A survey of the nutritional and haemagglutination properties of several tropical seeds

A survey of the nutritional and haemagglutination properties of several tropical seeds

G Grant, L J More, N H McKenzie, P M Dorward, J C Stewart, L Telek* and A Pusztai

The Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, Scotland, UK.
* The Mayagüez Institute of Tropical Agriculture, P.O. Box 70, Mayagüez, Puerto Rico.

The nutritional potential of a number of raw tropical seeds has been assessed in a series of feeding trials with rats. Seed lectin reactivity was also monitored.

Abelmoschus esculentus, Chenopodium quinoa, Delonix regia, Phaseolus calcaratus, Phaseolus lathyroides, Parkia biglandulosa, Papaver somniferum, Sesbania arabica, Terminalia catappa, Vigna sinensis and Voandzeia subterranea seeds supported moderate rat growth. The seeds contained only low levels of essentially non- toxic lectin and they have great potential as dietary protein sources for man and animals.

Artocarpus communis, Canavalia ensiformis, Canavalia maritima, Dioclea grandiflora, Phaseolus acutifolius, Phaseolus coccineus and Phaseolus vulgaris var. Processor, var. Rosinha G2 and var Carioca 80 seeds were toxic and contained high levels of potentially toxic lectins.

Albizia adinocephala, Albizia lebbeck, Bauhinia violacea, Cassia nodosa, Cassia tora, Dioclea sclerocarpa, Entada phaseoloides, Enterolobium cyclocarpum, Leucaena leucocephala and Moringa olifera seeds were also highly toxic but had only low levels of essentially non-toxic lectins suggesting that the toxicity was due to other antinutritional factors.

Bauhinia reticulata, Dolichos biflorus and Tamarindus indica proteins were poorly digested and utilised. The seeds contained low levels of lectins which agglutinated only rat and cattle erythrocytes which had been pre-treated with suitable proteases. Brownea macrophylla had a similar lectin reactivity.

Many seeds, which were once used in traditional human and animal diets, have now fallen into disuse due to the increased availability of commercial food products. However, since some of these seeds are native to and grow well in regions where food shortages and famine are endemic, their agricultural and nutritional potential needs to be re-assessed.

Seeds often contain factors, such as lectins, which are deleterious or indeed toxic to animals or man (Liener 1986; Liener 1989a; Cheeke and Schull 1985; Huisman et al 1989). These anti-nutritional factors need to be removed or inactivated by extensive washing and heat-treatment of the seeds or seed meal prior to their use in the diet. However, effective pre-treatment is difficult to achieve if there is limited availability of fuel for cooking (Greibel 1950; Korte 1972). This is a particularly serious problem in relation to the seed lectins since they are quite resistant to heat-treatment and some seeds, such as kidney bean, have to be heated for several hours at temperatures above 80 °C or boiled for 10-20 minutes to ensure the elimination of their lectin activity (Grant et al 1982; Liener 1986). It is therefore essential that any potential food sources should be examined for lectin content and oral toxicity.

A general survey of the nutritional and haemagglutinating properties of several tropical seeds, which are being considered for future development, has therefore been undertaken.

Seed samples of Abelmoschus esculentus (okra), Albizia adinocephala, Albizia lebbeck (kokko), Artocarpus communis (breadnut), Bauhinia reticulata, Bauhinia violacea, Brownea macrophylla, Canavalia ensiformis (jack bean), Canavalia maritima, Cassia nodosa (pink mohur), Cassia tora (sickle senna), Delonix regia (flamboyant tree seed), Dolichos biflorus (horse gram), Enterolobium cyclocarpum (elephants ear), Leucaena leucocephala, Moringa oleifera (horse radish bean), Phaseolus acutifolius (tepary bean), Phaseolus calcaratus (rice bean), Phaseolus coccineus (runner bean), Phaseolus lathyroides, Parkia biglandulosa (african locust bean), Sesbania arabica (sesbania) and Tamarindus indica (tamarind) were obtained from the Mayagüez Institute of Tropical Agriculture (Puerto Rico). Samples of Dioclea grandiflora (mucuna), Dioclea sclerocarpa (mucuna de batata) and Terminalia catappa (castanhola tree seed) were a gift kindly provided by Prof R. de Azevedo Moreira of the Federal University of Ceara (Brasil) as were the samples of Phaseolus vulgaris var. Rosinha G2 and var. Carioca 80 [Prof. V.C. Sgarbieri of the Universidade Estradual de Campinas (Brazil)], Voandzeia subterranea (bambarra groundnut) [Dr. R. Biswas of the University of Nigeria (Nigeria)], Entada phaseoloides (gila) [Mr. H.M. Rahman of Bangladesh Agricultural University (Bangladesh)] and Chenopodium quinoa (sweet quinoa) and Papaver somniferum (poppyseed) [Dr. V. Fowler, Scottish Agricultural Colleges (Aberdeen, UK). Vigna sinensis (cowpea) was purchased from Real Foods (Edinburgh, UK) and Phaseolus vulgaris var. Processor from Booker Seeds (Sleaford, Lincolnshire, UK). All the samples were visually sorted to ensure purity and then were ground in a Wiley Laboratory Mill fitted with a 425 mm mesh grid.

Casein and other dietary constituents were purchased from BDH Chemicals (Dorset, UK) and Sigma Chemical Company (Dorset, UK). Isocaloric diets were formulated as described by Palmer et al (1973). The basal diet comprised per kg: 500 g maize starch, 100 g potato starch, 150 g glucose, 150 g corn oil, 50 g mineral mixture and 50 g vitamin mixture. Casein and seed proteins, alone or as a 1+1 mixture, were added to this basal diet by substitution of the maize starch to give a total dietary protein content of 100 g/kg. The diets were not supplemented with any individual aminoacids.

Net protein utilisation (NPU) and nitrogen digestibility (DIG) were determined as described by Palmer et al (1973). Matched groups, each of 4 Hooded-Lister (Rowett strain) rats, 33 days old, were housed in Makralon cages which had wire mesh grids in their base to facilitate collection of food spillage and faeces. The rats were fed ad libitum exclusively on either the seed meal (test), casein (control) or protein-free (basal) diet for 10 days. Water was available ad libitum. The weight and food intake of the rats were monitored daily. Faeces were collected between day 5 and day 10 of the trial. The rats were killed by ether anaesthesia overdose. The carcass and faecal samples were freeze dried and ground with a coffee grinder. Total nitrogen was estimated by a semi-automated macrokjeldhal method (Davidson et al 1970). Food intake, weight change, NPU and DIG were expressed as per group of 4 rats.

Blood samples were collected into pre-heparinised tubes and immediately after collection were diluted 1+19 with saline (9 g sodium chloride/l). Rat erythrocytes were pre-treated with pronase (20 mg/ml of diluted blood for 30 min at 25 °C) and those from cattle with trypsin (10 mg/ml of diluted blood for 60 min at 25 °C) (Jaffe et al 1972).

Seed meals were extracted with 0.04M sodium borate buffer, pH 8.0, or 0.04M glycine-HCl buffer, pH 2.2, for 16h at +1 °C at a flour to buffer ratio of 1:20 (w/v). After centrifugation, the clear supernatants were stored frozen.

The haemagglutinating activity of the samples was evaluated against a range of animal and human erythrocytes by a serial dilution method (Grant et al 1983). One unit of haemagglutination activity (HU) was defined as the amount (mg) of sample per ml in the last dilution which caused agglutination of 50% of the blood cells. For comparison purposes, the activity of the seed samples against the various erythrocyte types was calculated as HU/mg original seed meal).

Abelmoschus esculentus, Chenopodium quinoa, Delonix regia, Papaver somniferum, Parkia biglandulosa, Phaseolus calcaratus, Phaseolus lathyroides, Sesbania arabica, Terminalia catappa, Vigna sinensis and Voandzeia subterranea seed proteins were digested and utilised quite well (Table 1). Thus, rats given these seeds as their sole source of dietary protein maintained body weight or gained weight slowly over the 10 day test period. Furthermore, when the seeds comprised only half of their dietary protein, rats grew rapidly.

Table 1: 1: Comparison of food intakes, weight changes, net protein utilisation (NPU) and nitrogen digestibility (DI) values obtained with rats fed on diets containing tropical seed meals (intake and weight changes are per group of 4 rats per day).

Diet	50g seed protein				100g seed protein/kg
	+50kg casein/kg
	Food	Weight			Food	Weight
	intake	change			intake	change
	(g)	(g)	NPU	DI	(g)	(g)	NPU	DI

Abelmoschus
Esculentus	27	+8.0	66	84	19	+1.7	26	68
Chenopodium
Quinoa	ND	ND	ND	ND	29	+1.2	45	78
Delonix
regia	35	+9.5	53	82	24	+2.3	42	63
Papaver
Somniferum	ND	ND	ND	ND	27	+2.2	40	77
Parkia
Biglandulosa	29	+5.0	62	78	ND	ND	ND	ND
Phaseolus
Calcaratus	36	+7.8	52	80	25	+0.3	34	62
Phaseolus
Lathyroides	29	+3.8	54	82	19	-0.1	25	66
Sesbania
Arabica	27	+4.4	61	84	20	-0.1	29	70
Terminalia
Catappa	38	+9.9	70	90	25	+2.5	53	88
Vigna
Sinensis	22	+3.3	56	90	21	+1.5	38	72
Voandzeia
Subterranea	ND	ND	ND	ND	27	+2.7	42	65

ND = not determined. For comparison, with the protein-free (basal diet) food intake was on average 14g and weight change -4.7g. With the casein control diet, the values were 31g and +12.7g respectively.

Extracts of these seeds agglutinated only pre-treated rat erythrocytes and to a lesser extent rabbit erythrocytes (Table 2). The lectin activity was however low even with these blood cell types. In addition, there was no consistent correlation between the lectin content and the nutritional performance of rats fed with these seeds (Table 2). It therefore seems likely that the constituent lectins were essentially non-toxic.

Table 2: Nitrogen contents (g/100g seed meal) and haemagglutination activities against various erythrocytes (HU/mg seed meal) of tropical seeds.

			Treated	Treated	Human	Human
		Rabbit	rat	cattle	blood	blood
		blood	blood	blood	cells	cells
	N	cells	cells	cells	O+	AB+

Abelmoschus
esculentus	4.05	0.1	1.2	0.1	0.1	0.1
Chenopodium quinoa	2.11	ND	ND	ND	ND	ND
Delonix regia	3.30	0.8	0.3	0.1	0.1	0.1
Papaver
somniferum	3.14	ND	ND	ND	ND	ND
Parkia
biglandulosa	2.63	1.3	83	0.1	0.1	0.1
Phaseolus
calcaratus	3.74	0.1	5.1	0.1	0.1	0.1
Phaseolus
lathyroides	4.56	0.1	2.6	0.1	0.1	0.1
Sesbania arabica	5.63	0.1	2.6	0.1	0.1	0.1
Terminalia catappa	7.75	0.1	5.1	0.1	0.1	0.1
Vigna sinensis	3.59	0.1	10	0.1	0.1	0.1
Voandzeia
subterranea	3.80	ND	ND	ND	ND	ND

The satisfactory nutritional performance of rats fed upon diets containing these seeds was achieved without expensive pre-treatment of the seeds or for supplementation of the diets with individual aminoacids. Therefore, these seeds have great potential for development as sources of dietary protein for man and animals.

Artocarpus communis, Canavalia ensiformis, Canavalia maritima, Dioclea grandiflora, Phaseolus acutifolius, Phaseolus coccineus and Phaseolus vulgaris var. Processor, var. Rosinha G2 and var. Carioca 80 were toxic (Table 3). Thus, rats given diets in which these seeds provided only 50% of the dietary protein had low food intakes and performed poorly. Furthermore, when the seeds were the sole source of dietary protein, the rats lost weight rapidly and many died within the 10 day experimental period.

Table 3: Comparison of food intakes, weight changes, net protein utilisation (NPU) and nitrogen digestibility (DI) values obtained with rats fed on diets containing various tropical seed meals (intake and weight changes are per group of 4 rats in g/day).

Diet	50g seed protein + 50g casein/kg				100g seed protein/kg
	Food	Weight			Food	Weight
	Intake	Changes	NPU	DI	intake	changes	NPU	DI

Artocarpus
communis	12	-5.1	-ve	40	ND	ND	died	ND
Canavalia
ensiformis	22	+1.4	27	78	ND	ND	died	ND
Canavalia maritima	23	+0.4	36	69	15	-6.1	-ve	51
Dioclea
grandiflora	18	-2.6	15	81	12	-9.5	-ve	68
Phaseolus
acutifolius	14	-4.2	10	55	ND	ND	died	ND
Phaseolus
coccineus	13	-4.8	-ve	52	ND	ND	died	ND
Phaseolus vulgaris
var. Rosinha G2	13	-3.0	26	62	12	-9.9	-ve	54
Phaseolus vulgaris
var. Carioca 80	18	-1.5	44	59	15	-4.5	17	38
Phaseolus vulgaris
var. Processor	11	-3.8	4	58	ND	ND	died	ND

Albizia
adinocephala	ND	ND	died	ND	ND	ND	ND	ND
Albizia lebbeck	17	-1.1	28	74	ND	ND	died	ND
Bauhinia violacea	16	-4.8	7	69	ND	ND	died	ND
Cassia nodosa	19	-0.2	40	57	ND	ND	ND	ND
Cassia tora	20	+0.8	39	67	15	-4.7	-ve	86
Dioclea
sclerocarpa	15	-1.5	36	65	17	-6.6	-ve	44
Entada
phaseoloides	15	-6.0	20	70	ND	ND	ND	ND
Enterolobium
cyclocarpum	25	-0.3	41	69	17	-6.7	-ve	34
Leucaena
leucocephala	9	-8.1	2	62	ND	ND	ND	ND
Moringa olifera	19	-0.1	42	91	9	-7.1	-ve	71

Bauhinia reticulata	27	+3.6	46	58	16	-1.9	18	20
Dolichos biflorus	35	+7.7	50	60	30	-1.0	15	38
Tamarindus indica	23	-1.1	18	10	ND	ND	ND	ND

ND = not determined; -ve, the NPU value was less than 0; died, the experiment was terminated within the 10 day experimental period because the rats died. For comparison, with the protein-free (basal diet) food intake was on average 14g and weight change -4.7g. On the casein control diet, the values were 31g and +12.7g.

Table 4: Nitrogen contents (g/100g seed meal) and haemagglutination activities against various erythrocytes (HU/mg seed meal) of tropical seeds.

			Treated	Treated	Human	Human
		Rabbit	rat	cattle	blood	blood
		blood	blood	blood	cells	cells
	N	cells	cells	cells	O+	AB+

Artocarpus
Communis	2.22	1250	10000	10	10	10
Canavalia
ensiformis	5.09	167	333	2.6	1.2	1.2
Canavalia
maritima	3.92	5.1	666	1.2	1.2	1.2
Dioclea
grandiflora	5.15	167	333	83	2.6	2.6
Phaseolus
acutifolius	5.13	167	666	5.1	10	42
Phaseolus
coccineus	3.05	83	10000	83	10	10
Phaseolus vulgaris
var Rosinha G2	4.80	42	83	5.1	42	10
Phaseolus vulgaris
var Carioca 80	3.45	2.6	20	2.6	2.6	2.6
Phaseolus vulgaris
var Processor	4.22	83	666	42	42	42

Albizia
Adinocephala	6.24	0.1	0.6	0.1	0.1	0.1
Albizia lebbeck	5.68	0.1	0.3	0.1	0.1	0.1
Bauhinia violacea	4.28	0.1	0.6	0.1	0.1	0.1
Cassia nodosa	2.14	0.1	0.6	0.1	0.1	0.1
Cassia tora	3.86	0.1	2.6	0.1	0.1	0.1
Dioclea
sclerocarpa	2.45	0.1	0.2	0.1	0.1	0.1
Entada
phaseoloides	3.85	ND	ND	ND	ND	ND
Enterolobium
cyclocarpum	4.17	0.1	2.6	0.1	0.1	0.1
Leucaena
leucocephala	4.60	0.1	42	0.1	0.1	0.1
Moringa olifera	5.40	0.1	1.2	0.1	0.1	0.1

Bauhinia
reticulata	3.15	0.1	2.6	1.2	0.1	0.1
Dolichos biflorus	2.10	ND	ND	ND	ND	ND
Tamarindus indica	1.99	0.1	0.3	10	0.1	0.1
Brownea
Macrophylla	1.06	0.1	0.3	10	0.1	0.1

This group of seeds generally had a high lectin activity (Table 4). Furthermore, the lectins were very reactive, agglutinated all of the erythrocyte types tested and exhibited particulary high activity against rabbit and pre-treated rat erythrocytes (Table 4). Legume lectins exhibiting this pattern of erythrocyte reactivity are toxic when fed to rats (Pusztai and Palmer 1977; Grant et al 1983). Thus, it seems likely that the constituent lectins are at least partly responsible for the high toxicity of these seeds to rats. These seeds would therefore require extensive heat-treatment before they could be safely incorporated into diets for animals or man. Canavalia ensiformis and Dioclea grandiflora seeds have been found to contain additional toxic factors which can only be eliminated by considerable pre-treatment of the seeds or seed meal (Grant et al 1986; Cheeke and Schull 1985; Liener 1989b).

Albizia adinocephala, Albizia lebbeck, Bauhinia violacea, Cassia nodosa, Cassia tora, Dioclea sclerocarpa, Entada phaseoloides, Enterolobium cyclocarpum, Leucaena leucocephala and Moringa olifera seeds were also highly toxic (Table 3). Thus, rats given diets containing these seeds had low food intakes, rapidly lost weight and had a high incidence of mortality.

Extracts of these seeds had a low content of lectin (Table 4). In addition, the lectins present only agglutinated pre-treated rat erythrocytes. This pattern of lectin reactivity and lectin content is similar to that observed in the essentially non-toxic first group of seeds (Table 1). It therefore seems unlikely that the lectins are, to any significant extent, responsible for the toxic effects of these seeds. Since the nature of the deleterious factors remains unknown, considerable caution should be exercised in the use of these seeds as dietary materials.

Bauhinia reticulata, Dolichos biflorus and Tamarindus indica seed proteins were poorly digested and utilised (Table 3). Thus, rats given Bauhinia reticulata or Dolichos biflorus as their sole source of dietary protein and those given Tamarindus indica as half of their dietary protein lost weight steadily over the 10 day test period.

Bauhinia reticulata and Tamarindus indica extracts exhibited weak agglutination with pre-treated rat erythrocytes, significant agglutination with treated-cattle erythrocytes but no haemagglutination with rabbit or human blood cells (Table 4). Brownea macrophylla exhibited a similar erythrocyte reactivity. Although the lectin activity in these seeds was relatively low, the lectins should be considered as potentially toxic, since bean lectins which agglutinate this specific type of erythrocytes are highly toxic when fed to rats (Jaffe et al 1972; Grant et al 1983; Jaffe 1980).

Abelmoschus esculentus, Albizia lebbeck, Cassia nodosa, Delonix regia, Dioclea sclerocarpa, Enterolobium cyclocarpum, Terminalia catappa and Vigna sinensis appear to contain little or no lectin if they are extracted at alkaline pH (Table 5). However, extraction of these seeds at pH 2.2 results in a significant increase in the amount of lectin released (Table 5), possibly because of the release of structurally bound lectin (Grant et al 1986). The erythrocyte reactivities of the agglutinins extracted at pH 2.2 and pH 8 were similar, suggesting that the increased activity at pH 2.2 reflects increased solubility rather than extraction of a different lectin; it is therefore unlikely that the lectin soluble at pH 2.2 contributes to the toxic effects of these seeds.

Table 5: Comparison of the haemagglutination activities (HU/mg seed meal) present in pH 2.2 or pH 8 extracts of tropical seeds.

	pH 8 extract			pH 2.2 extract
		Treated	Treated		Treated	Treated
	Rabbit	rat	cattle	Rabbit	rat	cattle
	blood	blood	blood	blood	blood	blood

Abelmoschus
esculentus	0.1	1.2	0.1	0.1	20	0.1
Albizia lebbeck	0.1	0.3	0.1	0.1	2.6	0.1
Cassia nodosa	0.1	0.6	0.1	0.1	2.6	0.1
Delonix regia	0.1	0.3	0.1	0.1	2.6	0.1
Dioclea
Sclerocarpa	0.1	0.2	0.1	0.1	20	0.1
Enterolobium
Cyclocarpum	0.1	2.6	0.1	0.1	10	0.1
Terminalia
catappa	0.1	5.1	0.1	0.1	20	0.1
Vigna sinensis	0.1	5.1	0.1	0.1	83	0.1

Rats given diets based upon Leucaena leucocephala seeds had progressive hair loss throughout the 10 day test period. This has been observed previously in animals fed Leucaena leucocephala forage and is thought to be caused by mimosine (Cheeke and Schull 1985).

Eleven of the seeds tested were found to be essentially non-toxic for rats and to contain low levels of lectins which agglutinated only rabbit or pre-treated rat erythrocytes or both.

Delonix regia (flamboyant), which is grown widely as an ornamental tree and Parkia biglandulosa (African locust tree), whose seeds are already used in small-scale oil production and as a coffee substitute, both produce numerous 25-40 cm long pods containing large numbers of seeds of moderately high N content. Although these seeds are widely available, they are apparently not utilised as foodstuffs at present. The results of the present nutritional studies with rats suggest that these could be put to far greater use. Similarly, Terminalia catappa (castanola tree), an ornamental tree and Sesbania arabica (sesbania), which is presently being used in gum and pulp production, produce seeds of high N content and no measurable toxicity and thus have good potential for development. Abelmoschus esculentus (okra), Chenopodium quinoa (sweet quinoa), Papaver somniferum (poppyseed), Phaseolus calcaratus (rice bean), Phaseolus lathyroides, Vigna sinensis (cowpea) and Voandzeia subterranea (bambarra groundnut) are already being used for human and animal feeding. However, because of their good N content and favourable nutritional properties in studies with rats, they could be more widely grown and utilised as dietary protein sources. Their potential for nutritional exploitation is further enhanced by the fact that they would not require prolonged and expensive heat- treatment prior to use.

The remainder of the seeds tested were highly deleterious or indeed toxic when fed to rats. However, it could not be clearly established whether the deleterious effects were due exclusively to lectins, to other anti-nutritional factors, such as toxic aminoacids and alkaloids, or to the combined action of a number of toxic or anti-nutritional factors.

The deleterious effects of some anti-nutritional factors are not readily eliminated by heat-treatment (Liener 1989). Thus, until the nature of the primary toxic factors has been established, as with Phaseolus vulgaris (kidney bean) whose toxicity is due primarily to the constituent lectins (Pusztai and Palmer 1977; Pusztai 1989), great caution should be exercised in the use of these seeds as dietary materials. This is particularly important since recent studies suggest that long-term exposure to relatively low levels of anti-nutritional or toxic factors may have deleterious effects upon body metabolism (Gumbmann et al 1985; Grant 1989).

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