Limonoids in citrus fruits

Citrus trees are capable of translocating limonoids from leaves to fruit tissues (12). When 20,000 cpm of XIV, randomly labeled, was fed to a leaf adjacent to a 5-g-size lemon, about 13.3% of the total activity was translocated to the fruit during 20 hrs. These findings of Hasegawa et al. (12,13) show that limonoids in citrus fruit tissues are synthesized in leaves and translocated to the fruit. 

This review paper summarizes the biochemical and biological aspects of limonoids in citrus and presents possible preharvest approaches to reduce bitter limonoids in citrus fruits. 

Ethylphenoxy)triethylamine and 2-(3,4-dimethoxyphenoxy)triethylamine markedly reduce the biosynthesis of limonoids in citrus leaves, presumably by inhibition of cyclase activity. Radio-tracer studies have revealed that limonoids are synthesized in the leaves of citrus and transported to the fruit. The fruit tissue does not appear to be capable of the de novo synthesis of limonoids from acetate or mevalonate. 

Apart from the more obvious benefits of fruit juice, such as being a source of potassium, it contains other substances that have or are claimed to have useful pharmacological activity. For example, limonin and other related limonoid substances present in citrus fruit are believed to have a role in inhibiting certain forms of cancer. Sorbitol, which occurs in many fruit juices, has a laxative effect. 

Several limonoids are known to be bitter principles of citrus (Rutaceae). A typical example is limonin. Although fresh juice does not elicit a bitter taste, sometimes it becomes bitter after heating or storage. This is explained by the formation of bitter-tasting limonin by deglycosylation and further cyclization from limonin glucoside, which is present in citrus fruit tissue and seeds and does not exhibit bitterness.146 Recently, it was reported that limonin had antitumor activity.147 Besides limonin, nomilin and obakunone, which are considered to be... 

Unlike most other citrus species in which limonin is predominant (over 50% of total limonoids) Citrus ichangensis accumulates ichangensin (11) as the predominant limonoid in its fruit tissues and seeds (26). Fruit tissues contain 50 times as much ichangensin as limonin. They also contain relatively high concentrations of deacetylnomilin (12). 

Hasegawa and Hoagland (1977) have recently carried out tracer studies on the biosynthesis of limonoids in citrus. These studies showed that young leaves were capable of synthesizing limonoids and su ested that the limonoids were translocated from this biosynthetic site to the fruits. 

Citrus limonoids are furanoid natural products found in citrus fruits. The most abundant limonoids, limonin and nomdin, have been determined to induce increased activity of GST and inhibit chemically induced carcinogenesis (6,7). These findings with furan containing natural products led to the investigation of mono and disubstituted furan and the corresponding sulfur containing thiophene compounds that are found in complex mixtures of beef aroma. 

Nomilin the general biosynthetic precursor of all limonoids, is biosynthesized via the terpenoid pathway from acetate and/or mevalonate in stems. Nomilin is then translocated to other plant tissues, including leaves, seeds, and fruit tissues, where it is converted to the other limonoids independently (Hasegawa et al. 1986 Ou et al. 1988). In most citrus species and hybrids, nomilin is converted to obacunone and then to obacunoate. Obacunaote is then converted to limonin probably via the intermediate ichangin. Nomilinic acid is the predominant acidic limonoid in citrus, but it does not seem to be directly involved in the biosynthetic pathways of major limonoids. 

The capability of Citrus to translocate limonoids from the fruit tissues to the seeds was demonstrated by administering 1 x 10 cpm of methyl- "deacetylnomilinate to the stem end of detached calamondin fruits. After 16 hrs of incubation, 7,500 cpm of activity were translocated to the seeds, and over 90% of the total activity in the seed extract was recovered as the original substrate (8). These results show that limonoids present in citrus seeds are translocated through the fruit tissue. 

While most of the limonoids of Citrus have been isolated from seeds, several occur in detectable amounts in other parts of the fruit. Minor amounts of deacetylnomilin, nomilin, obacunone, de-acetylnomilinic acid and nomilinic acid were identified in extracts of navel orange peel (37). 17-Dehydrolimonoate A-ring lactone was isolated from peel and juice of navel oranges (38) and nomilin has been reported to occur in grapefruit juice and juice vesicles (39). The only Citrus limonoids known to be bitter are limonin, VI, XII, obacunoic acid and IX (3). 

Control of Juice Bitterness. A number of advances have been reported in this field since it was last reviewed (3). A commercial application of the cellulose acetate adsorption technique for the removal of limonin from citrus juices was undertaken (49). New sorbent gel forms of cellulose esters for adsorption of limonin were developed (50). Knowledge was gained that limonoids are biosynthesized in citrus leaves and translocated to the fruit (12) and that specific bioregulators can inhibit accumulation of XIV in citrus leaves (15). Additional studies were carried out on the use of neodiosmin to suppress limonin and other types of bitterness (30,51). The influence of extractor and finisher pressures on the level of limonin and naringin in grapefruit juice was reported (34). Also, further studies were conducted on the microbial sources and properties of limonoate dehydrogenase (52), the enzyme that converts XIV to XV and can be used to prevent limonin from forming in freshly expressed citrus juices (53). 

Citrus ichangensis possesses an unusual distribution of limonoids (20). Unlike other species of citrus, fruit tissues and seeds of . ichangensis contain very high concentrations of nonbitter ichangensin and very low concentrations of bitter limonin. It also contains relatively high concentration of nonbitter deacetylnomilin. Quantitative analysis showed that a ratio of ichangensin to limonin in the fruit tissue is approximately 50 to 1. 

Stems are the major site of nomilin biosynthesis from acetate in citrus (26). Analysis of the phloem, the cortex and the inner core regions of the stem showed that the phloem region is the site of nomilin biosynthesis from acetate (27). Root tissues also have this capacity. Leaves, fruits and seeds are either incapable of biosynthesizing limonoids from acetate or have a very low capacity. However, these tissues are capable of biosynthesizing limonoids from nomilin. Nomilin is translocating from the stem to other locations, where it is further biosynthesized to other limonoids (26). 

Biochemical studies carried out at the Pasadena laboratory have shown how, where and when limonoids are biosynthesized and accumulate in citrus. The recent discovery of limonoid glucosides has cleared an unanswered question of how limonoid aglycones disappear at late stages of fruit growth and maturation. 

Certain limonoids have been shown to possess antifeedant effects on some insects and anticarcinogenesis activity in mice. Although further research is needed, limonoids appear to have potential as insecticides or chemopreventative agents against cancer. Citrus fruit and seeds are an excellent source of these compounds. 

Thirty-six limonoid aglycones have been isolated from Citrus and its hybrids. These aglycones are present in intact fruit tissues as open D-ring forms such as limonoate A-ring lactone (2), while in seeds they are present in both open and closed forms. It appears that these citrus limonoids are biosynthesized through at least four different limonoid pathways the limonin pathway, the ichangensin pathway, the calamin pathway and the 7-acetate limonoid pathway (12). The products of the limonin pathway are found in all citrus species. The products of the other three pathways are only found in certain species of citrus and its close relatives. 

No evidence of limonoid biosynthesis in fruit or seed tissues exists, despite the fact that most of the limonins are found in the seeds of mature fruits. Limonoid synthesis occurs in the leaves and limonoids are transported into the fruits (Maier, 1983). In citrus tissues, the naturally occurring precursor of limonin is a salt of limonoic acid A-ring lactone (60) (Fig. 25.13) in which the A ring is closed and the D ring is open. This tasteless compound is stable only in the salt form (Maier, 1983). In the presence of acid or the enzyme citrus limonoate D-ring hydrolase, the D-ring lacton-izes to form limonin (19). The rate of lactonization is accelerated by pasteurization of the juice. In the fruit, the precursor appears to be located in a compartment of the cell where the pH is neutral or alkaline, probably the cytoplasm (Maier, 1983). 

Bitterness is a major problem to the citrus industry. It is of economic importance because bitter juices have a lower market value. Bitterness is mainly caused by the accumulation of two different chemical compounds limonin from the limonoid terpine group and naringin from the flavonoid phenolic group in the fruit tissues. The highest concentrations of the bitter compounds are generally found in immature fruits. As the fruits mature, the concentrations of the bitter compounds decrease. 

The above HPLC methods are used for the analysis of limonin and/or nomilin in citrus juices. Herman et al. (1989) used a reverse phase HPLC method to quantify limonoids in fruits, seeds, leaves, stems and seedlings of Citrus ichangensis. The extraction procedure of Hasegawa et al. (1984) as described in Section 2.1.1 was followed. The dried extracts were dissolved in 50% methanol prior to injection. A reverse phase C-18 column (4.6 X 250 mm) was used. The column was eluted isocratically with water-methanol-acetonitrile (49 41 10) at a flow rate of 1 ml/min. Limonoids were detected by UV absorption at 210 nm. This... 

Figure 7.29 Citrus fruit are a rich source of terpenoids including the Cio diterpenes (+)- and (—)-limonene and a number of more complex limonoids which are distinctive in that the glucosides are tasteless while the aglycones have a bitter taste.

Bitterness, caused by naringin, is also removed by an enzyme, naringinase. Another possibility would be to eliminate one or more of the enzymes in the limonin biosynthetic pathway by using recombinant DNA techniques. Limonin, a triterpenoid, is probably synthesized via the mevalonate pathway, as are the monoterpenoid flavor compounds. It appears that nomilin, a precursor of limonin, is synthesized in the stems and roots of citrus and then the precursor transported to the fruit where it is converted by several enzymes to limonin and other bitter limonoids (46). 

Fruits of the genus Citrus constitute one of the world s most important horticultural crops. Production is at least 45 million tons, of which about 45% is converted into a variety of processed products (Maier, 1983). After expression of citrus juices, especially orange juice, precursors that occur in the juice are converted to bitter limonoids, primarily li-... 

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