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Food-science


Leucaena leucocephala A nutrition profile


Poonam Sethi and Pushpa R. Kulkarni

Abstract

Leucaena leucocephala is one of the fastest-growing leguminous trees. Its foliage is used as animal feed, and its leaves and seeds are used as human food in Central America, Indonesia, and Thailand. Mimosine, the toxic, non-protein amino acid in Leucaena, causes alopecia, growth retardation, cataract, goitre, decreased fertility, and mortality in non-ruminants. The mechanism of this toxicity is complicated. Mimosine probably exerts its toxic action by blocking the metabolic pathways of aromatic amino acids and tryptophan; by chelating metals; by antagonizing the action of vitamin B6; by inhibiting DNA, RNA, and protein synthesis; by exerting adverse effects on collagen biosynthesis; and by interfering in the metabolism of some amino acids, primarily glycine. Besides mimosine, other anti-nutritional and toxic factors in L. leucocephala add to its toxicity. Heat, moisture, chemical treatments, ensiling, rotation feeding, cutting management of the plant, new hybrids, introducing micro-organisms into the rumen of ruminants that are unable to detoxify mimosine, and preparing protein isolate from Leucaena seeds have all been used to overcome mimosine toxicity.

Introduction

Leucaena leucocephala, popularly known as kubabul or in India [1]. belongs to the Leguminosae family and is one of the fastest-growing leguminous trees [2]. It is an important crop encouraged under the social forestry schemes in drought-prone areas and semi-arid tracts in India, as it provides useful timber as well as leaves for fuel and energy and feed purposes [1, 3-6].

Leucaena foliage is used as animal feed, and the leaves and seeds are used as human food in Central America, Indonesia, and Thailand [7]. The seeds may also be used as concentrates for dairy animals [X], as manure [9-11], as a protein source [2], as an oil seed [4], and as a potential source of commercial gum [12, 13].

Leucaena is a genus of Central American shrubs and trees with 10 recognized species, although the botanical literature claims 55 species. The only valid ones appear to be L. collinsii, L. diversifolia, L. esculenta, L. Ianceolata, L. leucocephala, L. macrophylla, L. pulverulenta, L. retusa, L. shannoni, and L. trichodes. The rest are probably synonyms for these [14, 15]. Although all species probably have value for the tropics, only L. leucocephala (Lam.) de Wit has been exploited extensively so far [16-21].

Nutritive value of Leucaena forage

Leucaena foliage (leaflets plus stems) contain both nutrients and roughage, and make an almost complete ruminant feed, somewhat comparable to alfalfa forage [22]. Its amino acid pattern is comparable with that of soya bean and fish meal [23] and other animal feed sources available in developing nations [22-24].

The mineral composition and mimosine content of the Leucaena plant vary considerably in different species [25] and even within the same species, that is, L. leucocephala itself, for the various cultivars [25-32]. Further variations in composition have also been observed in different parts of the plant [23, 24, 27-30, 33] and at different stages of growth [30, 33-35].

Various species of Leucaena have been studied for its suitability as a forage plant [23, 24, 26-30, 32-42]. The aspects studied include crude protein, amino acid content of proteins, total nitrogen, free amino acids, and changes in free amino acid pools [43]; total ash; ether extractives; carbohydrates; crude fibre; neutral detergent fibre; acid detergent fibre; hemicellulose; cellulose; lignin nitrogen-free extract; in vitro dry matter digestibility; digestible crude protein [44]; total digestible nutrients [44]; minerals, including calcium, phosphorus [44], sodium, potassium, magnesium copper, zinc, iron, cobalt, manganese, and iodine; carotene; xanthophyll; vitamin K; mimosine; tannins and other phenolic compounds; oxalic acid; gums; ferredoxin; estrogenic activity; and volatile and organic solvent soluble mercury [9, 23-25, 27-37, 39, 45-52]. Among the various parts of the plant, seeds and immature leaves contain the highest amount of crude protein, and the stem and dry pods the lowest [24, 27, 29, 53]. Leaf protein concentrate (LPC) was prepared from L. leucocephala leaf meal (LLM). The recovery of LPC was 7.6% and it contained 65.9% crude protein, compared with 29.2% in LLM. Ash content was 17.6%, and the levels of lysine, histidine, arginine, isoleucine, and leucine were 5.6%, 2.3%, 5.9%, 5.4%, and 11% on a dry matter basis, respectively. The LPC had higher in vivo digestibility than the LLM, 63.2% and 48.8%, respectively. The LPC diet supported growth in rats but gave lower nutritive indexes than the control diet of soya bean and Guinea corn [54]. Glutamic acid, aspartic acid, leucine, and isoleucine are the major amino acids in the plant [53]. Depending on the soil minerals available to the root system, Leucaena forage can be an excellent source of calcium, phosphorus, and other dietary minerals [29, 32, 33, 39, 42, 55] but is deficient in sodium [29, 32, 42, 55]. Very limited data are available on the carbohycrates present in the leaves. Total carbohydrate (18.6%), reducing sugars (4.2%), oligosaccharide sucrose (1.2%), raffinose (0.6%), stachyose (by difference) (1.0%), total oligosaccharides (2.8%), and starch (1%) have been reported [24].

The gums (mucilage) isolated from the green leaves (2.5%) are acidic and complex in composition, as against the simple composition of the gum (galactomannan) from seeds. The gum from the leaves is similar to gum arabic and gum tragacanth in that it contains no mannose. In addition to galactose, the major constituent (36%), arabinose (17%), rhamnose (19%), xylose (8%), and hexuronic acid, tentatively identified as galacturonic acid (20%), are present. A water-soluble polysaccharide was isolated from the gum of L. leucocephala produced by gummosis due to fungal attack on the tree. The polysaccharide on hydrolysis gave galactose, arabinose, rhamnose, and glucuronic acid [56]. High proportions of hydroxyproline were found in L. leucocephala gum exudates, as are also seen among other Leguminosae.

Among the anti-nutritional factors, the alkaloid mimosine, beta-N(3-hydroxy-4-pyridone )- x-amino propionic acid, a toxic, non-protein amino acid (fig. 1) [23], is an important constituent. The values for mimosine vary among the different species of Leucaena [25, 57], being highest (5.4%) in L. macrophylla and lowest (1.2%) in L. diversifolia [58]. Among the various L. leucocephala cultivars it differed widely [2628, 30-32, 43], as it did in the different plant parts [23, 24, 27, 28, 30], being 12.3% in the yellow cotyledon and 0.5% in the empty green pod. Mature seeds are twice as rich in mimosine as young seeds, 6.2% and 3.2%, respectively, but the reverse is true of the leaves, 2.6% versus 5.1% [27, 30, 53, 59].

FIG. 1. Chemical structure of mimosine

The mimosine content also depends on the stage of plant growth [33, 35], being maximum on the thirtieth day (7.1%) and progressively decreasing by 45 days (6.0%) and 60 days (4.2%) of growth [33]. Cutting the plant had an effect as well, with mimosine content ranging from 2.5% to 4.9%, and from 1.4% to 3.4%, in the second and third cuttings, respectively [60].

Chemical composition and nutritive value of Leucaena seeds

The seeds are ovoid in shape and have brown hulls and yellow kernels. The hurl: kernel ratio is 50:50 by weight [61], although others reported a 60.5 :39.5 ratio [62]. The seed is low in oil, 5.1% to 10% [1, 2, 4, 24, 63-66], and rich in protein, 24.5% to 46% [1, 2, 4, 8, 23, 24, 27, 29, 63-65, 67]. The kernels have an oil content of 11.9% [62] to 15.3% [4, 62, 67] and a protein content of 52.5% to 66.4% [4, 62, 68, 69]. Thus the nutrients are concentrated in the kernels [4, 62, 68, 69], which can be used as a concentrate for dairy animals [8].

Within the L. leucocephala species itself, variations in composition are seen among the different cultivars [1-3, 23, 27, 29, 62-70]. The percentage in vitro digestibility of seed kernel meal was 25.3%, which increased appreciably to 89.2% on autoclaving the meal [70]. The other species of seeds studied for their proximate compositions are L. esculenta [71], L. pulverulenta, L. macrocarpa [72], L. Ianceolata, and L. retusa [65].

It is significant that the proteins of L. leucocephala [4, 23, 24, 67], L. pulverulenta, and L. macrocarpa [65] seeds are fairly rich in the essential amino acids isoleucine, leucine, phenylalanine, and histidine. Lysine and methionine are also present in moderate amounts. The in vitro digestibility of Leucaena protein isolate (LPI) is 76% [70]. Of the total free amino acids in L. leucocephala seed, 60% is mimosine.

The seeds contain a dark, green to brown, fatty oil with 5.1% to 10% yield [1, 2, 4, 24, 63-66, 73] containing approximately 26% to 29% saturated acids and 71% to 73% unsaturated acids [24, 66]. The oil is rich in linoleic acid (42.5-65%) [1, 24, 62, 63, 66, 74-78] and contains significant quantities of arachidic (0.8-1.6%) [1, 62, 74-78] and lignoceric acids (0.71.7%) [1, 13]. The principal constituents of the unsaponifiable fraction of the oil are sterol 35%, methylsterols 8%, triterpenoid alcohols 20%, tocopherols 17%, and total hydrocarbons and carotenoids 20% [79]. The main sterol is sitosterol, 55% [64, 66, 74, 79, 80], and the main tocopherol is a-tocopherol [79]. The glycolipid composition of subabul seed oil was studied [73]. The oil is the richest vegetable source of phosphatides [2].

Leucaena seeds have a total carbohydrate content of approximately 35 % to 45 % [24, 64, 72], with reducing sugars constituting 5.2% [24]. Starch is absent from a number of the species [61, 65], including L. leucocephala, although it was reportedly 1.3% in the seeds [24]. The total oligosaccharide content of L. leucocephala is 3.5% to 3.6% [24, 81], with sucrose 1.9% to 2.0%, raffinose 0.7% to 0.8%, and stachyose 0.7% to 0.8% [24, 81, 82]. DPinitol [83] and myoinositol have also been found in seeds [84].

The seeds contain a large amount of galactomannan mucilage (20-25% yield) located in the endosperm [12, 13, 52, 85-90]. Galactomannan, composed of 57% mannose and 43% galactose [85], is essentially similar in composition to guar gum. Leucaena seed gum was isolated and chemically modified, and its archeological characteristics were studied [91]. The polysaccharides have an average degree of polymerization of 150 [85]. The seed gum gives highly viscous solutions at low solute concentrations [52, 85, 90]. Thus, L. leucocephala seeds have the potential to be used as a laxative, in vegetable soups [89], and in other commercial products [92, 93]. L. glauca seed gum was modified and evaluated by graft copolymerization with acrylonitrile [94]. Also, graft copolymerization of various vinyl monomers onto the seed gum was performed using H202 as the initiator in aqueous slurry [95].

Leucaena seed has high calcium and phosphorus levels [24, 69]. Oxalic acid has been detected in these seeds [61]. Various vitamins, such as thiamine, riboflavin, niacin, betacarotenes, and ascorbic acid, are present in the hulled meal of L. esculenta seeds, a traditional food in Mexico [71].

Anti-nutritional factors of Leucaena seeds

Mimosine, the toxic, non-protein amino acid, contributes as much as 14.8% to the total nitrogen content of L. leucocephala seeds [24]. The mimosine content varies from 2.2% to 10% [23, 24, 28, 30, 61]. The concentration is higher in the seeds than in other parts of the plant, second only to the immature tender leaves [23, 24, 27, 28, 30]. The concentration of mimosine in the seeds varies widely among the various cultivars of the species. Mimosine accounts for ape proximately 60% (2,689.5 mg%) of the total free amino acids (4,885.8 mg%) in L. leucocephala seeds.

These seeds have a low tannin content (1.2%), in contrast to the high levels in the dry pods and the bark (16.3%). The various Leucaena species have different results to the tannin test [24, 27, 65].

Reports on inhibitory activity against bovine trypsin and a-chymotrypsin in L. leucocephala, L. pulverulenta, L. macrocarpa, and L. esculenta seeds are controversial [24, 61, 71, 72, 96].

Haemagglutinins in L. leucocephala seeds have been isolated, identified, purified, and characterized [23, 61, 97]. Haemagglutinating activity was also detected in L. esculenta seeds, but not in seeds of L. pulverulenta and L. macrocarpa [73].

L. leucocephala as an animal feed

Leucaena provides palatable, digestible, and nutritious forage for ruminants such as cattle, water buffalo, and goats, and thus helps to increase milk production in both the humid and the monsoonal tropics. Mimosine causes weight loss and ill health in non ruminants when Leucaena is fed at levels above 7.5% (dry mass) of the diet [7].

Under certain conditions, mimosine is readily conversed to another toxic compound, 3-hydroxy-4(1H) pyridone (DHP) (fig. 2) [58, 98, 99]. Although mimosine is directly toxic, DHP is only indirectly so, through its goitrogenic action. Thus, animals that break down mimosine to DHP can tolerate higher dietary levels of Leucaena than other animals, and animals that can degrade DHP even further can tolerate higher levels yet, perhaps diets consisting solely of Leucaena [100].

Ruminants in certain parts of the world, such as Papua New Guinea, Australia, some other Pacific islands, and African countries, lack the appropriate rumen bacteria and suffer nutritional disabilities after feeding on high levels of Leucaena. The appropriate bacteria are being introduced into these locations, so that ruminant animals all over the world will be able to eat Leucaena extensively and without harm [101].

The enzyme necessary to convert mimosine to DHP is contained in some (but not all) of the Leucaena cells that harbour mimosine. Conversion occurs when the enzyme comes in contact with substrate, for example, through maceration. The enzyme becomes inactive at pH levels lower than 4, when heated suddenly to temperatures higher than 70C, and when dried. These findings have enormous implications for past and future research. The conversions that take place under specific circumstances would explain many of the conflicting reports about toxicity from Leucaena in both ruminants and non ruminants [100].

FIG. 2. Transformation of mimosine into dibydroxypyridine by the rumen micro-organisms

Cattle feed

The in vivo digestibility of Leucaena forage is similar to that of other legumes and is estimated to be in the range of 50% to 70% [23]. Mimosine tends to reduce the activity of cellulolytic bacteria, but in about a week the rumen bacteria adapt and digestion improves considerably [58].

Leucaena leaf is equivalent to cottonseed cake [101 ] and superior to groundnut cake [102] when used in rations for fattening cattle. Very high live weight gains were recorded in Queensland, Australie [7, 23, 58,102], and in several other places [103].

Several reports show that L. leucocephala roughage could be a substitute for the imported protein supplements fed to dairy cows [104]. Dairy cattle produce well when fed Leucaena [7, 105, 106]. Annual milk production of 5,000 to 9,700 L/ha was recorded in Australia, Hawaii, and Indonesia [7, 58]. In India, when cows and buffaloes were fed L. leucocephala foliage at 10% of their diets, their milk yield was 20% higher than that of the control group fed no Leucaena [107].

However, the use of Leucaena for cattle feedings is not without its problems, due to the presence of mimosine. Symptoms of mimosine toxicity include decreased weight gain, cataract in young animals, infertility, goitre, and-the most striking feature loss of hair [99, 108, 109]. In Australia, cattle fed completely on Leucaena may lose some of their coarse hairs but they will not die. However, newborn calves have shown signs of enlarged thyroids, which may result in death within a few days if their mothers have signs of toxicity [25]. Also, thyroxine levels were reported to be higher in the group fed an exclusive diet of L. leucocephala for 23 months from 10 months of age, compared with the control group [110].

Mimosine itself is not goitrogenic, but its bacterial metabolite 3,4-dihydroxy pyridine (3,4-DHP) is [98]. Mimosine is hydrolysed by the gut microflora of ruminants to 3,4-DHP [58, 98, 99]. When DHP was detected in the urine of ruminants fed L. leucocephala, only a small amount of mimosine was present, and no DHP glucoside was present [111]. Later reports claimed that embryonic death and resorption in cattle may have been due to the anti-mitotic effect of mimosine from the plant or the goitrogenic effect of 3,4-DHP [110].

In cattle, rumen micro-organisms hydrolyse mimosine to 3,4-DHP so efficiently and rapidly that even when the animals are fed on a diet rich in Leucaena, their blood, meat, and milk are quite free of mimosine [7, 23]. It is possible to increase the efficiency of mimosine degradation by the rumen micro-organisms by changing their population through diet manipulation [112]. Substantial degradation of DHP was confirmed in studies on the excretion pattern of DHP, which can be affected by the diet including Leucaena [113]. Ruminal micro-organisms in cattle at a Florida agriculture research station did not have the ability to detoxify Leucaena by degrading 3,4-DHP, but a DHP isomer, 2,3-DHP, was degraded in some cattle [114].

Cattle in Australia and a few other countries do not degrade DHP, due to the absence of the required microorganisms. Even these animals can thrive on diets containing 30% to 40% Leucaena, however, without suffering from the effects of mimosine toxicity [7, 115]. When Leucaena accounts for more than 50% of the animals' intake, with feeding continued for more than six months, it results in mimosine toxicity symptoms [23]. Cattle can tolerate L. leucocephala intakes corresponding to 0.18 g mimosine/kg body weight without serious toxic symptoms [116]. Diets containing less than 1% mimosine on a dry matter basis have little adverse effect on thyroid function or feed intake, whereas above this level hypothyroidism and low feed intake may occur [117]. Under Australian conditions, Leucaena can be fed safely only as a supplement (<30%) to roughage diets, rather than as a major dietary component, until some solution to toxicity is found.

Cattle do not die from goitres resulting from feeding on Leucaena forage. The effects are mostly reversible and can be seen soon after the animals are removed from the pasture to recover [58]. Mimosine has no effect on the milk or meat of cattle feeding on the plant that can cause a health hazard to humans consuming these products [23].

Sheep and goats

Like cattle, sheep find Leucaena forage very palatable [23]. They can tolerate L. leucocephala intakes corresponding to only 0.14 g mimosine/kg body weight, however, whereas cattle can tolerate 0.18 g/kg without serious toxic symptoms [116]. High intakes by sheep, equivalent to a daily intake of 0.2 to 0.3 g mimosine/kg, cause remarkable shedding of fleece within 7 to 10 days [99]. The zinc contents of blood and wool of lambs fed L. leucocephala foliage for 70 days were lower than the levels at the start of the experiment. Toxicity was confirmed in these animals by the development of alopecia by days 7 to 10, as well as by other clinical symptoms [118].

It is established from intravenous, intra-abomasal, and intra-ruminal administration of mimosine [99] that sheep, like cattle, cannot detoxify mimosine after absorption. Instead, extensive degradation of mimosine to 2,3-DHP and 3,4-DHP takes place in the rumen [119].

Goats can tolerate L. leucocephala intakes corresponding to 0.18 g mimosine/kg, the same as for cattle, without serious toxic symptoms [116]. Experimental goats in India, exclusively fed L. leucocephala fodder for three months, maintained their body weights throughout the experimental period without any deleterious effect, suggesting that the plant is a fairly good fodder for stall-fed goats [120]. The goats were able to detoxify mimosine due to the degrading action of rumen micro-organisms [31, 115, 120-122]. However, in another report, the body weight of goats fed only the plant decreased in six weeks due to lower intake and decreased digestibility of crude protein. Also, the concentration of inorganic phosphorus was significantly lower in the blood of these animals [44].

Variations in the dietary constituents had an effect on the metabolism and excretion of mimosine by goats [123].

Non-ruminants

Leucaena should not be a major portion of the diet in nonruminant animals. Although most of these animals eat the plants with relish, they are less able to tolerate mimosine than are ruminants [7]. Non-ruminants generally do not tolerate rations that contain more than 5% to 10% Leucaena (dry weight). Mimosine toxicity symptoms disappear after a short time and leave no residual effects when the plants are removed from the diet.

Subabul up to 40% in camel rations depressed feed conversion efficiency but apparently did not have any adverse influence on palatability, growth performance, thyroid status, hair, and general health of camel calves [124].

Rats and mice

Reports of toxicity in rats and mice due to mimosine in Leucaena suggested reduction in hair growth [7, 30, 51]; growth depression [7, 30, 51] accompanied by appetite inhibition [7]; retardation of functional organs such as ovaries, testes, and thyroid gland; necrosis or degeneration of the liver, kidneys, and cutis; cataract; reduced fertility and reproductive failure; goitre, attributed mainly to 3,4DHP; neurotoxic effects manifested as paralysis of the hind limbs [48, 125]; short lifespan [7, 30, 51]; and mortality [51, 125].

The mimosine content of diets was reduced when Leucaena leaves were ensiled [45, 126]. Heating [61], other processing treatments [69, 70], and supplementation of diets with minerals (iron, zinc) [70] and amino acids (phenylalanine, tyrosine) [127] also reduced symptoms of mimosine toxicity. A few contrary reports are also available, however [71, 128, 129].

Leucaena as human food

Leucaena is consumed by humans in Central America, Indonesia, and Thailand. It can be eaten in processed and unprocessed forms. In Java the seeds are fermented into tempe or are eaten as sprouts or bean cake. Tempe lamtoro, a food made of fermented Leucaena seeds, lacks mimosine, probably owing to the combination of washing, soaking, boiling, drying, and fermenting to prepare it [7]. In Indonesia, Thailand, and Central America, people also eat the young leaves, flowers, and young pods, particularly in soups; the young dry seeds are popped like popcorn [7, 99]. The mature seeds can be ground and used as a flour, or roasted and used to prepare a coffeelike beverage. The young leaves can be blanched and eaten as a green vegetable, although a slight bitter taste persists. The young pods, also blanched, can be added to salads [130]. In Mexico Leucaena pods are eaten raw or in soups or tacos [7]. Seeds of L. pulverulenta and L. macrocarpa are eaten fresh and raw in Mexico [72]. Also, L. leucocephala and L. esculenta are being considered as non-conventional sources of protein, together with other leguminous seeds, in Mexico [131].

Leucaena is not recommended for extensive human consumption because, as mimosine causes loss of hair in non-ruminant mammals, it may have some adverse effects on humans, too [132]. In Indonesia, where L. leucocephala is consumed, from time to time, groups of children and adults developed alopecia involving the scalp and eyebrows. Localized oedema of the scalp has also been observed [133].

Yod kratin (leaves of the lead tree L. glauca) is a popular vegetable in Thailand. It contains a considerablef amount of iron-binding phenolic groups. It is consumed with the main meal year round, at least once a week, and sometimes every day. With a common portion of 20 g, iron absorption was reduced by almost 90%. As little as 5 g inhibited iron absorption by 75%. Adding 100 g ascorbic acid reduced this inhibition from 5 g yod kratin by half, and from 10 g by 25% [134]

Mimosine toxicity, however, is not always evident, possibly because Leucaena is too small a part of the diet. In addition, if leucaena-containing soups and stews are cooked in iron pots, the metal in the pots detoxifies the mimosine by complexing with it [58]. Simple processing can result in a leaf meal largely free of mimosine or can break down most of the mimosine to DHP in the pods. The problem then becomes the toxicity of DHP [135].

Various parts of L. leucocephala are reported to have medicinal properties ranging from control of stomach diseases to contraception and abortion [7]. The plant is reported to he a worm reppellent, and the stembark is taken in Assam, India, to relieve internal pain.

Mechanism of mimosine toxicity

Mimosine, b-N-(3-hydroxy-4-pyridone)-a-amino propionic acid, is one of the free amino acids in 1.. leucocephala. Reports relating to the biosynthesis [136-141], degradation [135, 142, 143], and biochemical effects of mimosine have been reviewed [23, 108, 144]; however, many aspects of the toxic mechanism(s) remain unrevealed. The mechanism is complicated and a number of theories have been put forward to explain it.

Mimosine may exert its toxic action by blocking the metabolic pathways of aromatic amino acids and tryptophan [145]. Due to its structural resemblance to Ltyrosine, it probably acts as a tyrosine analogue or antagonist that inhibits protein biosynthesis in the living body and causes toxic symptoms, including growth retardation [127, 146, 147]. The growth retardation of cattle consuming Leucaena is associated with lower serum thyrosine levels [148], which in turn could be related to the reduction in serum tyrosine level caused by mimosine [149].

The metal-chelating ability of the 3-hydroxy-4-oxo function of the pyridone ring in mimosine [150] could possibly disturb the action of metal-containing enzymes, especially those containing iron cations, and cause inhibition of some biological reactions [151, 152]. In experiments on iron-deficient rats, the bioavailability of iron (measured as blood haemoglobin, and liver and heart iron) and iron absorption from tempeh, a fermented food prepared from L. leucocephala seeds, was higher than that from whole seeds of the plant. Soaking, boiling, drying, and fermenting the seeds during tempeh preparation drastically decreased levels of the toxic amino acid mimosine and of the iron-absorption inhibitor phytate [153].

FIG. 3. Proposed complex formation between mimosine and pyridoxal-phosphate

Mimosine probably acts as a vitamin B6 antagonist [154], inhibiting the activities of a number of enzymes that require pyridoxal phosphate (fig. 3) [144, 155157], such as cystathionine synthetase and cystathionase from rat liver [108]. Inhibition of the system that forms cysteine from methionine [108] is significant, since the protein in hair contains an unusually large amount of cysteine. However, contrary reports are also available with regard to the interaction between mimosine and pyridoxal phosphate leading to mimosine toxicity [145, 149, 158, 159].

In various biological systems, DNA, RNA, and protein synthesis are inhibited 199, 147, 151, 160165]. Analysis suggests that the amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells [166].

The degradation pathways of mimosine and DHP have been studied by a number of workers [147, 151, 167]. In studies of rats, pigeons, and carp, the activity of enzyme(s) degrading mimosine or 3,4-DHP was higher in the kidneys than in the liver [167]. The urine of mice given mimosine contained mimosine, mimosinamine, mimosinic acid, and a trace amount of DHP. The possible metabolic pathways of mimosine are shown in figure 4 [167].

FIG. 4. Scheme of the proposed metabolic pathway of mimosine

Mimosine had adverse effects on the biosynthesis of collagen in embryonic cartilage from chick embryos, due to inhibition of the synthesis of hydroxyproline [168]. The reduction in collagen content, or the more fragile character of the collagen in various organs, might induce such symptoms as capillary haemorrhage, proteinuria, and uterine perforations in animals [132,168].

Mimosine had neurotoxic effects [23, 169] on young rats, which developed distinct paralysis of the hind limbs on a 25% Leucaena diet. These effects were reversible with a normal control diet [23].

The alkaloid seems to interfere with the metabolism of some amino acids, primarily with that of glycine. The abnormally high urinary excretion of glycine after mimosine ingestion is likely caused by mimosine interfering in glycine metabolism [149]. It is speculated that the bile acids conjugate mimosine instead of glycine, leading to the formation of atypical "mimocholic" and "mimochenodeoxycholic" conjugates in the living organism (fig. 5) [129]. Such atypical bile salts can infuence fat metabolism (for example, at the absorption stage) and may consequently also influence the absorption of fat-soluble vitamins [129].

FIG. 5. Two possible atypical bile acid conjugates that may be formed by conjugation of the respective bile acid with mimosine through an amide linkage (*)

Besides mimosine, haemagglutinins [23, 97], protease inhibitors [170], several phenolic compounds [24, 27, 65], and some unidentified toxic factors [171] present in L. leucocephala leaves and seeds might add further to the plant's toxicity.

Possible solutions to mimosine toxicity

Heat treatment of Leucaena leaf meal and drying by exposure to sunlight and high temperatures [30, 99, 172] cause considerable reductions in mimosine. Moist heat treatments such as cooking [172] dipping leaves in hot water [173], and autoclaving leaves and seeds [24, 61 l, reduce the content more than dry heat [23, 96]. A virtually complete degradation of mimosine was reported [174] in an aqueous slurry at pH 8.0 and 45C in 10 minutes. Heating the intact leaf at 70C resulted in 90% reduction of mimosine in 15 minutes.

Washing with water [69, 175] and soaking [23, 69] the leaves and seeds had a significant effect in lowering their mimosine contents. Prolonged soaking (48 hours) in 30C water was most effective in reducing virtually all the mimosine in the leaves [173].

One of the most effective reagents for extracting 95% mimosine is 0.05 N sodium acetate [176, 177]. Studies were carried out by treating L. leucocephala seeds with a number of reagents. Urea and sodium hydrogen carbonate completely removed mimosine. The protein content of the mimosine-free seed mass was reduced to 80% of the original after treatment with urea, and 88% after treatment with sodium hydrogen carbonate solution [178].

Supplementing Leucaena with mineral (iron, zinc) salts reduced mimosine toxicity [30, 171, 172, 179, 180] in rats, chicks, and goats [23, 169, 175, 179, 181], although this was not confirmed by all authors [61, 71]. Reduction of mimosine toxicity by ferrous ions [137] may be due to the formation of a ferric chelate of mimosine [150] after oxidation of ferrous ions to ferric ions.

The growth inhibition caused by mimosine in rats can be partially counteracted by supplementing the diet with phenylalanine and wholly reversed with tyrosine [127]. However, it has also been reported that tyrosine or glycine did not protect the rats from growth depression attributable to mimosine [129]. Adding various substances as detoxifying agents, such as methionine, cystine, glycine, tyrosine, sodium sulphate, riboflavin, nicotinic acid, pyridoxine, and calcium pantothenate plus i-inositol, had no appreciable effects.

Ensiling is an effective method for reducing mimosine content of Leucaena leaves [45]. Mimosine in the L. glauca plant is removed by fermentation with lactic acid bacteria. The mimosine-removed silage is useful as a component of livestock feeds and human foods [182].

By properly cutting the plant, the toxic content can be limited, thus enabling better use of this forage. The mimosine content was reduced progressively as the number of cuttings of the plant increased, from the second to the third cutting [60].

Development of new hybrids with low mimosine content [174] is a possible solution, with some good results already achieved along this line. Hybrids of L. leucocephala and L. pulverulenta have low mimosine and high protein content. Trials in goats showed that the adverse effects caused by mimosine are markedly reduced in animals fed the hybrids come pared with the strains available today [47].

In the most recent approach, micro-organisms from animals that have been adapted to the Leucaena diet and are capable of degrading mimosine and DHP are introduced into the rumen of ruminants that are unable to detoxify mimosine and/or DHP [7, 114, 183]. This could enable the animals to be maintained on even a 100% Leucaena diet with no toxic effects, thereby increasing the potential of the plant as forage. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of sheep before Leucaena was fed, suggesting that these bacteria were indigenous members of the rumen microflora of sheep in Venezuela.

Leucaena seed protein isolates with relatively low mimosine levels have been prepared successfully by isoelectric precipitation of seed kernel proteins [184]. Thus, protein isolate preparation could itself be considered as a means of using Leucaena proteins for food purposes, with minimal mimosine toxicity. The isolate compared favourably with commercial soy protein isolate in terms of in vitro digestibility [70] and functional properties, such as viscosity, gelation, oil and water absorption, foaming, and emulsification. They also were effectively used as an egg protein substitute in cakes and as an egg yolk protein substitute in mayonnaise-like products. This is of significance in view of the ever-increasing demand for egg proteins and the ever-growing population of vegetarians the world over.

Acknowledgements

We are grateful to Dyuman Bhai, trustee, Sri Aurobindo Ashram, and Mr. Manindra Pal of Gloria Land, Dairy Farm, Sri Aurobindo Ashram, Pondicherry, India, for gifts of the L. leucocephala seed samples. This research was supported in part by the R.D. Biria Smarak Kosh, Bombay Hospital, Bombay, India.

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