Stopping the progression towards type 2 diabetes mellitus : investigating the hypoglycaemic (glucose-lowering) potential of antioxidant-rich plant extracts : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science, Massey University, Albany, New Zealand
Background: Prediabetes is a condition where the blood glucose levels are high but not high enough to be classified as having type 2 diabetes mellitus (T2DM). It is also considered a high risk for developing T2DM. There is increasing evidence that demonstrates antioxidant-rich plant extracts exhibiting hypoglycaemic effects in humans. Therefore the extracts may improve glycaemic control in individuals with prediabetes and help prevent or delay the progression of prediabetes towards T2DM.
Overall Aim: To examine the acute hypoglycaemic potential of four antioxidant-rich plant extracts, namely the New Zealand pine bark, grape seed, rooibos tea and olive leaf extracts in humans.
Methods/Design: The hypoglycaemic effects of the New Zealand pine bark was examined in healthy participants (n=25) in an acute, placebo-controlled, single-blind, crossover, dose-response (50 and 400 mg), exploratory study (Pine Bark study). Blood samples were collected via finger pricking using disposable lancet to measure glucose levels at -20, 0, 15, 30, 45, 60, 90 and 120 min during an oral glucose tolerance test (OGTT) with 75 g of glucose. The hypoglycaemic effects of grape seed, rooibos tea and olive leaf extracts matched for antioxidant capacity were examined in an acute, placebo-controlled, crossover study (GLARE study) in participants with prediabetes (n=19). Blood samples were collected via cannulating the antecubital fossa region of the arm at -10, 0, 15, 30, 45, 60, 90 and 120 min during the OGTT with 75 g of glucose. Outcome glycaemic measures were analysed in both clinical studies (Pine Bark study and GLARE study). An in vitro mechanistic study investigating the potential inhibitory action of all four plant extracts (grape seed, rooibos tea, olive leaf and New Zealand pine bark) on digestive enzyme α-amylase and the dipeptidyl peptidase-4 (DPP4) enzyme were carried out using appropriate enzymatic assays of inhibition.
Results: Prior to secondary analysis in the Pine Bark study, a significant reduction in the primary outcome mean glucose incremental area under the curve (iAUC) was only observed for the 400 mg dose of pine bark (21.3% reduction, p=0.016) compared to control. After stratification in the monophasic glucose curve shape group (n=12), 50 and 400 mg of pine bark significantly reduced the mean glucose iAUC compared to control (28.1% reduction, p=0.034 and 29.5% reduction, p=0.012), respectively. In contrast, mean glucose iAUC was not significantly different in the complex glucose curve shape group (n=13). In the monophasic group, 400 mg dose further improved glycaemic indices by reducing mean percentage increment of postprandial glucose (%PG) (33.9% reduction, p=0.010), mean glucose peak (11.2% reduction, p=0.025), and mean 2h postprandial glucose (2hPG) (8.9% reduction, p=0.027) compared to control. Within the complex group, there were no other significant changes except for reductions in mean %PG after 50 mg and 400 mg dose (33.8% reduction, p=0.012 and 41.4% reduction, p=0.025) compared to control, respectively. There were no significant differences between treatments in both subgroups (p>0.05).
In the GLARE study, there were no overall significant changes in glucose and insulin responses between the extracts and control, or amongst the plant extracts (p>0.05). After secondary analysis, the less healthy subgroup (n=9), grape seed consumption showed significant reduction in mean glucose iAUC (21.9% reduction, p=0.016), mean 2hPG (14.7% reduction, p=0.034) and mean 2h postprandial insulin (2hPI) (22.4% reduction, p=0.029), whilst there was significant improvement in mean overall insulin sensitivity (ISIoverall) (15.0% increase, p=0.028) and mean glucose metabolic clearance rate (MCR) (16.7% increase, p=0.016) compared to control. Rooibos tea extract was shown to improve β-cell function measured by the mean oral disposition index (DI) (32.4% increase, p=0.031) in the less healthy subgroup compared to control. This was coupled with a non-significant improvement in insulin sensitivity measured by mean insulin-secretion-sensitivity-index-2 (ISSI-2) (18.3% increase, p=0.074). Olive leaf exhibited improved mean insulin sensitivity indices of insulinogenic index (IGI₃₀) (27.8% increase, p=0.078), Stumvoll first phase insulin sensitivity (ISIfirst) (17.8% increase, p=0.075) and Stumvoll second phase insulin sensitivity (ISIsecond) (15.6% increase, p=0.062) in the less healthy subgroup compared to control, although significance was not reached. Olive leaf extract was also consistently shown to elevate insulin levels in the study, with a higher mean 2hPI in the healthier subgroup (49.5% increase, p=0.030) and an elevated mean insulin iAUC in the less healthy (16.7% increase, p=0.040) subgroups. There were no significant changes in glucose and insulin responses in the healthier subgroup (n=10) compared to control nor between treatments in both subgroups (p>0.05).
The mechanistic study demonstrated that the New Zealand pine bark extract exhibited the greatest inhibitory effects against digestive enzyme α-amylase (IC₅₀ 3.98 ± 0.11 mg/mL) and DPP4 enzyme (IC₅₀ 2.51 ± 0.04 mg/mL) compared to the other extracts (p<0.001). Both grape seed and rooibos tea extracts showed good inhibition of both enzymes tested. Rooibos tea was able to inhibit DPP4 enzyme to a greater extent than grape seed (p=0.018). In contrast, olive leaf extract showed minimal inhibition on α-amylase and no inhibition action against DPP4 enzyme.
Conclusions: All four plant extracts (New Zealand pine bark, grape seed, rooibos tea and olive leaf) have shown acute hypoglycaemic potential in the Pine Bark study and the GLARE study by improving various indices of glucose and insulin responses in humans. The inhibitory action of the New Zealand pine bark, grape seed and rooibos tea extracts on DPP4 enzyme might have contributed to the hypoglycaemic effects observed in the clinical studies conducted. Whereas for olive leaf extract other underlying mechanisms on glycaemia remain to be elucidated. Our acute studies have indicated the need to investigate the chronic impact of these plant extracts in longer-term studies. Future studies in the prediabetes cohort should also look to target different metabolic profiles of varying degrees of dysglycaemia, as this may provide more meaningful results.
Chapter 4 was published under a Creative Commons Attribution 4.0 license (CC BY 4.0) as: Lim, W X J, Chepulis, L, von Hurst, P, Gammon, C S, & Page, R A. (2020). An acute, placebo-controlled, single-blind, crossover, dose-response, exploratory study to assess the effects of New Zealand pine bark extract (Enzogenol ®) on glycaemic responses in healthy participants. Nutrients,12(2):497. https://doi.org/10.3390/nu12020497