Plant Foods for Human Nutrition 37:201-228 (1987)

© Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands 201

Anti-nutritional and toxic factors in food legumes: a review

Y.P. GUPTA
Division of Agricultural Biochemistry, Indian Agricultural Research Institute, New Delhi-l lO012,
India

Received 4 November 1985; in revisedform 17 December 1986

Key words: proteolytic inhibitors, phytohemagglutinins,lathyrogens, cyanogeneticcompounds,

favism

Abstract. A comprehensivereviewon the presence of certain important anti-nutritionaland toxic

factors in food legumes has been conducted. These substances include proteolytic inhibitors,
phytohemagglutinins,lathyrogens,cyanogeneticcompounds, compoundscausing favism,factors
affecting digestibilityand saponins. These factors are shown to be widelypresent in leguminous
foods which are importantconstituentsof the diet of a large sectionof the world'spopulation, and
particularly, of people in the developingcountries.

Introduction

Food legumes constitute an important part of the diet of a large section of the
population in the developing world, as a good source of proteins, carbo-
hydrates, minerals and B-vitamins. But in the raw state, they contain certain
toxic substances which include trypsin inhibitors, phytohemagglutinins,
lathyrogens, compounds causing favism, cyanogenetic and goiterogenic factors,
saponins and alkaloids [142, 146, 228]. It is reported [86, 142] that these sub-
stances are generally eliminated by soaking and subsequent discarding of the
liquid and/or by heat treatment at relatively elevated temperatures. Some of
these important substances have been comprehensively reviewed in this paper.

Factors affecting digestibility

Pulse digestibility has been found to be affected by the methods of processing

and cooking, by the quantities consumed and by the state of the digestive tract.
The presence of saponins, glycosides, tannins, alkaloids, conjugates of protein
with phytin or hemi-cellulose and substances inhibiting the action of digestive
enzyme trypsin in different food legumes adversely affect their digestibility as
these substances are indigestible or are antagonistic to digestion [87].
Digestibility of food legumes has been consistently 15-20 per cent lower than
that of casein [214]. The lower apparent protein digestibility is attributed to the
quantities of tannins and other polyphenols present in the seed-coat [60]. The
unfavourable influence of tannins on the nutritional properties of dry beans has
202
been described earlier [193, 194]. Protein utilisation is also influenced by hemag-
glutinins and other inhibitors. Plant fibre interfered with protein digestibility
[128]. The physiological factors affecting protein digestibility have been des-
cribed by Kies [127].
Imbalance of amino acids and their availability also affected protein utilisa-
tion [87]. In vitro studies have shown that raw kidneybean is less digestible by
pepsin and trypsin than casein, and autoclaving did not improve its digestibility
by pepsin. But Chatterbuck et al. [33] observed that autoclaving markedly
improved in vitro digestibility of soy protein concentrates and isolates. It was
also found that different varieties of soybean showed wide variation in their in
vitro digestion with trypsin [81]. In vitro pancreatic digestion of proteins in
bengalgram, greengram, blackgram, redgram, lentil and pea was much slower
than that of meat meal and casein [88]. Earlier, Manage & Sohonie [157]
reported similar findings with horsegram that the rate of in vitro proteolytic
digestion was slower as compared to that of casein. It seems that trypsin
inhibitor has been interfering with proteolytic digestion. Similar. results were
obtained with doublebean and fieldbean [105, 188].
Protein digestibility of pea, fababean and lentil meals and their concentrates
and isolates (81 to 90 per cent) was found similar to that of casein (87 per cent),
and their poor growth-promoting ability was reported to be due to growth-
depressing factors such as tannins, trypsin inhibitors and hemagglutinins [17].
It was recently demonstrated [253] that the rate of protein digestibility in the raw
bean extract was less than that of casein and bovine serum albumin due to the
presence of hemagglutinins. It was suggested that lectins affected the activity of
digestive enzymes. Protein digestibility of cooked pigeonpea meal was also
found to be low [231]. In vitro proteolytic digestion of denatured protein
phaseolin from Phaseolus vulgaris seeds showed that it was fully digested and its
native form was partially digested [25]. The poor digestion in case of native form
was due to the presence of trypsin inhibitor. It was reported that trypsin
inhibitor and in vitro protein digestibility are related to nutritional quality [203,
223]. Deshpande et al. [47] also obtained poor in vitro proteolytic digestibility
of Phaseolus vuIgaris dry seeds. Studies by Hernandez-Infante et al. [99] showed
that in vitro digestibility of black beans when dried with broth was poor as
compared to that obtained with white beans. In vitro studies on the digestibility
of carbohydrates showed that greengram was digested at a faster rate than
bengalgram, blackgram and redgram [87].
Germination and cooking were reported to improve in vitro protein diges-
tibility of horsegram and mothbean [224]. E1Faki et al. [57] also observed
improvement in in vitro protein digestibility of horsegram and cowpea by
germination and cooking but there was no significant improvement in case of
chickpea. Phillips et al. [189] found that cooking initially increased in vitro
protein digestibility of cowpea and then decreased. Gross [79] reported that
cooking improved in vitro protein digestibility of raw wingedbean, which was
relatively low to that of other pulses (Table 1). Similar findings were reported
earlier [56]. Jaya & Venkataraman [114] observed that germination improved
 203
Table 1. Per cent Digestibility in vitro.

Raw seed Cooked seed

Psophocarpus tetragonolobus 68,9 79.8
(Wingedbean var. USP-47)
Glyeine max. (Soybean) 71, I 85.5
Phaseolus vulgaris 74.3 80.3
(var. castilla)
Phaseolus vulgaris 75,2 83.7
(var. canario)
Lupinus mutabilis 80,3 85.3
(Tarwi)
Source: Gross [79].

the in vitro carbohydrate digestibility of bengalgram and greengram but the

findings of E1Faki et al. [57] showed that germination did not cause an increase
in the in vitro carbohydrate digestibility of chickpea, horsegram and cowpea.

Saponins
Saponins occur in a wide variety of food plants [68, 177, 261]; bengalgram,
soyabeans, navybeans, haricotbeans and kidneybeans being relatively rich.
These seeds contained 56, 43, 21, 19 and 16g/Kg material, respectively (Table
2). The earlier reported values of saponin in soybean were quite low ranging
between 0.46 to 0.50 per cent [21] showing wide differences. Recently, Sodipo &
Arinze [234] have also reported low values of saponins in beans (Phaseolus
vulgaris) containing 245.0 mg per Kg dry material. It is difficult to explain the

Table 2. Saponin content of legumes.

Seeds Saponin content
(g/Kg dry material)
1. Bengalgram 56.0
2. Soybean 43.0
3. Navybeans
Australia 21.0
Canada 6,7
USA 4.5
4. Greenbeans I3.0
5. Red Kidneybeans I6.0
6. Greengram 5.7
7. Lentil 3.7~4.6
8. Fababeans (Viciafaba) 4.3
9. Broadbeans 3.5
10. Greenpeas 11.0
11. Limabeans 1.1
12. Haricotbeans 19.0
Source: Fenwick and Oakenfull [68].
204
reasons for the high values obtained by Fenwick & Oakenfull [68]. The presence
of saponins in potatoes when eaten in large quantity causes abdominal pain,
vomiting and diarrhoea. Soya products- tofu, textured vegetable protein and
protein isolate are found to be good sources of saponins [67]. Saponins are also
important in the human diet as they lower plasma cholesterol level in the
animals and reduce the risk of heart diseases [179, 190-191]. It has also been
reported [8] that they cause resistance among legume seeds against insect attack.
Saponins are not destroyed during cooking or processing. Raw navybeans or
canned baked beans or canned broadbeans did not exhibit differences in the
amount of saponins [21]. Fermentation was reported to reduce their level.
Fermented soya product-tempeh, contained half the amount from that of raw
soybean [190].

Proteolytic inhibitors

Protease inhibitors having ability to inhibit proteolytic activity of certain enzy-

mes are found throughout the plant kingdom particularly among food legumes
[134, 136, 146, 147, 213, 232, 242]. It has been demonstrated that the presence
of these inhibitors in raw or dry-heat processed food legumes cause depression
in growth and alteration in the pancreatic function of rats [17, 134, 232, 242],
but it has been shown that these are largely inactivated by moist-heat processing
[145]. These inhibitors have attracted the attention of nutritionists for the
possible role which these substances might play in determining the nutritive
value of plant proteins. But there is still uncertainty regrding their exact nutri-
tional significance since there does not seem to be a clear-cut correlation
between their amount and the beneficial effect which heat has on their nutrition-
al values. Kakade et al [120, 121] found no correlation between the trypsin
inhibitor activity and protein efficiency ratio of different varieties of soybean.
Sitren et al. [232] concluded that the levels of trypsin inhibitor and lectin in
soybean and peanut flours did not correlate with their overall biological impact
in feeding studies with the rat.
Trypsin inhibitors possessing growth inhibitory property have been identifed
in all food legumes in varying degrees [3, 44, 55, 80, 82-84, 90, 91,104, 120-122,
130, 133, 134, 147, 202, 204, 220, 221,233,235-237, 239, 245, 246, 258,259, 264].
These inhibitors have been isolated and characterised from different legume
seeds [146, 147]. These include soybean [19], limabean [20], doublebean and
fieldbean [206, 235, 237], navybean [263], adzukibean [268], bengatgram [14],
black eyed pea [73], wingedbean [32, 100, 130-132, 222, 249, 250], kintokibean
[250], guar seed [126] and blackgram [123]. These seeds contain one or more
inhibitors having a molecular weight of about 8,000-10,000 [213], but soybean
inhibitor has a molecular weight of 19,900. Soybean inhibitor has a unique
property of combining with trypsin to form an inactive complex and thereby
inhibit proteolytic activity. A comparative study on inhibitors from different
legumes [192] revealed that soybean, fieldbean, kidneybean and bengalgram
 205
were more active in inhibiting the bovine pancreatic proteinases, whereas cow-
pea and redgram were more effective in inhibiting the human chymotrypsins.
Trypsin inhibitor isolated from blackgram [123] has a molecular weight of
12,500. It has been characterised as non-competitive and stable at pH 2-10
within a temperature range of 30-80 °C for 30 minutes. It was identified as a
glycoprotein, comprising of about 18.2 per cent carbohydrate and its one
molecule containing 75 amino acid residues. Wingedbean contained two major
trypsin inhibitors [13] having a molecular weight of about 20,000 and four
cystine residues/molecule similar to soybean trypsin inhibitor. They have been
characterised to be homogenous proteins. Inhibitor from wingedbean is specific
for chymotrypsin. It inhibits bovine-chymotrypsin and not bovine trypsin. It
has a molecular weight of 20,900 and contains four half-cystine residues/-
molecule [131]. It is stable over the pH range 2.0-11.5 and to heating upto 70 °C
at pH 4.1 and 8.0. Kintokibeans are found to contain at least five trypsin
inhibitors having identical properties under gel filtration and ion-exchange
chromatography but they differed among themselves in their specificity having
two distinct sites of their activity towards trypsin and chymotrypsin [259]. Three
of them resembled that of limabeans [95], kidneybeans [196] and garden beans
[266] in respect of amino acid composition having higher amount of aspartic
acid, serine and half cystine residues and containing no tryptophan. Studies by
Tan-Wilson et al. [252] on Bowman-Birk proteinase inhibitors revealed that
eight soybean strains had four to seven isoinhibitors species in each strain. Kaur
& Bhatia [126] isolated three protein fractions from guar seed by Sephadex
G-200 gel chromatography and found that all the three had considerable trypsin
inhibitor activity. Hafez & Mohamed [90, 91] successfully separated non-protein
trypsin and chymotrypsin inhibitors from soybean containing active peptides.
They found that the inhibition of chymotrypsin is competitive and that of
trypsin is non-competitive.
Different studies do not support the view that these inhibitors help protein
breakdown during germination or prevent their degradation at seed maturation.
It has been found that these inhibitors isolated from soybean [18] or pea [102]
do not inhibit endogenous proteins of the same plant. Palmer et al. [185]
reported an increase in the trypsin inhibitor activity in kidney-beans during
germination but Collins and Sanders [40] and Noor et al. [176] did not observe
any appreciable change in the activity in soybean or greengram, whereas others
[58, 84, 126, 212] found reduced activity in greengram, blackgram, beans and
guar seed. Trypsin and chymotrypsin inhibitor activities in beans were reduced
by 62.9 and 67.1 per cent, respectively after five days of germination [212], while
in case of guar seed, the trypsin inhibitor activity was reduced by 30 per cent
after three days of germination [ 126]. Subbalakshmi et al. [243] reported similar
findings on the changes in the trypsin inhibitor activity due to germination.
Hobday et al. [102] and Baumgarten & Chrispeels [13] found that the protease
inhibitor in pea or greengram is located outside the protein bodies.
Trypsin inhibitors are greatly affected by heat, genetic variance and stage of
seed development [86]. Anti-tryptic activity among different food legumes
206

Table 3. Trypsin and chymotrypsin activity in different legumes.

Seeds Trypsin inhibition Chymotrypsin

(Units/mg protein) inhibition
(units/mg protein)
t. Hyacinth 16.97 17.59
(Dolichos lablab L.)
2. Lathyrus 11.88 11.25
3. Soybean 38.57 6.57
4. Cowpea 19.00 7.20
5. Bengalgram 8.57 2.79
6. Redgram 7.77 1.87
7. Blackgram 7.56 1.63
8. Greengram 5.36 --
9. Clusterbean 2.45 0.07
Source: Sumanthi and Pattabiraman [245].

(Table 3) showed that beans were optimum; greengram being the lowest [103,
245]. Studies on soybean [124] have revealed that cotyledon component con-
tained about 83 per cent of the total inhibitor activity of the seed while there was
no activity in the embryo portion. Stage of soybean seed formation had a
pronounced effect on its activity which gradually increased from early-milky to
mature stage (Table 4) [124]. Soybean plant did not contain any activity at any
stage of its growth. The rate of synthesis of the inhibitor was higher at the initial
stages of seed formation. Phosphorus fertilization did not seem to influence its
activity. It was concluded that the trypsin inhibitor was synthesised in the seed
with initiation of seed formation and it was not translocated from the plant
parts to the seed. Collins & Sanders [40] and Kute et al. [135] reported similar
findings. They observed an increase in the trypsin inhibitor activity in soybean
and wingedbean as the seed developed maturity.
These inhibitors in different legume seeds are inactivated by heat [3, 59, 124,
142, 146, 147, 183, 202, 220, 255]. Their inactivation is reported to greatly

Table 4. Trypsin inhibitor activity during developingsoyben seed (expressed as per cent inhibition/
mg protein).
Variety Level of P Activity at different stages of seed
Kg P205/ha development
Early Mid- Mature
milky milky
0 20.9 28.6 45.6
Bragg 56 20.9 28.9 45.7
112 20.6 29.0 45.6
0 2 t .4 27.9 46.0
Punjab-I 56 21.7 28.5 46.3
112 21.9 28.3 46,5
Source: Kapoor and Gupta [124].
 207

improve the nutritive value of their seed proteins [22, 55, 122, 141,202, 233]. The
presence of residual tannins in the cooked beans accounted for the inhibitor
activity remaining after heat treatment, which was hardly one per cent of the
total activity [44, 60, 240, 248]. Temperature, duration of heating, particle size
and moisture are found most beneficial in destroying the inhibitor activity [3,
147, 202, 204]. Antunes & Sgarbieri [7] reported that this inhibitor was a
heat-resistant antinutritional constituent. A high initial moisture content (20 per
cent) in soybean favoured rapid destruction of the inhibitor [3, 99]. Sitren et al.
[232] found that raw soybean caused the maximum (76 per cent) inhibition of
the trypsin enzyme, the dry-heated soybean caused 61 per cent inhibition, and
the moist-heated soybean caused the least inhibition as 11 per cent. Studies by
Kapoor & Gupta [124] showed that autoclaving of soybean seed for 30 minutes
or steaming for 60 minutes completely inactivated the inhibitor activity. How-
ever, soaking for 8 hours prior to steaming considerably reduced its time of
inactivation to 15 minutes. The inhibitor was completely inactivated by direct
injection of steam for two minutes followed by cooking for 20 minutes at
4.2 Kg/sg.cm. pressure [220]. Studies on the kinetics of the inhibitor activity in
relation to temperature and moisture [189] showed that the loss of activity was
of first order reaction. The first order rate constants for loss of activity in cowpea
flour containing 7.5, 19.4 and 26.5 per cent moisture when heated at 100°, 125°
and 150°C for periods of 0.5 to 120 minutes ranged from 1 x 10 -2 to 18 rain -l .
Buere et al. [28] also reported that the trypsin inhibitor activity loss in beans by
temperature followed first order reaction kinetics and the rate of loss greatly
increased with increasing moisture from 2-55 per cent. Dielectric heating [24,
230], infra-red cooking [66] and microwave radiation [267] were also reported to
inactivate the inhibitor, causing improvement in the nutritional quality of
soybean. Ellendrieder et al. [61] reported the presence of proteinous substances
which accelerated thermal inactivation of trypsin inhibitor. There was a loss of
90 per cent in the trypsin inhibitor activity in the crude extract from kin-
tokibeans at 100°C for 60 minutes but it was observed that the purified trypsin
inhibitor was heat-stable and was inactivated by heat in the presence of high
molecular weight proteins [258]. Doell et al. [49] found that most of the inhibitor
activity was lost during processing of soya foods and the remaining was elimi-
nated during cooking. It has also been found that soaking in one per cent salt
solution and sodium bicarbonate has considerably reduced the inhibitor activity
of common Indian pulses [239].
Different varieties of soybean are found to differ in their trypsin inhibitor
activity [80]. Singh & Eggum [231] reported large variation in the levels of
protease inhibitors of pigeonpea varieties. The concentration of these inhibitors
was higher in some of its wild species. Trypsin and chymotrypsin inhibitor
activity in wingedbean varied from 22.2 to 42.5 mg of trypsin inhibited and from
30.1 to 47.6mg of chymotrypsin inhibited per g of seed material, respectively
[133]. The inhibitor activity in the seed was also shown to be influenced by
location [82].
208

Lathyrogens

In India and other countries, people consuming lathyrus (Lathyrus sativus) for
prolonged periods suffer from a paralytic disease known as lathyrism, which
causes a public health hazard. The incidence of lathyrism in areas where lathy-
rus used to be consumed has been established. Various aspects of this problem
have been reviewed from time to time [85, 89, 94, 146, 219].
Human lathyrism in India is widely common where the crippling disease
afflicts the poorer sections of the people especially under conditions of famine
resulting from droughts when the field crops miserably fail, and lathyrus forms
the staple diet because this pulse is highly drought resistant, and as an alternate
crop, it is cultivated on lands subjected to drought, excessive rain or floods. In
India, this pulse occupies nearly 5 million acres under cultivation, which is 4 per
cent of the total area under pulse crop and constitutes 3 per cent of the total
pulse production; Madhya Pradesh produces more than 50 per cent of the total
produce in the country [89]. The various reports have confirmed that prolonged
consumption of this pulse for a period of three to six months afflicts the central
nervous system characterised by weakness and paralysis of the leg muscles and
death in extreme cases (neurolathyrism). This disease affects mostly young men
between 20 and 29 years of age. A 1958 epidemiological survey in India in-
dicated that in a single district of Rewa in Madhya Pradesh, there were as many
as 25,000 cases of neurolathyrism in a total population of 634,000 [53]. Another
survey in 1974 in Raipur (Madhya Pradesh) showed that the rate of prevalence
of lathyrism was 40 per 1,000 [54]. The number of cases were over 1,00,000 in
1975 [175]. Studies by Attal et al. [11] revealed that there were typical differences
and characteristics features in cases of lathyrism in Amgaon area (Distt.
Bhandara) of Maharashtra.
Various attempts have been made to identify the causative agent of human
lathyrism. The presence of certain phenolic compounds, a toxic alkaloid, a
water soluble toxic amine, excess quantity of manganese, an alkaline volatile
liquid, presence of selenium which interferes in methionine metabolism and
presence of pathogenic fungus which grows,on the pulse during wet and virus
infection have been attributed by different workers to be causative factors for
the toxicity [174, 244]. But none appeared to be conclusive.
Several groups of workers in India [1, 170, 215] isolated a neurotoxic com-
pound from the lathyrus seeds, and characterised it as fl(N)-L-e, fl-diaminopro-
pionic acid (BOAA or ODAP) to ~oe the causative principle of human neur-
olathyrism. Its injection in young chicks, rats and monkeys was shown to
produce severe neurotoxic symptoms [184, 215]~ The effect of BOAA in experi-
mental animals made its involvement in human lathyrism highly suggestive.
However, its oral feeding as well as parenteral administration of the seed
extracts to experimental animals even for prolonged periods did not induce
paralysis [173]. Monkeys fed on the pulse as the sole diet source for almost one
year did not show any external symptoms of neurological behaviour.
In addition to BOAA, another water soluble aliphatic amino acid glycoside
 209
with a nitrile group was isolated from the lathyrus seeds and shown to be toxic
to day-old chicks at a dose of 50mg per 100g body weight. This compound
produced paralysis of the limbs within 5 to 10 minutes of intraperitoneal
injection [216, 2t7]. It was designated as N-fl-D-glucopyranosyl-N-a-L-
arbinosyl-e, fl-diaminopropionitrile. This compound was reported to act syner-
gistically with BOAA.
The neurotoxic compound (BOAA) is reported to undergo transamination in
rat tissue giving to a keto acid product [36], which inhibits the growth of several
micro-organisms. It has been found [215] that a 30 per cent ethanol extract of
lathyrus meal caused neurological symptoms in day-old chicks, and intraperito-
neal administration of BOAA in two days-old rats caused typical convulsions
within 10 minutes [173]. It was concluded [38] that BOAA interfered with the
ammonia generation of fixing mechanism in the brain and led to chronic
ammonia toxicity. Biochemical studies [35, 37] with brain homogenates
prepared from the BOAA injected rats revealed an increase in transglutaminase,
protease, glutaminase, adenosine deaminase and transaminase levels, and a
decrease in the brain glucose, glycogen, ATP, phosphocreatinine and acetylch-
oline levels of the convulsing animals. It was concluded that protein bound and
free amide group could be the source of ammonia in BOAA treated rats, and
BOAA was a typical convulsant.
Earlier studies [209] on BOAA administered through the lumbar route in
monkeys supported the concept of a blood brain barrier but the later work [207]
did not support the concept of effective blood brain barrier to the toxin. The
existence of a blood brain barrier in human beings is yet to be established.
It was shown [208] that treatment of adult animals with drugs or chemicals
known to induce an acidotic condition would make them susceptible to the
toxin. It was interpreted that acidosis had favoured increased entry of the toxin
into the brain. BOAA was detected in the brain of young rats and acidotic rats
but not in adult rats [34].
Recent studies on the effect of BOAA on glutamate metabolism revealed [137]
that this compound could be a potential antagonist of glutamate. These inves-
tigations established that BOAA was neurologic and the amino group was
involved in causing neurotoxic symptoms.
Studies on the biosynthesis of this neurotoxin compound [154] revealed that
it was synthesised by the action of oxalyl-COA synthetase and oxalyl-L-e,
fl-diamino-propionic acid oxalyl transferase enzymes. Neurotoxin was syn-
thesised by the oxalylation of L-e, fl-diaminopropionic acid [155] as follows:
Mg++
Oxalate + ATP + COA . " Oxalyl-COA + AMP + PPi (i)
Oxalyl-COA-
synthetase

Oxalyl-COA + L-e, fl-diaminopropionic acid ~ BOAA + COA

synthetase
(ii)
A partial resolution of these two enzymes activities using C.M. Sephadex
column was reported [156]. Studies on the factors involved in the manifestation
210
of the paralytic disease and their mode of action, however, need further inves-
tigation.
There has been no effective medicine for the cure of lathyrism and the disease
could only be checked by feeding a nutritive diet to the affected person and by
discontinuing the consumption of this crippling pulse. A possibility of some
improvement by indigenous drugs was reported by Attal et al. [11] but it is
lacking confirmation.
This pulse wildly grows in a number of countries including India, and con-
tinues to cripple a large section of the poor people. Therefore, certain
procedures have been evolved to identify its adulteration among pulses employ-
ing visual, microscopic, chemical and chromatographic tests [48, 52, 93, 172].
Also simple domestic procedures for detoxification have been proposed [166].
These include steeping the dehusked seeds overnight followed by steaming for
30 minutes and subsequently rejecting the extract. In this process, many of the
water soluble nutrients are lost, and need supplementation. Roasting of the seed
at 140 °C for 15-20 minutes has also been advocated. Ramachand et al. [205]
suggested that the fermentation method with the use of bacillus species may be
exploited as they observed that neurotoxin is broken down by this bacillus
without affecting the nutritional quality of the seed.
The utility of such domestic procedures could not be adopted on a national
scale because of certain practical difficulties. As an alternate approach, some
attempts are being made to evolve genetically new strains of lathyrus to be low
or free from toxin. Analysis of a large collection of lathyrus seeds revealed a
wide variation in the amount of BOAA ranging from 0.1 to 2.5 per cent [171,
238]. These seeds have also been found to contain other compounds which could
be lathyrogenic. These compounds were identified to be e, 7-diaminobutyric
acid and/?-cyanoalanine producing neurotoxic effects when injected into ani-
mals. Thus, the role of these compounds have to betaken into consideration in
the pathogenesis of lathyrism for achieving a breeding programme successfully.
In India, this pulse is otherwise a boon to the poor people as an article of diet
especially in times of famine when lathyrus is the only available food in certain
regions of the country particularly as it is rich in proteins and lysine, and is
potentially useful as a protein and lysine supplement. Therefore, it is necessary
to take preventive steps to minimise or to eliminate the incidence of human
lathyrism.

Phytohemagglutinins
Substances possessing the property to agglutinate red blood cells are known as
phytohemagglutinins (lectins). These substances, protein in nature, are widely
distributed among food legumes [76, 106, 144, 146, 149, 255]. Except con-
canavalin A lectin from jackbean, these lectins in food legumes are glycopro-
teins containing 4 to 10 per cent carbohydrates. They constitute about 2 to 10
per cent of total protein in most leguminous seeds. Besides their ability to
 211
agglutinate red blood cells, Liener [146] has reported that they exhibit other
chemical and biological properties, which include interaction with specific blood
groups, mitogenesis, agglutination of tumor cells and toxicity towards animals.
Various microbiological and immunochemical studies [106, 129, 199, 201,
265] have provided evidence that the higher intake of lectins produce toxic
effects, inhibiting growth and causing death. It is believed that these lectins bind
intestinal mucosal cells, causing malfunction, disruption and lesion in the small
intestine and thereby interfere with the absorption of nutrients from the gut
[106, 143,201,253]. Pusztai et al. [200] have reported that a proportion of bound
lectin goes to the circulating system. Others [64, 149] have also supported the
view that binding of lectins to the cells lining the intestine may cause a serious
impairment in the ability of the cells to absorb nutrients from the gastrointesti-
nal tract and thus inhibiting growth and in extreme cases, death. Gatehouse et
al. [72] have suggested a similar mechanism of lectin toxicity in insects that the
ingested lectin causes disruption of the epithelical cells of the larvae midget
leading to breakdown of the transport of nutrients into these cells, and the
absorption of potentially harmful susbtances. Some others [12, 115, 116] have
advocated that bacteria also play an important role in causing toxicity by
lectins. Others [116(a), 211] indicated that these lectins interfere with the diges-
tive enzymes and reduce the availability of nutrients. Studies by Thompson et
al. [253] have shown that the addition of lectins at the same concentration
present in raw bean extract, decreased the rate of digestion of heat-treated bean
extract, casein and bovine serum albumin to levels close to that of raw bean
extract. They concluded that lectins affected the activity of digestive enzymes.
It was recently shown [78] that diets containing kidneybean lectins severely
disrupted the brush borders of duodenal and jejunal enterocytes of rats aged
between 30 and 123 days, and that oral immunisation did not protect the rats
from the effects of toxicity, and that the immune response was a result of
continuous absorption of lectin throughout the feeding period. The extent of
toxicity was not affected by the age of the animals.
Their nutritional significance and physiological functions were earlier review-
ed [143, 144]. In the biological system, they have a unique property of binding
saccharides and saccharides-containing proteins. It was speculated [254, 255]
that they (a) act as antibodies to counteract soil bacteria; (b) serve to protect
plants against fungal attack by inhibiting fungal polysaccharides; (c) serve to
transport or store sugars; (d) act as glue for attaching glycoprotein enzymes in
organised multi-enzyme systems; and (e) play a key role in the development and
differentiation of embryonic cells. It has also been proposed that they are
involved in the symbiotic relationship between leguminous plants and bacteria
in binding to the root nodules of the plant and in the interaction of legumes with
bacteria in the nitrogen fixing system as they were detected in the roots of
Phaseolus vulgaris [92], and were also found to be associated with bacteria
nodulating soybean [23]. It has been shown that kidneybean lectins protect
plants against insect [72, 113], fungal [165] and pathogenic bacterial attack [227].
Gatehouse et al. [72] have shown that both albumin and globulin protein
212
fractions from kidneybean containing hemagglutinating activity were toxic
when ingested by the developing larvae of the bruchid beetle (Caltosobruchus
maculatus), implicating the seed lectins as being involved in seed resistance.
Jayne-Williams & Burgess [116] have postulated that binding of lectins to the
cells lining the intestine may interfere with the normal defence mechanism of the
cells.
There are over 600 species of leguminosae family which exhibit hemaggluti-
nating activity [255]. These lectins are found to be localised in the cytoplasm of
the cotyledon and embryonic cells, and greatly reduce on germination. These
have been isolated and characterised from different sources which include
Phaseolus species--kidneybean, navybean, limabean, waxbean, etc. [6, 27, 46,
77, 107, 110, 111,158, 159, 197, 226, 229, 247]; lentil [69, 76, 117, 241]; pea [29,
30, 62, 63, 160, 186, 256, 257, 262]; horsegram [158, 187]; soybean [151];
fieldbean [206, 218]; broadbean [158]; j ackbean [182] and wingedbean [74, 108,
133, 195, 251].
Kidneybean lectins are of considerable nutritional significance as they con-
stitute an important source of dietary protein in several parts of the world but
they are toxic to both mammals and birds when ingested [65, 116, 198] and are
poorly digested by rats [129, 199]. Lectins from this source are found to differ
in their specificity depending on animal species, human blood group and pre-
treatment of cells with proteolytic enzymes [27, 107, 110, 229]. The presence of
five heterogenous proteins in kidneybeans [164] each consisting of isomeric
noncovalently bound tetramere is reported to be responsible for differences in
the lectin activity in its different species. Others [27, 111, 149] identified two
groups to distinguish specificity of lectin activity. One isolated from P. vulgaris
was non-specifc with human erythrocytes of all blood groups and the other
isolated from P. lunatus was specific for A blood group cells. Wingedbean
hemagglutinins showed strong reaction with human and rabbit bloods and
caused higher toxicity [74, 108, 133, 195] than that of soybean [79]. Gillespie et
al. [74] identified two distinct groups of wingedbean hemagglutinins (isolectins)
differing in specificity towards erythrocytes. Kortt & Cardwell [133(a)] isolated
acidic and basic lectins from six varieties of wingedbeans collected from different
regions of south-east Asia, and found no difference in their properties, par-
ticularly in respect of their amino acid composition. These isolated lectins
slightly differed in their specificity. The acidic lectins agglutinated trypsinised
human type A, B and O erythrocytes but not trypsinised rabbit erythrocytes,
whereas the basic lectins did not agglutinate trypsinised human type O eryth-
rocytes but were able to agglutinate trypsinised rabbit and human type A and
B erythrocyte. There was little varietal differences in the wingedbean hema-
gglutinin activity [133]. Its activity varied from 40 to 320 units per mg of
wingedbean [251]. Rasanen et al. [210] obtained a leuco-agglutinin from kidney-
bean in a crystaltised form having a molecular weight of 126,000 consisiting of
four identical units. Other lectins isolated from waxbean, navybean, limabean,
lentil, soybean and horsegram had a molecular weight of 30,000, 32,000,
124,000, 52,000, 122,000 and 120,000, respectively [6, 69, 77, 151, 187, 226].
 213
There were two principal lectins in pea [63, 160] and the third one which was
identified, was due to hybridization of the other two [62]. They were found to
require Ca + + and Mn + + for their activity [186] and a tryptophan residue played
an important role [29]. Pea also contained two mitogenic lectins having similar
properties to the other ones [256]. Pea lectins [257] were similar to that of
soybean [138] and lentil [241] having two binding sites for mannose and methyl
ct-D-glucoside.
Their growth inhibitory property in beans is well recognised [106, 146]. Lectin
isolated from soybean was shown to inhibit growth of rats [140]. It accounted
for 25 per cent of the growth inhibition which raw soybean produced in rats. On
the other hand, feeding trials with soybean [260] showed that its lectin had little
direct effect on the nutritive properties of its protein. Soybean lectin was first
isolated by Liener [139], and its characteristics were studied by Sharon and his
co-workers [150, 151]. Fieldbean tectin had a similar growth inhibiting effect on
rats. It caused zonal necrosis of the liver [206, 218]. Bhatty & Christison [17]
obtained similar findings with fababean and lentil. They concluded that lectins
were responsible for the poor growth obtained with fababean and lentil meals,
concentrates and isolates.
There were more than 200 Phaseolus species which exhibit hemagglutinating
activity in varying degrees. The larger group, represented by Phaseolus vulgaris,
reacts nonspecifically with human erythrocytes of all blood groups and the
smaller group, represented by Phaseolus lunatus, is specific for A blood group
cells [144]. Kidneybean and blackbean were found to display a significant level
of hemagglutinin activity of 3560 and 2450units/ml (Table 5) [103]. They
inhibited growth at levels as low as 0.5 per cent of the diet; kidneybean lectin
being more effective than the blackbean lectin. Kidneybean lectin caused 100 per
cent mortality at 0.5 per cent level which blackbean lectin produced a similar
mortality rate at 1.2 per cent level. A high level of hemagglutinin in kidneybeans

Table 5. Hemagglutinating and antitryptic activities of crude extracts* of raw legumes.

Legume Hemagglutinating Antitryptic

activity activity
(units/ml) (units/ml)
Phaseolus vulgaris
Black bean 2450 2050
Kidney bean 3560 1552
Cicer arietinum 0 220
(Bengalgram)
Cajanus eajan 0 418
(Redgram)
Phaseolus aureus 0 260
(Greengram)
* A 10 per cent suspension of the finely ground meal in one per cent NaC1, clarified by centrifuga-
tion.
Source: Honavar et al. [103].
214
Table 6. Effect of heat treatment* on the hemagglutinating activity in different legumes.

Species Common Hemagglutinin (units/g)

name
Raw Heat
treated

Phaseolus Natal round 1,55,000 50

vulgaris yellow bean
Phaseolus Umzumbi bean 45,000 0
vulgai"is
P. vulgaris Haricotbeans 40,000 20
Glyeine max. Soybean 30,000 0
Vicia sativa Common vetch 120 10
Vicia faba Broadbean 90 11
Pisum sativum Green pea 80 4
Phaseolus aureus Mungbean 78 0
Vigna sinensis Cowpea 6 2
* Heat treatment---autoclaving for 30 minutes at 121 °C.
Source: DeMuelenaere [46].

(over 10 per cent of total protein) was also found by Pusztai et al. [199]. Yellow
bean, umzumbibean, haricotbean and soybean contained very high hemaggluti-
nin; their activity being 155,000, 45,000, 40,000 and 30,000 units per gm,
respectively [46], whereas Vicia sativa, broadbean, greenpea, cowpea, mung-
bean, bengalgram and redgram were relatively either very low or almost free
(Tables 5 and 6). Lectins from limabean, bengalgram, greengram, redgram and
castorbean were non-toxic [46, 103, 158, 182]. However, Manage et al. [158]
observed that oral administration of limabean lectin had a depressing effect on
the growth of rats.
These lectins resist inactivation by dry heat [45, 251]. Moist-heat treatment
however eliminates the toxicity [109]. Similar findings were obtained by Sitren
et al. [232]. They observed that raw and dry-heated soybean contained 112.1 ktg/
g and 94.7/zg/g lectin, respectively, whereas moist-heated soybean was com-
pletely free of lectin. Autoclaving for a shorter period (5-30 minutes) completely
destroyed the bean activity [45, 46, 118, 133, 251] (Table 6). ManciniFilho et al.
[t 59] reported that ionizing radiations with a dose of 50 Krad destroyed 50 per
cent of the activity.

Favism

Favism is a disease characterised by hemolytic anaemia affecting certain in-

dividuals consuming raw or cooked broadbeans (Viciafaba) [15, 146, 155]. The
disease is common among the inhabitants of the Middle East, North Africa,
Italy and Greece [15]. The blood cells of the affected individuals exhibit a
number of biochemical abnormalities, the most significant of which are a
diminished level of reduced glutathione and glucose-6-phosphate dehyd-
 215
rogenase activity. These beans contain anti-nutritional compounds which are
thermolabile [31, 161] and thermostable [179, 180].
Various studies [16, 39, 43, 148, 152, 153, 168, 169, 179, 181] have implicated
vicine and convicine occurring naturally as glucosides and their respective
aglycons divicine and isouramil pyrimidines as the causative agents of favism.
It has been reported [70, 97] that these aglycones are produced in the large
intestines on consumption of fababeans, and are transported to the blood,
where they form certain products in the presence of oxygen [2, 39], causing
favism in sensitive individuals. Muduuli et al. [168] demonstrated that these
compounds elevated the levels of plasma lipid and lipid peroxides, liver
peroxides and glutathione and erythrocyte hemolysis in vitro and depressed
plasma vitamin E levels. In vitro studies revealed that divicine and isouramil
pyrimidines caused a rapid decrease in the glutathione content of glucose-6-
phosphate dehydrogenase deficient red blood cells, an effect which was accoun-
ted for the hemolytic activity exerted by broadbeans [142]. Divicine pyrimidine
was also found to be toxic when injected into experimental animals, and because
of its occurrence in Vicia sativa, it was, at one time, considered as the causative
factor of lathyrism. Divicine compound as the causative factor of favism is not
final as its occurrence is not confined to broadbeans.
The concentration of vicine and convicine compounds in raw fababeans has
been found to be 5.3 mg and 2.3 mg per gm material, respectively [9]. A new
method for quantitation of these compounds was developed [38(a)]. These
compounds were earlier isolated and their properties were studied [163]. Arbid
& Marquardt [10] have developed a procedure for the purification of these
compounds while a rapid reverse-phase liquid chromatography method was
developed for their determination [162].
Certain workers [26, 98, 112] have suggested that it may be possible to screen
fababean cultivars to effectively eliminate these compounds because except for
the toxic principle, these beans are of good nutritional quality [50, 51]. A
number of procedures have been proposed to reduce or eliminate these com-
pounds. These include development of cultivars free from these compounds,
selective destruction of these compounds, and extraction of the compounds
from the seeds. Gardiner et al. [71] screened a number of fababean genotypes
having variation in the concentration of these compounds, but no strain, free of
these compounds, has been identified. Hegazy & Marquardt [96] succeeded in
extracting these compounds completely, but the seeds after extraction were
rendered unpalatable. Mager et al. [153] have earlier demonstrated that these
compounds could be easily hydrolysed by/%glucosidase enzyme from almonds.
Recent studies by Arbid & Marquardt [t9] have shown that 88 to 89 per cent
of these compounds were hydrolysed when one gram of fababean paste was
mixed with 0.1 gm of almond powder and 0.1 ml of lemon juice, and incubated
at 30 °C for three hours. Their studies have revealed that the concentration of
these compounds in the fababean food preparation is greatly reduced by enzym-
ic hydrolysis.
216
Table 7. Cyanidecontent of certain plants.
Plant HCN yield
(rag/100g)
1. Limabean(Phaseolus lunatus) 210.~312.0
Samples incriminatedin
fatal human poisoning
Normal levels 14.4-16.7
2. Sorghum 250.0
3. Cassava 113.0
4. Linseedmeal 53.0
5. Black-eyedpea (Vignasinensis) 2.1
6. Garden pea (Pisum sativum) 2.3
7. Kidneybean(Phaseolus vulgaris) 2.0
8. Bengalgram(Cicer arietinum) 0.8
9. Redgram(Cajanus cajan) 0.5
Source: Liener [146] and Montgomery1167].

Cyanogenetic glycosides

Legumes also exhibit toxicity because of their cyanide producing potential.

They contain certain cyanogenetic glycosides which release H C N on hydrolysis
[41, 42, 142, 146, 167, 225]. Cassava and limabeans predominantly contain these
glycosides. Limabeans (Phaseolus lunatus) were reported to be responsible for
serious outbreak of human poisoning and cases of human intoxication. The
yield of H C N from limabeans varied from 210.0 to 312.0mg/100g (Table 7).
Such a high level of H C N has been the causative factor for fatal human
poisoning. However, the yield of H C N from bengalgram, redgram, peas and
kidneybean is negligible ranging from 0.5 to 2.3 mg/100 g, and therefore do not
exhibit toxicity. A recent report [107] showed that soy protein products and
soybean hulls contained 0.07-0.3 and 1.24/~g/g of HCN, respectively, whereas
in the earlier report [125], soybean was shown to be free of cyanogenetic
compounds. HCN in cereal grains and products is negligible ranging from 0.001
to 0.45 #g/gm [41] and is of little nutritional significance.
It has been shown that most of the liberated H C N is lost by volatilization
during cooking of the limabean, and cyanide is rapidly converted to thiocyana-
tes or other compounds, but cases of human intoxication and chronic neurologi-
cal effects occurred even with cooked limabeans [167].

Conclusion

Some other substaflces like toxic amino acids, goiterogens, anti-vitamin factors,
etc., which also interfere and cause toxicity, have not been covered. The present
write-up indicates that these substances are widely common in diferent food
legumes consumed by human beings, but their ill-effects are not often manifest,
 217

suggesting thereby that foods producing immediate ill-effects are either avoided
or ways and means have been devised to eliminate them. For instance, cooking
itself and other common means of preparation have proved effective in destroy-
ing many of the toxic constituents. However, in some cases, complete detoxifica-
tion does not take place. Inadequate commercial processing of soybean
products is an example. The incidence of lathyrism from consumption of
lathyrus in times of famine, and development of favism disease from consuming
broadbeans are common.

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