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United States Department of Agriculture

Agricultural Research Service

Phenolics:Introduction

Survey of Phenolic Compounds Produced in Citrus

Introduction

The phenolic compounds of Citrus are secondary metabolic products that are believed to be produced as a result of the plant's interaction with the environment (Beier and Oertli 1983, Afek et al. 1986, Zaat et al. 1987, Laks and Pruner 1989, Snyder and Nicholson 1990). The phenolics are derived from phenylalanine and absorb light in the low ultraviolet range. In general, many absorb light around 285 nm (see figure 1 and figure 2). The phenolics that occur in Citrus include the flavonoids (flavanones, flavones, and flavonols) the anthocyanins, the coumarins, and the psorolens, among others.

Flavonoids may act as inducers (Zaat et al. 1987) and as phytoalexins (Laks and Pruner 1989, Snyder and Nicholson 1990)—that is, low-molecular-weight antimicrobial compounds that are both synthesized and accumulated in plant cells as a defense mechanism after exposure to microorganisms (Dixon 1986, Laks and Pruner 1989). Coumarins, acting as phytoalexins, are reportedly produced in response to pathogens' attacks on Citrus (Feldman and Hanks 1968, Afek et al. 1986, Nakatani et al. 1987).

Psoralens (linear furocoumarins) are toxic to insects, especially in the presence of ultraviolet light (Nahrstedt 1990, and references therein), and have been identified as phytoalexins in celery (Beier and Oertli 1983). Evidence indicates that someCitrus species may contain one or more flavedo compounds that confer insect resistance on their fruits. In particular, the Mediterranean fruit fly (Ceratitis capitata, a tephritid fruit fly) does not survive in lemons (Back and Pemberton 1918, Anonymous 1990). Caribbean fruit fly (Anastrepha suspensa, also a tephritid) pupae do not mature in lemons or limes (Nguyen and Fraser 1989, Anonymous 1990). These two observations could be related to a particular hydroxylation pattern of a flavonoid compound, the importance of which has been demonstrated for larval growth inhibition (Elliger et al. 1980).

In addition, phenolics appear to have desirable medicinal properties. Some have been reported to be antitumor agents and to exhibit antiviral and antimicrobial activities (Robbins 1980), hypotensive effects (Matsubara et al. 1985), and antioxidant properties (Robak and Gryglewski 1988). Psoralens are used in conjunction with ultraviolet light to treat psoriasis and other human skin disorders (Stolk and Siddiqui 1988). Both psoralens and coumarins are found in citrus oils (Lawrence 1982).

Recent evidence suggests that phenolics may play an important role in the regulation of plant metabolism. For example, flavonoids have been shown to be naturally occurring auxin transport regulators (Jacobs and Rubery 1988).

In short, the plant phenolics play a major role in both plant and animal health. Although much basic research still remains to be done, is possible that many of these compounds, either as isolates or in conjunction with other compounds, may be used in both agricultural and pharmaceutical roles. Developing an understanding of the distribution of phenolics in Citrus and its related species will give an assessment of the diversity present in this important group of plants.

Citrus flavanones play an important role in citrus fruit and juice quality, contributing to juice cloud, as hesperidin does in lemons and oranges (Mizrahi and Berk 1970), and bitterness, as naringin does in grapefruits and pummelos (Horowitz and Gentili 1961, Guadagni et al. 1973, Horowitz 1986).

Different plant species, even different cultivars within a species, accumulate different flavonoids, a face that can be of use in establishing taxonomic relationships between different citrus species and relatives. There are qualitative differences in the flavanones (and other secondary products) detected, as opposed to quantitative differences in the primary products, which are influenced by environmental stresses.

The biosynthesis of flavonoids in plant tissues has been extensively studied in many plants, and several of the biosynthetic steps have been elucidated as shown in figure 3. The control mechanisms for complex modifications in flavonoid biosynthesis (such as B-ring hydroxylations, methylation, or glycosylation) have also been studied in a small number of cultured cell suspensions from plants such as Glycine max, Haplopappus gracilis, and Petroselium hortense (Hahlbrock and Grisebach 1979, Heller and Forkmann 1988, Hahlbrock and Scheel 1989). However, these systems have not focused on flavanone modification enzymes, and many final steps are postulated only with unproven intermediates or responsible enzymes. Determining the flavanone distribution in citrus tissues could point to possible tissue-specific enzymatic activity. For example, in Haplopappus, the enzyme flavanone synthase has a different optimal pH for naringenin production than for eriodictyol production (Hahlbrock and Grisebach 1979). Therefore, the pH at the site of the reaction (that is, in a particular tissue) could help to determine substrate specificity or the major reaction product. In addition, in some plant systems flavonoids are accumulated preferentially in the epidermis, while in others they occur in the mesophyll (Wiermann 1981). The results presented in table 1 demonstrate that in citrus species the albedo and juice vesicles generally have a greater number of flavanone peaks than the leaf tissue.

A number of surveys have identified and quantified Citrus flavanones based on separations performed by extractions and recrystallizations, paper chromatography and column chromatography, and preparative thin-layer chromatography (Hattori et al. 1952, Mizelle et al. 1965, Hagen et al. 1966, Maier and Metzler 1967, Mizelle et al. 1967, Fisher 1968, Albach and Redman 1969, Albach et al. 1969, Nishiura et al. 1969, Coffin 1971, Nishiura et al. 1971a,b, Tomas et al. 1978, Kamiya et al. 1979, Grieve and Scora 1980, Anis and Aminuddin 1981, Albach and Wutscher 1988). These techniques have also been used to isolate flavones, psoralens, and coumarins (Maier and Metzler 1967, Brunet and Ibrahim 1973, Dreyer and Huey 1974, Tatum and Berry 1979, Afek et al. 1986, Mizuno et al. 1987). The column chromatography and thin-layer chromatography surveys usually quantitate only one or two of the phenolics in citrus fruits. Several studies identifying naringin in grapefruit and hesperidin in lemon have been performed (Maier and Metzler 1967, Albach et al. 1969, Tomas et al. 1978, Mizuno et al. 1987, Albach and Wutscher 1988). Unfortunately, these studies are of limited quantitative value.

The influence of polyploidy on the chemical composition of leaves and fruit is unknown. The occurrence of polyploidy in Citrus and related genera has been reported since the 1920's (Cameron et al. 1964, Cameron and Soost 1968, 1969, Soost and Cameron 1981). Tetraploidy is the most common natural polyploidy. The degree of polyploidy in Citrus plants is manifested with various physical effects, but no work relating the degree of polyploidy to the levels of plant phenolics has been done.

Past studies of citrus phenolics using high-performance liquid chromatography (HPLC) have been confined a few flavonoids or to a few Citrus cultivars, mainly to individual species or individual phenolic compounds. Frequently this technique has been used to isolate individual compounds and not to quantitate them (Brunet and Ibrahim 1973, Dreyer and Huey 1974, Ting et al. 1979, Beier and Oertli 1983, Park et al. 1983, Jourdan et al. 1985, Gaydou et al. 1987, McHale et al. 1987, Mizuno et al. 1987, Rousseff et al. 1987, Tisserat et al. 1989, Vandercook and Tisserat 1989). Such studies are also of limited quantitative value. Investigations identifying or quantifying a few flavanones have generally been performed on lemon, orange, and grapefruit cultivars. (Hattori et al. 1952, Mizelle et al. 1965, Hagen et al. 1966, Maier and Metzler 1967, Mizelle et al. 1967, Fisher 1968, Albach et al. 1969, Nishiura et al. 1971a, Lawrence 1982, Mizuno et al. 1987, Rousseff et al. 1987, Albach and Wutscher 1988, Vandercook and Tisserat 1989). A few qualitative surveys of flavonoids in a large number of citrus cultivars have been published. The results of these studies are summarized in appendix 1.

This publication is the first to provide direct quantification of all major flavonoids in the leaves and fruit for a large number of Citrus species and cultivars. This information can be used to assess taxomonic classifications, evaluate potential sources of phenolic compounds for agricultural and pharmaceutical uses, and evaluate breeding program results.


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United States Department of Agriculture
Agricultural Research Service

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Original posting: April 1, 1999.

Last Modified: 2/6/2002
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