Phenolic Patterns in Citrus
The overall tissue composition of phenolics has not been previously studied in the members of the Citrus subtribe. These compounds can readily be divided into classes according to their absorbance spectra (figure 2). Table 2 lists the percentages obtained for each sample of flavone/ol, flavanone, coumarin/cinnamic acid derivatives, and psoralen classes which absorb at 285 nm, as well as the concentrations (in milligrams per gram of fresh weight) of flavone/ols, flavanones, and coumarins. Void and unidentified peaks may contain chromones as well as other compounds.
Chromatograms for each sample were monitored at two wavelengths: 285 nm, which detects mainly flavanones and to a lesser extent flavones, and 325 nm, which detects coumarins and to a lesser extent psoralens and flavones. The chromatograms detected at 285 nm generally have higher total absorbances (excluding the void volume) than those detected at 325 nm. Furthermore, when the UV spectra of the peaks were examined they were readily classified, and the upslope, apex, and downslope spectra generally matched, indicating the peaks were those of a single compound (figure 2). Typical chromatograms monitored at 285 nm and 325 nm are shown in figure 5. Peaks identified at 285 nm usually also occur at 325 nm (figure 5 and table 5 and table 6). The peaks are well separated, and the peak shapes are very similar at these detection wavelengths.
For most citrus taxa, only 13 percent of the peaks detected at 325 nm, on average, were not detected at 285 nm. Exceptions occur in C. medica (citron) 'Citron of Commerce' and 'India Sour', C. paradisi (grapefruit) 'Camulos', and C. sinensis (orange) 'Thomson': these cultivars had over 20 percent dissimilar total peak areas (table 5 and table 6). The citron's phenolic profiles do not resemble those of other species in table 5 and table 6 since the overall phenolic concentrations of citrons are extremely low and are dominated by coumarins. For most samples, few significant peaks (consisting of a single peak with a percentage area over 10 percent of the total area calculated for all peaks of a chromatogram) are detected at 325 nm. Grapefruit and orange produce larger numbers of unknown peaks with relatively high percentage areas at 285 nm. This causes a higher value for dissimilar (or unique) peaks. The average total proportional area per chromatogram of peaks detected at 285 nm but not detected at 325 nm is almost twice as high (24.5 percent versus 13.5 percent).
We believe that the number of nonvoid volume peaks can be used to describe a chromatogram's complexity and the complexity of the phenolic profile for a particular cultivar. Table 5 and table 6 summarize the average number of phenolic peaks occurring in several cultivars in various species. A wide range in the number of phenolic peaks (5 to 50 at a detection level of 0.01 mg/g fresh weight sample) occurs in Citrus depending on the species and the type of tissue examined.
The general trend indicates that hybrid taxa (including lemon, lime, tangelo, orange, grapefruit, and tangor) have more complicated spectra than the primary species (pummelo, citron, and mandarin). The most complicated chromatograms are found for their wild, presumably somewhat hybridized relatives: papeda, New Guinea lime, large-leaf wild lime, and red pulp finger lime. However, this observation is somewhat limited since in some cases only a small number of cultivars from a particular species were employed in this study.
Summarizing table 2, the highest concentrations and usually the highest percentages of flavone/ols occur in the leaf in citrus. The levels of flavone/ols in the flavedo are slightly lower. The concentration of flavone/ols is much less in the albedo and juice sacs of the fruit. The concentration of flavanones is greatest in the fruit albedo. The leaf or the flavedo has the highest concentrations and percentages of coumarins. It has been reported that the flavedo of 'Washington' navel orange contains about 24 times the concentration of flavonoids found in the albedo (Brunet and Ibrahim 1973). However, our results fail to verify this in other cultivars of orange.
In 7 of the 35 species and hybrids tested, the leaves contain the highest concentration (mg/g fresh weight) of flavanones. Some of these species may be related. Citron and a Microcitrus species such as Australian desert lime or the Australian large-leaf wild lime may be ancestors of lime (Swingle and Reece 1967). Rough lemon has citron and mandarin as ancestors (Hodgson 1967). Tangor seems less related, except that its flavonoid pattern resembles that of lime and rough lemon and it also has mandarin as an ancestor (Swingle and Reece 1967). Citron has only slightly higher flavanone concentrations in its leaves than in the fruit tissues; however, the overall flavanone levels in all tissues of the citron are very low, so this observation is somewhat tentative. Citron is believed to be one of the primary species and a parental source of lemon, lime, and rough lemon (Hodgson 1967, Mizelle et al. 1967, Swingle and Reece 1967).
In another nine, the flavanone concentrations were highest in the albedo (calamondin, C. sinensis × (C. sinensis × P. trifoliata) (citrangor), citrumelo, grapefruit, lemelo, Natsudaidai orange, pummelo, sour orange, and tangelo). Grapefruit, lemelo, Natsudaidai orange, and tangelo are believed to be pummelo hybrids (Mizelle et al. 1967, Swingle and Reece 1967); calamondin is a Fortunella × C. reticulata hybrid; and citrangor and citrumelo cultivars are believed to be Poncirus hybrids (Hodgson 1967, Swingle and Reece 1967, Albach and Redman 1969).
In three commercially important citrus specieslemon, mandarin, and orangethe flavanone concentrations tend to be roughly equal in leaf, flavedo, and albedo tissues. Mandarin and orange generally have slightly higher concentrations of flavanones in the leaf than in the other tissues. Lemon tends to have slightly higher flavanone concentrations in the albedo. Juice vesicles in general contain the lowest overall concentrations of phenolics when compared to other parts of the fruit or the leaves, and their main phenolic constituents (based on phenolic percentages) are flavanones (table 2).
Fruit samples contain the same individual phenolic compounds as the leaf tissues, although the concentrations may differ dramatically. An evaluation of the leaf composition and concentration generally finds the same phenolics as in the juice vesicles, except that more flavanones are found in the fruit tissues than the leaf of many cultivars. In pummelo and grapefruit, however, the flavedo contains a larger number and higher levels of coumarins and psoralens than the other tissues. Such analyses could prove useful in genetic and taxonomic characterization of seedlings without the necessity of growing trees to fruiting age.
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Original posting: April 1, 1999.