Michael J. C. Rhodes

Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver β-glucosidase activity

Flavonoid and isoflavonoid glycosides are common dietary phenolics which may be absorbed from the small intestine of humans. The ability of cell‐free extracts from human small intestine and liver to deglycosylate various (iso)flavonoid glycosides was investigated. Quercetin 4′‐glucoside, naringenin 7‐glucoside, apigenin 7‐glucoside, genistein 7‐glucoside and daidzein 7‐glucoside were rapidly deglycosylated by both tissue extracts, whereas quercetin 3,4′‐diglucoside, quercetin 3‐glucoside, kaempferol 3‐glucoside, quercetin 3‐rhamnoglucoside and naringenin 7‐rhamnoglucoside remained unchanged. The K m for hydrolysis of quercetin 4′‐glucoside and genistein 7‐glucoside was ∼32±12 and ∼14±3 μM in both tissues respectively. The enzymatic activity of the cell‐free extracts exhibits similar properties to the cytosolic broad‐specificity β‐glucosidase previously described in mammals.

Quercetin Glucosides Interact With the Intestinal Glucose Transport Pathway

Flavonols are efficient antioxidants with the potential to protect biological macromolecules from oxidative damage in vivo, and if absorbed into the circulation they may protect against cardiovascular disease. Although flavonol aglycones are present in foods at low concentrations, their glycosides are abundant in onions, apples, beans and tea, and are thought to be stable under the conditions of the human stomach and small bowel. There is, however, recent evidence to suggest that intact glycosides of quercetin may be absorbed from the small intestine by a mechanism involving the glucose transport pathway. In the present study we tested this hypothesis by measuring the effect of quercetin glycosides on the rate of efflux of galactose from the jejunal mucosa. Everted sacs of rat jejunum preloaded with 14C-galactose were exposed to quercetin glycosides isolated from onions. Quercetin mono- and diglucosides were shown to accelerate the carrier-mediated efflux of galactose via a sodium-dependent pathway. HPLC analysis confirmed the stability of the glycosides under conditions simulating those in the upper alimentary tract. These studies suggest that purified quercetin glucosides are capable of interacting with the sodium dependent glucose transport receptors in the mucosal epithelium and may therefore be absorbed by the small intestine in vivo.

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes.

Abstract‘Hairy root’ cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of ‘in vitro’ plant tissue culture for the production of valuable plant secondary products is identified and confirmed.

Analysis of the Major Flavonol Glycosides Present in Four Varieties of Onion (Allium cepa) and Changes in Composition Resulting from Autolysis

The major flavonoids of mature onion bulb were confirmed as the 3,4′-O-diglucoside (Qdg) and 4′-O-monoglucoside (Qmg) of quercetin using a combination of chromatographic comparisons, mass spectrometry and nuclear magnetic resonance spectroscopy. These two components account for over 85% of the total flavonoids in three varieties of onion with Qdg as the main component. Quercetin is detected in these long stored onions but only at low levels of less than 2% of the total. The remaining flavonoid fraction (approx 15%) comprises upto 17 different components of which quercetin-3-O-glucoside and isorhamnetin glucoside are prominent members although each contribute less than 1% of the total flavonoid fraction. There are significant differences in the levels of Qdg and Qmg between the four different onion varieties analysed; Qdg varying from 50–1300 mg kg-1 fresh onion tissue and Qmg from 36–394 mg kg-1. Maceration of the tissue for the three varieties tested led to a loss of Qdg and the appearance of Qmg and free quercetin. In the variety Rijnsburger 50% of the Qdg was degraded in 5 h and had completely disappeared after 24 h. These changes in Qdg can be quantitatively explained by increases in Qmg and free quercetin. The possible significance of quercetin glycosides in the diet is discussed.

Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.

Laparoscopic gastric banding for morbid obesity. Surgical outcome in 335 cases.

AbstractBackground: Morbid obesity occurs in 2–5% of the population of Europe, Australia, and the United States and is becoming more common. Open surgical techniques, such as vertical banded gastroplasty and other divisional procedures in the stomach, have led to long-term weight reduction as well as an amelioration of the attendant medical problems in approximately two-thirds of patients. Materials and methods: A total of 335 patients with a median age of 41 years underwent gastric banding. We emphasized the need for long-term maintenance and follow-up. The indications for surgery comprised a body mass index >35, a stated desire to undergo the procedure, and a full understanding of all possible complications. Results: All patients have needed band adjustments of 1–4 ml over the course of their follow-up. No patient had increased his or her weight during the follow-up, and only three patients have not enjoyed sustained weight loss. Conclusions: Laparoscopic gastric banding has much to recommend it. Certainly in the short term, its results in terms of effectiveness of weight loss are at least as good as those of any open procedure. Longer follow-up will show whether this weight loss is maintainable. The procedure is technically demanding, and the major prerequisite of satisfactory performance of this surgery is laparoscopic experience.

The use of the polymerase chain reaction in plant transformation studies.

SummaryTransformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR.For tobacco species the PCR reaction worked efficiently with up to 2 μg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.

Composition and content of flavonol glycosides in broccoli florets (Brassica olearacea) and their fate during cooking

The two main flavonol glycosides present in broccoli florets were identified as quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside. Three minor glucosides of quercetin and kaempferol were also detected, namely isoquercitrin, kaempferol 3-O-glucoside and a kaempferol diglucoside. The sophorosides of quercetin and kaempferol were present in raw florets at a level of 65 mg kg−1 and 166 mg kg−1 fresh weight, respectively. The total content of quercetin and kaempferol glycosides expressed as aglycone was 43 and 94 μg g−1 fresh weight, respectively, and these agree with other recently published data. During the cooking process only 14–28% of the individual glucosides were retained in the cooked tissue, the remainder being largely leached into the cooking water with only a small loss attributed to the formation of the respective aglycones.

Induction of the anticarcinogenic marker enzyme, quinone reductase, in murine hepatoma cells in vitro by flavonoids

Some flavonoids induce phase II enzymes both in vivo and in vitro. We have determined the structural requirements for this activity by examining the ability of naturally-occurring flavonoids to induce the phase II enzyme, quinone reductase (NAD(P)H:quinone oxidoreductase; EC 1.6.99.2), in murine Hepalclc7 cells. Hydroxylation of the B ring is not essential for induction, since galangin and kaempferol (with 0 and 1 hydroxyl in the B ring, respectively) are better inducers than quercetin (2 B ring hydroxyls). A 2,3 double bond in the C ring is essential for induction, since taxifolin, which has the same substitution pattern as quercetin but lacks the 2,3 double bond, is not an inducer. This is supported by catechin and epicatechin, which do not possess the 2,3 double bond and are also not inducers. A 3-hydroxyl group increases the activity but is not essential for induction, since apigenin is an inducer but kaempferol (which has the same structure as apigenin but possesses a 3-hydroxyl group) is more effective. The data show that, of the flavonoids, the flavonols are the most effective inducers of quinone reductase activity in Hepa1c1c7 cells (kaempferol approximately galangin > quercetin > myricetin approximately apigenin (a flavone)) and that flavanols and flavans are ineffective.

Analytical problems in the study of flavonoid compounds in onions

Flavonoids, although potentially mutagenic, are widely thought to have beneficial effects in the diet resulting from their antioxidant and metal binding properties. Epidemiological evidence correlates diets rich in flavonoid compounds with a low risk of coronary heart disease. In the present work flavonoids (principally flavonols and anthocyanins) are studied in the onion, a major dietary source of flavonoids. This work concentrates on the development of methods to study flavonoids in their natural form in plant foods as conjugates. Two major components quercetin monoglucoside and quercetin diglucoside account for 80% of the total flavonoids in onions. Anthocyanins are only minor components of the flavonoid spectrum in the edible portion of red varieties. A preliminary study of flavonols in onions, which had been finely chopped, suggests that in most varieties there is only a small loss in total flavonol but that there is a progressive loss of the diglucoside component with an accompanying quantitative accumulation of the monoglucoside and the aglycone.