b 5.7 ± 0.5b 55.5
For each treatment, the means in a row followed by different letters were significantly different at p < 0.05.
2.78 2.0 5.63 0.14 2.52 1.90 2.44 8.77
Mean
RSD (%) b
0.67 ± 0.01 0.13 ± 0.04b 0.11 ± 0.01c 0.37 ± 0.01b 8.38 ± 0.4c 3.20 ± 0.3c 15.1 ± 3.0c 5.6 ± 0.27c 33.6
1.49 3.77 9.09 2.7 4.77 9.38 9.87 4.82
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though peel had higher carotenoid content, it had lower antioxidant activity. A moderate correlation between carotenoids and bcarotene bleaching activity (Table 3) suggests that not only carotenoids but other compounds, such as xanthophyll derivatives and phenolic compounds, which were not determined in the present study, might also participate.
A
Antioxidant activity coefficient
and b-carotene. This method is based on the fact that lipid radicals as auto-oxidation products of linoleic acid attack double bonds of b-carotene, but the presence of an antioxidative substance can prevent the attack and retain the yellowish-orange colour of b-carotene and thus reduce their bleaching activity (Krinsky, 1989). Pulp exhibited an excellent antioxidant activity coefficient of 2611 ± 12.7 at a concentration of 40 mg/l, significantly higher (p < 0.05) than peel and seed. Peel and seed exhibited moderate activity while the standard b-carotene exhibited lower activity (Fig. 2A). The activity of all the extracts was concentration-dependent and moderate correlation was observed between various carotenoid isomers and b-carotene bleaching assay (Table 3). Even
Peel
3000
Pulp
a
a
a
2000
b
a
1500 1000
c
500
b
b
b
b
b
c
c d
c
d
0 20 30 Concentration (µg/mL)
40
Peel
100 ABTS scavenging activity (%)
β-carotene
Seed
2500
10
B
3.2.2. ABTS assay The method described gives a measure of antioxidant activity for carotenoids and phenolic acids determined by decolourisation of ABTS, through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm. Fig. 2B illus-
*
Pulp 80
*
Seed
*
β-carotene
60 40 20 0
C
DPPH scavenging activity (%)
0
10
Peel
20 Concentration (µg/mL) Pulp
Seed
30
β-carotene
40
*
30 * *
20
10
0 0
10
20
30
40
Concentration µg/mL) Fig. 2. Antioxidant activity coefficient values of carotenoids obtained from peel, pulp and seed extracts of Canarium odontophyllum as determined by b-carotene bleaching assay (A), For each treatment means in a row followed by different letters are significantly different at p < 0.05; ABTS scavenging activity of carotenoids obtained from peel, pulp and seed extracts of Canarium odontophyllum (B), *denotes significant difference at p < 0.005; DPPH radical-scavenging activity of carotenoids obtained from peel, pulp and seed extracts of Canarium odontophyllum (C), *denotes significant difference at p < 0.005.
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Table 3 Correlation among carotenoid isomers and antioxidant activity using DPPH, ABTS, beta-carotene bleaching (BCB) and haemoglobin oxidation (HO) methods. Carotenoids
all-trans-lutein 9-cis-lutein 13-cis-lutein di-cis-b-carotene 15-cis-b-carotene 9-cis-b-carotene all-trans-b-carotene cis-b-carotene
Table 4 Haemoglobin oxidation inhibitory activity of carotenoids from peel, pulp and seeds of Canarium odontophyllum.
Antioxidant activity DPPH
ABTS
BCB
HO
0.004 0.0850 0.0916 0.0273 0.3058 0.0777 0.2372 0.0497
0.6142 0.7197 0.8870 0.7833 0.9959 0.8707 0.9801 0.8269
0.5675 0.4555 0.2518 0.3829 0.0599 0.2738 0.1017 0.3267
0.3364 0.4457 0.658 0.5192 0.8848 0.6341 0.8311 0.5777
Samples
Concentration (mg/l)
MDA (lM)
Inhibition (%)
Non-induced haemoglobin H2O2-induced haemoglobin Peel
– – 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20
0.313 4.33 2.13 1.81 1.91 1.95 2.37 1.64 2.14 2.21 2.16 1.74 1.87 1.99 2.00 2.14 2.16 2.21
– – 50.5 ± 0.1c 58.0 ± 1a 54.3 ± 2.0b 53.8 ± 0.8b 45.3 ± 1.1d 58.5 ± 2.4a 53.2 ± 3.8b 48.7c 50.1 ± 0.05c 59.7 ± 0.03a 56.8 ± 0.17b 51.1 ± 1.7c 53.8 ± 0.5a 51.4 ± 0.12b 50 ± 1.1b 47.2 ± 1c
Pulp
Seed
trates the effect of the C. odontophyllum extracts against ABTS radicals. Peel exhibited a significantly (p < 0.05) higher scavenging activity of 84.5 ± 0.9% at a concentration of 40 mg/l, higher than beta carotene (74.6 ± 0.4) standard. Pulp exhibited moderate activity ranging from 18.3 ± 2.8% to 44.5 ± 0.3%, whereas seed extracts exhibited low activity. A very high correlation was observed between b-carotene and its isomers and ABTS assay (Table 3), indicating that b-carotene and its isomers are effective ABTS scavengers. Lutein and its isomers had a lower correlation than b-carotene and its isomers. Thus, b-carotenoids and their isomers exert a major antioxidant activity using this assay. Our results are supported by similar findings of high scavenging activity of carotenoid extract of Lycium barbarum against ABTS radicals reported by Wang et al. (2010). Zanfini, Corbini, Rosa, and Dreassi (2010), have also reported a strong correlation between ABTS scavenging activity and b-carotene content. 3.2.3. DPPH radical-scavenging activity DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule, which is widely used to investigate radical-scavenging activity. In DPPH radical-scavenging assay, antioxidants react with DPPH, and convert it to yellow coloured a,a-diphenyl-b-picryl hydrazine. The degree of discolouration indicates the radical-scavenging potential of the antioxidant (Blois, 1958). All the extracts exhibited moderate scavenging activity against DPPH radicals (Fig. 2C). Among them, pulp extract exhibited the highest scavenging activity (30.5 ± 1%), significantly (p > 0.05) higher than peel (26.4 ± 0.9%) and seed (17.2 ± 0.34%) extracts at a concentration of 40 mg/ml, and was higher than the scavenging activity of beta carotene (18.6 ± 0.2%). All the sample extracts showed a very low correlation to scavenge DPPH radicals and thus, a lower scavenging activity was noticed. Carotenoids like zeaxanthin have a very low scavenging activity against DPPH radicals due to the presence of a b-ionone ring, which might decrease the resonance effect of pi electrons due to steric hindrance and hence lower free radical-scavenging activity (Wang et al., 2010). Hu, Lin, Lu, Chou, and Yang, 2008 have reported parallel results of lower scavenging activity of all-translutein and all-trans-carotene against DPPH radicals. 3.2.4. Haemoglobin oxidation assay The reliability of chemical assay for antioxidant activity determination is doubtful and, hence, haemoglobin oxidation assay was chosen because it mimics the human biological system (Filipe et al., 2009). Oxidative damage to haemoglobin by exposure to hydrogen peroxide is a primary mechanism to induce specific structural changes and might contribute to haemoglobin-mediated toxicity in diseases linked to oxidative stress (Vallelian et al., 2008). The results showed that all the extracts had a good protective effect against peroxide-induced haemoglobin oxidation (Table 4). The percentage inhibition of haemoglobin oxidation varied signif-
b-carotene
a–d
For each treatment, the means in a column followed by different letters were significantly different at p < 0.05.
icantly (p < 0.05) in all the tested fractions, with the highest activity (59.7 ± 0.03%) observed in seed extract, while the lowest (45.3 ± 1.1%) was in pulp fraction. Malondialdehyde (MDA) is a by-product of peroxidation of phospholipids and is generally regarded as a marker for oxidative stresses, rendering its determination in biological samples particularly interesting (Rodríguez et al., 2006). The MDA produced from haemoglobin oxidation treated with seed extract was lower than pulp and peel extract at 10 mg/l. The MDA levels of H2O2-induced haemoglobin oxidation treated with different concentrations (5, 10, and 15 mg/l) of the sample extracts decreased compared to H2O2-induced haemoglobin alone. However, all the extracts had protective effect against H2O2-induced oxidation only at lower concentration. A similar finding was reported by Polyakov, Leshina, Konovalova, and Kispret, 2001, and also by Sthal and Sies (2003), where at higher concentrations carotenoids had a pro-oxidant effect. Recently, we have observed pro-oxidant activity of carotenoids at high concentrations (2 ppm), while antioxidant activity was noticed at 1 ppm concentration using haemoglobin oxidation assay (unpublished data). Carotenoids were effective inhibitors of lipid peroxidation, as measured by MDA formation, by blocking the auto-oxidation of liposomes (Krinsky, 1989). 4. Conclusions This study has examined the carotenoid content and antioxidant capacity of C. odontophyllum peel, pulp and seed extracts. Peel and pulp extracts exhibited high concentration of carotenoids with acceptable level of antioxidant capacity in the studied assays. The evidence from this study suggests C. odontophyllum fruit can be used as a natural antioxidant and also used in production of functional food. This is the first report about existence of carotenoids from C. odontophyllum. Further investigations are needed to determine polyphenolic compounds from this fruit and to determine their antioxidant activities. Acknowledgements We would like to acknowledge Agriculture Research Centre at the Department of Agriculture, Sarawak, Malaysia for providing funding under consultant project on ‘‘Foodcomponents and antiox-
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idant properties of Canarium odontophyllum Miq. fruit”. The authors are grateful to the Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia for laboratory facilities.
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Lin, C. H., & Chen, B. H. (2003). Determination of carotenoids in tomato juice by liquid chromatography. Journal of Chromotography A, 1012, 103–109. Polyakov, N. E., Leshina, T. V., Konovalova, T. A., & Kispret, L. D. (2001). Carotenoids as scavengers of free radicals in a Fenton reaction: antioxidants or prooxidants? Free Radical Biology & Medicine, 31, 398–404. Rao, A. V., & Rao, L. G. (2007). Carotenoids and human health. Pharmacological Research, 55, 207–216. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26(9/10), 1231–1237. Rodriguez-Amaya, D. B., Bobbio, P. A., & Bobbio, F. O. (1983). Carotenoid composition and vitamin A value of Brazalian fruit Cyphomandara betacea. Food Chemistry, 12, 61–65. Rodríguez, J., Pierro, D. D., Gioia, M., Monaco, S., Delgado, R., Coletta, M., et al. (2006). Effects of a natural extract from Mangifera indica L, and its active compound, mangiferin, on energy state and lipid peroxidation of red blood cells”. Biochimica et Biophysica Acta, 1760, 1333–1342. Solovchenko, A. E., Avertcheva, O. V., & Merzlyak, M. N. (2006). Elevated sunlight promotes ripening-associated pigment changes in apple fruits. Postharvest Biology and Technology, 40, 183–189. Sthal, W., & Sies, H. (2003). Antioxidant activity of carotenoids. Molecular Aspects of Medicine, 24, 345–351. Vallelian, F., Pimenova, T., Pereira, C. P., Abraham, B., Mikolajczyk, M. G., Schoedon, S., et al. (2008). The reaction of hydrogen peroxide with hemoglobin induces extensive a-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways. Free Radical Biology & Medicine, 45, 1150–1158. Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agriculture and Food Chemistry, 46, 4113–4117. Voon, B. H., & Kueh, H. S. (1999). The nutritional value of indigeneous fruits and vegetables in Sarawak. Asia Pacific Journal of Clinical Nutrition, 8, 24–31. Wang, C. C., Chang, S. C., Inbaraj, B. S., & Chen, B. H. (2010). Isolation of carotenoids, flavonoids and polysaccharides from Lycium barbarum L. and evaluation of antioxidant activity. Food Chemistry, 120, 184–192. Zanfini, A., Corbini, G., Rosa, C. L., & Dreassi, E. (2010). Antioxidant activity of tomato lipophilic extracts and interactions between carotenoids and alpha tocophenols in synthetic mixtures. LWT-Food Science and Technology, 43, 67–72. Ziegler, R. G. (1989). A review of epidemiologic evidence that carotenoids reduce the risk of cancer. The Journal of Nutrition, 119, 116–122.
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Carotenoids and antioxidant capacities from Canarium odontophyllum Miq. fruit K. Nagendra Prasad a, Lye Yee Chew a, H.E. Khoo a, Bao Yang b, Azrina Azlan a, Amin Ismail a,c,⇑ a
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China c Laboratory of Analysis and Authentication, Halal Products Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b
a r t i c l e
i n f o
Article history: Received 14 May 2010 Received in revised form 12 July 2010 Accepted 3 August 2010
Keywords: Antioxidant activity Canarium odontophylluim Carotenoids Haemoglobin oxidation
a b s t r a c t Carotenoids were isolated and identified from peel, pulp and seed fractions of Canarium odontophyllum Miq., and their antioxidant capacities were evaluated. all-trans-b-carotene was present in a large amount in peel (69.5 ± 1.0 mg/kg), followed by pulp (31.1 ± 0.76 mg/kg) and seed (15.1 ± 3.0 mg/kg). Additionally, 15-cis-b-carotene, 9-cis-b-carotene and 13-cis-b-carotenes were also major contributors to carotenoid contents in peel, pulp and seed fractions. Pulp exhibited excellent b-carotene bleaching activity, significantly higher than peel and seed; high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, whereas peel exhibited significantly higher scavenging activity of 2,2’-azino-bis(3-ethylbenzthiazoline6-sulphonic acid) (ABTS) radicals. All the extracts exhibited good inhibitory effect against hydrogen peroxide-induced haemoglobin oxidation, ranging from 45.3 to 59.7%. This is the first report about carotenoids and antioxidant capacities from C. odontophyllum fruit, and indicates that this fruit can be explored and promoted as a potential source of natural antioxidants. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Carotenoids are lipophilic compounds, responsible for red, orange and yellow hues of fruits and vegetables (Rao & Rao, 2007). Many studies have proven certain cancers, coronary heart disease and other degenerative diseases could be reduced through eating carotenoid-rich fruits and vegetables (Ziegler, 1989). Recently, carotenoids from underutilised fruits have gained much attention in the Malaysian diet, especially among rural communities (Khoo, Ismail, Mohd-Esa, & Idris, 2008). Canarium odontophyllum Miq. belongs to Burseraceae family and is classified as an underutilised fruit, found in the tropical rainforest of Sarawak, Malaysia (Lau, 2009). The fruit is called ‘dabai’ and eaten by the native people. The fruits are oblong in shape, measuring 3–4 cm in length and weighing 10–13 g. The peel of the fruit is dark purple in colour with yellow pulp and a single seed in the centre. Peel constitutes 10% of the fruit weight, while pulp and seed contribute 46 and 44%, respectively. The fruit is considered rich in minerals, proteins, carbohydrates and fat (Voon & Kueh, 1999). The objective of the present study was to make a systematic comparison, and to investigate the carotenoid composition of peel, pulp and seed fractions from C. odontophyllum fruit fractions, and to demonstrate their antioxidant capacities.
2.1. Plant material
⇑ Corresponding author at: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +603 89472435; fax: +603 8942679. E-mail address: [email protected] (A. Ismail). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.08.010
Fresh fruits of C. odontophyllum Miq. (20 kg) at the commercially mature stage were provided by Agriculture Research Centre, Department of Agriculture, Kuching, Sarawak, Malaysia. The fruits were packed in an ice-box and transported by air on the same day to Universiti Putra Malaysia, Serdang, Selangor, Malaysia. Fruits free from pest and physical damage were chosen, and washed with tap water and air dried. The fruit peel, pulp and seeds were manually separated, freeze-dried (Virtis, New York, NY) and were ground into powder using a blender then sieved. 2.2. Chemicals and standards Carotenoid standards (all-trans-b-carotene and all-trans-lutein, all HPLC grade), 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2’(azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), Tween 20, and linoleic acid, were obtained from Sigma–Aldrich Co. (St. Louis, MO). Ethanol, acetonitrile, butanol (HPLC grade), and n-hexane were purchased from Fisher Scientific (Pittsburgh, PA). Hydrogen peroxide (H2O2) was obtained from Merck (Darmstadt, Germany). All other chemicals and solvents used were of analytical grade. 2.3. Extraction of sample Extraction of carotenoids was carried out according to the method developed by Chen, Tai, and Chen (2004). Lyophilised peel,
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pulp and seed samples (10 g) were mixed with 50 ml of hexane:acetone:ethanol (70:15:15, v/v/v) containing 0.1% magnesium carbonate and 0.05% BHT. The mixture was stirred for 1 h using an orbital shaker (Heidolph, Schwabach, Germany). Later, 5 ml of 40% methanolic KOH were added and the solution was saponified at 25 °C in the dark for 2 h. Then, 30 ml of hexane were added, the mixture shaken vigorously and the upper layer was collected. The lower layer was extracted twice and the supernatant was also collected and filtered through sodium sulphate powder to remove traces of water. The supernatant obtained was pooled, concentrated and evaporated using a rotary evaporator (Büchi, Flawil, Switzerland). The resultant extract obtained was dissolved in methanol:methylene chloride (50:50, v/v) and filtered through a 0.2-lm membrane filter for HPLC analysis. The extraction yield obtained for peel, pulp and seeds were determined to be 0.213, 0.113 and 0.015 g/100 g of sample, respectively.
of Tween 20. The chloroform in the mixture was evaporated to dryness under vacuum and 100 ml of deionised water was added. The mixture was shaken vigorously to form an emulsion. Emulsion (1 ml) and 100 ll of the samples at different concentrations were taken in different test tubes and incubated at 45 °C for 2 h. Control samples received only the emulsion without any sample, while blank consisted of emulsion without b-carotene and the sample. The absorbance of the solutions was monitored at 470 nm. The rate of bleaching of b-carotene was calculated as antioxidant activity coefficient (AAC) and calculated using the equation:
AAC ¼ ½Asð120Þ Acð120Þ =Acð0Þ Acð120Þ 1000 where As (120) is the absorbance of the sample at time 120 min, Ac (120) is the absorbance of the control at 120 min, Ac (0) is the absorbance of control at 0 min, Ac (120) is the absorbance of control at 120 min. The higher the AAC values are, the higher the antioxidant activity.
2.4. HPLC determination of carotenoids Identification and quantification of carotenoids in C. odontophyllum peel, pulp and seed fractions were carried out using an HPLC method as described by Lin and Chen (2003) with some modifications. HPLC analysis was carried out using a Hewlett Packard 1100 system (Agilent Technologies, Santa Clara, CA) coupled with diode array detector. A 150 4.6 mm, 3 lm C30 analytical column (Waters Corporation, Milford, MA) was used in this analysis. The mobile phase consisted of acetonitrile:1-butanol (7:3, v/v, A) and methylene chloride (B). Separation was carried out with a flow rate of 1 ml/min by gradient elution, with 99% A and 1% B initially, increasing to 4% B in 20 min and then returned to 1% B in 22 min. The injection volume was 20 ll and the column temperature was maintained at 25 °C and detection was carried out at 476 nm. 2.5. Identification of carotenoids The carotenoids from peel, pulp and seeds of C. odontophyllum were identified using the HPLC system software by comparing retention time (RT) and absorption spectra of unknown peaks with reference standards. Furthermore, the cis-isomers of carotenoids were assigned based on the spectral characteristics and Q-ratio values reported in the literature. Quantification was done by external standard method based on peak area. 2.6. Preparation of standard curve and recovery Different concentrations of all-trans forms of b-carotene (10– 50 mg/l) and lutein, were injected into the HPLC, and the linear regression equation for each standard curve was acquired by plotting the quantity of standard compound injected against the peak area. The correlation coefficient r2 for all standards was greater than 0.99. The limit of detection and quantification were measured based on the method described by Wang, Chang, Inbaraj, and Chen (2010). Three concentrations of lutein, and b-carotene were prepared for recovery determination. 2.7. Antioxidant activity determination 2.7.1. Beta-carotene bleaching assay Beta-carotene bleaching assay was performed according to the method of Velioglu, Mazza, Gao, and Oomah (1998) with some modifications. For a typical assay, 1 ml of b-carotene solution, (2 g/l dissolved in chloroform), was added into an amber-coloured, round-bottomed flask containing 0.02 ml of linoleic acid and 0.2 ml
2.7.2. ABTS radical-scavenging activity The radical-scavenging activity of the samples against ABTS was carried out according to the method described by Re et al. (1999). ABTS radical cation was produced by reacting ABTS stock solution (7 mM) with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 16 h. The ABTS solution was diluted to get an absorbance of 0.70 at 734 nm and equilibrated at 30 °C. ABTS solution (1 ml) was mixed with the sample solution (100 ll) at different concentrations and the decrease in absorbance after 6 min of incubation was monitored using a spectrophotometer. Control samples received only the ABTS solution, while distiled water was used as blank. The inhibition of ABTS radicals by the test samples was calculated as, scavenging activity (%) = ((control ODsample OD)/control OD) 100where OD is optical density. 2.7.3. DPPH radical-scavenging assay DPPH radical-scavenging activities of peel, pulp and seed extracts of C. odontophyllum were determined according to Blois (1958) with some modifications. Different concentrations (10, 20, 30 and 40 mg/l) of the sample extracts and b-carotene were placed in different test tubes and were mixed with 1 ml of 0.2 mM DPPH (dissolved in methanol). The reaction mixture was shaken vigorously and incubated at 28 °C in a dark room for 40 min. The control was prepared as above without any extract, and methanol was used as blank. The changes in the absorbance of the samples were measured at 517 nm using a spectrophotometer. The inhibition of DPPH radicals by the test samples was calculated as, scavenging activity (%) = ((control ODsample OD)/control OD) 100. 2.7.4. Haemoglobin oxidation assay Fasting venous blood (10 ml) from healthy volunteers (aged 20– 25 years) was collected in EDTA tubes (0.4 g/l) and centrifuged at 1600g for 20 min at 4 °C, to separate plasma and red blood cells (RBC). RBC were washed with phosphate buffered saline (PBS) three times. Haemoglobin oxidation was performed on the same day of blood withdrawal as described by Rodríguez et al. (2006), with some modification. The RBC were suspended with PBS to obtain 5% of RBC and pre-incubated at 37 °C for 10 min in the presence of 1 mM sodium azide. Subsequently, 1.6 ml of RBC was transferred to test tubes for experimental analysis. All test tubes except control were added with 10 mM of H2O2 and with or without the sample extracts (5, 10, 15, 20 mg/l, 0.2 ml). Following 60min incubation at 37 °C, the mixture was kept for 60 s in an ice bath and then centrifuged at 1853g for 10 min at 4 °C. Malondialdehyde (MDA) levels of haemoglobin oxidation were measured
K.N. Prasad et al. / Food Chemistry 124 (2011) 1549–1555
using TBA assay as described by Buege and Aust (1978). The percentage inhibition of the sample extracts against haemoglobin oxidation was also calculated using the following equation
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percentage inhibition ¼ ððabsorbance of H2 O2 -induced haemoglobin sample absorbanceÞ=absorbance of H2 O2 induced haemoglobinÞ 100
Fig. 1. HPLC chromatogram of carotenoids of Canarium odontophyullum peel, pulp and seed extracts. The peak identification of carotenoids are (1) all-trans-lutein; (2) 9-cislutein; (3) 13-cis-lutein; (4) di-cis-b-carotene; (5) 15-cis-b-carotene; (6) 9-cis-b-carotene; (7) all-trans-b-carotene and (8) 13-cis-b-carotene.
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2.8. Statistical analysis Data were expressed as means ± standard deviations (SD) of three replicate determinations and then analysed by SPSS V.13 (SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) and Duncan’s new multiple-range test were used to determine the differences among the means. Values with p less than 0.05 were considered to be significant. 3. Results and discussion 3.1. HPLC analysis of carotenoids in C. odontophyllum peel, pulp and seed extracts HPLC analysis was performed to determine the carotenoids in C. odontophyllum peel, pulp and seed extracts. The percent recoveries for lutein and b-carotene were in the range of 90–105%. The detection limits for all-trans-lutein, and all-trans-b-carotene were 0.22 and 5.46 mg/l, while quantification limits were 0.66 and 16.38 mg/l, respectively. A total of 8 carotenoids were effectively resolved from peel, pulp and seed (Fig. 1) with peak purities higher than 90%. The separation factor (a) values were greater than 1 for all the peaks, showing that a good selectivity of mobile phase to carotenoid composition was attained (Table 1). Moreover, the retention factor (k0 ) values for all peaks ranged from 1.47 to 6.13, which also signifies that a proper solvent strength was maintained. Carotenoids were mainly identified based on comparison of retention time, absorption spectra from the standards and Q-ratio. The UV absorption spectra and Q-ratios obtained in the present investigation are in good agreement with various authors (Lin & Chen, 2003; Rodriguez-Amaya, Bobbio, & Bobbio, 1983). Peel exhibited the highest concentration of all the carotenoids identified (Table 2). All-trans-b-carotene content (69.5 ± 1.0 mg/ kg) was the highest in peel, followed by pulp (31.1 ± 0.76 mg/kg) and seed (15.1 ± 3.0 mg/kg). In addition b-carotene isomers, namely 15-cis-b-carotene, 9-cis-b-carotene and 13-cis-b-carotene isomers were the other main contributors to carotenoid composi-
tion. Carotenoid composition in pulp was 31.1 ± 0.76 mg/kg being the highest for all-trans-b-carotene and 0.10 ± 0.02 mg/kg being the lowest for 9-cis-lutein. Seed contained lower levels of all the carotenoids, compared with peel and pulp. The relative standard deviation (RSD) between 4 and 10% is acceptable, showing a good precision (AOAC, 2002). Most of the results obtained in the present investigation are within this range and this shows the method we have followed is a valid one. Additionally, our results are in good agreement with other reports where the carotenoid composition of peel is higher than pulp and seeds. Kreck, Kurbel, Ludwig, and Paschold (2006) have reported higher content of carotenoids in pumpkin peel, compared to pulp and juice. Gross, Gabai, and Lifshitz (1973) have reported higher concentration of b-carotene (10.8%), and lutein (55.8%) in avocado peel, compared to pulp (4.0 and 25%, respectively). A similar observation was also noticed in Cyphomandra betacea fruits where the peel contained high concentrations of b-carotene and zeaxanthin than the pulp (Rodriguez-Amaya et al., 1983). Normally, fruits are exposed to sunlight during their growth. Sunlight favours the breakdown of chlorophyll and induction of carotenogenesis. Specific changes in carotenoids patterns could be mediated by redox signals, probably from reactive oxygen species generated under excessive sunlight and by ethylene, the formation of which is intensified under photooxidative conditions (Gross et al., 1973). Solovchenko, Avertcheva, and Merzlyak (2006) have also reported that detachment of fruit from the tree after ripening accelerates breakdown of chlorophyll and synthesis of carotenoids, due to a shift in the hormonal balance in favour of ethylene, resulting in an increase in the carotenoid content during the postharvest storage period. 3.2. Antioxidant activity of chemical assay 3.2.1. Beta-carotene bleaching assay Beta-carotene bleaching method is widely used to measure antioxidant activity of plant extracts. It is an in vitro assay that measures the inhibition of coupled auto-oxidation of linoleic acid
Table 1 Retention time (min), factor (k0 ), and separation factor (a) and other tentative identification data for all-trans and cis forms of carotenoids from Canarium odontophyllum.
a
Peak no.
Compound
Retention time (min)
k0
a
kmax (nm)
1 2 3 4 5 6 7 8
all-trans-lutein 9-cis-lutein 13-cis-lutein di-cis-b-carotene 15-cis -b-carotene 9-cis-b-carotene all-trans-b-carotene 13-cis-b-carotene
1.27 1.87 2.17 2.54 3.14 3.67 4.70 5.29
1.47 2.16 2.51 2.94 3.64 4.25 5.45 6.13
1.47 1.16 1.17 1.23 1.09 1.28 1.12 1.12
418 424 422 426 426 426 430 428
442 448 444 450 452 450 456 452
470 476 470 478 478 478 482 478
Q-ratio
Q-ratio reported
0.16 0.34 0.64 0.34 0.16 0.19
0.19 0.34 0.68 0.37 0.10 0.20
a
Ref: Lin and Chen (2003).
Table 2 Carotenoids from Canarium odontophyllum peel, pulp and seed extracts. Carotenoids (mg/kg fresh weight)
Peel
Pulp
Mean all-trans-lutein 9-cis-lutein 13-cis-lutein di-cis-b-carotene 15- cis - b-carotene 9-cis-b-carotene all -trans-b-carotene 13-cis-b-carotene Total a–c
RSD (%) a
1.62 ± 0.03 0.32 ± 0.01a 0.62 ± 0.01a 0.69 ± 0.01a 18.3 ± 2.4a 39.6 ± 0.28a 69.52 ± 1.0a 19.4 ± 1.2a 149
1.85 3.12 1.61 1.45 1.12 0.71 1.44 6.18
Seeds
Mean
RSD (%) b
0.36 ± 0.01 0.10 ± 0.02b 0.16 ± 0.009b 0.35 ± 0.0005b 11.9 ± 0.3b 5.8 ± 0.69b 31.1 ± 0.76b 5.7 ± 0.5b 55.5
For each treatment, the means in a row followed by different letters were significantly different at p < 0.05.
2.78 2.0 5.63 0.14 2.52 1.90 2.44 8.77
Mean
RSD (%) b
0.67 ± 0.01 0.13 ± 0.04b 0.11 ± 0.01c 0.37 ± 0.01b 8.38 ± 0.4c 3.20 ± 0.3c 15.1 ± 3.0c 5.6 ± 0.27c 33.6
1.49 3.77 9.09 2.7 4.77 9.38 9.87 4.82
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though peel had higher carotenoid content, it had lower antioxidant activity. A moderate correlation between carotenoids and bcarotene bleaching activity (Table 3) suggests that not only carotenoids but other compounds, such as xanthophyll derivatives and phenolic compounds, which were not determined in the present study, might also participate.
A
Antioxidant activity coefficient
and b-carotene. This method is based on the fact that lipid radicals as auto-oxidation products of linoleic acid attack double bonds of b-carotene, but the presence of an antioxidative substance can prevent the attack and retain the yellowish-orange colour of b-carotene and thus reduce their bleaching activity (Krinsky, 1989). Pulp exhibited an excellent antioxidant activity coefficient of 2611 ± 12.7 at a concentration of 40 mg/l, significantly higher (p < 0.05) than peel and seed. Peel and seed exhibited moderate activity while the standard b-carotene exhibited lower activity (Fig. 2A). The activity of all the extracts was concentration-dependent and moderate correlation was observed between various carotenoid isomers and b-carotene bleaching assay (Table 3). Even
Peel
3000
Pulp
a
a
a
2000
b
a
1500 1000
c
500
b
b
b
b
b
c
c d
c
d
0 20 30 Concentration (µg/mL)
40
Peel
100 ABTS scavenging activity (%)
β-carotene
Seed
2500
10
B
3.2.2. ABTS assay The method described gives a measure of antioxidant activity for carotenoids and phenolic acids determined by decolourisation of ABTS, through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm. Fig. 2B illus-
*
Pulp 80
*
Seed
*
β-carotene
60 40 20 0
C
DPPH scavenging activity (%)
0
10
Peel
20 Concentration (µg/mL) Pulp
Seed
30
β-carotene
40
*
30 * *
20
10
0 0
10
20
30
40
Concentration µg/mL) Fig. 2. Antioxidant activity coefficient values of carotenoids obtained from peel, pulp and seed extracts of Canarium odontophyllum as determined by b-carotene bleaching assay (A), For each treatment means in a row followed by different letters are significantly different at p < 0.05; ABTS scavenging activity of carotenoids obtained from peel, pulp and seed extracts of Canarium odontophyllum (B), *denotes significant difference at p < 0.005; DPPH radical-scavenging activity of carotenoids obtained from peel, pulp and seed extracts of Canarium odontophyllum (C), *denotes significant difference at p < 0.005.
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Table 3 Correlation among carotenoid isomers and antioxidant activity using DPPH, ABTS, beta-carotene bleaching (BCB) and haemoglobin oxidation (HO) methods. Carotenoids
all-trans-lutein 9-cis-lutein 13-cis-lutein di-cis-b-carotene 15-cis-b-carotene 9-cis-b-carotene all-trans-b-carotene cis-b-carotene
Table 4 Haemoglobin oxidation inhibitory activity of carotenoids from peel, pulp and seeds of Canarium odontophyllum.
Antioxidant activity DPPH
ABTS
BCB
HO
0.004 0.0850 0.0916 0.0273 0.3058 0.0777 0.2372 0.0497
0.6142 0.7197 0.8870 0.7833 0.9959 0.8707 0.9801 0.8269
0.5675 0.4555 0.2518 0.3829 0.0599 0.2738 0.1017 0.3267
0.3364 0.4457 0.658 0.5192 0.8848 0.6341 0.8311 0.5777
Samples
Concentration (mg/l)
MDA (lM)
Inhibition (%)
Non-induced haemoglobin H2O2-induced haemoglobin Peel
– – 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20
0.313 4.33 2.13 1.81 1.91 1.95 2.37 1.64 2.14 2.21 2.16 1.74 1.87 1.99 2.00 2.14 2.16 2.21
– – 50.5 ± 0.1c 58.0 ± 1a 54.3 ± 2.0b 53.8 ± 0.8b 45.3 ± 1.1d 58.5 ± 2.4a 53.2 ± 3.8b 48.7c 50.1 ± 0.05c 59.7 ± 0.03a 56.8 ± 0.17b 51.1 ± 1.7c 53.8 ± 0.5a 51.4 ± 0.12b 50 ± 1.1b 47.2 ± 1c
Pulp
Seed
trates the effect of the C. odontophyllum extracts against ABTS radicals. Peel exhibited a significantly (p < 0.05) higher scavenging activity of 84.5 ± 0.9% at a concentration of 40 mg/l, higher than beta carotene (74.6 ± 0.4) standard. Pulp exhibited moderate activity ranging from 18.3 ± 2.8% to 44.5 ± 0.3%, whereas seed extracts exhibited low activity. A very high correlation was observed between b-carotene and its isomers and ABTS assay (Table 3), indicating that b-carotene and its isomers are effective ABTS scavengers. Lutein and its isomers had a lower correlation than b-carotene and its isomers. Thus, b-carotenoids and their isomers exert a major antioxidant activity using this assay. Our results are supported by similar findings of high scavenging activity of carotenoid extract of Lycium barbarum against ABTS radicals reported by Wang et al. (2010). Zanfini, Corbini, Rosa, and Dreassi (2010), have also reported a strong correlation between ABTS scavenging activity and b-carotene content. 3.2.3. DPPH radical-scavenging activity DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule, which is widely used to investigate radical-scavenging activity. In DPPH radical-scavenging assay, antioxidants react with DPPH, and convert it to yellow coloured a,a-diphenyl-b-picryl hydrazine. The degree of discolouration indicates the radical-scavenging potential of the antioxidant (Blois, 1958). All the extracts exhibited moderate scavenging activity against DPPH radicals (Fig. 2C). Among them, pulp extract exhibited the highest scavenging activity (30.5 ± 1%), significantly (p > 0.05) higher than peel (26.4 ± 0.9%) and seed (17.2 ± 0.34%) extracts at a concentration of 40 mg/ml, and was higher than the scavenging activity of beta carotene (18.6 ± 0.2%). All the sample extracts showed a very low correlation to scavenge DPPH radicals and thus, a lower scavenging activity was noticed. Carotenoids like zeaxanthin have a very low scavenging activity against DPPH radicals due to the presence of a b-ionone ring, which might decrease the resonance effect of pi electrons due to steric hindrance and hence lower free radical-scavenging activity (Wang et al., 2010). Hu, Lin, Lu, Chou, and Yang, 2008 have reported parallel results of lower scavenging activity of all-translutein and all-trans-carotene against DPPH radicals. 3.2.4. Haemoglobin oxidation assay The reliability of chemical assay for antioxidant activity determination is doubtful and, hence, haemoglobin oxidation assay was chosen because it mimics the human biological system (Filipe et al., 2009). Oxidative damage to haemoglobin by exposure to hydrogen peroxide is a primary mechanism to induce specific structural changes and might contribute to haemoglobin-mediated toxicity in diseases linked to oxidative stress (Vallelian et al., 2008). The results showed that all the extracts had a good protective effect against peroxide-induced haemoglobin oxidation (Table 4). The percentage inhibition of haemoglobin oxidation varied signif-
b-carotene
a–d
For each treatment, the means in a column followed by different letters were significantly different at p < 0.05.
icantly (p < 0.05) in all the tested fractions, with the highest activity (59.7 ± 0.03%) observed in seed extract, while the lowest (45.3 ± 1.1%) was in pulp fraction. Malondialdehyde (MDA) is a by-product of peroxidation of phospholipids and is generally regarded as a marker for oxidative stresses, rendering its determination in biological samples particularly interesting (Rodríguez et al., 2006). The MDA produced from haemoglobin oxidation treated with seed extract was lower than pulp and peel extract at 10 mg/l. The MDA levels of H2O2-induced haemoglobin oxidation treated with different concentrations (5, 10, and 15 mg/l) of the sample extracts decreased compared to H2O2-induced haemoglobin alone. However, all the extracts had protective effect against H2O2-induced oxidation only at lower concentration. A similar finding was reported by Polyakov, Leshina, Konovalova, and Kispret, 2001, and also by Sthal and Sies (2003), where at higher concentrations carotenoids had a pro-oxidant effect. Recently, we have observed pro-oxidant activity of carotenoids at high concentrations (2 ppm), while antioxidant activity was noticed at 1 ppm concentration using haemoglobin oxidation assay (unpublished data). Carotenoids were effective inhibitors of lipid peroxidation, as measured by MDA formation, by blocking the auto-oxidation of liposomes (Krinsky, 1989). 4. Conclusions This study has examined the carotenoid content and antioxidant capacity of C. odontophyllum peel, pulp and seed extracts. Peel and pulp extracts exhibited high concentration of carotenoids with acceptable level of antioxidant capacity in the studied assays. The evidence from this study suggests C. odontophyllum fruit can be used as a natural antioxidant and also used in production of functional food. This is the first report about existence of carotenoids from C. odontophyllum. Further investigations are needed to determine polyphenolic compounds from this fruit and to determine their antioxidant activities. Acknowledgements We would like to acknowledge Agriculture Research Centre at the Department of Agriculture, Sarawak, Malaysia for providing funding under consultant project on ‘‘Foodcomponents and antiox-
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idant properties of Canarium odontophyllum Miq. fruit”. The authors are grateful to the Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia for laboratory facilities.
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