Pharmaceutical Biology, 2011; 49(2): 211220 2011 Informa Healthcare USA, Inc.

. ISSN 1388-0209 print/ISSN 1744-5116 online DOI: 10.3109/13880209.2010.504732

REVIEW ARTICLE

Phytochemical and pharmacological potential of Medicago sativa: A review

Kundan Singh Bora1 and Anupam Sharma2
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L.R. Institute of Pharmacy, Solan, Himachal Pradesh, India, and 2University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
1

Abstract Context: Many herbal remedies have so far been employed for the treatment and management of various ailments since the beginning of human civilization. Medicago is an extensive genus of the family Leguminosae, comprising about 83 different species. Medicago sativa (Linn.) has long been used as traditional herbal medicine in China, Iraq, Turkey, India and America for the treatment of a variety of ailments. Objectives: The aim of this review was to collect all available scientific literature published and combine it into this review. The present review comprises the ethnopharmacological, phytochemical and therapeutic potential of M. sativa. Methods: The present review includes 117 references compiled from major databases as Chemical Abstracts, Science Direct, SciFinder, PubMed, Dr. Dukes Phytochemical and Ethnobotany, CIMER, and InteliHealth. Results: An exhaustive survey of literature revealed that saponins, flavonoids, phytoestrogens, coumarins, alkaloids, amino acids, phytosterols, vitamins, digestive enzymes and terpenes constitute major classes of phytoconstituents of this plant. Pharmacological reports revealed that it is used as neuroprotective, hypocholesterolemic, antioxidant, antiulcer, antimicrobial, hypolipidemic, estrogenic, and in the treatment of atherosclerosis, heart disease, stroke, cancer, diabetes and menopausal symptoms in women. Conclusion: M. sativa seems to hold great potential for in-depth investigation for various biological activities, especially their effects on central nervous and cardiovascular system. Through this review, the authors hope to attract the attention of natural product researchers throughout the world to focus on the unexplored potential of M. sativa, and it may be useful in developing new formulations with more therapeutic value. Keywords: Antidiabetic, antiatherosclerotic, hypocholesterolemic, Medicago sativa, neuroprotective

Introduction
The use of plants for medicines or tonic properties goes back to prehistoric times and has attracted the interest of scientists for centuries. The Vedas give the earliest written record about the science of healing. The reference to medicinal plant is also found in the Ebers papyrus (16th century B.C.) which lists in detail over 7000 herbal remedies, for example poppy, castor oil, caraway etc. The Indians were the first to use chaulmoogra fruits for its antileprotic activity. The Brazilians employed Ipecacuanha for the treatment of dysentery and diarrhea. The root of Rauwolfia serpentine (Benth.), an indigenous plant, has since time immemorial been widely

used in India and Malaysia as an antidote for insect and snake bites and mental derangement. The utility of plants as therapeutic agents in traditional medicine system is still prevalent today. For example, the Middle Eastern civilization developed the Greco-Arabic system of medicine (Unani system of medicine), which is practiced in the Indian sub-continent. The Ayurvedic and Sidha system of medicines were contributed by Indians. All these systems procure more than 80% of their medicaments from plants (Bhavna et al., 2007). Medicago sativa Linn. (Leguminosae), commonly known as the father of all foods (al-fal-fa), is a perennial herbaceous leguminous plant species that originated

Address for Correspondence: Kundan Singh Bora, L.R. Institute of Pharmacy, Solan, Himachal Pradesh 173212, India. Tel.: +91-1792 252854. Fax: +91-1792 252851. E-mail: kundanresearch1381@gmail.com (Received 11 June 2010; revised 24 June 2010; accepted 24 June 2010)

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Kundan Singh Bora, and Anupam Sharma found in Argentina (primarily grazed), Australia, South Africa, and the Middle East (FAO, 2006). Within the USA, the leading M. sativa growing states are California, South Dakota, and Wisconsin. The upper Midwestern states account for about 50% of US production, the Northeastern states 10%, the Western states 40%, and the Southeastern states almost none. M. sativa has a wide range of adaptation and can be grown from very cold northern plains to high mountain valleys, from rich temperate agricultural regions to Mediterranean climates and searing hot deserts.

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in Asia (Ehsanpour & Razavizadeh, 2005; Duke, 1985; BHMA, 1996). This is the most ancient plant, cultivated throughout the world as a fodder plant. In America, M. sativa has been extensively cultivated since the arrival of Europeans. M. sativa has been grown for a variety of purposes such as soil improvement, animal feed and medicinal uses (Steppler, 1987). M. sativa has a long tradition of use as Ayurvedic and homoeopathic medicine in central nervous and digestive system disorders, and for the treatment of various other ailments. However, only limited research has been conducted on this plant species. The present review emphasizes the traditional uses and phytopharmacological potential of M. sativa. Additionally, sporadic pharmacological work has so far been carried out to prove its traditional claims. Through this review, the authors hope to attract the attention of natural product researchers throughout the world to focus on the unexplored potential of M. sativa. No review so far has been compiled on this species, and therefore it was considered important to take stock of what has been done in the past, in order to provide a solid foundation and direction to any future research that may be conducted on this plant.

Ethnopharmacological uses
Traditionally, M. sativa is used to improve the memory, to cure kidney pain, cough, sore muscles, as a rejuvenator, antidiabetic, antioxidant, anti-inflammatory, antifungal, anti-asthmatic, antimicrobial, diuretic, galactagogue and in central nervous system (CNS) disorders (Finkler, 1985; BHMA, 1996; Inamul, 2004; DerMarderosian et al., 2005). M. sativa has a long tradition of use as Ayurvedic and homoeopathic medicine in CNS disorders (Inamul, 2004). M. sativa has been used by the Chinese since the sixth century to treat kidney stones, fever, gravel, dysuria and to relieve fluid retention and swelling. Ancient Indian Ayurvedic physicians used M. sativa to treat ulcers, arthritis pain and fluid retention. In Mexico M. sativa is believed to improve the memory, to cure sore muscles and inflammation. Early Americans used M. sativa to treat arthritis, boils, cancer, scurvy, and urinary and bowel problems. In Iraq M. sativa is used in arthritis. In Turkey it is used as cardiotonic and to treat scurvy and arthritis (Michael et al., 2007; Finkler, 1985; Inamul, 2004; DerMarderosian et al., 2005). Moreover, it is considered beneficial in bladder disorders, blood clotting disorders, boils, cough, diuresis, gastrointestinal tract disorders, breast cancer, cervical cancer, kidney disorders, prostate disorders, appetite stimulation, inflammation, increasing breast milk, asthma, indigestion, insect bites, jaundice, menopausal symptoms, allergies, increasing excretion of neutral steroids and bile acids in fecal matter, nutritional support, stomach ulcers, skin damage from radiation, galactagogue, increasing peristaltic action of the stomach and bowels, thrombocytopenic purpura, uterine stimulant, rheumatoid arthritis, scurvy, vitamin supplementation (vitamins A, C, E, K) and wound healing (Gray & Flatt, 1997a, 1997b; BHMA, 1996; DerMarderosian et al., 2005; Swanston et al., 1990).

Scientific classifications (Heywood & Ball, 1968)

Kingdom: Plantae; Division: Magnoliophyta; Class: Magnoliopsida; Order: Fabales; Family: Fabaceae; Subfamily: Faboideae; Tribe: Trifolieae; Genus: Medicago; Species: sativa; Binomial name: M. sativa Linn.

Description: general morphology

M. sativa is a cool season perennial legume living from three to twelve years, depending on variety and climate. The general morphology of M. sativa plant was considered by Teuber and Brick (1988), and Barnes and Sheaffer (1995). The mature M. sativa plant is characterized by a strong taproot. This taproot may eventually surpass 6 m or more in length with several to many lateral roots connected at the crown when M. sativa is grown in deep, well drained, moist soils. The crown, a complex structure near the soil surface, has perennial meristem activity, producing buds that develop into stems. Tri- or multi-foliolate leaves form alternately on the stem, and secondary and tertiary stems can develop from leaf axils. A plant in a typical forage production field has between 5 and 15 stems and can reach nearly 1 m in height. Flowers vary in color yet purple, variegated, yellow, cream and white are the most common. After pollination, these flowers most commonly produce spiral-shaped seed pods.

Phytochemical reports
M. sativa has been reported to contain a variety of phytochemicals. It has following different classes of phytoconstituents. Alkaloids: asparagines, trigoneline, stachydrine, l-homostachydrine (Duke, 1985; Mills, 1994; Tamsyn et al., 2009). Amino acids: medicanine, lysine, arginine,
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Worldwide production
M. sativa is the most cultivated legume in the world. Worldwide production was around 436 million tons (436000000 metric tons) in 2006. The USA is the largest M. sativa producer in the world, but considerable area is

Medicago sativa: A review histidine, tyrosine, phenylalanine, methionine, aspartic acid, glutamic acid, asparagine, serine, alanine, threonine (Worthington & Breskin, 1983; Fushiya et al., 1984; Lihu & Pedak, 1979; Mego & Erdelsky, 1977). Carotene (Gupta et al., 1981). Coumarins: myrsellinol, scopoletin, esculetin, 4-coumaric acid (Duke, 1985; El-Khrisy et al., 1994; Orr et al., 1993). Digestive enzymes (Duke, 1985). Enzymes: isoflavone reductase, vestitone reductase, iminopeptidase, and two aminopeptidases (Xiaoqiang et al., 2006; Shao et al., 2007; Biagioni et al., 1990). Flavonoids: quercetin, myricetin, luteolin, apigenin, chrysoeriol, tricin, 4'-O-[2'-O-E-feruloyl-O-glucuronopyranosyl(12)- O--lucuronopyranoside] apigenin,7-O--glucuronopyranosyl-4'-O-[2'-O-E-feruloylO--glucuronopyranosyl(12)-O-- glucuronopyranoside] apigenin, 7-O--glucuronopyranosyl-4'-O-[2'O-p-E-coumaroyl-O--glucuronopyranosyl(12)-O-glucuronopyranoside] apigenin, 7-O-{2-O-E-feruloyl[-glucuronopyranosyl (13)]-O--lucuronopyranosyl (12)-O--glucurono-pyranoside} apigenin, O-glycosyltransferases, medicarpin, coumestrol, sativan, vestitol, formononetin (Bickhoff et al., 1964; Stochmal et al., 2001; Kowalska et al., 2007; Stochmal & Oleszek, 2007; Parry & Edwards, 1994; Dutu et al., 2002; Dixon & Steele, 1999; Sumner et al., 1996; Piretti et al., 1982). Minerals: Ca, K, P, Mg, Fe, Zn, Cu, Al, B, Cr, Co, Mn, Mo, Se, Si, Na, Sn (Worthington & Breskin, 1983; Potgieter, 2005). Non-protein amino acids: l-canaverin (Albourine et al., 1991; Tamsyn et al., 2009). Organic acids: citrate, malate, malonate, succinate, fumarate, lactate, benzoate (Francoise et al., 1991; Tamsyn et al., 2009). Phenolic compounds: p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, ferulic acids, salicylic acid, sinapic acids, caffeic acid, hesperetin, naringenin, chlorogenic acid, tannic acid, heterosides (Newby et al., 1980; Volynets et al., 1979; Dutu et al., 2002). Phytoestrogens: coumestrol, genistein, formometin, diadzein, biocanine A (Adler, 1962; Bickhoff et al., 1960). Phytosterols: -sitosterol, stigmasterol (Timbekova et al., 1996). Polyamines: norspermidine, norspermine (Rodriguez-Garay et al., 1989). Protein: ferritin, protein phosphatase 2A holoenzyme, -amylase (Doehlert et al., 1982; Toth et al., 2000; Barcelo et al., 1997). Saponins: soyasapogenols, hederagenin, medicagenic acid (Oleszek & Jurzysta, 1986; Massiot et al., 1988; Oleszek, 1988; Wieslaw et al., 1990), 3-Glc-GlcRha-28Glc-medicagenic acid, 3-Glc-28-Glc-medicagenic acid, 3-Glc-malonyl-28-Glc-medicagenic acid, AraGlc-Ara-hederagenin, 3-Glc-Ara-28-Glc hederagenin, hex-hex-bayogenin, 3-Glc-Glc-medicagenic acid, 3-RhaGal-Glc-soyasapongenol B (soyasapogenol I), 3-GlcAra-28-Glc-hederagenin, 3-Glc-medicagenic acid, 3-Glcmalonyl-medicageinc acid, Rha-Gal-GlcA soyasapogenol E, Rha-hex-hex-soyasapogenol E, Ara-hex-hederagenin, pen-hederagenin (Huhman & Sumner, 2002), zahnic acids, soyasapogenol A, soyasapogenol B, soyasapogenol E, bayogenin glycoside, 3-O-[-d-glucopyranosyl(13)-d-glucopyranosyl]-28-O--d-glucopyranoside medicagenate, 3-O-[-l-rhamnopyranosyl(12)- -d 2011 Informa Healthcare USA, Inc.

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glucopyranosyl(1 2)- -d-glucopyranoside] medicagenic acid, 3-O-[-d-glucopyranosyl(12)--dglucopyranosyl(12)--d- glucopyranosyl]-28--dglucopyranoside medicagenate, 3-O-[-d- glucuronopyranosylmethylester]-28-O-[-d-xylopyranosyl(14)--lrhamnopyranosyl(12)--l-arabinopyranoside] medicagenate, 3-O-[-l-rhamnopyranosyl(12)--dgalactopyranosyl(12)--d-glucuronopyranosyl]-21O--l-rhamnopyranoside soyasapogenol A, 3-O-[-dglucopyranosyl(12)--d-glucopyranosyl(12) glucopyranosyl]-28-O-[-d-xylopyranosyl(14)--lrhamnopyranosyl(12)--l-arabinopyranoside] medicagenate,3-O-[-d-glucopyranosyl(12)--d-glucopyranosyl (1 2)glucopyranosyl]-28-O-{ -d-xylopyranosyl (1 4)-[ -d-apiofuranosyl-(13)]- -l-rhamnopyranosyl(12)--l-arabinopyranoside} medicagenate, 3-O-[-d-glucopyranosyl(12)--d-glucopyranosyl(12)-d-glucopyranosyl]-28-O-[-d-xylopyranosyl(14)--lrhamnopyranosyl(12)--l-arabinopyranoside] zahnic acid, 3-O-[-d-glucopyranosyl(12)--d-glucopyranosyl (12)--d-glucopyranosyl]-28-O-{-d-xylopyranosyl(14)-[-d-apiofuranoside-(13)]--l-rhamnopyranosyl (12)--l-arabinopyranoside} zahnic acid, 3O-[-d-galactopyranosyl(12)--d-glucuronopyranosyl]28-O--d-glucopyranoside bayogenin, 3-O-[-l- rhamnopyranosyl (12)--d-galactopyranosyl(12)--dglucuronopyranoside] soyasapogenol E (Bialy et al., 1999), 3-O-[-d-glucopyranosyl (1-2)--d-glucopyranosyl(1-2)-d-glucopyranosyl]-2,3,16-trihydroxyolean-12en-23,28-dioic acid-23-O--l-arabinopyranosyl-28-O[-d-apiofuranosyl(1-3)--d-xylopyranosyl(14) -l-rhamnopyranosyl(1-2)- -l-arabinoside] (Oleszek et al., 1992), 28-O-[-d-xylopyranosyl-(14)-l-rhamnopyranosyl(12)--l-arabinopyranosyl] medicagenate,28-O-[-d-xylopyranosyl(14)--l- rhamnopyranosyl (12)--l-arabinopyranosyl]-3-O--dglucopyranosylmedicagenate, 28-O-[-d- xylopyranosyl (1 4)- -l-rhamnopyranosyl(1 2)- -larabinopyranosyl]-3-O-[-d-glucopyranosyl(12)--dglucopyranosyl]medicagenate, 28-O-[-d-xylopyranosyl (1 4)- -l-rhamnopyranosyl(1 2)- -larabinopyranosyl]-3-O--d- glucuronopyranosylmedicagenate, 3-O-[-l-rhamnopyranosyl(12)--dglucopyranosyl(12)--d-glucuropyranosyl]soyasapogenol B (Massiot et al., 1991), dehydrosoyasaponin I, soyasaponin I, azukisaponin II, azukisaponin V (Kitagawa et al., 1988). Sterols: -spinasterol, -sitosterol, stigmasterol, myrsellinol, scopoletin, esculetin (El-Khrisy et al., 1994; Abdel-Hafez, 1993), dihydrospinasterol, 24-methylcholest-7-enol, stigmasterol, campesterol (Li-Shar & Claus, 1988). Thyrotropin-releasing hormone analog (Worthington & Breskin, 1983). Vitamins: A, B1, B6, B12, C, D, E, K, niacin, pantothenic acid, biotin, folic acid (Worthington & Breskin, 1983; Horst et al., 1984). Volatile components: terpenes, limonene, linalool, transocimene, furanoids, nonadienal, 2-methyl 4-pentenai, benzaldehyde, ethyl benzaldehyde, alcohols, butanol, hexanol, octanol, pentan-3-ol, 3-methylbutanol, trans-

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Kundan Singh Bora, and Anupam Sharma intestinal absorption of cholesterol, increased fecal excretion of endogenous and exogenous neutral steroids and bile acids, and decreased the percentage distribution of fecal deoxycholic and lithocholic acids (Malinow et al., 1981a). Khaleel et al. (2005) performed a study and showed that highest saponin content extract just before fruiting stage (free from both coumestrol and canavanine) of M. sativa exhibited significant hypocholesterolemic and antiatherosclerotic activity. This study proved that M. sativa was found to safely reduce natural cholesterol and to possess a strong anti-atherosclerotic activity. The extracts produced the most significant decrease in total cholesterol and LDL-cholesterol by 85.1 and 88%, respectively, of the corresponding levels in hypercholesterolemic rabbits. This decrease was more significant than that produced by gemfibrozil (73 and 74%) upon concomitant administration with a cholesterol enriched diet using the same animal model at the tested dose level. Moreover, it was also observed that all M. sativa preparations produced significant antioxidant properties. M. sativa when supplied in the diet (6.25% by weight) and infusion (1 g/400 mL) has been reported to reduce the level of hyperglycemia in streptozotocin-induced diabetes (Swanston et al., 1990). Aqueous extract of the plant (1 mg/mL) stimulated 2-deoxy-glucose transport (1.8-fold), glucose oxidation (1.7-fold) and incorporation of glucose into glycogen (1.6-fold) in mouse abdominal muscle. In acute, 20 min tests, 0.25-1 mg/mL aqueous extract of M. sativa evoked a stepwise 2.5-6.3-fold stimulation of insulin secretion from the BRIN-BD11 pancreatic beta cell line. This effect was abolished by 0.5 mM diazoxide, and prior exposure to the extract did not affect subsequent stimulation of insulin secretion by 10 mM l-alanine, thereby negating a detrimental effect on cell viability. The effect of the extract was potentiated by 16.7 mM glucose and by 1 mM 3-isobutyl-1methylxanthine. l-Alanine (10 mM) and a depolarizing concentration of KCl (25 mM) did not increase the insulin-releasing activity of M. sativa. Sequential extraction with solvents revealed insulin-releasing activity in both the methanol and water fractions, indicating a cumulative effect of more than one constituent (Mohamed et al., 2006). The manganese content of M. sativa (45.5 mg/kg) is reported to be the active principle responsible for a hypoglycemic effect documented for M. sativa. A diabetic patient, treated with soluble insulin but poorly controlled, found that an M. sativa extract adequately controlled his diabetes. When administered separately, only small doses of manganese chloride (5-10 mg) were required to have a hypoglycemic effect. However, no effect was seen on the blood sugar concentrations of non-diabetic controls or of other diabetic patients, who were also administered manganese. It was concluded that manganese lowered the blood sugar concentration in this particular diabetic patient because he was unable to utilize manganese stored in his body (Rubenstein et al., 1962).
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2-pentenol, trans-2-hexenol, trans-3-hexenol, pent-1en-3-ol, ott-I-en-3-ol, octa-1,5-dien-3-ol, benzyl alcohol, 2-phenylethanol, ketones, pent-1-en-3-one, pentan-3one, octan-3-one, methyl phenyl ketone, esters, trans-3hexenylacetate, trans-3-hexenylbutanoate, aldehydes, hexanal, trans-2-pentenal, trans-2-hexenal, trans-2nonenal, trans-2,4-hexadienal, furane-2-ethyl (Aldo & Luciano, 1997). Figure 1 shows the chemical structures of common saponin aglycones present in M. sativa.

Pharmacological/clinical reports
Several studies indicate that the ingestion of M. sativa reduces cholesterol absorption and atherosclerotic plaque formation in animals (Wilcox & Galloway, 1961; Malinow et al., 1977a, 1981a; Cohen et al., 1990). M. sativa top (stem and leaves) saponins have been reported to decrease plasma cholesterol concentrations without changing high-density lipoprotein cholesterol concentrations, decrease intestinal absorption of cholesterol, increase excretion of neutral steroids and bile acids, prevent atherosclerosis and induce the regression of atherosclerosis (Malinow et al., 1982a). Hypocholesterolemic activity has been reported for root saponins, when given to monkeys receiving a high-cholesterol diet (Malinow et al., 1977b) In a study, the ability of M. sativa plant to reduce liver cholesterol accumulation in cholesterol-fed rats was enhanced by the removal of saponins. Therefore, M. sativa saponins appear to play an important role in neutral steroid excretion, but are not essential for increasing bile acid excretion (Story et al., 1984). In an experiment with prairie dogs, the lowest incidence of cholesterol gallstones was served with the diet of the higher fiber content (85% alfalfa) (Cohen et al., 1990). In a short-term study involving three normolipidemic individuals given M. sativa seeds (80-60 g daily), serum cholesterol concentrations were reported to be reduced. In another small study in which heat-treated M. sativa seeds (40 g three times daily for eight weeks) were taken by eight type-IIA hyperlipoproteinemic patients and three type IIB patients, a significant decrease was noted in total serum cholesterol concentrations, low-density lipoprotein (LDL) cholesterol and apolipoprotein B. The LDL cholesterol concentration fell by less than 5% in two of the 11 patients (Molgaard et al., 1987). Cholestaid, a product available in the USA containing 900 mg of M. sativa extract with 100 mg citric acid, is said to neutralize the cholesterol in the stomach before it reaches the liver, thus facilitating the excretion of cholesterol from the body with no side effects or toxicity (Levy, 1999; Dewey, 2001). M. sativa top saponins have been shown to decrease cholesterolemia without changing the levels of high density lipoprotein-cholesterol; hence, they reduced the total cholesterol/high density lipoprotein-cholesterol ratio in Macaca fascicularis. Furthermore, they decreased

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Medicago sativa: A review

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OH

HO

CO2H

HO

CH2OH Soyasapogenol B

HO

CH2OH Soyasapogenol E

HO

CO2H Medicagenic acid

OH H Pharmaceutical Biology Downloaded from informahealthcare.com by St Johns University on 08/27/11 For personal use only. HO COOH COOH OH

HO

CH2OH Bayogenin

HO

CH2OH Hederagenin

HO

CH2OH Soyasapogenol A

OH OR2

O HO O OH OCH3 Formononetin HO CO2H Zahnic acid CO2H

R 1O

CH2OH

R1 = GlcAP R2 = H

Galp

Rhap

Soyasaponin I

Figure 1. Chemical structures of some common saponin aglycones present in M. sativa.

Methanol extract of M. sativa has been shown significant estrogenic activity using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. The extract showed significant competitive binding to estrogen receptor (ER). The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extract, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of extract were examined using transiently transfected human embryonic kidney (HEK 293) cells. Methanol extract exhibited preferential agonist activity toward ER. Phytoestrogens of the extract were determined to be responsible for estrogenic activity (Boue et al., 2003). Extract of the leaves from M. sativa has been shown to be used in treatment of neurovegetative menopausal symptoms in women. Hot flushes and night sweating completely disappeared with the treatment of M. sativa extract. The plant product induced a significant increase in prolactin and thyroid stimulating hormone response to thyroid releasing hormone. Basal levels of estradiol, luteinizing hormone, follicle stimulating hormone,
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prolactin, and thyroid stimulating hormone were unchanged. Thus, M. sativa suggested having a central slight antidopaminergic action without side effects (Minerva, 1998). Seed extract of M. sativa has been reported to exhibit significant reduction of total cholesterol, phospholipid, triglyceride, LDL-cholesterol and VLDL-cholesterol in chicks, where clofibrate was used as a standard drug (Dixit & Joshi, 1985). In an in vitro experiment, polysaccharides isolated from M. sativa exhibited immunopotentiating activity by increasing mouse lymphocyte uptake of [3H] thymidine (Zhao et al., 1993). The antimicrobial activity of saponins isolated from M. sativa against selected medically important yeasts, Gram-positive and -negative bacteria has been investigated. Increasing antibiotic activity was observed going from the saponin extracts to the sapogenin samples, suggesting that the sugar moiety is not important for the antimicrobial efficacy. Activity was found especially high against Gram-positive bacteria (Bacillus cereus,

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Kundan Singh Bora, and Anupam Sharma Recently, the authors have shown that M. sativa exhibited significant antioxidant and cerebroprotective effects against ischemia and reperfusion insult in mice (Kundan & Anupam, 2010). Pretreatment with M. sativa extract (100 or 200 mg kg1, p.o.) markedly reduced cerebral infarct size, xanthine oxidase, superoxide anion and thiobarbituric acid-reactive substance levels, significantly restored reduced glutathione, superoxide dismutase and total tissue sulfhydryl levels and attenuated impairment in short-term memory and motor coordination. In addition, M. sativa directly scavenged free radicals generated against a stable radical 1,1-diphenyl-2-picrylhydrazyl and, superoxide anion radicals generated in phenazine methosulfate-nicotinamide adenine dinucleotide systems, and also inhibited xanthine dehydrogenase/xanthine oxidase conversion and resultant superoxide anion production. In addition to the above mentioned activities, M. sativa is also considered beneficial in arthritis (Hall, 1981), cardiovascular complaints (Reilly, 1989), convalescence, debility (Mills, 1994) and dysuria (Duke, 2002). The plant is said to promote weight gain (Meyer, 1954), and can be useful for people with protein allergies (Hall, 1981), GI cramping and colic (Reilly, 1989), gravel (Duke, 2002), hyperacidic stomach (Hall, 1981), hypoestrogenism/ hyperestrogenism (Reilly, 1989). It also improves appetite (Lust, 1974), and intellect (Duke, 2002). l-Canaverine isolated from the plant has been shown to have antitumor activity against certain types of leukemia cells in mice and selective toxicity in dog cancer cells grown in vitro (Hendler, 1995). M. sativa has been reported to be beneficial in the treatment of peptic ulcers (Lust, 1974), polycystic ovaries (Reilly, 1989), prolactin excess (Reilly, 1989), hemorrhage, as a tonic after blood loss and during anemia (Mills, 1994), urinary and bowel problems (Lust, 1974), scurvy (Duke, 2002), secondary hypothyroidism (Reilly, 1989) and Herpes simplex (Hendler, 1995).

B. subtilis, Staphylococcus aureus, and Enterococcus faecalis). Discrete antifungal activity was also observed, mainly against Saccharomyces cerevisiae. The observed antimicrobial properties of M. sativa were related to the content of medicagenic acid (Avato et al., 2006). An investigation into the effect of various herbs on hepatic drug metabolizing enzymes in the rat, showed that M. sativa potentiated the activity of aminopyrine N-demethylase but had no effect on glutathione S-transferase or epoxide hydrolase activities (Garrett et al., 1982). M. sativa can be used as an alternative natural carotenoid instead of the synthetic apo-ester in goldfish diets (Yanar et al., 2008). Medicagenic acid isolated from M. sativa exhibited significant nematicidal activity against the plant-parasitic nematode Xiphinema index (Argentieri et al., 2008). Refined components of M. sativa polysaccharides have been shown to inhibit the activities of reverse transcriptase of HIV and protease of HIV (Zhang et al., 2006). Health beverage manufactured from M. sativa buds was found beneficial in maintaining normal digestive function and nutrition balance in human body, reducing cholesterol, and preventing osteoporosis, arteriosclerosis and aging (Song & Wei, 2007). The saponin extract of M. sativa containing hederagenins and saponin compounds I, II and III was reported to be used in manufacturing of hypolipidemic agents and antiatherosclerotics (Yu et al., 2007). The dried powder of M. sativa has been shown to inhibit the germination and growth of Echinochloa crus-galli Wight (Gwon & Kim, 2006). Xiong (2003) demonstrated that extracts prepared from M. sativa roots may be used to prepare medical preparations like powder, pill, or decoction for lowering the levels of cholesterol and lipid in blood, improving the liver function and the control and transmission of nerve tissue, and treating calculus. Among M. sativa secondary metabolites, saponins and phytoestrogens offer interesting medicinal and nutraceutical prospects. Phytoestrogens, mainly coumestrol, apigenin and quercetin exhibit strong estrogenic activity and have potential for use in treatment of hormone-related cancers (Huyghe et al., 2007). The extracts prepared from M. sativa flowery aerial parts and seeds exhibited significant in vitro and in vivo antimitotic activities (Dutu et al., 2002). Saponins isolated from the aerial parts of M. sativa have been shown to stimulate lipolytic activity without influencing amylolytic or proteolytic activity of NeoPancreatinum (Sroka et al., 1997). Triterpenoid glycosides of the roots and leaves of M. sativa have shown antibacterial activity against Corynebacterium michiganense, Corynebacterium insidiosum, and Agrobacterium tumefaciens. The inhibitory effects of these glycosides on cotton and cabbage seed germinations have also been tested (Timbekova, 1995).

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Toxicity/side-effects
Both M. sativa seed and herb have been reported to induce a systemic lupus erythematosus (SLE)-like syndrome in female monkeys (Barnes et al. 2007; Boon & Smith, 2004; Brinker, 2001; Malinow et al., 1982a, 1982b). This activity has been attributed to canavanine, a nonprotein amino acid constituent, which has been found to have effects on human immunoregulatory cells in vitro (Jorge et al., 1985). Re-activation of quiescent SLE in humans has been associated with the ingestion of M. sativa tablets which, following analysis, were found to contain canavanine (Roberts & Hayashi, 1983). It was not stated whether the tablets contained seed or herb material. Canavanine is known to be toxic to all animal species because it is a structural analogue of arginine and may interfere with the binding of this amino acid to enzymes and its incorporation into proteins. M. sativa seeds are reported to contain substantial quantities of canavanine (8.33-13.6 mg/kg), whereas the herb is stated
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Medicago sativa: A review to contain amounts that are considerably less (Natelson, 1985). Pancytopenia has been associated with human ingestion of ground M. sativa seeds (80-160 g/day), which were taken to lower plasma cholesterol concentrations (Malinow et al., 1981a). Dietary studies using alfalfa top saponins (ATS) in the diet of rats and monkeys showed no evidence of toxicity and serum lipid concentrations were lowered (Malinow et al., 1982a, 1981a). In addition, when ATS were given to cholesterol-fed animals, a reduction in serum lipid concentrations was observed. ATS are reported to be free of the SLE-inducing substance that is present in the seeds (Malinow et al., 1982c). Negative results were documented for M. sativa when tested for mutagenicity using Salmonella strains TA98 and TA100 (White et al., 1983). Consultation is a necessity for a health care practitioner prior to prescribe use of M. sativa if undergoing hormone replacement therapy or taking birth control medication or using blood thinners (Barnes et al. 2007; Boon & Smith, 2004; Brinker, 2001).

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Doses
Preparations equivalent to 930 g dried aerial parts, per day (Mills, 1985; BHMA, 1983) Dried aerial parts: 510 g, 3 times per day (BHMA, 1983). 310 g, 3 times per day (Mills, 1985) Infusion: 510 g dried aerial parts, 3 times per day (BHMA, 1983) Fluid extract: 510 g dried equivalent, 3 times per day (BHMA, 1983) (1:1, 25% alcohol, 510 mL) Tablets: A dose of two tablets (1 g each) of Cholestaid (esterin processed M. sativa) taken by mouth 3 times daily for up to two months, then one tablet 3 times daily, has been recommended (Farnsworth, 1995). Seeds: For treating high cholesterol, a dose of 40 g of heated seeds taken by mouth 3 times daily has been recommended (Farnsworth, 1995).

Contraindications
People with a history of lupus or a family history of systemic lupus erythematosus should avoid M. sativa supplements (Barnes et al. 2007; Boon & Smith, 2004; Brinker, 2001). M. sativa seeds should not be ingested during pregnancy or lactation (Brinker, 2001).

Discussion
Plants have played a significant role in maintaining human health and improving the quality of human life for thousands of years, and have served humans well as valuable components of medicines, seasonings, beverages, cosmetics and dyes. Herbal medicine is based on the premise that plants contain natural substances that can promote health and alleviate illness. In recent times, focus on plant research has increased all over the world and a large body of evidence has collected to show
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immense potential of medicinal plants used in various traditional systems. The present review emphasizes the phytochemical, traditional, pharmacological, clinical and toxicological reports on M. sativa. Saponins, flavonoids, phytoestrogens, coumarins, alkaloids, amino acids, phytosterols, vitamins, digestive enzymes and terpenes constitute major classes of phytoconstituents of the plant. M. sativa has been used since centuries as homoeopathic and Ayurvedic medicine for a variety of ailments. The plant is widely used in foods and is listed by the Council of Europe as a source of natural food flavor (category N2 and N3). These categories indicate that M. sativa can be added to foodstuffs in small quantities with a limitation on the concentrations of an active ingredient in the final product. In the USA, M. sativa is listed as GRAS (generally recognized as safe). Recent research carried out indicates its uses such as neuroprotective, hypocholesterolemic, antioxidant, antiulcer, antimicrobial, hypolipidemic, immunopotentiating, estrogenic, digestive, nutritive for human body, and in the treatment of atherosclerosis, heart disease, stroke, cancer, diabetes and neurovegetative menopausal symptoms in women. The presence of a wide range of chemical compounds indicates that the plant could lead the way for the development of novel agents having good biological activity. Exploration of the chemical compounds of the plant will provide the basis for developing such a lead. Many chemical compounds are present in the plant but isolation of active constituents by using various appropriate chromatographic techniques should be carried out. Further studies can be carried out using different extraction methods such as microwave extraction, isolation and identification of active compounds, pharmacological screening of extracts as well as isolated compound(s) so that accuracy of results can be obtained and use of extracts can be made in different herbal formulation. Despite a long tradition of use of M. sativa for treatment of various ailments, only sporadic pharmacological work has so far been carried out to prove its traditional claims. Additionally, the plant has been included in a number of herbal and homoeopathic formulations, which are in clinical use for the treatment of various ailments. M. sativa seems to hold great potential for in depth investigation for various biological activities. Therefore, it is necessary to exploit its maximum potential in the field of medicinal and pharmaceutical sciences for novel and fruitful application Currently, the authors are involved in evaluating the CNS effects of traditionally used medicinal plants, including M. sativa, with a view to isolating bioactive phytoconstituent(s).

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Declaration of interest
Financial support from the L.L.R. Educational Trust, Solan, Himachal Pradesh, India, which runs the L.R. Institute of Pharmacy, is gratefully acknowledged.

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The authors alone are responsible for the content and writing of the paper.

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