Geoffrey W. Plumb

Properties of quercetin conjugates: Modulation of LDL oxidation and binding to human serum albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3′-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2 μM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3′-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide>quercetin>quercetin-3-glucuronide=quercetin-3-glucoside>catechin>quercetin-4′-glucuronide>isorhamnetin-3-glucuronide>quercetin-3′-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3′-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3′-sulfate>quercetin-7-glucuronide>quercetin-3-glucuronide>quercetin-4′-glucuronide=isorahmnetin-3-glucuronide). Kq values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3′-sulfate (∼4 μM)=quercetin≥quercetin-7-glucuronide>quercetin-3-glucuronide=quercetin-3-glucoside>isorhamnetin-3-glucuronide>quercetin-4′-glucuronide (∼20 μM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.

Antioxidant properties of flavonol glycosides from green beans

We have examined the antioxidant activity of one class of polyphenolic compounds in green beans: two novel flavonol glycosides (quercetin 3-O-[xylosyl(1-->2)]-rhamnosyl (1-->6)-glucoside and the corresponding kaempferol analogue), quercetin 3-O-glucuronide, kaempferol 3-O-glucuronide, quercetin 3-O-rutinoside and kaempferol 3-O-rutinoside. The Trolox equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the glucuronide and rutinoside of quercetin were good antioxidants, but not as effective as the quercetin aglycone. TEAC values for the glucuronide and other glycosides of kaempferol were much lower than the corresponding quercetin species but similar to that of the kaempferol aglycone. Quercetin 3-O-glucuronide and quercetin 3-O-rutinoside were both potent inhibitors of lipid peroxidation, in contrast to the those of kaempferol. The compounds described herein demonstrate the antioxidant activity of the flavonols present in green beans and indicate the effect on antioxidant activity of sugar substitutions in the phenolic C ring.

Development of a rapid dipstick immunoassay for the detection of peanut contamination of food

Polyclonal antisera were raised to conarachin, the 7S globulin of peanut, Arachis hypogea. The antisera were of high titre and were specific for conarachin, showing no significant cross‐reaction with proteins from a range of nuts and legumes, as determined by immunoblotting and ELISA. A dipstick ELISA was developed using these antisera as both the capture and detector elements of the assay. The final steps utilized an avidin‐biotin detection system and tetramethylbenzidine as the substrate. The dipstick assay was highly sensitive, and employed a simple one‐step extraction method. It was able to detect as little as 0.01% (w/w) of peanut in marzipan and 0.1% (w/w) of peanut in chocolate. Roasted nuts were also detected, down to a concentration of 0.1% (w/w) in both foods. The dipstick assay also functioned with a range of foodstuffs, and readily indicated any that contained peanut. This method enables analysts to test, for the first time, for the presence of peanuts in food in a fast and easy‐to‐use manner....

Antioxidant properties of flavonol glycosides from tea.

We have determined the antioxidant activity of the major flavonols found in tea: a monoglycoside, a diglycoside and two triglycosides of kaempferol and three monoglycosides, a diglycoside and two triglycosides of quercetin. The Trolox equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid peroxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the quercetin monoglycosides and diglycoside were approximately half as effective as quercetin aglycone. The quercetin triglycosides were much less effective than the monoglycosides and the diglycoside. The kaempferol glycosides were 32-39% less effective in the aqueous phase antioxidant assay compared to the kaempferol aglycone. Quercetin monoglycosides and diglycoside were potent inhibitors of lipid peroxidation, in contrast to the triglycoside which was much less effective. All the kaempferol glycosides were significantly less potent inhibitors of lipid peroxidation compared to the kaempferol aglycone. The compounds described herein demonstrate the antioxidant activity of the major flavonols in tea and indicate the effect of substituting a range of sugar moieties in the phenolic C ring.

A study of the trypsinolysis of pea 11 S globulin

Abstract The trypsinolysis of hexameric pea 11 S storage globulin (legumin) in high and low ionic strength conditions has been studied in relation to changes observed in subunit composition and quaternary/tertiary structure. Gel filtration, N-terminal analysis and SDS-PAGE revealed details on the probable method of enzyme attack. In high ionic strength a stable intermediate was formed, termed legumin-T. However, in low ionic strength, trypsinolysis proceeded further, completely degrading the tertiary/quaternary structure, with fragmentation to peptides. The site of initial enzyme attack was shown to be at the C-termini of the acidic polypeptides, eventually reducing their molecular mass to 33 000. The resulting hexameric legumin-T complex was shown by circular dichroism studies to possess a similar secondary structure of that of native legumin. The significance of 11 S globulin proteolysis to structure-function relationships in food systems and its relevance to nutritional value are discussed, together with the application of limited proteolysis for probing structural features of 11 S globulins and proteins in general.

Influence of fruit and vegetable extracts on lipid peroxidation in microsomes containing specific cytochrome P450s

Abstract We have examined the effect of 12 food extracts on iron/ascorbate-induced lipid peroxidation using microsomes enriched with specific cytochrome P450 isoenzymes, namely 1A1 and 3A4, prepared from human lymphoblastic cells. As a comparison, we also studied control microsomes, which contained negligible amounts of cytochrome P450. We observed antioxidant effects with both control and P450-containing microsomes in the case of grapefruit, green tea, coffee, tarragon and rosemary extracts. Pro-oxidant effects were observed for the brassica extracts (cabbage, cauliflower and Brussels sprouts) for all three microsome groups. Differences in the degree of lipid peroxidation between microsomes containing P450s 1 A 1 3 A 4 and control microsomes were seen for apple, tomato and parsnip peel extracts. The antioxidant properties of some of the food extracts were therefore modified by the presence of specific P450s. The results demonstrate the potential of P450-enriched microsomes in determining the antioxidant properties of food extracts and components.

Application of high-performance liquid chromatography to the assessment of subunit heterogeneity in plant 11S storage globulins

Plant 11S storage proteins from a wide range of species have been resolved into their component subunits by ion-exchange fast protein liquid chromatography under denaturing conditions. The complex profiles obtained were reproducible and characteristic for each plant species analysed. The technique is shown to have distinct advantages over the more conventional electrophoretic approaches used to assess 11S subunit heterogeneity. The potential for the fast protein liquid chromatographic method as a simple screening system is discussed.

Human cytochrome P450's are pro-oxidants in iron/ascorbate-initiated microsomal lipid peroxidation.

We have examined the effect of human cytochrome P450s (1A1,1A2,3A4,2A6,2B6,2D6,2E1) on ascorbate/iron-induced lipid peroxidation. Using microsomes prepared from human lymphoblastic cells enriched in recombinant cytochrome P450 isoenzymes, we have shown that the degree of peroxidation is a function of the amount of P450 present rather than the presence of any specific isoenzyme. Incorporated P450 increased the amount of peroxidation products by up to 2.1-fold compared to the control microsomes with no P450. It is therefore concluded that cytochrome P450s play a significant role in ascorbate/iron peroxidation.

Characterisation and crystallisation of an 11S seed storage globulin from coconut (Cocos nucifera)

Abstract Cocosin, an 11S seed storage globulin from coconut, has been purified and characterised by HPLC gel filtration and anion-exchange chromatography. At least eight different subunit species have been identified. Despite this heterogeneity, cocosin crystallises very readily into forms typical of this class of protein. The molecular weight of the protein was in the range 250 000–300 000, higher than the value of 208 000 previously ascribed. A subunit molecular weight of approximately 54 000, together with the crystal symmetry, suggests that cocosin is a typical hexameric 11S globulin, and not a tetramer as was previously postulated.

A comparison of the trypsinolysis products of nine 11S globulin species

Abstract The trypsinolysis of nine different US globulin species in high ionic strength conditions has been studied in relation to changes observed in subunit composition and quaternary/tertiary structure. HPLC gel filtration and SDS-PAGE analyses provided some information that was used to postulate the method of enzyme attack and categorize these proteins into two groups. One group of IIS globulins possessed a greatly disrupted subunit structure, the acidic polypeptides being highly degraded and the hydrolysis of the basic polypeptides being initiated. The other class of globulins, however, possessed intact basic polypeptides and only slightly cleaved acidic polypeptides (M r = 33 000) after trypsinolysis. Gel filtration analysis demonstrated that both groups of globulins still possessed much of their original structure, forming a common intermediate at M r ~ 200 000.