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Title: Influence of lactase on the in vitro and in vivo antiglycaemic effects of onion flavonoids
Author: Ranjbar, Golnaz
ISNI:       0000 0004 6422 461X
Awarding Body: University of Hertfordshire
Current Institution: University of Hertfordshire
Date of Award: 2017
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Introduction: Lactase, in addition to its role in the digestion of lactose available in milk and dairy products, is implicated in the metabolism of a range of phenolic phytochemicals in the gut. Experiments with Caco-2 cells have shown that these cells which mimic the intestinal mucosa indicate that quercetin glucosides and quercetin aglycone (widely consumed in onions and apples) block glucose uptake from the gut by competing with glucose for the sodium-dependent SGLT-1 and sodium-independent GLUT-2 transporters respectively (Johnston et al., 2005a, Schulze et al., 2015). It has been suggested that dietary phenolics that block glucose uptake from the gut may reduce the risk of type 2 diabetes. However, the ability of quercetin glucosides to block SGLT-1 is lost or reduced when the glucoside moiety is cleaved off during lactase hydrolysis. It is currently unknown if lactose-tolerant individuals deglycosylate quercetin to a greater extent than lactose-intolerant individuals and therefore are less able to reduce glucose uptake from the intestine. The aim of in vitro study was to model human gut condition for glucose transport by using Caco-2 cell models and to model role of human intestinal LPH by incubation of Caco-2 cells with quercetin flavonoids and purified β-galactosidases and in vivo was to investigate whether lactose-tolerant and lactose-intolerant subjects show differences in the uptake of glucose. Methods: Caco-2 cells were cultured in DMEM full medium in 24 well plates. Thereafter, glucose uptake assay was conducted by using 3H-glucose in the presence and absence of sodium, to assess the effect of flavonoids such as phloridzin, quercetin 4'-glucoside, quercetin 3,4'-diglucoside, quercetin 3'-glucoside, and quercetin aglycone on glucose uptake. Transwell inserts were also used to demonstrate the bidirectional permeability through Caco-2 monolayers, transport of glucose from apical (SGLT-1) to basolateral side (GLUT-2). β-galactosidase enzyme assay was conducted by using β-galactosidase from Aspergillus oryzae, Caco-2 cells were treated with 100 μM quercetin glucosides, 25% w/v onion extract and β-galactosidase in order to model the hydrolysis of flavonoids by lactase in the small intestine. HPLC was carried out to determine if quercetin glucosides are found in onion extract and test whether β-galactosidase is active and result in deglycosylation of substrates such as individual quercetin glucosides and quercetin glucosides in onion extract. For the clinical study, lactose intolerance was identified by the hydrogen breath test (Gastrolyzer), and blood glucose levels were measured by taking finger-prick blood samples in several intervals (0, 15, 30, 60, 90, 120) minutes using an EKF glucose analyser. Results: Findings from the current in vitro research confirm that phloridzin is an inhibitor of sodium-dependent conditions (SGLT-1) transporter with 80% reduction, this therefore was used as a positive control. Quercetin 4'-glucoside and quercetin 3,4'-diglucoside at (100πM) significantly decreased the uptake of glucose in the presence of sodium with up to 75% reduction compared to control p < 0.01. However, no significant glucose inhibition was found from these quercetin glucosides in the absence of sodium condition (p > 0.5), whilst quercetin aglycone significantly inhibited the glucose uptake with 50% reduction compared to control at significance levels of (p = 0.02). HPLC data identified quercetin 3,4'-diglucoside and quercetin 4'-glucoside with RT = 4.082 min and 11.392 min in the onion extract by showing peaks at similar ranges with RT= 4.114 min and 11.385 min with their standards, and the concentration of quercetin 4'-glucoside was measured as the highest level (42μg/ml) in onion extract compared to 3,4'-glucoside and quercetin 3-glucoside. Further HPLC illustrated that, after incubation of quercetin glucosides and onion extract with β-galactosidase Aspergillus oryzae for 20, 40 and 60 minutes, the peaks occurred at similar RT =16.453 min and 16.441 min respectively in accordance with standard quercetin (RT=16.239 min), suggesting the deglycosylation of these compounds with β-galactosidase from Aspergillus oryzae. According to findings from the clinical study, reduction of peak glucose levels by an onion meal was higher in lactose-intolerant people than lactose-tolerant people (44.2% versus 19.3%, p = 0.042). Also, the area under the blood glucose curve was reduced more in lactose-intolerant people compared to lactose-tolerant people, however was not statistically significant (54.5% versus 42.1%, p = 0.425). Discussion: Our result suggests that quercetin 4'-glucoside and quercetin 3,4'-diglucosides and onion extract were the main inhibitors of glucose uptake in sodium-dependent conditions (SGLT-1). Whereas, quercetin aglycone inhibited GLUT-2 glucose transport on Caco-2 cell monolayers under sodium independent conditions. Our findings were in accordance to several previous studies (Boyer et al., 2005, Kwon et al., 2007, Schulze et al., 2015). Notably, in vitro studies were conducted to model whether the in vivo study is likely to succeed or not. Findings from our human study showed that glucose uptake was blocked by the onion solution and a diet containing quercetin glucosides (onion meal) may be of greater benefit for glycaemic control in lactose-intolerant people than in lactose-tolerant people.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Flavonoid ; quercetin ; glucose ; lactase