Biosci. Biotech. Biochem.

, 57 (9), 1606-1608, 1993

Note
Screening for Antioxidative Activity of Edible Pulses
Takanori TSUDA, YUKA'MAKINO, * Hiroshi KATO, Toshihiko OSAWA, **.t and Shunro KAWAKISHI**
Food Research Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji.cho, Nishi-ku, Nagoya 451, Japan
* Japan Food Research Laboratories, Nagoya Branch, 4-5-13 Osu, Nakaku, Nagoya 460, Japan
** Department of Food Science and Technology, Nagoya University, Nagoya 464-01, Japan
Received April 6, 1993

Screening for anti oxidative activity of edible pulses was
investigated. Thirty-five species of pulses were extracted with
methanol, and an anti oxidative assay was done by the thiocyanate
and thiobarbituric acid (TBA) methods. Eleven species showed
strong activity. An ethyl acetate extract of tamarind also had
strong activity. The amounts of tocopherol were low in these
antioxidative pulses, suggesting that the antioxidants in these
pulses were not tocopherols.
Lipid peroxidation is a major factor in deterioration during the
storage and processing of foods. It is also thought to induce
physiological obstruction causing cell aging and carcinogenesis;
The addition of antioxidants has become popular as a means of
increasing the storage period of food products and improving
the stability of lipids and lipid-containing foods without loss of
sensory and nutritional qualities.
In the past effective synthetic antioxidants were used by the
food industry, but these days consumers, assuming that natural
compounds are safer, prefer natural antioxidants to synthetic
antioxidants. Hence, much research has been conducted to find
natural antioxidants with high antioxidative activity. 1 - 6)

Table I. Antioxidative Activity of 1.0mg and 0.2mg of Methanol Extracts from Edible Pulses as Measured by the Thiocyanate Method

Common name Scientific name 1.0mg 0.2mg

1. Azuki bean Vigna angularis (Willd.) Ohwi et Ohashi +++

2., Kidney bean (Honkintoki) Phaseolus vulgaris L. +++ ++
3. Kidney bean (Yamashirokurosando) Phaseolus vulgaris L. +++ ++
4. Kidney bean (Uzuramame) Phaseo/us vulgaris L. +++
5. Kidney bean (Taisyokintoki) Phaseolus vulgaris L. +++
6. Kidney bean (Ofukumame) Phaseolus vulgaris L. +++ +
7. Kidney bean (Toramame) Phaseo/us vulgaris L. +++
8. Kidney bean (Ohtebo) Phaseolus vulgariS L. +++ +
9. Black soy bean (Hikarikuro) Glycine max (L.) Mex:r. +++
10. Scarlet runner bean (M urasakihanamame) Phaseolous cosinneus L. +++
11. Scarlet runner bean (Shirohanamame) Phaseolous cosinneus L. +++ +
12. Lima bean Phaseolous lunatas L. +++
13. Green gram Vigna radiata (L.) Wilczek +++
14. Black matpe Vigna mungo (L.) Hepper +++
15. Rice bean Vigna umbellata (Thunb.) Ohwi et Ohashi +++
16. Cowpea (Kintokisasage) Vigna unguiculata (L.) Walp. var.. unguiculata (L.) Ohashi
17. Black eyes Vigna unguiculata (L.) Walp. var . .unguiculata (L.) Ohashi
18. Yard-long bean Vigna unguiculata ssp. sesquipeda/is (L.) Verde. +
19. Lentil Lens culinaris Medik
20. Chick pea Cicer arietinam L.
21. Maple peas Pisum Sativum L. +++
22. Marrowfat peas Pisum Sativum L. +++
23. Winter peas Pisum Sativum L. +++
24. ,Bro.ad bean (small type) Vicia faba L. +++
2S. Broad bean (large type) Vicia faba L.
26. Sword bean Can(lvalia gladiata (Jacq.) DC. +
27. Hyacinth bean (Black seed) Lablab purpureus (L.) Sweet +++ +
28. Hyacinth bean (Red seed) Lablab purpureus (L.) Sweet +++ +
29. Winged bean Psophocarpus tetragonolobus (L.) DC. +++ +
30. Fenugreek .. Trigonella foenumgraecum L. +
31. Bambarra groundnut Vigna subterranea (L.) Verde
32. Pigeon pea Cajanus cajan (L.) Millsp. ++ +
33. Guar Cyamopsis tetragonoloba (L.) Taubert +++ ++
34. Horse gram Macrotylama uniflorum (Lam.) Verde. +++ +
3S. Tamarind Tamarindus indica L. +++ +++

The strength of antioxidative activity was expressed by the induction period (days) when absorbance increased to 0.1 at O.D. SOOnm were
- < IS, + IS':"30, + +30-45, + + '+ >4~ in the thiocyanate metho?

t To whom correspondence should be adressed.

Abbreviations: HPLC, high pressure liquid chromatography; TBA, thiobarbituric acid; BHA, butyl hydroxyanisole.

NII-Electronic Library Service

 Antioxidative Activity of Edible Pulses 1607

Pulses are cultivated throughout the world and consumed in
various 'dishes. There are a few reports about the anti oxidative
activity of pulse extracts. Antiqxidative activity of procyanidins
B-1 and B-Jfrom azuki be3.;n has been r:eported by Ariga et al. 7 S );
the effects ofn~vy bean hull ex~ract on the oxidativ~ damage of
edible oil have been reported by Onyeneho andHettiarachchy.9)
Since extensive screening for anti oxidative activity of edible
pulses has never been done, we decided to examine the activity
and to isolate the active principles. This' paper d~als with the
screening for anti oxidative activity of 35 species of edible pulses.
Each pulse (5 g) was extracted twice with 50 ml.of methanol at
room temperature, filtered, and concentrated in vacuo; concen-
trated samples were then .lyophilized and used for the antio-
xidative assay. The pulses with, antioxidative activity in methanol
extracts were extracted with ethyl acetate and' treated in the
same as described above.
Each sample (1.0 mg or 0.2 mg) was added to a mixture of
linoleic aci<;l-99.0% distilled ethanol-O.2Mphosphate buffer (pH
7.0) system, 10) then the degree of oxidation was measured by the
thiocyanate 11 ) and TBA methods. 12) Butyl hydroxyanisole (BHA)
(0.2 mg) and (X-tocopherol (0.2 mg) were used as standards for
assay.
Antioxidative activities of the methanol extracts (1.0 mg and
0.2 mg) of 35 species of edible pulses as measured by the thiocy-
anate method are shown in Table I. Among the pulses, 28 species
showed anti oxidative activity by the thiocyanate method. With
the 0.2 mg of methanol extract, eleven species of the pulses showed
anti oxidative activity as measured the thiocyanate method. The
results of the TBA method had the same tendency as the thiocy-
anate method both at 1.0 mg and 0.2 mg addition. (Results not
shown.) Among the pulses tested,' honkintoki (Phaseolus vulgaris
L., No.2), yamashirokurosando (Phaseolus vulgaris' L., No.3),
guar (Cyamopsis tetragonolobus .(L.) Taubert, No. 33), and
tamarind.(Tamarindus indica L., No. 35) had strong activity.
Since it is not clear if the antioxidants in the methanol extracts
are polar or nonpolar substances, the pulses ,showing antioxida-
tive activity in the methanol extract were again extracted with
ethyl acetate and theantioxidative activity were measured by ,the
thiocyanate and TBA methods. Only the tamarind extract had
strong antioxidative activity with the addition of 1.0 mg. The
induction period was over 45 and 50 days respectively using the
thiocyanate and TBA methods. (Results not shown). '
Since tocopherols are believed to be important in antioxidative
activity, tocophetol contents in the active pulses were 'measured
by the method by Su et al. previously described. 13 ) High pres-
sure liquid chromatography (HPLC)' analysis was done with ~
4.6mm x 250mm Zorbax SIL column (DuPont, U.S ..f\,.):with a
mobile phase of n-hexane (500), dioxane (10), and ethanol (2)
at a flow rate of 1.5 ml/ini~. To'copherols were detecteq, by a fiuo-
resence spectrometer at the excitation wavelength of 295 nm and
emission wavelength of 325 nm. Table II shows the measurement
of tocopherols in the pulses that had exhibited antioxidative activ-
ity in methanol extracts (with 1.0 mg). As shown in this table, the
level was relatively low. The strong activity of methanol extracts
could not be explained by tocopherols. In tamarind, which had
antioxidiative activity in ethyl acetate extract, the tocopherol
content was also low (2.5 mg/IOO g dry weight). These results
indicate that the antioxidants in tamarind are not tocopherols.
In general, seeds contain a great variety of natural antioxidants
that are thought to inhibit lipid peroxidation, resulting in protect

Table II. Measurement of Tocopherols in Edible Pulses

Common name Scientific name p y 0 Total

mg/IOO g dry weight

1. Azuki bean Vigna angularis (Willd.) Ohwi et Ohashi ND 0.1 3.4 8.0 12.5
2. Kidney bean (Honkintoki) Phaseolus vulgaris L. ND ND 2.4 0.1 2.5
3. Kidney bean (Yamashirokurosando) Phaseolus vulgaris L. ND ND 3.2 0.1 3.3
4. Kidney bean (Uzuramame) Phaseolus vulgaris L. ND 0.2 1.5 Trace 1.7
5. Kidney bean (Taisyokintoki) Phaseolus vulgaris L. ND 0.1 1.6 Trace 1.7
6. Kidney bean (Ofukumame) Phaseolus vulgaris L. ND 0.1 2.6 0.2 2.9
7. Kidney bean (Toramame) Phaseolus vulgaris L. ND 0.1 5.4 0.2 5.7
8. Kidney bean (Ohtebo) Phaseolus vulgariS L. ND ND 3.2 0.2 3.4
9. Black soy bean (Hikarikuro) Glycine max (L.) Merr. 0.1 0.2 6.7 8.2 15.2
to. Scarlet runner bean (M urasakihanamame) Phaseolous cosinneus L. ND Trace 2.1 0.2 2.3
11. Scarlet runner bean (Shirohanamame) Phaseolous cosinneus L. ND ND 1.2 0.1 1.3
12. Lima bean Phaseolous lunatas L. ND ND 6.6 0.8 7.4
13. Green gram Vigna radiala (L.) Wilczek 0.1 ND 1l.S 0.8 12.4
14. Black matpe Vigna mungo (L.) Hepper ND ND 7.5 0.1 7.6
15. Rice bean Vigna umbel/ala (Thunb.) Ohwi et Ohashi ND 0.1 3.0 7.1 10.2
18. Yard-long bean Vigna unguiculata ssp. sesquipedalis (L.) Verdc. ND Trace 3.9 8.1 12.0
21. Maple peas Pisum Sativum L. ND ND 5.2 0.1 5.3
22. Marrowfat peas Pisum Sativum L. ND 0.1 5.4 0.2 5.7
23. Winter peas Pisum Sativum L. ND ND 6.1 0.5 6.6
24. Broad bean (small type) Vida faba L. 0.1 0.1 3.5 0.1 3.8
26. Sword bean Canavalia gladiata (Jacq.) DC. ND ND 3.3 1.3 4.6
27. Hyacinth bean (Black seed) Lablab purpureus (L.) Sweet ND 0.1 4.0 0.3 4.4
28. Hyacinth bean (Red seed) Lablab purpureus (L.) Sweet ND 0.1 3.9 0.3 4.3
29. Winged bean Psophocarpus tetragonolobus (L.) DC. ND ND to.5 ND to.5
30. Fenugreek Trigonella foenumgraecum L. 4.9 ND 0.4 ND 5.3
32. Pigeon pea Cajanus cajan (L.) Millsp. 0.1 ND 6.5 0.4 7.0
33. Guar Cyamopsis tetragonoloba (L.) Taubert 1.4 Trace 4.0 0.3 5.7
34. Horse gram Macrotylama uniflorum (Lam.) Verde. ND ND 6.1 0.5 6.6
35. Tamarind Tamarindus indica L. 0.1 ND 2.4 Trace 2.5

ND = none determined.
The results were average of three determinations.

NII-Electronic Library Service

1608 T. TSUDA
against damage to membrane functions, and play an important
role in protecting the ability to germinate during storage. 14)
Therefore, it is interesting that the extracts of many species of
pulses show antioxidative activity. One of the species, tamarind,
had marked activity in both the methanol extract and the ethyl
acetate extract, indicating that tamarind would have both polar
and specific nonpolar antioxidative substances. On the other
hand, species other than tamarind would have polar active
substances. Isolation and identification of such antioxidative
substances cont(;!.ining these seeds are being done.
Acknowledgments. We wish to thank Professor K. .Maeda of Kochi
University for providing horse gram and guar seeds. We also thank
Dr. K. Hanada of the Tropical Agriculture Research Center, Okinawa
Branch, and Mr. K. Sekiya of Dainippon Pharmaceutical Co., Ltd.,
Osaka, Japan, for supplying winged bean and tamarind seeds.
References
1) T. Osawa and M. Namiki, J. Agric. Food Chem., 33, 777-780 (1985).
2) Y. Fukuda, T. Osawa, M. Namiki, and T. Osaki, Agric. Bioi. Chem.,
e( al.
49, 301-306 (1985).
3) J. D. Su, T. Osawa, S. Kawakishi, and M. Namiki, Phytochemistry,
25, 1315-1319 (1988).
4) N. Ramarathnam, T. Osawa, M. Namiki, and S. Kawakishi, J.
Agric. Food Chem., 37, 316-319(1989).
5) A. Nishina, K. Kubota, H. Kameoka, and T. Osawa, J. Am. Oil
Chem. Soc., 68, 735-739 (1991).
6) T. Osawa, H. Katsuzaki, Y. Hagiwara, H. Hagiwara, and T.
Shibamoto, J. Agric. Food Chem., 40, 1135-1138 (1992).
7) T. Ariga, I. Koshiyama,and D. Fukushima, Agric. Bioi. Chem., 52,
2717-2722 (1988).
8) T. Ariga and M. Hamano, Agric. Bioi. Chem., 54, 2499 (1990).
9) S. N. Onyeneho and N. S. Hettiarachchy, J. Agric. Food Chem., 39,
1701-1704 (1991).
10) T. Osawa and M. Namiki, Agric. Bioi. Chem., 45, 735-739 (1981).
11) H. Mitsuda, K. Yasumoto, and K. Iwami, Eiyo To Shokuryo, 19,
210-214 (1966).
12) A. Ottolenghi, Arch. Biochem. Biophys., 79, 355-363 (1959).
13) J. D. Su, T. Osawa, and M. Namiki, Agric. Bioi. Chem., SO, 199-203
(1986).
14) T. Osawa, N. Ramarathnam, S. Kawakishi, M. Namiki, and T.
Tashiro, Agric. Bioi. Chem., 49, 3085-3087 (1985).
NII-Electronic Library Service