White clover had been reported to be high in protein and minerals (Anonymous 2005; Frame and Newbould 1986; Frame et al. 1998), containing 22–28 % crude protein, 2.7–3.3 % crude fat, 9.4–11.9 % ash, 6.6–7 % lignin and a crude fibre content of 15.7–21.1 % (Anonymous 2005). White clover was reported to contain per kg dry matter: 26.6–5.3 g N, 1.9–4.7 g P, 15.4–38.0 g K, 12.0–23.1 g Ca, 1.4–2.9 g Mg, 2.4–3.6 g S, 0.5–4.6 g Na, 3.4–25.6 g Cl, 102–448gm Fe, 40–87 mg Mn, 22–32 mg Zn, 5.4–9.7 mg Cu, 0.10–0.38 mg Co, 0.14–0.44 mg I, 1.3–14.2 mg Mo, 0.005–153 mg Se and 26–50 mg B (Frame and Newbould 1986). As animal forage, white clover contributes optimally as a 10–20 % component when grown in conjunction with other grasses (Anonymous 2005). It was found to be more digestible than other temperate forage legumes and therefore the ingested nutrients could be utilized more efficiently.

Healthy and stressed white clover plants contained many types of secondary metabolite such as flavonols, flavones, condensed tannins, isoflavones, isoflavanones, pterocarpans, coumestans, cyanogenic glucosides and saponins in various plant tissues (Carlsen and Fomsgaard 2008). Many of these bioactive compounds from white clover could be exploited for suppressing weeds and soil-borne diseases. The oil yield for T. repens was small 0.021 % (weight/fresh weight basis) (Tava et al. 2009). Several classes of compounds were found in the oil, including alcohols, aldehydes, ketones, terpenes, esters, hydrocarbons, phenolics and acids.

HE strains of white clover in New Zealand were reported to contain the cyanogenetic glucosides lotaustralin and linamarin, the glucosides of methylethylketone cyanhydrin and acetone cyanohydrin, respectively (Butler and Butler 1960). The isoflavones genistein, biochanin A and formononetin were found in glycosidic forms in white clover and insignificant amount of coumestrol (Francis et al. 1967). The cyanogenic glucosides linamarin and lotaustralin were isolated as a mixture from Trifolium repens (Maher and Hughes 1971). α-Hydroxy-methylbutyronitrile-β-d-glucoside was found in white clover (Hughes and Conn 1976). Linamarin and lotaustralin were identified in T. repens cv. Armena in concentrations of 1.29 and 3.13 mg/g of dry matter, respectively (Stochmal and Oleszek 1994). The linamarin/lotaustralin ratio ranged from 0.4 to 0.8 and was inversely correlated with the total cyanogen content; cultivars with higher cyanogen level had a lower linamarin/lotaustralin ratio (Stochmal and Oleszek 1997). At temperatures below 15 °C all varieties contained the highest cyanogen content; an increase in temperature during the summer time resulted in a drastic decrease in cyanogen synthesis.

In leaves, petioles, roots and nodules of white clover, pinitol (3-O-methyl-chiro-inositol) was found to be the predominant sugar, with sucrose present in lower amount (Davis and Nordin 1983). Significant amounts of α-methyl glucoside and β-methyl glucoside, linamarin, glucose and fructose were found in the leaves and petioles. In the nodules, glucose was rarely present at detectable levels. Malonic acid did not appear to be present in unusually high concentrations in either leaves or nodules.

From white clover plant, sapogenins soyasapogenols A, B and C were isolated (Walter et al. 1955). From the whole plant of white clover, five triterpenoid saponins, designated cloversaponins I–V, were isolated together with four known saponins, β-d-glucuronopyranosylsoyasapogenol B, soyasaponin I, soyasaponin II, azukisaponin II as their methyl esters and astragaloside VIII (Sakamoto et al. 1992).

Phytoestrogens isolated from ladino clover included genistein, biochanin A, formononetin and most significantly coumestrol (Bickoff et al. 1957, 1958, 1960, 1962; Guggolz et al. 1961). Sachse (1974) reported five estrogenic isoflavones—biochanin A, formononetin, pratensein, genistein, daidzein—and the estrogenic coumarin derivate coumestrol in 32 white clover varieties. Other phenolic compounds isolated from ladino clover included 3′,4′,7-trihydroxyflavone (Livingston and Bickoff 1964), daphnoretin (2-hydroxy-6-methoxy-3-(2-oxochromen-7-yl)oxychromen-7-one) (Livingston et al. 1964a), trifoliol (3,7-dihydroxy-9-methoxy-6H-benzofuro(3,2,-c)-1-benzopyran-6-one) (Livingston et al. 1964b; Bickhoff et al. 1965a) and 7,4′-dihydryoxyflavone (Bickhoff et al. 1965b). An acetylated isoflavone, genistein 7-(2″-p-coumaroylglucoside), genistein (Saxena and Jain 1986), 2″-O-acetylated formononetin and formononetin (Saxena and Jain 1989) were identified in Trifolium repens. Saloniemi et al. (1993) found the following isoflavones daidzein, formononetin, genistein and biochanin A in white clover varieties.

Flavones 4′,5,6,7,8-pentahydroxy-3-methoxyflavone and 5,6,7,8-tetrahydroxy-3-methoxyflavone, as well as two flavones 3,7-dihydroxy-4′-methoxyflavone and 5,6,7,8-tetrahydroxy-4′-methoxyflavone, were isolated from white clover shoots (Ponce et al. 2004). The known quercetin, rhamnetin, acacetin, 7-hydroxy-4′-methoxyflavonol; 3,5,6,7,8-pentahydroxy-4′-methoxyflavone; 2′,3′,4′,5′,6′-pentahydroxy-chalcone; 6-hydroxykaempferol; 4′,5,6,7,8-pentahydroxyflavone; and 3,4′-dimethoxykaempferol were also obtained.

Two bicoumarins, named repensin A and B, were isolated from Trifolium repens and their structures established as 7-methoxy-7′,8′-dihydroxy-8,6′-bicoumarinyl and 7,5′-dihydroxy-3,6′-bicoumarinyl, respectively (Zhan et al. 2003). Shoots of T. repens were found to contain cyanogenic glycosides, mostly linamarin (α-hydroxyisobutyronitrile-β-d-glucopyranoside) and a smaller proportion of lotaustralin (2-hydroxy-2-methylbutyronitrile-β-d-glucopyranoside) that breaks down to release toxic hydrogen cyanide when damaged (Gleadow et al. 2009).

In all tissues of clover seedlings not infected by the stem nematode, Ditylenchus dipsaci, isoflavonoids occurred predominantly as their glycosidic conjugates, in the order roots > meristems > leaves with formononetin-7-0-glucoside-6″-O-malonate (FGM) and medicarpin-3-O-glucoside-6″-O-malonate (MGM) as the major metabolites (Cook et al. 1995). The conjugates accumulated with age in all tissues and no differences were observed between the isoflavonoid content of healthy susceptible and resistant plants. Infection with either D. dipsaci race (oat and clover) elicited the accumulation of medicarpin, MGM and FGM in the meristems to a similar degree in resistant and susceptible seedlings. However, formononetin accumulated only in the infected meristems of the resistant plants. Johnson et al. (2005) found that different flavonoids increased in concentration in plants without rhizobial nodules than in plants with active or inactive nodules. The concentration of 4′,7-dihydroxyflavone was higher in plants without rhizobial nodules than in plants with active or inactive nodules. The content of formononetin was higher in roots with active rhizobial nodules than in inactive nodules and roots alone. Flavonol contents of white clover seeds (2.8–2,000 mg/g), leaves (<2–1,700 mg/g) and total above-ground material (20–2,210 mg/g) were higher than in roots (n.d.–208 mg/g) and flowers (66–481 mg/g) (Carlsen et al. 2008).

l-pipecolinic acid (piperidine-2-carboxylic acid) was isolated from the leaves (Morrison 1953) and a leaf protease (Brady 1961). Four coumestans detected in white clover infected with various foliar pathogens and were identified as coumestrol, 12-O-methylcoumestrol, trifoliol and 7,10,12-trihydroxycoumestan (repensol) (Wong and Latch 1971a; 1971b); other phenolic compounds found in diseased white clover included isorhamnetin; 4′,7-dihydroxyflavonol; kaempferol; quercetin; 4′,7-dihydroxyflavone; 3′ 4″,7-trihydroxyflavone; geraldone; luteolin; formononetin; daphnoretin; and a trihydroxycoumestan (Wong and Latch 1971b). White clover contained the phytoalexin medicarpin (Gustine 1981). Isoflavonoids medicarpin and demethylmedicarpin were detected in the droplet of white clover leaflets inoculated with Monilia fructicola; several minor isoflavonoids detected in the extracts were identified as formononetin (7-hydroxy-4′-methoxyisoflavone), 2′-hydroxyformononetin and vestitone (7,2′-dihydroxy-4′-methoxyisoflavanone) (Woodward 1981). The following isoflavonoid phytoalexins were detected in fungus-inoculated leaflets: 7,2′,4′-trihydroxyisoflavanone, vestitone, medicarpin, demethylmedicarpin, variabilin, sativan, vestitol, genistein, formononetin, glycitein and daidzein (Ingham 1978, 1982).

In white clover, decreased foliar protein coincided with an increased number of protease isoforms (Kingston-Smith et al. 2003). Major flavonoids in the leaves were found to be derivatives of quercetin and kaempferol (Hofmann et al. 2000). Total leaf flavonoids in Trifolium repens were found to be low only 1 mg/g compared to 50–65 mg/g for T. dubium and Lotus corniculatus and up to 15 mg/g for T. pratense (de Rijke et al. 2004) In T. pratense and T. repens, the main constituents were flavonoid glucoside-(di)malonates, while T. dubium and L. corniculatus mainly contained flavonoid (di)glycosides.