Limonoid tissue

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. 

Other studies show the presence of a diversity of compounds other than limonoids as defenses in the tissues of Meliaceae. Woody tissues of Trichilia trifolia afforded three novel dolabellanes with flexible Curing structures. These substances were very active antifeedants in the Sitophilus bioassay (Ramirez et al., 2000). T. martiana seeds yielded large amounts of 2-((Z,Z)-6,9-heptadecadienyl)furan. T. hirta and T. americana bark have yielded novel steroids by insect bioassay-guided isolation and application of a nanoprobe nuclear magnetic resonance (NMR) technique for structure elucidation (Chaurest etal.,1996). Compounds isolated included hydroxyandrosta-l,4-diene-3,16-dione (Fig. 1.4) and derivatives. However, studies by Wheeler et al. (2001) suggest that other unidentified compounds may also be... 

When 5-g-size navel oranges were fed with 44 x 10 cpm of acetate-2- 4C at the rate of 4.4 x 10 cpm per day for 10 consecutive days either by injection of the fruit or by application on the peel, no labeled XIV was detected in the fruit. Also, no 4C was incorporated into XIV when 5-g-size navel orange was similarly fed with 20 x 10 cpm of mevalonate-2--1-4C. When labeled acetate was fed to leaves adjacent to a 5-g-navel orange, radioactivity was incorporated into XIV in the fruit (12). These results suggest that limonoids are most likely not synthesized in the fruit tissues (12). However, they do not rule out the possibility that a precursor(s) of limonoids beyond acetate or meva-lonate is synthesized in leaves and translocated to fruit and there converted to limonoids. 

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. 

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. 

In the specificity tests, it was observed that only deoxy-limonin (27%) and deacetylnomilin (66%) cross reacted with the antibody. However, previous studies (28,29) on the relative concentration of these two compounds in citrus tissues showed that deoxylimonin is present at only 0.47% that of limonin and, therefore, will not be detected in diluted samples. Deacetylnomilin is also one of the minor constituents of the total limonoid fraction and will not contribute to limonin values. 

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... 

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. 

In further research on the limonoid glucosides, we found that they appear to be present only in fruit tissues and seeds. Radioactive tracer work showed that the fruit tissue itself is... 

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). 

Radioactive tracer work also demonstrated that NAA inhibits the accumulation of limonoids in fruit tissues of lemon trees (32). 

Fmit tissues do not normally contain bitter limonin, but instead contain the nonbitter precursor of limonin, limonoate A-ring lactone (2). Limonoate A-ring lactone is the predominant limonoid aglycone present in fmit tissues of most citms species, and it is gradually converted to limonin after the juice is extracted. This conversion proceeds under acidic conditions below pH 6.5 and is accelerated by the... 

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. 

Radioactive tracer work has shown that nomilin (3) is biosynthesized by the terpenoid pathway from acetate in the phloem region of stems and translocated to other parts of plants such as leaves, fruit tissues and seeds (13,14). At those locations, nomilin is further biosynthesized to other limonoids. Limonoid biosynthesis occurs at each location independently, thus the composition of limonoids in fruit tissues, seeds and leaves are different from each of the others. Limonin is biosynthesized from nomilin via obacunone (4), obacunoate (5) and ichangin (6) (15-17) (Fig. 2)... 

In fruit tissues and seeds, the limonoid aglycones are converted to 17- 3-D-... 

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). 

Limonoid UDP-D>glucose transferase Limonin bitterness is a problem in juices extracted from early-season to mid-season winter fruit, but not a problem in juice extracted from the late season fruit. As the fruit ripens, the concentration of limonoate A-ring lactone (2) decreases (23,24). This natural limonin debittering process has been known for over a century, but the mechanism of this metabolism was not understood until the recent discovery that limonoid glucosides are present in mature fruit tissues and seeds. We observed that in navel orange the initial... 

Other possible enzymes. Limonol (9) is a nonbitter limonoid (21). This compound is a minor limonoid and the pathway from limonin to limonol has not been investigated. This enzyme gene may be useful as specific target enzyme gene, but we assume that the activity is very low and it may not be practical to isolate the enzyme from citrus tissues. Deoxylimonin (8) is also a nonbitter limonoid present in Citrus. (20). The conversion of limonin to deoxylimonin requires a multienzyme system. Therefore, it is not practical to utilize this pathway for genetic engineering manipulation. 

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. 

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. 

Sample preparation for citrus tissues is different from citrus juices. Monolactones are the predominant form of limonoids in most intact citrus tissues, but most of the analytical methods can measure only the dilactone forms. To analyze the total of both forms in citrus tissues, the monolactones must first be extracted from the tissues and converted to the dilactones during sample preparation. Acidification and the use of heat are the two main methods of converting monolactones into dilactones. 

TLC is the easiest and most economical technique of detecting limonoid aglycones. Unlike other methods, TLC can be used for a variety of tissue samples. TLC solvent systems were well developed and an appropriate system can be found for a particular type of tissue sample with just a few trials. The major disadvantage is the use of subjective visual comparison with standards for qualitation and quantitation. 

In the food industry, high-performance liquid chromatography (HPLC) is the most widely used technique for the determination of limonin levels in citrus juices, since it is accurate and reliable. Most of the HPLC methods developed are for the analysis of limonin in citrus juices. These methods may not work for other bitter limonoid aglycones and/or tissues. 

Limonoids Citrus fruits Provide protection to lung tissue... 

The most highly oxidized limonoids in neem are modified in both the C and D rings. Azadirachtin, 15, (25, 2d), the most studied member of this structural class, contains 16 oxygen atoms and has 16 chiral centers. In addition to their own unique structural modifications, members of the class have side-chain substitutions similar to those observed in compounds already discussed. Other representative examples of this class are shown in Figure 6 25-27), These compounds, although found in various plant tissues, are predominantly in the neem kernel. Azadirachtin is the most... 

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