Herbs: Challenges in Chemistry and Biology - ACS Publications

Chapter 17

Moringa, a Novel Plant Rich in Antioxidants, Bioavailable Iron, and Nutrients 1,3

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Ray-Yu Y a n g , Samson C. S. Tsou , Tung-Ching Lee , Leing-Chung Chang , George K u o , and Po-Yong L a i 1

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Downloaded by CORNELL UNIV on July 6, 2012 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch017

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AVRDC, The World Vegetable Center, Shanhua, Tainan, Taiwan, Republic of China Department of Food Science and Center for Advanced Food Technology, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901 Institute of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan, Republic of China

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A strong food-based approach is critical to alleviate nutritional deficiencies in the tropics. Our survey of over 120 species of Asian indigenous vegetables for nutrient contents, antioxidant activities (AOA), and indigenous knowledge of their medicinal uses indicated that Moringa oleifera was among the most promising species. We conducted additional studies to evaluate four Moringa species for A O A , antioxidant contents and nutritional quality, and to investigate M. oleifera's A O A and iron as affected by freezing, boiling, and in vitro digestion. We concluded that the four Moringa species are high in A O A , antioxidant and nutrient contents, low in oxalate content. Boiling in water enhanced aqueous A O A , and the A O A was maintained after simulated digestion. Cooking Moringa increased available iron and raised total available iron o f mixtures with mungbean. Moringa, an easily grown perennial, have tremendous potential to improve diets and health.

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© 2006 American Chemical Society

In Herbs: Challenges in Chemistry and Biology; Wang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


225 Moringa oleifera (L.) is a perennial tree that is widely grown in the tropics (7). Its foliage is consumed as a vegetable (2,3) and various parts of the tree can be used for industrial oil, medicinal preparations (4), and water purification (5). Our survey of over 120 species of Asian indigenous vegetables for nutrient contents, antioxidant activities (AOA), and indigenous knowledge of their medicinal uses indicated that Moringa oleifera was among the most promising species (6). The property of high A O A for Moringa was also reported in other studies (7-9). The multiple attributes of Moringa have tremendous potential to improve nutrition and health for the developing world. The wide-spectrum nutrients and phytochemicals it contains are natural, acceptable, affordable, and accessible. Leaves harvested from kitchen gardens and school gardens can be sold in local markets, dried leaves can be stored and taken as supplements during periods of low vegetable and fruit availability and under-nutrition status (10,11). M. oleifera gains more research attentions recently. However, M. oleifera is only one of 13 currently known species of Moringa (4). Little is known about the nutrient content, A O A and beneficial properties of the other Moringa species. Moringas are clustered into three species groups according to their morphological differences. The first group including four species are slender trees and are principally Asian; the second group including three species are the largest Moringas with the trunks shaped like bottles or elephant feet; the third group including 6 species are tuberous shrubs trees and are confined in a few African countries (4). In our study we explored the A O A and nutrition potential of four Moringa species: M. oleifera and M. peregrina of group 1, and M. stenopetala and M. drouhardii of group 2. M. stenopetala is the most economically important species after M. oleifera; Among Moringas, M. drouhardii has the most pungent odor similar to mustard oil; and M. peregrina has the widest habitat range and the only one of the slender trees extended out of Asia (4). The objectives of this study were to (1) study the variation of A O A and nutrient contents among the four Moringa species; (2) investigate the contribution of antioxidants including vitamin A , C, Ε and phenolics to A O A ; (3) examine the effects of various temperatures and simulated digestion on A O A and available iron of M. oleifera leaves.

Materials and Methods Plant materials and preparation. Seeds of the four Moringa species (Table I) were sown in November 2000 and transplanted to an A V R D C field in April 2001. One to two kg of leaflets from each tree were sampled during 2003 - 2004. No senescing leaflets were included in the study. Fresh leaflets extracted in water or methanol were stored at -70 °C and subjected to A O A analyses. Dried leaflet


226 samples were analyzed for dry matter, protein, calcium and iron contents. Frozen leaflets at -70 °C were used for measuring vitamin antioxidant contents and other nutritional components.

Table I. Four Moringa Species Used in This Study Species


oleifera

Plant Age 3yr

peregrina 3 yr stenopetala 3yr drouhardii 3yr

Leaf, seed Leaf, Leaf, Leaf,

Origin

Group

Part stem,

Slender tree

India

stem stem stem

Slender tree Bottle tree Bottle tree

Arabia, Red Sea area Kenya, Ethiopia Madagascar

AOA methods. The A B T S / H 0 / H R P decoloration method was carried out as described in Arnao et al. (72) with some modifications (75). The capacity of different components to scavenge the A B T S radical cation ( A B T S ) was compared to a standard antioxidant Trolox ( 0 - 4 mM) in a dose response curve. The D P P H assay was performed as described in Goupy et al. (14) with some modifications (75). The radical form of D P P H has an absorbance at 517 nm, which disappears upon reduction by H ' pulled from antioxidant compounds. The SOS was measured by the X A / X O D system as described by Murakami et al (15). Superoxide is generated from the oxidation of xanthine to uric acid by xanthine oxidase. It reacts with N B T to cause a color change from light yellow to dark purple, which can be measured at 560 nm. The A O A against lipid peroxidation by ferric thiocyanate method was tested using linoleic acid and the free radical generator A A P H (16). 2

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AO contents. Total soluble phenolics were extracted with methanol from frozen samples and determined using Folin-Ciocalteu reagent (77). The determination of total ascorbic acid is on the basis of coupling 2,4dinitrophenylhydrazine (DNPH) with the ketonic groups of dehydroascorbic acid through the oxidation of ascorbic acid by 2,6-dichlorophenolindophenol (DCPIP) to form yellow-orange color in acidic condition (18). Contents of β-Carotene and α-tocopherols were measured by H P L C as described in Hanson et al. (13). Nutritional quality. The protein content was measured with micro-Kjeldahl digestion followed by distillation method (19). The determination of calcium and iron contents were performed by ashing procedure, strong acid washing and then detected with Atomic Absorption Spectroscopy (19). The contents of oligo saccharides and oxalate were determined using H P L C method. Simulated digestion. The in vitro assay, described by Yang et al. (20) involves simulated gastrointestinal digestion using commercial available


227 enzymes including pepsin and pancreatine, with subsequent measurement of the soluble/permeable iron or antioxidant molecules released by the digestion.

Results and Discussion


A O A of the Four Moringa Species Ranges of AOA. Moringa leaves showed high antioxidant capacity in all four of the antioxidative mechanisms: scavenging of superoxide, A B T S and D P P H radicals and inhibition of lipid peroxidation. The A O A values were the lowest for ILPm (52 TE/g) and highest for A B T S m (411 TE/g) on a dry weight basis, which was around 1,300 - 10,280 TE/100g on a fresh weight basis (Figure 1). These values were I X - 5X higher compared to O R A C values based on FW of selected high A O A vegetables and fruits including kale (1,770 TE/100g), spinach (1,260 TE/100g), prunes (5,770 TE/100g), raisins (2,830 TE/100g) and blueberries (2,400 TE/100g) (21). Ranking by averaged AOA. Among the four species, M. peregina had the highest A O A values by all the A O A assays except ILPw. The ranking of the four species averaged over A O A (TE/g DW) was: peregina (234) >stenopetala (121)> oleifera (90)= drouhardii (90). Although drouhardii ranked the last with M. oleifera, it showed the second highest value of SOSw next to peregina. AOA methods and W/M extractions. Among the four species, the uppermost two A O A values were found in the methanol extract of peregina by A B T S and D P P H methods, which were about 2.1 times higher compared to water extractable A O A . However, ILP and SOS methods measured higher activities from water extracts. The top ILPw and SOSw values were found in stenopetala and peregina, respectively. These results indicated that water extracted antioxidants of Moringa were superior to methanol extracted antioxidants in superoxide scavenging and inhibition of lipid peroxidation, whereas methanol extracted antioxidants exerted stronger capacity in scavenging of A B T S radical. Compositions of W/M extracts. Our previous unpublished study on composition of water and methanol extracts of Chinese cedar (Toona sinensis) indicated that methanol extract contained four main phytochemical categories based on H P L C peak area and U V - V I S spectra: hydrophilic phenolics (such as gallic acids and chlorogenic acids), less hydrophilic phenolics (such as flavonoids and their glycosides), xanthophylls (such as lutein) and chlorophylls; and water extract contained three main clusters: ascorbate, hydrophilic phenolics, and less hydrophilic phenolics. Despite the high solubility of ascorbate in methanol, the crude methanol extract only contained about 10% of total ascorbate, which was confirmed by using colorimetric methods to measure


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ascorbate content of water and methanol extracts of Chinese cedar and sweet pepper. Tocopherols were determined from methanol extracts; however, they were not distinguished in the H P L C profile due to their conjugation with fatty acids in general and lower density compared to phenolics. The peak area of less hydrophilic phenolics in water extract was about 10% of that in methanol extract which indicated that a great portion of "less-hydrophilic phenolics" were not water extractable, and thus did not contribute to water extractable A O A .

Figure 1. AOA of water and methanol extracts offour Moringa species by four AOA methods. AOA methods followed by subscripts w or m refer to water or methanol extracts, respectively.

Antioxidant Contents of Moringa Content ranges. Concentrations of four natural antioxidants (total phenolics and antioxidant vitamins A , C and E) were measured for the four species. The content ranges on a dry weight basis were 7 4 - 2 1 0 μηιοΐ/g for phenolics, 70 - 100 μπιοΐ/g for ascorbate, 1.1 - 2.8 μπιοΐ/g for β-carotene and 0.7 - 1.1 μιηοΙ/g for α-tocopherol (Figure 2). Antioxidant content of Moringas are high even compared to vegetables and fruits known for high antioxidant contents such as strawberries high in phenolics (330 mg gallic acid equivalent (GAE)/100 g FW, or 190 μηιοί G A E / g D W ) (22); hot pepper high in ascorbate (200 mg/100 g FW, or 110 μηιοΐ/g DW), carrot high in β-carotene (10 mg/100 g FW, or 1.8 μπιοΙ/g DW) and soybean which is high in α-tocopherol (0.85


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mg/100 g F W , or 1.8 μιηοΐ/g DW) (23). Moringas are an excellent source of a wide spectrum of dietary antioxidants.

Figure 2. Antioxidant contents of the four Moringa species

Contribution of AO to AOA The concentrations of phenolics and ascorbate were about 25 - 300 times greater than the α-tocopherol and β-carotene contents. From the density point of view, phenolics and ascorbate were the dominant antioxidants in Moringa and they were principally contributive to A O A in both water and methanol extracts. However, only phenolics content showed a linear correlation with methanol extractable A O A (Figure 3). Ascorbate content was independent of either water or methanol extracted A O A . These results suggest that A O A of methanol extracts could be inferred or estimated from phenolics contents, but not from ascorbate content. This conclusion was supported by the evidence that only 10% of ascorbate content was detected in methanol extract. The aqueous phase may contain enzymes and other bioactive molecules, in addition to ascorbate and phenolics, and that may have caused synergisms and/or antagonisms among antioxidants that did not allow prediction of A O A simply by the summing the individual antioxidant contents. However, the crude water extracts of vegetables are more like the food matrix and the actual environments for food intake compared to methanol extracts.



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Herbs: Challenges in Chemistry and Biology - ACS Publications

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