ISOFLAVONOIDS

In contrast to most other flavonoids, isoflavonoids have a rather limited taxonomic distribution, mainly within the Leguminosae. Most of our knowledge about the biosynthesis of isoflavonoids originates from studies with radioactive isotopes, by feeding labelled ¹⁴C cinnamates.

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Example of a BIOLOGICALLY ACTIVE ISOFLAVONOID

Rotenone comes from Derris root and Lonchocarpus species leaf (Family: Leguminosae)
It is an insecticide and also used as a fish poison.

* (blue): carbons derived from methionine.
(red): carbons derived from PRENYL (isoprenoid). 

Biochemical pathway to the formation of rotenone.

Six rotenoid esters occur naturally and are isolated from the plant Derris eliptica found in Southeast Asia or from the plant Lonchocarpus utilis or L. urucu native to South America.

Rotenone is the most potent. It is unstable in light and heat and almost all toxicity can be lost after two to three days during the summer. It is very toxic to fish, one of its main uses by native people over the centuries being to paralyze fish for capture and consumption. Crystalline rotenone has an acute oral LD50 of 60, 132 and 3000mg/kg for guinea pigs, rats, and rabbits (Matsumura, 1985). Because the toxicity of derris powders exceeds that of the equivalent content of rotenone, it is obvious that the other esters in crude preparations have significant biologic activity.

Acute poisoning in animals is characterized by an initial respiratory stimulation followed by respiratory depression, ataxia, convulsions, and death by respiratory arrest (Shimkin and Anderson, 1936). The anesthetic-like action on nerves appears to be related to the ability of rotenone to block electron transport in mitochondria by inhibiting oxidation linked to NADH₂, this resulting in nerve conduction blockade (O'Brien, 1967; Corbett, 1974). The estimated fatal oral dose for a 70kg man is of the order of 10 to 100g.

Rotenone has been used topically for treatment of head lice, sacbies, and other ectoparasites, but the dust is highly irritating to the eyes (conjunctivitis), the skin (dermatitis), and to the upper respiratory tract (rhinitis) and throat (pharyngitis).

Biosynthesis of Flavonoid

Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA. This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones, which contain two phenyl rings. Conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield flavanones → dihydroflavonols → anthocyanins. Along this pathway, many products can be formed, including the flavonols, flavan-3-ols, proanthocyanidins (tannins) and a host of other various polyphenolics.

Flavonoids are synthesized via the phenylpropanoid pathway. Phenylalanine ammonia lyase (PAL) catalyzes the conversion of phenylalanine to cinnamate. PAL also shows activity with converting tyrosine to p-coumarate, albeit to a lower efficiency. The cinnamate 4-hydroxylase (C4H) catalyzes the synthesis of p-hydroxycinnamate from cinnamate and 4-coumarate:CoA ligase (4CL) converts p-coumarate to its coenzyme-A ester, activating it for reaction with malonyl CoA. The flavonoid biosynthetic pathway starts with the condensation of one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, yielding naringenin chalcone. This reaction is carried out by the enzyme chalcone synthase (CHS). Chalcone is isomerised to a flavanone by the enzyme chalcone flavanone isomerase (CHI). From these central intermediates, the pathway diverges into several side branches, each resulting in a different class of flavonoids. Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific 3ß-hydroxylation of (2S)-flavanones to dihydroflavonols. For the biosynthesis of anthocyanins, dihydroflavonol reductase (DFR) catalyzes the reduction of dihydroflavonols to flavan-3,4-diols (leucoanthocyanins), which are converted to anthocyanidins by anthocyanidin synthase (ANS). The formation of glucosides is catalyzed by UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT), which stabilize the anthocyanidins by 3-O-glucosylation (Harborne 1994, Bohm 1998). The overview of the flavonoid pathway is presented in Fig 1B. There is evidence that the enzymes involved in flavonoid metabolism might be acting as membrane-associated multienzyme complexes, which has implications on the overall efficiency, specificity, and regulation of the pathway (Stafford 1991, Winkel-Shirley 1999, 2001).

Studies of the flavonoid pathway range from classical genetic analysis of flower color inheritance patterns by Mendel, through the establishment of their chemical structures, to efforts to understand the factors involved in their biochemical synthesis (Bohm 1998). Basic knowledge of the flavonoid biosynthesis was gained from experimental studies using radio-labeled precursors at the end of 1950’s. The development of more sophisticated methods in analytical chemistry and enzymology, and later in gene technology, has produced a vast number of studies and detailed information of the flavonoid biosynthesis in several plant species. The flavonoid biosynthetic pathway has been comprehensively reviewed (e.g. by Dooner & Robbins 1991, Koes et al. 1994, Holton & Cornish 1995, Mol et al. 1998, Weisshaar & Jenkins 1998, Winkel-Shirley 2001).

The first gene isolated from the flavonoid biosynthetic pathway was a CHS gene from parsley (Petroselinum hortense) (Kreuzaler et al. 1983). Transcriptional control of the structural genes of the flavonoid biosynthetic pathway has been most intensively studied in relation to the biosynthesis of anthocyanins. Groundbreaking research concerning the expression of the structural and regulatory genes of the flavonoid pathway has been done with maize (Zea mays) (Goff et al. 1990, Taylor et al. 1990, Tonelli et al. 1991), arabidopsis (Arabidopsis thaliana) (Shirley et al. 1992) and with ornamental plants like snapdragon (Antirrhinum majus) (Martin et al.1991), petunia (van der Krol et al. 1988) and gerbera (Elomaa et al. 1993, Helariutta et al. 1993, 1995). Naturally occurring flavonoid mutants and variants, or genetically transformed mutant plants have been important tools in several investigations clarifying the functions of the flavonoid pathway genes (Shirley et al. 1995, Tanaka et al. 1998).

The expression of flavonoid pathway genes in fruit tissues has been studied on grape (Vitis vinifera) (Boss et al. 1996, Kobayashi et al. 2001), citrus (Citrus unshiu Marc.) (Moriguchi et al. 2001), and strawberry plants (Fragaria spp.) (Manning 1998, Aharoni et al. 2001). The scarcity of studies in this area may be due to a difficulty caused by the special features of the fruit tissues, e.g. the richness of different secondary metabolites and RNases, which may hinder the easy application of the molecular biological research methods.