Biosynthesis of limonoids in Citrus

Analyses of citrus leaves and fruits, and radioisotope tracer work showed that the leaves could synthesize limonoids and that the trees could translocate limonoids from leaves to the fruit. These results suggest that limonoids in citrus fruit tissues are synthesized in leaves and translocated to the fruit. No evidence was found to support the presence of limonoid biosynthetic systems in the fruit tissues.     

Metabolism of limonoids via a deoxylimonoid pathway in Citrus

Methyl deacetylnomilinate was metabolized in leaves of calamondin (Citrus microcarpa) to form a deoxy derivative. This reaction indicated the presence in citrus of an epoxidase, which is required for the first step of the deoxylimonoid pathway. The product of the second step, deoxylimonic acid, was isolated from grapefruit seeds. Deoxylimonate A-ring lactone hydrolase, which is involved in the third step of the deoxylimonoid pathway, was also found in grapefruit seeds. These results and the previously reported presence of the first metabolite of this pathway, deoxylimonin, show that limonoids are metabolized in citrus, not only via the 17-dehydrolimonoid pathway as previously established, but also via the deoxylimonoid pathway.     

Biosynthesis of obacunone from nomilin in Citrus limon

Radioactive tracer work showed that [¹⁴C]nomilin was converted to at least four metabolites in Citrus limon. One metabolite was identified as obacunone, showing that obacunone is biosynthesized from nomilin in C. limon.     

Distribution of limonoids in the rutaceae

Chemical studies on Choisya arizonica, C. mollis, C. ternata, Esenbeckia hartmanii, Hesperethusa crenulata, Eremocitrus glauca, Triphasia trifolia, Coleonema album and Pamburus missionis are described with special reference to the limonoid bitter principles. The distribution of limonoids in the Rutaceae and especarlly the subfamily Aurantioideae is discussed. Two new coumarins, 7-isopentenyloxy-8-isopentenylcoumiain from Choisya arizonica and C. mollis and 2′,3′-dihydroxydihydrosuberosin from Coleonema album have been isolated and their structures determined.     

Limonin biosynthesis from obacunone via obacunoate in Citrus limon

Radioactive tracer work showed that [¹⁴C]obacunone was converted to at least four metabolites in Citrus limon. Two were identified as obacunoate and limonin. When [¹⁴C]methyl obacunoate was fed, limonin was found to be one of the metabolites. Based on these results and data accumulated thus far, biosynthetic pathways of limonoids in citrus are proposed.     

Activity,extraction and stability of enzymes involved in polysaccharide biosynthesis in Citrus

The effects of EDTA, 2-mercaptoethanol and bovine serum albumin on the extraction and stability of hexokinase, phosphoglucomutase, UDPglucose pyrophosphorylase and 1,3-β-glucan synthase from Mexican lime bark have been examined. The activity of these enzymes was generally increased and stability was tested in refrigerated and frozen extracts. Bovine serum albumin was the best stabilizing agent for phosphoglucomutase and 1,3-β-glucan synthase, but the former was more stable in refrigerated and the latter in frozen extracts. UDPglucose pyrophosphorylase stability was strongly dependent on the presence of 2-mercaptoethanol. The fact that the activity of 1,3-β-glucan synthase is the lowest of the four enzymes even in the presence of optimal concentrations of its activator strongly suggests that it is the key enzyme in the regulation of the metabolic pathway.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Isofraxinellone,a limonoid lactone from the bark of Fagaropsis glabra

Two lactones, both degraded limonoids, have been isolated from the trunk bark of Fagaropsis glabra. Isofraxinellone is a new natural compound and fraxinellone has previously been characterized in plants of the same family. The structure of isofraxinellone was determined by physical and chemical methods.     

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Hydrocarbons from epicuticular waxes of Citrus peels

The hydrocarbon fraction of the epicuticular wax layer of peel from 5 cultivars of Citrus was monitored at various stages of fruit development. GLC     

17-dehydrolimonoate A-ring lactone: A possible metabolite of limonoate A-ring lactone in Citrus fruits

A new limonoid, 17-dehydrolimonoate A-ring lactone (III), was isolated from orange peel, juice and lemon seeds and seedlings. This compound which is non-bitter appears to be an initial product of limonoate A-ring lactone (II) metabolism. As such it may be the initial intermediate in at least one debittering pathway in citrus fruits.     

Biosynthesis of oleanolic acid glycosides by subcellular fractions of Calendula officinalis seedlings

The synthesis of oleanolic acid 3β-d-glucuronoside from oleanolic acid and UDPGlcA has been demonstrated in cell-free preparations from C. officinalis seedlings. Moreover, the formation of more complex glycosides by successive additions of galactose and glucose to oleanolic acid glucuronoside was observed when cell-free preparations were incubated with UDPGal or UDPGlc. The consecutive steps of oleanolic acid glycosylation are localized in three different cellular compartments. The biosynthesis of the 3-glucuronoside takes place in the microsomes, the elongation of the sugar chain at C-3 of the aglycone proceeds in heavy membrane structures which are probably fragments of the Golgi complex while a cytosol enzyme(s) is involved in glucosylation of the C-17 carboxyl group of oleanolic acid.     

Distribution of limonoids in citrus seeds

Absolute concentrations of the major limonoids in the seeds of 18 cultivars representing eight species in the subgenus Citrus have been determined.     

Fatty acids of triglycerides from Citrus juice sacs

The fatty acid composition of trigylcerides from oranges, grapefruit, lemons and limes, was determined by GLC. Each species possessed its own intrinsic fatty acid pattern which might be used to differentiate it from the other species. The five major acids in all species were palmitic, palmitoleic, oleic, linoleic and linolenic. Collectively these acids comprised greater than 92% of the total acid content. Lemons were distinguished from all other species by their higher 16/16:1 ratios while grapefruit showed the highest total percentage of 16 and 16:1 acids. Lemons and limes contained higher percentages of branched-chain acids than oranges and grapefruit.     

Isolation and characterization of limonoate and nomilinoate A-ring lactones

A method combining solid-phase extraction and reversed-phase high-performance liquid chromatography is described for the isolation of two key metabolites in the limonoid biosynthetic pathway critical to citrus quality. Potassium salts of limonoate A-ring lactone and nomilinoate A-ring lactone were isolated from young Chandler pummelo seedlings and characterized on the basis of proton and carbon NMR data.     

7α-Oxygenated limonoids from the rutaceae

Limonol, deoxylimonol, and 7α-obacunol were isolated from Citrus paradisi seeds. Limonyl acetate and 7α-obacunyl acetate were isolated from fru     

Fatty acids of monogalactosyl diglycerides from Citrus

Fatty acids in vesicular and leaf monogalactosyl diglycerides (MGDG) of citrus were studied. Vesicular MGDG contained front 94.4 to 97.3% C₁₆, C_16:1, C_18:1, C_18:2, and C_18:3; whereas leaf MGDG contained ca 90% C_18:3, 3% C₁₆ and 1.8 to 9.5% C_18:2. Species varied considerably in their percentages of vesicular C_18:2, C1_8:3 and to a lesser degree, C_16:1 and C_18:1 fatty acids with lemons being the most distinctive. Branched fatty acids were present to the extent of 5.6% in vesicular and to only 0.1% in leaf MGDG.     

Alkaloids,limonoids and furocoumarins from three Mexican Esenbeckia species

The chemical constituents of three Mexican Esenbeckia species have been determined. Rutaevin was the main limonoid present in the seeds of all three species, E. litoralis, E. flava and E. berlandieri. The husks, leaves, wood and bark contained a wide array of known furocoumarins and furoquinoline alkaloids. In addition, 1-hydroxy-3-methoxy-N-methylacridone was obtained from E. litoralis bark and a new natural 2-quinolone alkaloid, formulated as 3,3-diisopropyl-N-methyl-2,4-quinoldione, was obtained from E. flava wood. The structure was assigned from spectroscopic considerations and conversion to N-methylhaplofoline.     

Six new flavonoids from Citrus

Valencia orange (C. sinensis) and Robinson tangerine [(C. paradisi × C. reticulata) × (C. reticulata)] were examined for flavonoids. Thirteen flavonoids were isolated, six of which are new constituents of citrus peel. These are: 3,5,6,7,3′,4′-hexamethoxyflavone, 3,5,7,8,3′,4′-hexamethoxyflavone, 5-hydroxy-3,7,8,3′,4′-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 5,7,8,4′-tetramethoxyflavone and 5,7,8,3′,4′-pentamethoxyflavone. The latter three flavonoids are reported for the first time as natural products. A method is described for readily obtaining small quantities of 5,7,8,4′-tetramethoxy and 5,7,8,3′,4′-pentamethoxyflavones from their 5,6,7-trimethoxy analogs.     

In vivo Biosynthesis of isopentenylacetophenones in Eupatorium rugosum

The biosynthesis of dehydrotremetone in Eupatorium rugosum has been investigated by feeding radioactive precursors to intact plants. The carbon atoms of acetate-[1-¹⁴C] and acetate-[2-¹⁴C] were identified in dehydrotremetone by degradation of the molecule. From the pattern of labeling it was concluded that the acetophenone moiety was derived from acetate via the polyacetate pathway. From the incorporation of mevalonate it appeared that the furan ring and its side chain were formed from an isoprenoid compound. Potential aromatic intermediates were chemically synthesized and also fed to plants but only tremetone was found to be efficiently incorporated into dehydrotremetone. Neither 4-hydroxyacetophenone nor 4-hydroxy-3[isopenten-(2)-yl]-acetophenone were efficiently incorporated into dehydrotremetone.