Lucerne (Medicago sativa L.) in the human diet ...

0 downloads 0 Views 468KB Size Report
Jun 20, 2017 - diseases, such as tuberculosis, HIV, and malaria. ..... Haemolysis may result from an ecogenetic disorder known as favism, a condition related ...

Accepted Manuscript Title: Lucerne (Medicago sativa L.) in the human diet − Case reports and short reports Authors: Eliza Gaweł, Mieczysław Grzelak, Magdalena Janyszek PII: DOI: Reference:

S2210-8033(17)30052-0 http://dx.doi.org/doi:10.1016/j.hermed.2017.07.002 HERMED 188

To appear in: Received date: Revised date: Accepted date:

12-2-2016 20-6-2017 31-7-2017

Please cite this article as: Gaweł, Eliza, Grzelak, Mieczysław, Janyszek, Magdalena, Lucerne (Medicago sativa L.) in the human diet − Case reports and short reports.Journal of Herbal Medicine http://dx.doi.org/10.1016/j.hermed.2017.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Lucerne (Medicago sativa L.) in the human diet - Case reports and short reports Eliza Gaweła, *, Mieczysław Grzelakb, Magdalena Janyszekc a

Department of Forage Crop Production, Institute of Soil Science and Plant Cultivation, State Research Institute, Puławy. Poland b Department of Grassland and Natural Landscape Sciences, Poznań University of Life Sciences, Poznań, Poland c Department of Botany, Poznań University of Life Sciences, Poznań, Poland Corresponding author Dr Eliza Gaweł Department of Forage Crop Production, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich Street 8, 24-100 Puławy, Poland E-mail: [email protected], tel. + 48 81 86 794, fax. +48 81 47 86 800

ABSTRACT Aim: The ever-increasing growth in the size of the human population is causing a growing demand for animal protein. This demand has spurred an interest in cheaper, plant-derived proteins that have an equally rich protein composition as alternatives to proteins of animal origin. A long record of attempts to substitute proteins originating primarily from legumes, cereals, cassava, leaf proteins and whole plant lucerne proteins for animal proteins in the human diet exists. Lucerne Medicago (L.) is the most popular and widespread proteinyielding crop which is grown in cool-climate regions. Extracts and concentrates of lucerne contain, along with proteins, also many vitamins, nutritive substances and secondary metabolites. Due to valuable chemical constituents which show phytobiotic action on humans lucerne is used in folk medicine and phytotherapy. The health-promoting impact of a lucerne-derived protein extract in animals is well-known and well-documented. It is interesting therefore to describe the human body’s response to protein supplementation of lucerne. Materials and Methods: The literature in scientific journals and reports regarding the lucerne protein in human nutrition was reviewed. Results: A series of studies have demonstrated that a lucerne concentrate may be a dietary supplement that supports humans in combatting malnutrition, ischaemia, and various digestive tract disorders. The lucerne concentrate also increases the resistance of the immune system, improves the content of haemoglobin in the blood by supplying large amounts of iron and promotes a beneficial intestinal microbial population. The concentrate aids in the recovery from severe cancer-related disorders and supplements the treatment of certain diseases, such as tuberculosis, HIV, and malaria. Conclusions: This study presents a short review of the literature on the properties of lucerne and the secondary metabolites it contains and also on a protein concentrate produced from the lucerne juice. The case reports and short reports described in this review indicate that the lucerne protein concentrate can be used as a dietary supplement for improving the health and quality of human life.

2

Keywords: supplement in the human diet, content of amino acids, minerals and vitamins, concentrate from lucerne, protein, phytochemically active substances, secondary metabolites 1. Introduction Lucerne is native to warmer temperate regions. It has been grown as a fodder crop for animals since the ancient Greek and Roman times. At present, perennial and multi-harvest lucernes are of major importance in Europe because they have a deep root system capable of extracting water and nutrients from the deep layers of the soil. The deep root system also makes lucerne resistant to drought. Lucerne should be grown in a fertile soil at a pH close to neutral. Similar to other legumes, lucerne roots are colonized by bacteria that fix nitrogen from the air. The nitrogen in the vegetable residues is retained in the soil, thereby enriching the soil environment or it is transferred to other non-legume crops. Lucerne is harvested three, four, or sometimes as many as five to six times a year. Juvenile intensively mowed plants have the highest nutritional value. Apart from proteins, minerals and vitamins, lucerne also contains secondary metabolites which have phytobiotic activity in humans and animals. The size of the world’s human population has continually increased with a concomitant growth in the demand for animal protein and other protein-based foodstuffs. The production of animal protein relies on the development of animal farming and on an increased demand for animal feed, which severely taxes the available resources of land, capital and energy required to grow fodder plants, hurts the environment (e.g., by animal droppings, the emission of nitrogen and greenhouse gases, and the warming of the climate) and compromises biodiversity. These are the reasons behind an on-going search for new, cheaper, safer and more environmentally friendly protein sources and for other valuable food additives and secondary metabolites of plant origin (Aiking, 2011). Legume and cereal seeds provide the main source of non-animal protein for humans (Day, 2013). Plant protein has also been obtained from the aerial parts of lucerne (Davys et al., 2011; Mathur et al. 2013), and from cassava (Fasuyi and Aletor, 2005), sea algae (Dewanji and Matai, 1996) and even from some other vegetable and cereal crops (Dewanji and Matai, 1996; Hussein et al. 1999). Easily produced by simple technology, cassava meal and leaf protein extract (CLPC), are attractive protein sources for local food production and malnutrition mitigation systems in the majority of developing regions (Fasuyi and Aletor, 2005). An idea also emerged from those regions to produce protein to feed humans and animals from the plant biomass that is used to manufacture bioethanol in bioethanol refineries (Chiesa and Gnansounou, 2011). Food shortages and the resulting undernutrition are very serious issues in developing countries both in terms of the wellbeing of local communities and of their economic development. An undernourished population is often in poor health and prone to different disorders and unable to profit from education and to perform well at their jobs (Davys et al., 2011). Prolonged undernutrition seriously affects the physical development and the nervous systems of the affected youths. Therefore, in many African, South American countries, in India and other Asian countries, and even in Europe, possibilities are being explored for the production of plant proteins which have phytobiotic activity and which could be extracted from locally grown crops well suited to the local habitat. Furthermore, approximately 5% of the total human population do not tolerate gluten, a protein which is present in the grains of rye, barley and oats and is the predominant protein in wheat grain. Such people are inclined to give up gluten-containing food altogether (Podolska et al. 2006) and to use alternative protein sources. Nourishment with natural products is currently coming in vogue in some highly developed countries. Abstention from meat and its replacement with vegetable products in the human

3

diet is essential to this new trend. Protein extracts and concentrates of vegetable origin are a valuable source of amino acids and other life-sustaining nutrients for all such people. Lucerne is a plant that can be used to produce a protein concentrate (Bertin et al., 2008; Day, 2013; Davys et al., 2011; Gaweł, 2012; Gaweł and Grzelak, 2014; Zanin, 1998). The plant supplies amino acids and other life-sustaining nutrients in areas where for cultural reasons and tradition, little or no meat is consumed. Lucerne is a very valuable small-seed legume that can produce an annual crop of 45-90 t∙ha-1 of fresh herb which, when dried, yields 15-22 t∙ha-1 of protein-rich fodder (Gaweł and Żurek, 2003). The protein and crude fibre content of the crop is closely related to its developmental stage at harvest. Therefore, the fodder thus obtained is quality-manageable and, depending on its crude fibre content, can be fed to different monogastric and ruminants according to their nutritional needs. The high fodder value of lucerne is testified by the widespread use of major lucerne species on all continents. The nutritional qualities of lucerne have been known for a long time in folk medicine in Iraq, Turkey and America, where the plant has been used to treat various ailments, stimulate the appetite, assuage abscesses and boils, maintain proper blood circulation, and increase disease resistance (Bora and Sharma, 2011). Even the traditional Chinese medicine regards it as a medicinal plant useful for human nutrition especially to aid the treatment of alimentary tract diseases (e.g., bloats and ulcers) and because of its oestrogenic properties (APEF Newsletter,2007; Davys et al., 2011). Lucerne leaves contain substantial amounts of ßcarotene, vitamins B, C, D, E and K, and salts of potassium, calcium, iron, and phosphorus. Due to its high chlorophyll content, lucerne has detoxifying and anticancerogenic activities, especially in the alimentary tract (Furgał and Milik, 2008). Lucerne is mainly used as an animal feed. It is less frequently used as part of the human diet. In Poland, folk medicine uses this species because of its rich chemical composition and applies it in concert with pharmacological substances to treat diseases of the digestive tract, circulatory system and the immune system (Bertin et al., 2008; Gaweł, 2012). Due to the positive impact of lucerne on the human body, the literature on the phytochemical and therapeutic potential of lucerne and its uses in human nutrition was compiled. The aim of the review was to assess the impact of compounds from lucerne as a human diet supplement and to describe its use in particular cases. 2. Methodology This literature survey investigates various nutrients, secondary metabolites, protein and vitamins present in lucerne and evaluates their impact on human health. The relevant data were retrieved using popular search engines such as PubMed, Google, and Firefox and by searching the content of numerous indexed journals related to human nutrition, herbal medicine, and the chemical composition of plants. The keywords used to search for relevant literature included lucerne as related to nutritional impact on humans, chemical composition of lucerne, lucerne protein concentrate in human nutrition, health responses in humans to lucerne protein concentrate. 3. Obtaining extracts and protein concentrates of vegetable origin The earliest available studies on the possibility of substituting animal-derived proteins in the human diet with proteins of plant origin were published in the 1970s. According to Pirie (1975) leaf protein, which was characterized with high nutritive value because of its high content of amino acids, vitamins, lignin and polyphenols, was recommended as food for

4

humans in 1960 for the first time. The downside of leaf protein is the content of small amounts of some antinutritional secondary metabolites (phytinians, tannins, saponins) which may negatively affect the human body when consumed in large amounts (Pirie, 1975). The aerial parts of many plant species have been used in human nutrition for a long time as a source of leaf protein, sugars, mineral salts and vitamins. Different technologies have been exploited for their utilization based on pulping, juice extraction, drying and plant dehydration (Badar and Kulkarni, 2011). Initially, human nutrition relied on freshly expressed juice, frozen juice, coagulates obtained by heat treatment, dehydrated matter and extracts from various protein, mineral salts and lipid yielding plants (Badar and Kulkarni, 2011). Leaf proteins are extracted by treating the plant with heat, acid, organic solvents or polyelectrolytes (Baraniak and Baraniak, 1989). It is important when processing that the ready product should be free of extract residue. Various simple devices have been constructed to obtain the juice and protein extracts such as presses, screw expellers and extruders to be used by small rural communities or in individual homesteads (Raji and Olofin, 2011). The high nutritive value of amino acids in the green juice expressed from the aerial parts of lucerne and prepared by spray-drying was found as early as in the 1970’s (Hartman et. al., 1967). A ground-breaking process of obtaining the “white” edible Pro-Xan II protein fraction from expressed lucerne juice that had been heat treated at 80°C was described by Edwards et. al. (1975). In France, a method was developed to produce a leaf concentrate from lucerne on a commercial scale by coagulation and fluidized bed technology (Scientific Opinion2009; Zanin, 1998). This product, enriched with vitamin C as an antioxidant or kept in cold storage, has been recommended for the treatment of people who have developed health issues related to undernutrition or a deficiency of some nutrients in the diet (Commission decision , 2009). The concentrate contained many amino acids, mono- or polyunsaturated fatty acids, vitamins, minerals, organic acids whose activity was previously described by Andurand et al. (2012), Gaweł (2012), Gaweł and Grzelak (2014), Zanin, (1998). 4. Type Medicago (L.) - Lucerne (L.) Lucerne (Medicago sativa L.) comprises approximately 100 species, of which 90 have been studied closely and for which descriptions are available. The genus Medicago (L.) encompasses annuals, biennials and perennials grown for hay, haylage and dried silage. Available cultivars also include, along with those suitable for hay, genotypes adapted for use for pasture and for grazing. Lucerne also provides a raw material from which extracts and protein concentrate are produced. Lucerne fodder is used as a food for ruminants and monogastrics. Its nutritional value depends on many factors, and the growth stage and harvest date are of major importance. Nutritional quality of lucerne fodder varies substantially within a season. In Poland, farmers call lucerne “the queen of fodder crops” because of its high nutritional value. Fodder is composed of the aerial parts of the lucerne plant but herbal medicine also utilizes the roots, e.g., because of their high saponin content, as well as seeds and sprouts which contain phytosterols. Some of lucerne compounds are utilized by cosmetics industry. In the human diet, lucerne provides a source of protein, mineral salts, vitamins, macro and micro nutrients and secondary metabolites. Iran formerly Persia is regarded as the area in which the genus Medicago (L.) originated and from which it rapidly spread to all continents. Greece was the first European country to which lucerne arrived from ancient Persia (Media). The ancient Greeks called the plant “the medic grass” and the Latin version of the name “herba medica” gave rise to “Medicago”, the name under which Linnaeus introduced lucerne into the botanical lexicon. In antiquity, lucerne was the principal fodder for horses and it spread along the caravan routes (as well with cavalry and chariot horses of invading armies) of the Persians, Greeks and Romans. It

5

was carried as far afield as China, India, Greece, North Africa and Spain. In the beginning of the 16th century lucerne cultivation began in France and, by the 18th century, it reached Russia, Austria, Germany and Switzerland. Lucerne is grown between the 30 and 60th parallels of the northern and southern hemisphere and the area covers eastern Asia, the Middle East, Europe, the northern part of Africa, the USA, the central and southern part of Canada, northern Mexico, the central part of South America, Australia and new Zealand. At present, lucerne Medicago (L.) is the world’s major leguminous fodder. In 2009, it was grown in an area of 30 million hectares all across the world, and 11.9 million hectares was grown in North America, 7 million hectares in South America, 7.12 million hectares in Europe, 2.23 million hectares in Asia, and 1.75 million hectares in Africa and Oceania (https://en.wikipedia.org/wiki/Alfalfa). Common lucerne (Medicago sativa ssp. sativa (L); sand lucerne (Medicago sativa ssp. media (M x varia); yellow lucerne/alfalfa (Medicago sativa ssp. falcata (L.) are the major lucerne species grown in Europe. They are perennials with considerable persistence. Common lucerne has a straight, strongly ramose stalk that reaches 90 cm in height. The leaves are trifoliate, although multifoliate forms have been developed. Lucerne has a deep tap root (as deep as 3 – 5 m, in some cases even 15 m), flowers that range from violet to purple. Lucerne’s inflorescence is cephaloid, the fruit is a polyspermous pod filled with small oval seeds. Yellow lucerne, according to the current taxonomy, is a subspecies of common lucerne. Yellow lucerne’s stalks are strongly ramose, hairy, erect or prostrate, measuring 40-60 cm in length, with a long ramose main tap root, trifoliar leaves that are slightly puberulent, yellow flowers, and an aciniform inflorescence. Yellow lucerne’s fruit is a falcate pod filled with dark brown seeds. Yellow lucerne is remarkable for its resistance to drought and freezing and these qualities have been exploited in breeding. Sand lucerne (Medicago sativa ssp. media (M x varia) (Martyn)) is a hybrid of common and yellow lucerne and thus shows traits intermediate between the two. It is very variable, very drought and freeze-tolerant, and its flower heads may be yellow, dark blue, violet red to violet or a mixture of these colours. The fruit is a polyspermous pod with oval seeds, renniform or oblong in shape, and yellow to yellow-brown in colour. 5. Content of natural substances in lucerne and their impact on humans The composition of lucerne features various phytochemically active substances: carotene, chlorophyll, cumarines, organic acids, amino acids (see Table 1), lutein and other secondary metabolites such as isoflavones, alkaloids, saponins, phytinians and L-canavanine which show phytobiotic activity even in small quantities (Scientific Opinion of the Panel on Dietetic Products….,2009). Amino acids In 2009, lucerne concentrate was allowed to be used in human nutrition due to its rich and valuable chemical composition as it has a high amino acid content, especially of the so-called essential and semi-essential amino acids. Those amino acids occur in amounts much higher than those found in eggs or wheat in the lucerne concentrate (Scientific Opinion of the Panel on Dietetic…., 2009; Zanin, 1998) (Table 1). Table 1. L- canavanine

6

The presence of L-canavanine, a potent anti-cancer agent, in lucerne juice is much higher than in the lucerne concentrate but less than in soybean or onions (Scientific Opinion, 2009) (Table 2). Tests performed on autoimmune-prone MRL-lpr/lpr mice showed that the lucerne sprout extract contained other phytoestrogens that positively affected their bodies, in addition to coumestrol, which increased the survival rate (Hong et al., 2011). The toxic amino acid L-canavanine occurs in lucerne in larger quantities in the leaves (0.91.2 mg/g) than it does in the shoots (0.6-0.9 mg/g). The highest amounts have been identified in the seeds and sprouts (80-150 mg/ka). A leaf concentrate, however, contains only 4.3 mg/kg of L-canavanine (Scientific Opinion,2009). L-canavanine ingested in a large amount shows cytotoxic and antimetabolic activity which disturbs DNA and RNA synthesis. It may also cause auto-immune disorders in humans. For that reason, caution should be exercised when using a lucerne protein concentrate in the human diet. Alcocer-Varela et al. (1985) demonstrated that L-canavanine-containing lucerne sprouts induced auto-immune haemolytic anaemia and exacerbated the development of systemic lupus erythaematosus (SLE) in monkeys, whether they were healthy or already suffering from that condition. Table 2. Saponins Saponins (water soluble, soap-like foaming glycosides) occur in much larger quantities in the roots (up to 5%) of lucerne than they do in the aerial parts (1.5 to 3%), which are used to produce animal feed and the protein concentrate. The saponin content in the lucerne protein concentrate ranges from 0.5 to 1.5% (Commission decision, 2009). Saponins are classified as anti-nutritional substances but in the human body they show an anti-arteriosclerotical activity by inhibiting the deposition of cholesterol in the blood system (Hwang et al., 2001). Malinow et al. (1978) observed the reduction of arteriosclerotical plaques after monkeys had been fed a feed containing cholesterol and lucerne meal for 6 months. Similar results were obtained from experiments with rabbits. Following a 4-month period of the ingestion of lucerne meal and lucerne seeds, the rabbits showed a reduced concentration of cholesterol in the aortic sudanophilia, in the aortic infima-plus-media and in the liver (Malinow et al., 1980). In a later study, a decrease in the cholesterol concentration was also observed in Macaca fascicularis (Malinow et al., 1981). In the human body, saponins form insoluble complexes with cholesterol which are subsequently excreted in the faeces. The anti-sclerotical activity of a lucerne extract which contained flavonoids, phytoestrogens, and ascorbic acid is effected by inhibiting the formation of cholesterol, thereby protecting people against arteriosclerosis and preventing the development of coronary heart disease in postmenopausal women (Hwang et al., 2001). An important property of the saponin-like group of substances is related to their ability to precipitate cholesterol, which is an amphipathic molecule having a lipophilic and hydrophilic portion (Barbosa, 2014). A body of evidence suggests that bioactive substances (secondary metabolites) may be used as an aid in the treatment of diabetes. A study on rats showed the inhibition of postprandial glycaemia in individuals with type 1 and type 2 diabetes who had been administered a water extract of lucerne seeds (Winiarska et al., 2007). Because of their high content of phenolic compounds and high antioxidant activity lucerne sprouts have been reported to show a nutraceutical effects in humans (Zinca and Vizireanu, 2013). Saponins also show antibacterial, antifungal and antiviral properties, restrict the development of protozoa, lower the digestibility of protein, negatively affect the reproductive potential of humans and animals and inhibit the development of carcinogenic cells (Francis et al., 2002). Sadowska et al. (2014), while looking into the effect of the aerial parts and roots of lucerne on the development of Candida albicans, an opportunistic fungal pathogen of humans

7

and animals, showed a reduction of hyphal growth, yeast adhesion and biofilm formation both in humans and in animals. Those authors stressed the role of vegetable saponins in enhancing the activity of curative and disinfecting drugs in antifungal therapy. In addition, saponins also control the gram-positive bacteria Bacillus cereus, B. subtilis, Staphylcoccus ureus and Enterococcus faecalis and some yeast-like fungi (Avato et al., 2006). Other negative and positive effects of saponins on the human body have been described by Gaweł (2012) and Sadowska (2014). Relatively rarely, haemolysis followed a long period of ingestion of substantial amounts of saponin-containing lucerne products, and the result was that haemoglobin, which is otherwise present in erythrocytes, permeated into the blood serum. Haemolysis may result from an ecogenetic disorder known as favism, a condition related to the deficiency of glucose-6-phosphate dehydrogenase which is crucial for the carbohydrate metabolism of erythrocytes and leads to the production of glutathione, a strongly oxidizing enzyme that protects erythrocytes against disintegration. In a damaged digestive tract, saponins may penetrate into the bloodstream and cause haemolysis, which caused Śmiechowska (2000) to express the opinion that saponins should be used with caution and in small quantities in the treatment of diseases. Hemolysis is caused only by certain saponins according to a review by Barbosa (2014). Oda et al. (2000) studied the haemolytic activity of 47 saponins and found no relationship between their immuno-stimulating and haemolytic properties. These researchers showed that the chemical structure of saponins was involved in their haemolytic activity. Oda et al., (2000) also asserts that saponins which have a glycine moiety and a sugar chain as part of their structure also show a high blood-hemolysing activity. Studies on resistance of red blood cells and their breakdown due to saponins revealed the permanent disruption of the erythrocyte membrane, which failed to repair itself once the contact with saponins was discontinued (Baumann et al., 2000).

Phytoestrogens Phytoestrogens, which occur in protein products of vegetable origin have a beneficial effect on humans and animals if ingested in small quantities. Improper or excessive intake of phytoestrogens may have a negative impact on organisms but all of the side-effects of their use have not been currently recognized (Rishi, 2002). Lucerne contains phytoestrogens, which are classified as isoflavones, and coumestrol. Both phytoestrogens and coumestrol show oestrogenic activity similar to that of the female hormones responsible for the development of the female reproductive organs (Bora and Sharma, 2011; Zanin, 1998). Phytoestrogens mitigate menopause-related ailments in women, help keep osteoporosis in check, and restrain the development of hormone-dependent cancers of the breast and prostate gland in men (Hwang et al., 2001; Rishi, 2002; Zanin, 1998). Menopause-related diseases can be treated with clover extracts which show oestrogenic activity and are rich in therapeutic substances such as phenolic acids, isoflavones and other flavonoids (Kołodziejczyk-Czepas, 2014). Indian studies describe an improvement of memory and of cognitive brain functions, an increase in bone density, protection against cancers of the breast, colon and prostate in older persons who have been given a treatment with plant isoflavones (Anklesaria, 2011). Other chemical substances in lucerne (vitamins, iron) Proteins, vitamins and mineral constituents have been extracted from the above-ground parts of various plants for a long time and given to persons suffering from health problems related to malnutrition or to the absence of some nutrients from their diet. Apart from protein, the concentrate contains a number of different nutrient constituents: carotene, B vitamins, calcium and iron which improve human health (Table 3). A method was developed to extract the valuable natural pigments carotene and lutein from the leaf protein to be used in foodstuffs

8

(Favati et al., 1988). Plant herbage-based protein formulas were substituted for iron and folic acid in the treatment of anaemia in women and adolescent girls in India, where the condition is widespread due to a low intake of animal protein (Vyas et al., 2010). Table 3.

6. Use of lucerne protein concentrate in treatment of human disorders and in diet supplementation– case reports and short reports Lucerne sprouts provide a valuable addition to salads and to mixed fresh vegetables in the human diet, thereby improving their palatability and nutritive value. Occasionally, dried lucerne is added to baked goods (i.e., cookies, bread) as well as to sauces and soups. In some countries, it may even be an ingredient in various wafers. Leaves, dried herbage as well as lucerne juice concentrate is used to treat various disorders of the alimentary tract, rheumatism, skin diseases, to improve general fitness, to enhance resistance, to cleanse the body from toxins and to combat malnutrition (Badar and Kulkarni, 2011; Bhalja and Subhadra, 2011; Bora and Sharma, 2012; Lodha et al., 2009; Rishi, 2002). The extraction of bioactive constituents conducted by Caunii et al., (2012) showed that the lucerne extract contained a number of free hydroxyl groups (hydrogen donors) which impart to the product a potent antioxidizing activity in the human body. The presence of oxidizing compounds in the human body leads to damage to cells (e.g., DNA mutation) and to development of many chronic diseases such as arteriosclerosis, arthritis, diabetes, or cancer. Synthetic and natural antioxidants are added to the diet to prevent and mitigate these conditions. Natural antioxidants are of particular value because they do not produce sideeffects (Sarmadi and Ismail, 2010). Lucerne protein concentrate is a natural antioxidant. Tests in rabbits by Hwang et al. (2001) showed a strong antioxidizing and anti-arteriosclerotic effect of flavonoids and phytoestrogens from the concentrate, especially in the presence of an acerole cherry extract. In their review on the phytochemical and pharmacological potential of lucerne Bora and Sharma (2011) emphasized the protective significance of substances contained in the plant. The authors of that study expressed the hope that medicinal substances occurring in lucerne will be exploited to develop natural pharmacological products with much therapeutic benefit, including antioxidant properties, the prevention of arteriosclerosis and aiding the function of the nervous and cardio-vascular systems. However, various extracts made from lucerne contain less protein than does a protein-xanthophylls concentrate obtained through dehydration. That novel method consists of processing the plants within 2 hours after harvest by passing them through a press, separating the juice from the fibrous residue and subjecting it to high temperature to coagulate and dehydrate the protein. As a result, proteolytic processes are disrupted and more nutritive compounds pass to the pressed-out juice compared to conventional grinding of the plant material. Dehydration of lucerne results in a product that contains 60-65% more chlorophyll pigments: phyto compounds which are important because of their anti-cancer properties and in the treatment of alimentary tract diseases, vitamin U (not actually a vitamin but a substance called S. Methylmethionin found in green vegetables notably in green cabbage) which is essential in the treatment of ulcers, stomach and large intestinal disorders and carotene (Andurand et al., 2010). To obtain a good quality concentrate with a high pigment content (i.e.,chlorophyll and carotenoids) and Omega 3 fatty acids which are sensitive to light and prone to oxidation, it is essential to harvest lucerne at an early growth stage and to properly protect the concentrate with antioxidants e.g., high amounts of vitamin C (Zanin, 1998).

9

A study was conducted for three months on two groups of adolescent women: one group was given daily doses of 60 mg iron and 500 mcg of folic acid, and the other group was administered leaf protein (LC; 5 mg iron, 13 mcg of folic acid). The study showed that the leaf protein was just as effective against anaemia as the drug treatment (Vyas et al., 2010). Chronic myeloid leukaemia (CML) patients require a special diet to support the organism in its struggle to contain the disease and to increase the wellbeing of the diseased person. The diet must contain the micro nutrients, protein and vitamins necessary to maintain the body weight and food parameters at a stable level. In India, CML patients receive leaf protein for reinforcement and recuperative purposes (Lodha et al., 2009). Those authors showed a positive effect in maintaining and increasing body weight, haemoglobin and red blood cell count (RBC) after three months of supplementing the patients’ diet with a daily dose of 10 grams of lucerne protein extract (Table 4). An additional benefit was the improved tolerance to chemotherapy, which the patients were undergoing at that time. Table 4. A deficiency of micro nutrients, and particularly of iron, results in serious health disorders, such as anaemia, and deterioration of the quality of life, especially in poor countries with low consumption of animal protein. Children are especially vulnerable to vitamin shortages. Iron and iodine if in short supply, negatively affect the physical and mental development of the children, and in the worst cases, their survival. In India, where children of up to 5 years of age account for 14% of the population, the government has launched a special programme to fight malnutrition in children, pregnant women and lactating mothers. The programme aims at increasing the caloric load of the meals and simultaneously increasing the micro nutrients by adding 3% lucerne concentrate (Matur et al., 2013). An experiment was performed on a control vs. a treated group of children 3 to 6 years of age which lasted 24 months (Table 5). At the end of the experiment, moderate anaemia was found in significantly fewer children and severe anaemia did not occur at all in the lucerne concentrate-treated group compared to the control group (Table 5). Morbidity in children (i.e., fever, diarrhoea, feeling unwell) in the lucerne concentrate-treated group was similar to that in the control group. However, according to Mathur et al. (2013) during the following 24 months of administering lucerne concentrate, the percentage of children who complained of no ailment was higher in the group with lucerne concentrate supplementation than in the control (non-supplemented) group. An obvious inference is that their well-being improved and the higher haemoglobin level was related to the supplementation of lucerne protein which also supplied the children with micro nutrients required to boost their resistance to various ailments.

Table 5. In another study, nutrients, amino acids, phytocompounds, β-carotene, vitamin A and iron contained in lucerne leaf concentrates and extracts were found to enhance the therapeutic efficiency for HIV, malaria and malnutrition-related anaemia (APEF Newsletter, 2007; Bhalja and Subhadra, 2011; Davys et al., 2011 Lodha et al., 2009; Taky et al., 2006; Vyas et al., 2010; Zanin, 1998). A protein-xanthophylls concentrate (PX) manufactured in France contains 55-60% protein, 9-10% crude fat including 6.4% polyunsaturated fatty acids, n-3.314% mineral constituents, 8% water and 1-2% of crude fibre (Andurand et al., 2010; Bora and Sharma, 2011). Improvement in the wellbeing, blood parameters and in general health conditions was shown in humans fed a diet supplemented with protein concentrate (Bhalja and Subhadra, 2011; Rambourg and Monties, 1983). A beneficial effect on the human body of the French protein concentrate liberally enriched with vitamin C was instrumental in the European Commission’s decision to allow its use in human nutrition (APEF Newsletter,

10

2007; Commission decision,2009). Vitamin C was added to the protein concentrate to prevent its oxidation during storage. In the human diet, a lucerne concentrate is used as a preventive and adjunctive agent in the treatment of various ailments. Chlorophyll contained in the lucerne aids detoxification in the human body and its alkalizing properties are exploited in the prevention of tumours’ of the alimentary system, in the elimination of bloat and in the treatment of ulcers (Zanin, 1998). Moreover, a lucerne concentrate boosts the immune system, especially in older people, and increases the haemoglobin content of the blood. Lucerne concentrate saponins lower the cholesterol level, thereby protecting against arteriosclerosis of the blood vessels (Rambourg and Monties, 1983). The oestrogenic activity of the protein concentrate should also be emphasized because it mitigates menopause symptoms in women (Anklesaria, 2011; Badar and Kulkarni, 2011). A lucerne concentrate administered to humans mitigates shortages of protein, vitamins and minerals in the diet (Tables 6 and 7). It is also a cheap source of those constituents for lowincome people and has been frequently distributed by missionary centres. Interested parties can familiarize themselves with the cultivation of the species and methods for extracting valuable juice. Beta-carotene is responsible for many vital functions in the human body. Ten grams of lucerne protein xanthophyll concentrate supplies twice to 1.5 times the ß-carotene daily requirement in juveniles and adolescents and 70% of the daily requirement for adults (Zanin, 1998) (Table 6). The vitamin E in the concentrate covers half of the daily requirement of children and one-fourth of adults. Excessive blood clotting may occur due to a high content of vitamin K in the concentrate (Davys et al., 2011; Zanin, 1998). To avoid this, 2 – 3 months after receiving the concentrate, it is recommended to discontinue its intake for three to four weeks (Table 6). Breaks in taking the formulation are also indicated because some chemical compounds and fat-soluble vitamins are stored in the liver and there is a hazard that the threshold limits of their content in the human body can be exceeded. Table 6. All minerals present in the lucerne concentrate and listed in Table 7 have important bodily functions and must be supplied to humans in the diet. Iron, iodine and calcium are considered as the most important (Zanin, 1998). Ten grams of the concentrate secures half of the daily requirement for children and one-third of the daily requirement for adults. A dose of 10 g of the concentrate contains 7 mg of iron, which covers 70% of the daily requirement for that important constituent for children and 39% for adolescents and adults. Other studies also confirmed the efficacy of the concentrate for the treatment of chronic leukaemia (Lodha et al., 2009). Iodine present in the concentrate covers only 2.5% of the requirement for children and 1.5% of the requirement for adults (Table 7). Blood haemolysis is a rarely observed sideeffect of supplementing the human diet with a lucerne concentrate. Table 7. The saponins and flavonoids in the lucerne concentrate favourably affect human health and strengthen the human immune system. Saponins prevent arteriosclerosis because they form insoluble complexes with cholesterol which are subsequently excreted in the faeces (Dong et al., 2007; Dong et al., 2011; Zanin, 1998; Khaleel et al., 2005; Reshef et al., 2006). Saponins also have antifungal activity. Some flavonoids in the lucerne concentrate have an activity similar to that of oestrogens (hormones) in that they mitigate neurovegetative complaints during menopause. Likewise, they protect against hormone-dependent breast cancer and inhibit osteoporosis (Anklesaria, 2011; Gaweł, 2012; Rishi, 2002). Preclinical trials carried out in India on a group of adolescent girls 14 – 18 years old suffering from anaemia showed an increase in the haemoglobin level and the enhancement of

11

other blood parameters following the administration of 10 g/day of a lucerne concentrate (Bertin et al., 2008). Likewise, an increase in the amount of haemoglobin and of other red corpuscle indices was obtained by Taky et al., (2006). In China, India and Romania, a lucerne concentrate administered to older persons at a daily rate of 15 g increased the level of haemoglobin, retinol, calcium and magnesium in the blood (Bertin et al., 2008). In young persons, it improved their sense of wellbeing and psycho-physical condition (Bertin et al., 2008). It also increased exercise tolerance in young Polish athletes (Furgał and Milik, 2008). In India, girls diagnosed with anaemia were given 5 to 10 g of a lucerne concentrate per day to increase their blood haemoglobin level and improve red corpuscular parameters (Matur and Ramani, 2006). A 180-day long study performed in Cameroon investigated the effect of daily rates of 10 and 15 g of a lucerne concentrate on body weight increases, the lymphocyte and haemoglobin content of the blood, the sense of wellbeing, improved appetite, general health, the occurrence of fever and diarrhoea, and a body weight increase index in HIV-seropositive and undernourished persons and compared it to the performance of similarly afflicted persons who received no treatment (APEF Newsletter, .2007). In general, after 60 days of the treatment with a lucerne concentrate an improvement in the treated over the non-treated group was evident and included an increased sense of wellbeing, a reduction of bacterial infections which were less acute and lasted for a shorter time. After 180 days, the body weight continued to decline in the non-treated control group, the blood content of haemoglobin and lymphocytes did not change significantly nor did that of the CRP (C reactive protein) while fever and infections continued to occur (Table 8). In the group that received 10 or 15 g of a lucerne concentrate, a radical improvement of appetite accompanied by body weight and body weight index increases as well as an increase of haemoglobin content by 7.6% was seen. A slow decline in CRP was also observed over the last 60 days of the study and an irreversible remission of fever before the 120th day (Table 8). Thus, the results confirmed the positive effect of a lucerne concentrate on wellbeing and resistance in AIDS-diseased persons, which may be of substantial significance in improving the welfare of people with this affliction. Lucerne is a natural product with high therapeutic value showing entopharmacological, phytochemical and therapeutic importance in human nutrition, primarily due to the presence of secondary metabolites (Bora and Sharma, 2011). In addition to all of the positive effects on humans as described above, a methanol extract of alfalfa also showed a significant antidepressant effect in mice as compared to other extracts (Bora and Sharma, 2012). 7. Conclusions In 2009, a protein-xanthophyll concentrate of lucerne received official permission for use in human nutrition. This concentrate’s composition included vitamins B, E, A, K U and Omega 3 fatty acids and numerous essential and semi-essential amino acids. The constituents of the concentrate were found to increase human resistance to infections and various ailments and, because of the iron content, to improve the blood content of haemoglobin, thereby helping to control anaemia caused by haemoglobin shortage. In addition, the concentrate enhanced the beneficial intestinal microflora, alleviated alimentary tract diseases and stopped diarrhoea, as well as improving the general sense of wellbeing. After prolonged intake, the vitamin K in the concentrate can result in excessive blood clotting. Likewise, if saponins are ingested for a long time in a human diet enriched with lucerne protein concentrate, in some extreme cases haemolysis may occur (Davys et al., 2011). Therefore, it is advisable to stop treatment for one month after the preparation has been taken for 2 – 3 months. The body uses that time to cleanse itself of the excess of accumulated vitamins and chemical compounds which, if present in inordinate amounts, can adversely affect health. A lucerne concentrate

12

was found to aid in the recovery from cancer radio- and chemotherapy as well as those suffering from HIV, tuberculosis and malaria. It controls malnutrition, hunger, anaemia and alimentary tract diseases in developing countries. The oestrogenic effect of the concentrate is of no small significance, especially its alleviation of menopause-related complaints in women. The low price of the concentrate makes it affordable to impoverished people and allows it to be used in the diet of persons who eat small amounts of animal protein for cultural reasons. Conflict of interest The authors declare that there are no conflicts of interest

REFERENCES 1. Aiking H. Future protein supply. Trends in Food Science & Technology 2011; 22: 112120. 2. Alcocer-Varela J, Iglesias A, Llorente L, and Alarcón-Segovia D. Effects of L-canavanine on T cells may explain the induction of systemic lupus erythematosus by alfalfa. Arthritis Rheum 1985; 28(1): 52-57. 3. Andurand J, Coulmier D, Despres JL, and Rambourg JC. Extraction industrielle de protéines et de pigments chez la luzerne: état des lieux et perspectives. Innovations Agronomiques 2010; 11:147-156. (in French) 4. Anklesaria B. The promise of phytoestrogens. Indian Journal of Pharmacology 2011; 43(4):369-370. 5. APEF Newsletter, 2007. APEF Newsletter, Association for the promotion of leaf concentrate … (2007). Lucerne Leaf Concentrate. A simple and effective solution in the fight against malnutrition. Possible help for people with Aids? Report of the lucerne leaf concentrate (LLC/HIV trial. Nogousso Clinic, Yacundé [Online]. Available http://nutritionluzerne.org/anglais/a_newsletter.htm (Accessed: 04. May. 2013). 6. Avato P, Bucci R, Tava A, Vitali C, Rosato A, Biały A, Jurzysta M. (2006). Antimicrobial activity of saponins from Medicago sp.: structure-activity relationship. Phytotherapy Research 2006; 20(6): 454-457. 7. Badar KV, Kulkarni AU. LPC is novel source of protein for human health and nutrition: A review. Current Botany 2011; 2(1):5-7. 8. Baraniak B, Baraniak A. Protein concentrate from alfalfa and cocksfoot by polyelectrolyte precipitation. Nahrung 1989; 33: 491-495. 9. Barbosa AP. Saponins as immunoadjuvant agent: A review. African Journal of Pharmacy and Pharmacology 2014; 8(41): 1049-1057. 10. Baumann E, Stoya G, Völkner A, Richter W, Lemke C, Linss W. Hemolysis of human erythrocytes with saponins affects the membrane structure. Acta Histochemica 2000, 102: 21-35. 11. Bertin E, Matur B, Rama SV. A comparative study of impact of leaf concentrate and iron and folic acid supplementation on blood profile of anaemic adolescent girls. In Grela (eds) Alfalfa in human and animals nutrition. Dzierdziówka –Lublin, Poland: Wydawnictwo Stowarzyszenia Rozwoju Regionalnego i Lokalnego “Progress” 2008; 3 pp 59-63. (in Polish, summary in English)

13

12. Bhalja P, Subhadra M. Effect of nutritional counselling and alfalfa supplementation on exercise performance and nutritional anaemia of overweigh and obese adult females. Journal of Human Ecology 2011; 35(3):55-159. 13. Bora KS, Sharma A. Phytochemical and pharmacological potential of Medicago sativa: a review. Pharmaceutical Biology 2011; 49(2):211-220. 14. Bora KS, Sharma A. Evaluation of anxiolytic effect of Medicago sativa in mice. Pharmaceutical Biology 2012; 50(7):878-882. 15. Caunii A, Pribac G, Grozea I, Gaitin D, Samfira I. Design of optimal sol vent for extraction of bio-active ingredients from six varieties of Medicago sativa. Chemistry Central Journal 2012; 6(1):123. http://journal.chemistrycentral.com/content/6/1/123 (accessed 21 11 2012) 16. Chiesa S, Gnansounou E. Protein extraction from biomass in a bioethanol refinery – Possible dietary application: Use as animal feed and potential extension to human consumption. Bioresource Technology 2011; 102(2): 427-436. 17. Commission decision, 2009. Commission decision of 13 October 2009 authorizing the placing on the market of a leaf extract from Lucerne (Medicago sativa) as novel food on novel food ingredient under Regulation (EC) No 258/97. 2009. European Parliament and of the Council. Available online: http://eur-lex.europa.ru/LexUri Serv.do?uri=OJ:L:2009:294:0012:0013:FR:PDF (Accessed:13 December 2012). 18. Davys MNG, Richardier F-C, Kennedy D, de Mathan O, Collin SM, Subtil J, Bertin E, Davys MJ. Leaf Concentrate and Other Benefits of Leaf Fractionation. In: Thompson, B., and Amoroso, L. (eds) Combating Micronutrient Deficiencies: Food-based Approaches. Rome, Italy: CAB International and FAO 2011; pp 338-365. 19. Day L. Protein from land plants – Potential resources for human nutrition and food security. Trends in Food Science & Technology 2013; 32: 25-42. 20. Dewanji A, Matai S. Nutritional evaluation of leaf protein extracted from three aquatic plants. Journal of Agricultural and Food Chemistry 1996; 44: 2162-2166. 21. Dong XF, Gao WW, Tong JM, Jia HQ, Sa RN, Zhang Q. (2007). Effect of Polysavone (alfalfa extract) on abdominal fat deposition and immunity in broiler chickens. Poultry Science 2007; 86:1955-1959. 22. Dong XF, Gao WW, Su JL, Tong JM, Zhang Q. Effects of dietary polysavone (Alfalfa extract) and chlortetracycline supplementation on antioxidation and meat quality in broiler chickens. British Poultry Science 2011; 52:302-309. 23. Edwards RH, Miller RE, De Fremery D, Knuckles BE, Bickoff EM, Kohier GO. Pilot plant production of an edible white fraction leaf protein concentrate from alfalfa. Journal of Agricultural and Food Chemistry 1975; 23(4): 620-626. 24. Fasuyi AO, Aletor VA. Varietal composition and functional properties of cassava (Manihot esculenta, Cranzt) leaf meal and leaf protein concentrates. Pakistan Journal of Nutrition 2005; 4(1): 43-49. 25. Favati F, King JW, Friedrich JP, Eskins K (1988). Supercritical CO2 extraction of carotene and lutein from leaf protein concentrates. Journal of Food Science 1988; 53(5): 15321536. 26. Francis G, Kerem Z, Makkar HP, Becker K (2002). The biological action of saponins in animal systems: a review. British Journal Nutrition 2002; 88: 587-605. 27. Furgał W, Milik K. Application a protein-xanthophyll lucerne concentrate as a diet supplement for humans - a case study. Alfalfa in human and animals nutrition. Dzierdziówka –Lublin, Poland: Wydawnictwo Stowarzyszenia Rozwoju Regionalnego i Lokalnego “Progress” 2008; 3 pp 49-58. (in Polish, summary in English)

14

28. Gaweł E. Chemical composition of lucerne leaf extract (EFL) and its applications as a phytobiotic in human nutrition. Acta Scientiarum Polonorum Technologia Alimentaria 2012; 11(3) 2012:303-310. [Online] Available at: http://www.food.actapol.net/volume11/issue/10_3_2012.pdf 29. Gaweł E, Grzelak M. Protein from lucerne in animals supplement diet. Journal of Food Agriculture and Environment 2014; 12(2): 314-319. 30. Gaweł E, Żurek J. Nutritional Value of selected lucerne cultivars. Biuletyn IHAR 2003; 225:167-174. (in Polish, summary in English) 31. Georgescu A, Nanu R. (1997). Leaf protein as human food. Report pp. 97. Institute for the Protection of Mother and Child Bucharest Romania 1997. 32. Hartman GH, Walter JR, Akeson R, Stahmann MA. Leaf protein concentrate prepared by spray-dring. Journal of Agricultural and Food Chemistry 1967; 15(1): 74-79. 33. Hong YH, Wang SC, Hsu C, Lin BF, Kuo YH, Huang C. Phytoestrogenic compounds in alfalfa sprout (Medicago sativa) beyond coumestrol. Journal of Agricultural and Food Chemistry 2011; 59(1):131-137. 34. Hussein L, El-Fouly MM, El-Baz FK, Ghanem SA. Nutritional quality and the presence of antinutritional factors in leaf protein concentrates (LPC). International Journal of Food Sciences and Nutrition 1999; 50(5): 333-343. 35. Hwang J, Hodis HN, Sevanian A. Soy and alfalfa phytoestrogen extracts become potent low-density lipoprotein antioxidants in the presence of acerola cherry extract. Journal of Agricultural and Food Chemistry 2001; 49(1): 308-3014. 36. Khaleel A, Gad MZ, El-Maraghy SA, Hifnawy MS, Abdel-Sattar E. Study of hypocholesteroemic and antiatherosclerotic properties of Medicago sativa L. cultivated in Egypt. Journal of Food and Drug Analysis 2005; 13(3):212-218. 37. Kołodziejczyk-Czepas J, Nowak P, Kowalska I, Stochmal A. Biological activity of clovers - Free radical scavenging ability and antioxidant action of six Trifolium species. Pharmaceutical Biology 2014; 52(10): 1308-1314. 38. Lodha A, Verma K, Malhotra H, Mathur B, Agrwal A. Effect of lucerne leaf concentrate supplementation on the nutritional status of chronic myeloid leukemia (CML) patients. Nutrition & Food Science 2009; 39(1):46-49. 39. Malinow MR, McLaughlin P, Naito HK, Lewis LA, McNulty WP (1978). Effect of alfalfa meal on shrinkage (regressin) of atherosclerotic plaques during cholesterol feeding in monkeys. Atherosclerosis 1978; 30(1): 27-43. 40. Malinow MR, McLaughlin P, Stafford C, Livingston AL, Kohler GO. (1980). Alfalfa saponins and alfalfa seeds. Dietary effects in cholesterol-fed rabbits. Atherosclerosis 1980; 37(3): 433-438. 41. Malinow MR, Connor WE, McLaughlin P, Stafford C, Lin DS, Livingston L, Kohler GO, McNulty WP. Cholesterol and bile acid balance in Macaca fascicularis. Journal of Clinical Investigation 1981; 67: 156-162. 42. Mathur B, Romani SV (2006). A comparative study of impact of leaf concentrate and iron and folic acid supplementation on blood profile of anemic adolescent girls. (Association Pour la Promotion, Des Extraits Foliaires en Nutrition, A.P.E.F. Paris December 20042005) 2006;7. [www.nutritionluzerne.org/.../3%20Rap%20Pr%20Mathi%20mai%2006.pdf -Windows Internet Explorer] 43. Mathur B, Pallavi J, Agarwal A. Efficacy of leaf concentrate as micronutrient fortifier in the supplementary nutrition of Integrated Child Development Services (ICDS). International Journal Nutrition Metabolism 2013; 5(6): 98-104.

15

44. Oda K, Matsuda H, Murakami T, Katayama S, Ohgitani T, Yoshikawa M. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biological Chemistry 2000, 381(1): 67-74. 45. Pirie NW (1975). Leaf protein. In Pirie (eds) Food Protein Source, Cambridge University Press 1975; : pp.133-141. 46. Podolska G, Konopka I, Dziuba J. Response of grain yield, yield components and allergic protein content of winter wheat to drought stress. Proceedings of the 9th European Society of Agronomy Congress (ESA) 2006; September 4-11, Warsaw Poland pp. 473-474. 47. Raji AO, Olofin AF. Design and development of leaf protein juice extraction machine. Journal of Industrial Research and Technology 2011; 3(1): 69-77. 48. Rambourg JC, Monties B. Determination of polyphenolic compounds in leaf protein concentrates of lucerne and their effect on the nutritional value. Plant Foods Human Nutrition 1983; 33:169-172. 49. Reshef G, Gestetner B, Birk Y, Bondi A. Effect of alfalfa saponins on the growth and some aspects of lipid metabolism of mice and quails. Journal Science of Food and Agriculture 2006; 27(1):63-72. 50. Rishi RK. Phytoestrogens in health and illness. Indian Journal of Pharmacology 2002; 34:311-320. 51. Sadowska B, Budzyńska A, Więckowska-Szakiel M, Paszkiewicz M, Stochmal A, Moniuszko-Szajwaj B, Kowalczyk M, Różalska B. New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Conidida albicans. Journal of Medical Microbiology 2014; 63: 1076-1086. 52. Sarmadi BH, Ismail A (2010). Antioxidative peptides from food proteins: A review. Peptides 2010; 31: 1949-1956. 53. Scientific Opinion, 2009. Scientific Opinion of the Panel on Dietetic Products Nutrition and Allergies on a request from the European Commission on the safety of “Alfalfa protein concentrate” as food. (2009): The EFSA Journal 997: 1-19. [Online] Available at: [www. efsa.europa.eu/de/scdocs/doc/997.pdf (Accessed:15. June 2012] 54. Śmiechowska M. Naturalne substancje antyodżywcze. In Łysiak-Szydłowska (eds) Żywienie kliniczne.Via medica Gdańsk 2000: 217-231. (in Polish) 55. Taky EE, Kido Y, Rikimaru T, Kennedy DO (2006). Possible use of alfalfa (Medicago sativa) as supplement in infant nutrition: Comparison of weight gained by rats fed on alfalfa and a popular weaning diet. Journal of the Science of Food Agriculture 2006; 59(1):109-115. 56. Varma K, Singh N, Boolchandani R, Mathur B, Lodha A, Malhotra H. Effect of lucerne concentrate on nutritional status of chronic myeloid leukemia patients. 2014; Availeble online at:http://www.ssmrae.com/admin/images/2a8c6c48f5efb8c2f66f9dada96dece7.pdf 57. Vyas S, Collin SM, Bertin E, Davys GJ, Mathur B. Leaf concentrate as an alternative to iron and folic acid supplements for anaemic adolescent girls: a randomised controlled trial in India. Public Health Nutrition 2010; 13(3):418-423. 58. Winiarska H, Dworacka M, Borowska M, Bobkiewicz-Kozłowska T, Gorecki P, Nścisz A, Mrozikiewicz PM. The effect of plant extracts of Medicago sativa and Trigonella foenum-graecum on postprandial glucose levels in type 2 diabetic rates. Herba Polonica 2007, 53(4): 34-44. (in English, summary in Polish) 59. Zanin V. (1998). A new nutritional idea for man, Lucerne leaf concentrate Association for the Promotion of Leaf Concentrate in Nutrition. Association pour la Promotion des Extraits Foliaires en Nutrition – “APEF”, Paris, France. 1998. [Online] Available at: www.nutrition-luzerne.org/anglais/pdf/EtudeZaninenglish.pdf (Accessed: 12. April 2012) http://cluster013.ovh.net/~nutritioh/wp-content/uploads/2014/05/Etude-Zanin-english.pdf

16

60. https://en.wikipedia.org/wiki/Alfalfa 61. Zinca G, Vizireanu C. Impact of germination on phenolic compounds content and antioxidant activity of alfalfa seeds (Medicago sativa L.). Journal of Agroalimentary Processes and Technologies 2013, 19(1): 105-110.

Table 1. Percentage amino acid content in lucerne (FRANCE LUZERNE) (Scientific Opinion ….2009; Zanin, 1998) Amino-acids

Eggs Wheat Lucerne LC Mg per 10 g (g/%) (g/%) (g/%) Glycine 3.4 4.1 4.8-6.3 Alanine 5.6 3.4 5.9-7.1 Valine* 5.6 4.4 5.8-6.7 308 Leucine* 8.1 6.9 8.5-10.6 443 Isoleucine* 4.8 3.5 4.3-6.7 242 Methionine* 3.4 1.6 1.5-2.6 112 Cysteine** 2.7 2.4 0.6-3.0 59 Phenylalanine* 6.5 5.0 5.8-7.0 250 Tryptophan* 1.7 1.2 1.6-3.4 100 Proline 3.5 10.0 4.4-5.7 Serine 7.3 5.2 4.1-5.6 Threonine* 5.1 3.0 4.6-5.8 239 Tyrosine** 4.1 3.2 3.7-5.2 242 Aspartic acid 10.5 5.0 9.3-10.7 Glutamic acid 12.4 31.0 10.6-12.5 Lysine* 6.7 2.7 5.6-7.4 321 Arginine 6.1 5.1 4.4-4.6 Histidine 2.5 2.3 1.8-2.5 * Essential amino acid ** Semi-essential amino acid synthesised only if their precursor is adequately present in the diet Table 2. L- canavanine concentration in fresh alfalfa seeds, leaves, APC and in comparison with other vegetables consumed by humans (in ppm or mg/kg) (Source: Scientific Opinion,.....2009) Lucerne Lucerne APC Lucerne Soy flour Lentil Onions seeds leaves Protein Concentrate mg/kg flour mg/kg Mg/kg ppm mg/kg mg/kg 80-150 10 4.3 2.1 2 800 10 000

17

Table 3. Analysis of a lucerne concentrate (Source: ……Zanin, 1998; Georgescu and Nanu, 1997; Varma et al., 2014) Nutrients Value/100 gms Energy 344 Kcal Protein 60 gm Fat 22.5 gm Carbohydrate 12.5 Fibre 1.0 gm Carotene 86700 mcg Thiamine 0.5 mg Riboflavin 0.5 mg Niacin 24.2 mg Folic acid 330.0 mcg Ascorbic acid 2.2 mg Calcium 1865.0 mg Iron 99.0 mg Table 4. Effect of an alfalfa concentrate on blood parameters in patients with leukaemia. Effect of the treatment of CML patients with lucerne concentrate, Baseline & post intervention nutritional assessment (Source: Lodha et al., 2009) Specification Baseline Post intervention % change Significant (p 12; lymphocytes CD4 = 350-1600; CRP (C reactive protein)< 6 Fatigue: none = 0, mild = 1; moderat e= 2; severe = 3 Appetite: poor = 0; average = 1; good = 2; very good = 3 general health: very poor = 0; poor = 1; usual = 2; good = 3; excellent = 4 1 fever - number of cases; 2diarrhoea – number of days IMC (body weight index)