Plant Foods Hum Nutr (2016) 71:204–210 DOI 10.1007/s11130-016-0548-3
ORIGINAL PAPER
Comparison of Phytochemicals and Antioxidant Capacity in Three Bean Varieties Grown in Central Malawi Gong-Jian Fan 1,2 & Victoria U. Ndolo 1 & Mangani Katundu 3 & Rachel Bezner Kerr 4,5 & Susan Arntfield 1 & Trust Beta 1,6
Published online: 17 May 2016 # Springer Science+Business Media New York 2016
Abstract The aims of the current work were: (1) to study the influence of variety and geographical production area on the total phenolic content, total anthocyanin content, total flavonoid content, total carotenoid content and antioxidant activity in bean varieties (Dimeta, Napirira and Nanyati) from different growing areas in central Malawi, and (2) to evaluate the possibility of establishing a classification based on the geographical areas of the growing regions. A total of 47 bean samples were collected from Makowe, Mphathi, ChumaChitsala and Khulungira Zone. These four locations were segregated based on altitude, latitude and longitude. Principal component and hierarchical cluster analysis were used to distinguish and classify among these samples. Significant differences (P < 0.05) in total phenolic content (2.92–4.97 mg/g), total anthocyanin content (14.52–152.31 μg/g), total flavonoid content (2.01–6.38 mg/g) and oxygen radical absorbance capacity (16.75–24.51 μmol/g) were found among the different sampled villages, showing a significant effect of the
* Trust Beta
[email protected] 1
Department of Food Science, University of Manitoba, Winnipeg, MB, Canada
2
College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing, Jiangsu, China
3
Department of Human Ecology, Faculty of Science, University of Malawi, Chancellor College, Zomba, Malawi
4
Department of Development Sociology, Cornell University, Ithaca, NY, USA
5
Department of Geography, University of Western Ontario, London, ON, Canada
6
Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
producing region on these parameters. The beans in Makowe had lower polyphenols than in other locations. Results of principal component analysis indicate that phytochemicals and antioxidant capacity could serve as parameters to establish a bean classification according to the geographical area of production. Keywords Bean varieties . Phytochemicals . Antioxidant capacity . Principal component analysis . Hierarchical cluster analysis
Introduction Horticulture concerned with plants that are used by people for food, either as edible products, or for culinary ingredients, for medicinal use or ornamental and aesthetic purposes. They are genetically very diverse group and play a major role in modern society end economy. Fruits and vegetables are an important component of traditional food, but are also central to healthy diets of modern urban population [1, 2]. Common beans (Phaseolus vulgaris L.) are a good source of phenolic compounds and other phytochemicals. Health benefits of legumes are related to the dietary fiber, phenolic compounds, saponins, phytosterols, proteins and peptides [3]. Phenolic compounds in common beans are found in the seed coats with lower amounts in the cotyledons. The antioxidant capacity of plant foods is derived from the cumulative synergistic action of a wide variety of antioxidants such as vitamins C and E and polyphenols, mainly phenolic acids and flavonoids, carotenoids, terpenoids, maillard compounds and trace minerals. The content of phytochemicals depends both quantitatively and qualitatively on plant genotype and on environmental factors including water and mineral nutrition [4, 5]. These phenomena have been observed in other food products such as pear and
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cherry wine, in which the chemical composition is influenced by changes in the environmental conditions [6, 7]. Nonetheless, to date, the ideal geographical climate conditions for the production of high quality common beans, in terms of chemical composition and organoleptic properties remain unknown. Principal component analysis works based on transformation of the original variables into new axes or principal components. It is possible to express maximum total variation in data set in only a few principal components and represent the decrease of the variation [8]. Hierarchical cluster analysis is also very useful to cluster food samples regarding dissimilarities without considering information about the class membership [7]. The objectives of the current work were to study the influence of variety and geographical production area on the total phenolic, total anthocyanin, total flavonoid, total carotenoid content and the antioxidant activity of whole beans (with the seed coats and the cotyledons) in three local landrace bean varieties from different growing areas in central Malawi, and to evaluate the possibility of establishing a classification based on the geographical areas of the growing regions. This study is part of a broader research project on agroecological methods to improve food security and nutrition for smallholder farm households in Malawi.
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Results were expressed as milligrams of gallic acid equivalent per gram of bean flour. Total flavonoid content (TFC) was determined as described by Biney and Beta [10] with some modifications. Results were expressed as milligrams rutin equivalent per gram of bean flour. Total anthocyanin content (TAC) was assayed by using spectrophotometry method [11]. The anthocyanin content was estimated as cyanidin-3-glucoside at 530 nm using a molar absorptivity coefficient of 26,900 and was expressed as microgram per 1 kg of dry weight. Total carotenoid content (TCC) was extracted according to the method as described by Ndolo and Beta [12] with some modifications. Briefly, 200 mg of samples were mixed with 2 mL of water-saturated butanol in tubes. All manipulations were carried out under dim light to avoid light-induced changes. Total carotenoid content was calculated using the following equation and expressed as microgram lutein equivalent per gram of bean flour. The antioxidant activity was evaluated by using the oxygen radical absorbance capacity (ORAC) method as previously reported [13]. Determinations were carried out in triplicate and results were expressed as μmol of Trolox equivalents per gram of bean flour. Statistical Analysis
Materials and Methods Materials A total of 47 bean samples (Phaseolus vulgaris L.) were collected from central Malawi in 2014 (Table 1). These samples are classified into three bean varieties, Dimeta (16), Napirira (18) and Nanyati (13). The dried beans were subsequently milled into powder with an electric mill, sieved (40 meshes) and stored at 4 °C prior to extraction. All chemicals and reagents used in the experiment were of analytical grade purchased from Sigma-Aldrich Company and Fisher Scientific Company. Chemical Analysis The extraction of antioxidant compounds from 47 bean samples were done as described by Limon et al. [9], with slight modifications. To 0.500 g of sample, 5 mL of acidified methanol (HCl : methanol : water, 1 : 80 : 20) was added in 10 ml centrifuge tubes and shaken at room temperature for 2 h using a horizontal rotary shaker. The mixture was then centrifuged at 5 °C for 10 min at 10,000 rpm and the supernatant collected and used for total phenolic content, total flavonoid content and oxygen radical absorbance capacity. Total phenolic content (TPC) was determined by the FolinCiocalteu colorimetric method with minor modification.
All tests were performed in triplicate. Data are presented as means ± SD. The statistical significance of differences between groups was evaluated by one-way analysis of variance. To classify and discriminate between the bean samples, principal component analysis and hierarchical cluster analysis was performed with SPSS for Windows version 20.0 (SPSS, Chicago, IL, USA).
Results and Discussion Total Phenolic Content TPC ranged from 3.14 to 3.86 mg/g, 3.56 to 4.79 mg/g to 2.92 to 4.18 mg/g, respectively (Fig. 1). Some studies reported higher and lower concentrations of total phenolic compounds in different kinds of beans [14, 15]. In addition, TPC was significantly lower in Makowe zone than in other three locations. These results were in accordance with the findings of Hedychium spicatum [16]. Total Flavonoid Content Napirira bean and Dimeta bean presented a higher TFC ranging from 4.25 to 6.38 mg/g and 3.87 to 5.51 mg/g, while Nanyati bean had a lower content ranging from 2.05 to
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Plant Foods Hum Nutr (2016) 71:204–210 Bean producer and geographic location from different bean-growing regions
Table 1 Zone
Village
Makowe
Mphathi
Chuma-Chitsala
Khulungira
Code
Latitude
Longitude
Altitude
Variety
Kainga
A1
S 14°22.945
E 034°04.266
1209 m
Dimeta, Napirira
Kaunyolo
A3
S 14°24.955
E 034°03.874
1270 m
Dimeta, Nanyati
Gaga Khomani
A4 A6
S 14°25.412 S 14°24.926
E 034°03.430 E 034°04.480
1299 m 1257 m
Nanyati Nanyati
Manase Kamala
A8 A9
S 14°24.609 S 14°24.156
E 034°03.500 E 034°04.157
1291 m 1285 m
Dimeta, Napirira Dimeta
Nsolo
A10
S 14°24.008
E 034°03.731
1247 m
Dimeta
Mchenga Kalumba
B1 B2
S 14°27.489 S 14°27.357
E 034°03.341 E 034°04.950
1300 m 1320 m
Dimeta Napirira
Dzinyemba Bisimeni
B3 B5
S 14°27.100 S 14°26.197
E 034°04.912 E 034°04.773
1315 m 1291 m
Napirira, Nanyati Nanyati
Uzayo
B7
S 14°27.592
E 034°03.451
1296 m
Dimeta, Nanyati
Majamanda Mabeka
B8 B9
S 14°27.679 S 14°28.052
E 034°03.480 E 034°03.337
1295 m 1301 m
Nanyati Napirira, Nanyati
Mkakaula Jonasi
B12 B13
S 14°27.308 S 14°28.311
E 034°04.139 E 034°04.862
1306 m 1329 m
Dimeta, Napirira, Nanyati Dimeta, Napirira
Chuma-chitsala
C3
S 14°23.341
E 034°08.535
1359 m
Napirira
Gunduze Kamenya Langwani Litcheto
C5 C6 C7 C8
S 14°21.828 S 14°22.816 S 14°20.907 S 14°21.570
E 034°07.495 E 034°08.010 E 034°07.986 E 034°21.570
1343 1379 1319 1205
m m m m
Nanyati Dimeta, Napirira Dimeta Napirira
Mtengowangwa Mkokamasa Chigona Gumbo Khulungira1
C10 C11 D1 D2 D4
S 14°22.564 S 14°23.261 S 14°23.962 S 14°24.635 S 14°24.590
E 034°08.009 E 034°08.160 E 034°11.144 E 034°10.278 E 034°08.876
1373 1370 1418 1405 1370
m m m m m
Napirira, Nanyati Dimeta, Napirira, Nanyati Dimeta Napirira Dimeta, Napirira
Khulungira 2 Manyenje Mfutso 2 Sefasi
D5 D6 D7 D8
S 14°23.958 S 14°24.982 S 14°23.904 S 14°24.619
E 034°08.699 E 034°10.910 E 034°09.381 E 034°10.042
1377 1439 1366 1401
m m m m
Napirira Dimeta, Napirira Napirira, Nanyati Dimeta, Napirira
Bean samples were distributed into four clusters of 30 villages: a Makowe Zone (sample number = 10), b Mphathi Zone (sample number = 15), c ChumaChitsala Zone (sample number = 11) and d Khulungira Zone (sample number = 11)
Makowe Chuma-Chitsala
Total phenolic content (mg/g)
6 5
a a
4
a a
b
ab
Mphathi Zone Khulungira a
b
b
ab ab a
3 2 1
pinto beans, black-eyed peas and soybeans ranging from 0.6 to 1.0 mg/g [17]. In addition, the three bean varieties had 8 Total flavonoids content (mg/g)
2.92 mg/g (Fig. 2). However, the three bean varieties in central Malawi were rich in TFC compared with red kidney beans,
6 b
Makowe Mphathi Zone Chuma-Chitsala Khulungira a a a a a a b
4 b
ab
a ab
2
0
0 Dimeta
Napirira
Nanyati
Fig. 1 Effect of variety and location on total phenolic content
Dimeta
Napirira
Nanyati
Fig. 2 Effect of variety and location on total flavonoid content
Plant Foods Hum Nutr (2016) 71:204–210
150 b
120
b
90
c
60 30
50
Mphathi Khulungira a
oxygen radical absorbance capacity(µmol/g)
Makowe Chuma-Chitsala
180 Total anthcoyanin content (mg/kg)
207
b a b b
a a a a
Makowe Chuma-Chitsala
Mphathi Zone Khulungira
40 a a
a a
30 b
ab
a
b
b
abab a
20 10 0
0 Dimeta
Napirira
Dimeta
Nanyati
Napirira
Nanyati
Fig. 3 Effect of variety and location on total anthocyanin content
Fig. 5 Effect of variety and location on oxygen radical absorbance capacity
significantly lower TFC in Makowe which has the lowest altitudes among the four locations. This was in agreement with earlier reports which stated that the flavonol concentrations of V. uliginosum berries increased with rising altitudes in those locations [18].
Oxygen Radical Absorbance Capacity
In these three bean varieties, TAC ranged from 14.52 to 31.22 mg/kg, 67.67 to 152.31 mg/kg and 32.88 to 47.70 mg/ kg, respectively (Fig. 3). Napirira bean has a higher TAC than Dimeta bean and Nanyati bean. Statistical analyses showed that Napirira bean had a significant difference (p < 0.05) in TAC among the four locations. The highest TAC was detected in Khulungira zone, which has a higher altitude. Some research suggested that TAC in grapes rise with increased altitude, distance from sea from east to west [19]. Total Carotenoid Content TCC of three varieties of bean is shown in Fig. 4. TCC ranged from 0.17 to 0.32 μg/kg in Dimeta bean, 0.20 to 0.39 μg/kg in Napirira bean and 0.13 to 0.23 μg/kg in Nanyati bean. TCC in the four locations increased with the altitude rising. Similar results were also reported in the fresh K. littledalei [20] and Polylepis tarapacana (Rosaceae) [21].
Total carotenoid content (µg/kg)
0.5
Makowe Chuma-Chitsala
0.4
a a
0.3 d
c
a
Mphathi Khulungira
b
b
a
c
0.2
ab ab
b
0.1
Principal Component Analysis Principal component analysis was applied in order to evaluate the data of total phenolic, total anthocyanin, total flavonoid, total carotenoid content and the antioxidant activity (Fig. 6). Principal component 1 explained up to 43.73 % of total variance and Principal component 2 explained 42.7 %, totaling 85.91 %. Samples were separated along the first principal 3 Dimeta D4
Nanyati
D2 C8
C3 C6 D5 A1 D61 B5 A10 B3 B13 A3 A4 C11 B7 A8 C11 B9 D8 A6 C10 A8 B9 0 A1 D1 0 D4 -1 1 A9 B1 B12 C6 C11 B13 C10 -1 B7 C5 B12 D7 B8 B12 -2 B3
A3
-2
C7
0.0 Dimeta
Napirira
Nanyati
Fig. 4 Effect of variety and location on total carotenoid content
Napirira
2
Second princilpal compement
Total Anthocyanin Content
These three bean varieties had higher ORAC values ranging from 19.88 to 28.68 μmol/g, 20.95 to 35.77 μmol/g and 18.14 to 30.54 μmol/g (Fig. 5). Statistical analysis showed significant differences among the four locations. The bean in Makowe zone had significantly lower ORAC. Malik et al. [22] suggested that Mentha species raised at higher altitude had much higher content of total phenols and flavonoids as well as antioxidant potential than the respective species raised in plains of Punjab during same period. These findings are in agreement with our results.
D7 D6 2
B2
-3 First princilpal compement
Fig. 6 Principal component analysis scores for three varieties beans
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Fig. 7 Dendrogram for bean samples obtained from hierarchical cluster analysis
component by differences observed in TAC, TFC and TCC. The second principal component classified the samples related to ORAC and TPC. Although principal component analysis itself cannot be used as a classification tool, it may indicate a trend that is relevant for visualizing the dimension space. The initial two factors, which were related to phytochemicals and antioxidant capacity and are indicated as positive or negative, are used to make the differentiation clearer. Dimeta bean and Nanyati bean grouped together, and Napirira bean varieties formed a group separate from the Dimeta and Nanyati. Hierarchical Cluster Analysis Hierarchical cluster analysis dendrogram is shown in Fig. 7, in which three clusters were suggested. The first group consisted of Napirira bean, which were all sourced from Mphathi Zone,
Chuma-Chitsala Zone and Khulungira Zone. The second group consisted of Dimeta bean and Nanyati bean, which were sourced from Mphathi Zone, Chuma-Chitsala Zone and Khulungira Zone. The third group consisted of all the three bean varieties, which were sourced from Makowe Zone and Mphathi Zone. This result suggests that variety had more influence on the phytochemicals and antioxidant capacity of beans than geographical production area. Kimura et al. [23] characterized onions produced from different varieties and cultivars. Their results suggested that the optimal usage of onions as processing ingredients varied with varieties and cultivars. In the dendrogram of hierarchical cluster analysis, all the Napirira bean except in Makowe zone were clustered away from other bean samples, visually representing the difference in the phytochemicals and antioxidant capacity. Overall, the 47 bean samples were successfully divided into
Plant Foods Hum Nutr (2016) 71:204–210
three categories based on our determination and chemometrics analysis.
209 6.
7.
Conclusion In conclusion, statistical analysis showed significant differences (p < 0.05) in TPC, TFC, TAC and TCC as well as ORAC of beans among the different sampled villages, showing a significant effect of the producing region on these parameters. Napirira bean had the highest TAC and TFC among the three bean varieties. The beans in Makowe Zone had lower polyphenols than in other locations. Results of Principal component and Hierarchical cluster analysis indicate that phytochemicals and antioxidant capacity could serve as parameters to establish a bean classification according to the geographical area of production. Acknowledgments This work was supported by the University of Ontario through the Global Affairs Canada-funded Malawi Farmer-toFarmer Agroecological (MAFFA) project and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Equipment used for analysis of phytochemicals and oxygen radical absorbance capacity assay was generously funded by the Canada Foundation for Innovation (New Opportunities Fund and Leaders Opportunities Fund).
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14. Conflict of Interest The authors declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects.
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