Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

A sensitive method using capillary electrophoresis for the separation, detection, and quantification of dihydrokaempferol (1) is reported. Well-resolved, sharp symmetrical peaks were obtained in grapefruit leaf extracts for 1, naringenin (2), and the internal standard, naringin (3). Long columns were required to resolve 1 from 2 in crude enzyme reactions and this resulted in run times of 60 min. The limit of detection for 1 was found to be 1.44 ng/μL (4.2 pg). The method showed excellent linearity and reproducibility. The method was used to determine the activity of flavanone 3-hydroxytransferase (F3H) in leaf tissue of grapefruit by quantification of the production of dihydrokaempferol in controlled time course reactions. The sensitivity of the method makes it adaptable to assaying F3H activity in individual young seedlings and/ or in small tissue samples and requires only 100 mg of tissue.

Limonin and naringin are the two major bitter compounds in Citrus paradisi (grapefruit), and tissue-specific patterns on their distribution are well-established. This study was undertaken to determine the distribution of these compounds within Duncan, Marsh, and Thompson Pink tissues using three-dimensional fruit dissection (600-900 samples per fruit) and highly specific radioimmunoassays for limonin and naringin quantification. Results from a GLM ANOVA showed that there was no radial distribution pattern of limonin or naringin accumulation in these fruit. There were statistically significant differences in the axial distribution of these compounds within the fruit tissues. The limonin concentration in flavedo, albedo, outer segment memberane, and juice vesicles increased toward the distal end of the fruit. Naringin concentrations in flavedo, albedo, and outer segment membranes tended to be higher in the center portion of the fruit. Limonoate A-ring monolactone levels are also reported.

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The health maintaining properties of citrus consumption are attributed to the wide assortment of bioactive compounds. Consumption of grapefruit along with certain medications, however, is posing a risk of drug toxicity and side reactions. The first study involved isolation of bioactive furocoumarins with a combination of chromatographic techniques and synthesis. Five furocoumarins namely, dihydroxybergamottin, paradisin A, bergamottin, bergaptol and geranylcoumarin were isolated from grapefruit and series of furocoumarin monomers and paradisin A were synthesized. The second study involved influence of pre- and post-harvest factors on the levels of furocoumarins in grapefruit juice. Considerable differences were observed in the levels of these compounds in different grapefruit cultivars. Ray Red showed the lowest levels of all three furocoumarins and Duncan contains the highest amount of DHB and bergamottin, where as the highest levels of paradisin A was observed in Star Ruby. The highest levels of DHB and bergamottin were found in Flame cultivar grown in California. The changes in the levels of these furocoumarins during the season in Rio Red and Marsh White grapefruit cultivars were evaluated. The third study investigated biological activities of grapefruit juices and furocoumarins. Grapefruit and Pummelo juices were found to be potent inhibitors of cytochrome CYP3A4 and CYP2C9 isoenzymes at 5% concentration while CYP2D6 was less affected. Among the five furocoumarins tested, the inhibitory potency was in the order of paradisin A>dihydroxybergamottin>bergamottin>bergaptol>geranylcoumarin at 0.1 µM to 0.1 mM concentrations. A fourth study investigated the effect of furocoumarins on bacterial auto-inducer signaling, and found that furocoumarins are potent inhibitors of AI-1 and AI-2 activities at 0.01% concentration. In a fifth study, involving synthesized furocoumarin monomers and dimer on anti-proliferative activities on normal and cancer cell lines, furocoumarins found to be non-toxic to normal cells. However, bergamottin showed a significant anti-proliferative activity in HT-29 and MCF-7 cell lines. This dissertation indicates that furocoumarins are bioactive compounds from grapefruit juice with potent inhibitory property of major drug metabolizing cytochrome P450 isoenzymes. Furocoumarins show a considerable variation between varieties, location and season. These results corroborate the involvement of furocoumarins in grapefruit drug interaction.

This dissertation examines the effects of postharvest treatment and processing on biologically active compounds of orange juice, and ??Rio Red?? grapefruit and their ability to prevent chemically induced colon cancer in rat model. The first study evaluated the differences in flavonoid content of commercial ??made from concentrate?? (MFC) orange juices and ??not from concentrate?? (NFC) orange and grapefruit juices. Total flavonoid content of MFC orange juices (53 mg/100 mL; n = 12) was significantly (P ≤ 0.05) higher than NFC orange juices (36.5 mg/100 mL; n = 14). The second study investigated the ionizing radiation and storage effects on bioactive compounds and quality of ??Rio Red?? grapefruit. Results showed that storage and irradiation significantly (P ≤ 0.05) affected the bioactive compounds in grapefruit, however, the effect of storage was prominent. The third study examined the influence of irradiation and freeze drying on bioactive compounds of grapefruit. Irradiation of grapefruit prior to freeze drying resulted in enhanced (P ≤ 0.05) flavonoid content (naringin and narirutin). Freeze drying markedly reduced (P ≤ 0.05) lycopene content. Freeze drying and irradiation reduced (P ≤ 0.05) volatile compounds (d-limonene and myrcene), with the exception of ethanol. In the fourth study suppression of colon cancer development in Sprague Dawley rats by natural and irradiated grapefruits and their functional compounds, naringin and limonin, were evaluated.The total number of aberrant crypts (AC; P = 0.02), number of high multiplicity AC foci (ACF; P = 0.01), and proliferative index (P = 0.02) were lower and apoptosis (P = 0.02) was higher in azoxymethane (AOM) injected rats on experimental diets. However, only natural grapefruit and limonin only suppressed AOM induced expansion (P = 0.008) of proliferative zone and also enhanced apoptosis more effectively than other experimental diets indicating that natural grapefruit and limonin may serve as better chemopreventive agents compared to IGFPP and naringin. The present study indicates that postharvest quarantine doses of irradiation slightly alter composition of bioactive compounds and in turn marginally reduce the chemopreventive ability of grapefruit against the promotion stage of colon cancer. These results warrant the necessity of testing the impact of post harvest treatments on fruits and vegetables chemopreventive ability.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (Kmapp = 67 μ M; Vmax = 20.45 pKat/μg), kaempferol (Kmapp = 12 μ M; Vmax = 11.63 pKat/μg), and myricetin (Kmapp = 33 μ M; Vmax = 12.21 pKat/μg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu2+, Fe2+, and Zn2+ as well as UDP (Kiapp = 69.5 μ M), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4o C was optimal for removing tags from CP3GT. Storage at 4o C for 2 hours resulted in approximately 70% retention of activity. The effect of thrombin treatment on CP3GT activity was tested. Purified CP3GT protein with and without tags was tested for activity with the flavonol quercetin. Data showed no significant difference in overall activity between tagged and native protein.