Life Sciences,Vol. 63, No. 2, m.

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USE OF THE TROLOX ASSAY TO ESTIMATE THEi ANTIOXIDANT

CONTENT OF SEVENTEEN EDIBLE WILD PLANTS OF NIGER

J.A. Cook’, D.J. VanderJagt’, A. Dasgupta’, G. Mounkaila3, R.S. Glew4, W. Blackwell’,

and R.H. Glew’. 5

‘Department of Biochemistry and Molecular Biology, University of New Mexico School of

Medicine, Albuquerque, New Mexico, U.S.A.
*Department of Pathology, University of New Mexico School of Medicine, Albuquerque,
New Mexico, U.S.A.
3Division of Life Sciences, University of Niamey, Niamey, Republic of Niger.
4Department of Anthropology, Michigan State University, East Lansing, Michigan, U.S.A

(Received in tinal form Aprd 17,1998)

Summary

Although wild edible plants of the western Sahel and other parts of sub-Saharan
Africa are consumed to some extent at all times of the year, greater amounts are
consumed when cereal harvests are insu&ient to support the populations living in
these areas. The purpose of this study was to use a recently reported Trolox-based
assay to measure the total a&oxidant capacity of aqueous extracts of 17 plants that
we gathered from southern Niger. The antioxidant contents of the aqueous extracts
were compared to those of spinach and potato. Of the 17 plants, 11 had a greater
antioxidant content than spinach and 14 had a greater antioxidant content than
potato. The leaves of Tqinanth globiferus had the greatest antioxidant content,
and the fruit of Padari macrophylla had the lowest. In general, leaves contained
more antioxidants than either fiuits or seeds. The total antioxidant capacity of the
aqueous extracts was relatively high, indicating that the wild plants of the western
Sahel may contain substantial amounts of water-soluble flavonoid glywsides, which
are potent antioxidants and have been shown to have anticancer properties.

Key Word: antioxidant, Trolox assay, wild edible plants, Niier

‘To whom all wrrespondence should be addressed: Robert H. Glew, PhD, Department of
Biochemistry and Molecular Biology, Room 249 BMSB, University of New Mexico School of
Medicine, Albuquerque, NM 87131-5221. Telephone: (505) 272-2362; FAX: (50.5) 272-6587;
e-mail: rglew@salud.unm.edu
106 Antioxidants in Sahelian Edible Wild Plants Vol. 63, No. 2, 1938

Although wild edible plants, commonly referred to as ‘famine foods’, are consumed to some extent at
all times of the year by people who inhabit the western Sahel and other parts of sub-Saharan Africa,
greater amounts are consumed when the harvest of staple cereals is insufficient to support those
populations. These plant foods include primarily the leaves, fruits, flowers, and seeds of spontaneous
trees and shrubs. A number of studies (I-5) have reported the content of particular essential nutrients
in these uncultivated plant foods of the western Sahel, including essential amino acids, fatty acids,
metals and trace minerals, ascorbic acid and fat-soluble substances, such as vitamin E and P-carotene,
the precursor to vitamin A.

In addition to the need to learn more about the content of specific nutrients in famine foods, it would
be advantageous to be able to assess certain aggregate properties of these plants. For example, in
light of the widely recognized importance of free radicals and reduced oxygen metabolites (e.g.,
superoxide anion, hydrogen peroxide, hydroxy free radical) as mediators of cellular and tissue
damage in many diseases, there is interest in knowing the overall fimctional antioxidant content of
edible plants. Apart from the presence of well-established antioxidants such as ascorbic acid and
vitamin E, plants also contain phenolic compounds, such as flavonoids, which can neutralize oxidizing
agents generated during normal metabolism in humans (6-S). There is considerable medical interest in
the flavonoid content of diets because of the growing body of evidence which indicates that the
antioxidant property of such substances may explain, in large part, their apparent cardioprotective and
antitumor properties (9,lO). We therefore sought an assay that might allow us to compare the total
antioxidant content of the many different wild plant foods from the western Sahel that we have been
studying (11,12). Ideally, such an assay should be relatively inexpensive and simple to perform, and
one that might be conducted using instruments that are likely to be available in lesser developed
regions of the world where this type of analysis would be particularly uset%l.

Recently, a two stage Trolox-based assay was described which allows one to compare the total
antioxidant capacity of different serum specimens by assessing the ability of one or more antioxidants
in the serum to quench a limiting and fixed quantity of oxidant (13, 14). Trolox is an a-tocopherol
analogue with enhanced water solubility. In order to determine if this assay could be applied to
aqueous extracts of wild plant foods of the western Sahel, we prepared water extracts of 17 famine
foods that grow in Niger and used the Trolox assay to compare their relative content of water-soluble
antioxidants.

Methods

Samples of 17 plant foods were collected in March and July, 1996 from various markets in southern
Niger, the area from Niamey to Zinder. Scientific and local (Hausa) names were obtained by two
members of the research team (RSG and GM, an agronomist in Niger), and were continned with a
plant name index (15). One of the plant specimens, Cupsiam up., is not indigenous to the area (16)
but does grow wild in the Sahel. The samples were air dried immediately following field collection
and transported to the United States in zip-lot” bags. In addition, in order to provide reference
points for comparison purposes, we analyzed similar extracts of spinach and potato for their
antioxidant capabilities. Spinach leaves were purchased fresh, at a grocery in Albuquerque, NM,
washed with water, and then dried in a vacuum at 25°C. The sample of potato was purchased in
dehydrated (flake) form and dried in the same manner.

Samples were ground to a fine powder and dried to constant weight in a vacuum at 25°C. Portions
(approximately 0.5 g) were weighed, resuspended in distilled water (8 ml water/g dry wt) and
vortexed vigorously and repeatedly. Samples were allowed to sit capped for 2 days at 4”C, after
Vol. 63, No. 2, 1998 Antioxidants in Sahelian Edible Wild Plants 107

which they were centrifuged at 3500 rpm at 1500 G’s for 20 min to remove large particles and then
again at 14,000 rpm for 5 min to obtain a clear supematant. A 6 pL aliquot of the resulting
supematant was subjected to analysis. The assay kit was purchased from Randox Laboratories
(Antrim, UK), and the assay was performed using a Syva-30R automated analyzer (Behring, San
Jose, CA). A two-point calibration was used as recommended by the manufacturer. In the first
stage of the assay, peroxidase (metmyoglobin) was reacted with hydrogen peroxide to produce
ferrimyoglobin, a free radical. Ferrimyoglobin was then incubated with a chromagen, 2,2’-
amino-di-(3-ethylbenzthiazole sophonate), to produce ATBSR’, a radical cation with a blue-
green color measured at 604 nm. Antioxidants in the added plant extract suppressed the blue-
green color to a degree that was proportional to their concentration. The absorbance of the
resulting oxidized solution was compared to that of the calibration standard, Trolox (6-hydroxy-
2,5,7-tetramethylchroman-2-carboxylic acid), and was inversely proportional to the amount of
antioxidant in the aqueous extract. Results were expressed in terms of Trolox equivalent
antioxidant capacity (TEAC) per total antioxidant unit (mm01 Trolox/ g dry wt). Analyses were
performed in duplicate and mean values are reported.

Results

In order to provide a point of reference using plants familiar in Europe and North America, we first
determined the antioxidant content of aqueous extracts of spinach and potato. We found that spinach
had an antioxidant capacity of 14.3 umol Trolox equivalent/ g dry wt, and potato contained 7.1 umol
Trolox equivalent/ g dry wt.

TABLE 1

Latin Names, Local Names, and Collection Areas of the Plant Species Analyzed in this Report

Scientific Name Local Name Plant Part Collection Site

Adansonia digitata dan kuka leaves Zinder (h4iria market)

Balanites aegyptiaca aduwa fruits and seeds Zinder (Zinder market)
Bombax costatum kuriga fruits Zinder (Zinder market)
Boscia senegalensis anza leaves Chikal (Filingue)
Capsicum spp. tattasai fruits Maradi
Cyperus esculentus haya fruits Zinder (Miria market)
Entaak africana tawarsa leaves Zinder (field)
Gynanakopsis gynandra gasaya leaves Tanout (field)
Hibiscus esculentus kubewa seeds Maradi
Hyphane thebaica goriba fruits Zinder (Miria market)
Leptadenia hastata yadya leaves Loga
Parinari macrophylla gawasa fruits and shells Margo
Parkia biglobosa dorowa seeds Tillabery
Phoenix dactyI$era dabino fruits Zinder (Zinder market)
Sesbania pacycarpa tchetcheko leaves Tanout (field)
Tapinanthus globverus kawshyi leaves Tanout (field)
unknown ataruhu fruits Zinder (Miria market)
108 Antioxidants in Sabelian Edible Wind Plants Vol. 63, No. 2, 1998

With regard to the 17 edible plants that were collected in Niger (Table l), the antioxidant content
ranged from 3.9 - 26.3 umol Trolox equivalent/ g dry wt (Table 2). The leaves of Tapinanthus
globiferus had the greatest antioxidant content, followed by the shell of the fruit of Hyphnce
thebaicu (24.0 umol Trolox equivalent/ g dry wt). The fruit of Parinari macrophylk had the lowest
antioxidant content (3.9 pm01 Trolox equivalent/ g dry wt) of the plants included in this study. In
general, leaves contained more antioxidants than either fruits or seeds. The fiuit with the greatest
antioxidant capacity was Bombax costatum (23.0 umol Trolox equivalent/ g dry wt.). The aqueous
extracts of the two seeds that we analyzed, namely Hibiscus esculentus and Parkia biglobosa,
showed a significant difibrence in their content of antioxidants (21.6 and 9.3 umol Trolox equivalent/
g dry wt, respectively). Of the 17 plants from Niger that we analyzed, 11 showed greater antioxidant
capacity than spinach and 14 showed greater antioxidant capacity than potato. The shell of the fruit
of Hyphaene thebaica had a greater antioxidant content than both spinach and potato, but the 6uit of
Hrphaen thebaica had a lower antioxidant content than either spinach or potato. All of the
antioxidant values obtained were within the calibration limits of the assay.

TABLE 2

Comparison of the Total Antioxidant Capacity of Aqueous Extracts of Spinach,

Potato and 17 Wild Edible Plants from the Republic of Niger.

Plant Antioxidant Capacity

(u mol Trolox equivalent/ g dry wt)

Adansonia dip’tata 7.7

Ataruhu* 20.4
Balanites aegyptiaca (fruit and seed) 14.5
Balanites aegyptiaca (tiuit) 15.5
Bombax costatum 23.0
Boscia senegalensis 22.0
Capsicum spp. 22.3
Cyperus esculentus 5.3
Entada africana 20.4
Gynandropsis gwandra 23.1
Hibiscus esculentus 21.6
Hrphaene thebaica (fruit) 5.1
Hyphaene thebaica (shell) 24.0
Leptadenia hastata 19.1
Parinari macrophylla 3.9
Parkia biglobosa 9.3
Phoenix dactyrifera 8.2
Sesbania pachycarpa 22.0
Tapinanthus globryerus 26.3

spinach 14.3
potato 7.1

*No scientific name available

Vol. 63, No. 2, 1998 Antioxidants in Sahelian Edible Wild Plants 109

Discussion

The main finding of this study was that the content of antioxidant in all 17 of the edible Sahelian wild
plants we analyzd was relatively high. Furthermore, in this regard, they compared favorably with
spinach and potato. This tinding is significant in light of the fact that oxidative stress is a secondary
e%ct of many human diseases (17), and that consumption of antioxidant-contaming foods can
potentially reduce oxidative damage to cells and tissues of the body. In addition, malnutrition
depresses cellular immune function and consumption of these antioxidant-containing plants could
potentially protect the immune system of malnourished individuals. Further, HIV infection is
prevalent in Sub-Saharan A&a. Available data indicate that antioxidants are deficient in HIV
infected populations due to increased utilization of antioxidant micro-nutrients (18) and
observational studies suggest that increased intake of antioxidsnts may delay progression of HIV
infection to AIDS (19). Consequently, consumption of these edible plants with high levels of
antioxidants may delay the progression of HIV infection in populations where resources are scarce
and antiretroviral agents are not available for the treatment of HIV infection. However, although the
level of natural antioxidants in these plants appear to be high, we don’t know to which specific natural
antioxidant substances these values can be attributed. Possibilities include ascorbic acid and
flavonoids, both of which are water-soluble. Because similar plant foods have been shown to contain
flavonoid glycosides (20,21), it is reasonable to expect that the plants reported herein may also
contain substantial levels of these compounds.

Based on comparative TEAC values reported by Miller and colleagues for naturally occuning
antioxidants (13), one can calculate the ascorbic acid content of potato, for example, assuming that all
of the antioxidant in the water extract is ascorbic acid. When this is done, one arrives at a value of
120 mg ascorbic acid/ 100 g dry wt. The ascorbic acid content of potato prepared in a variety of
ways (22) ranges f?om 65 to 100 mg/ 100 g dry wt. Thus, it appears that approximately 213 of the
antioxidant in the potato is attributable to ascorbic acid.

Future experiments will be aimed at identi@ing the specific antioxidants in the famine foods of Niger
that are responsible for their relatively high antioxidant content. It would be interesting if such a
study reveals the presence of appreciable quantities of flavonoids, for flavonoids are potent
antioxidants and have been shown to have sign&ant health promoting properties including
cardioprotective, anti-cancer and bone-protective effects (g-10).

In summary, we have shown that the Trolox-based assay, developed originally for serum
specimens, can be applied to aqueous plant extracts and it is an effective and efficient method for
measming the overall antioxidant capacity of plant foods. The method is simple, inexpensive and
should be adaptable to laboratories in many parts of the world where agronomists and other plant
scientists would value having a procedure available that they could use to screen edible plants for
their relative antioxidant content.

Acknowledgements

This work was supported by a grant from the Fogarty International Center of the National Institutes
of Health.
110 Antioxidants in Sahelian Edible Wild Plants Vol. 63, No. 2, 1998

References

1. B. BECKER Agroforestry Systems 1257-267 (1983).

2. T.R. KlM, A. PASTUSZYN, D.J. VANDERJAGT, R.S. GLEW, M. MILLSON, and R.H.
GLEW, J. Food Comp. Anal 10 73-81(1997).
3. O.M. SALIH, A.M. NOUR, andD.B. HARPER, J. Sci. Food Agric. 57 367-377 (1991).
4. G.C. SMITH, M.S. CLEGG, C.L. KEEN, and L.E. GRIVETTI, Int J. Food Sci. Nutr. 47 41-
53 (1996).
5. G.C. SMITH, S.R. DUEKER A.J. CLIFFORD, and L.E. GRIVETTI, Ecol. Food Nutr. 30 l-
16 (1995).
6. N.P. DAS, Biochem. Pharmac. 20 3435-45 (1971).
7. B. HALLIWELL, Annu. Rev. Nutr. 16 33-50 (1996).
8. M.G.L. HERTOG, E.J.M. FESKENS,P.C.H. HOLLMAN,M.B. KATAN, andD.
KROMHOUT, LANCA 234 1007-l 1 (1993).
9. J. KANNER, E. FRANKEL, R. GRANIT, B. GERMAN, and J.E. KINSELLA J. Agric. Food
Chem. 42 64-69 (1994).
10. B. STAVRIC, Clin Biochem. 27 245-48 (1994).
11. R.H. GLEW, D.J. VANDERJAGT, C. LOCKETT, L.E. GRIVETTI, G.C. SMITH, A.
PASTUSZYN, and M. MILLSON, J. Food Comp. Anal. 10 in press. (1997).
12. D. YAZZIE, D.J. VANDERJAGT, A. PASTUSZYN, A. OKOLA, and R.H. GLEW, J. Food
Comp. Anal. 7 189-193 (1994).
13. N. J. MILLER C. RICE-EVANS, M.J. DAVIES, V. GOPINATHAN, and A. MILNERA,
Clinical Science 84 407-412 (1993).
14. A. DASGUPTA and C. MAHLE, Life Sci. 4 437-443 (1997).
15. R.K. BRUMMIT and C.E. POWELL (Eds.), Index Kewensis, Royal Botanic Gardens, Kew,
Oxford University Press, Oxford (1992).
16. STURTEVANT, E.L., Sturtevant ‘s Notes on Bible Plants, HEDRICK, U.P. (Ed.), J.B. Lyon
Company State Printers, Albany, New York (1919).
17. J.M.C. GUTTERIDGE, FreeRad. Res. Commun. 19 141-58 (1993).
18. B.M. DWORKIN, G.P. WORMSER, F. AXELROD, N. PIERRE, E. SCHWARZ, E.
SCHWARTZ, and T. SEATON, J. Parenter. Nutr. 14 605-609 (1990).
19. B. ABRAMS, D. DUNCAN, and I. HERTZ-PICCIOTTO, J. Acquir. Immune Defic. Syndr. 6
949-958 (1993).
20. G. BLOCK and K. REINLI, Nutr.Cancer 26 123-148 (1996)
21. J.KUHNAU,WldRev.Nutr. Diet, 24 117-191 (1976).
22. U.S. DEPARTMENT OF AGRICULTURE, Agriculture Handbook No. 8-l 1, Composition of
Fooak: Vegetables and Vegetable Products; Raw, Processed, Prepared, 294-299, 327.
Agricultural Research Service, Washington, DC. (1984)