title: Assessment of citrus bioflavonoid and phytosterol supplements for dipeptidyl peptidase-4 inhibitory activity
Creators: name: Gupta, A
Publisher: name: University of Tasmania
Year: 2020

description: Background Diabetes is a chronic condition characterised by abnormal hyperglycaemic activity with type 2 diabetes accounting for 90% of diabetes cases. Prediabetes is a condition in which blood sugar levels are higher than normal, but do not fall in the range of diabetes. People with prediabetes suffer from either impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) and are at high risk of developing diabetes or cardiovascular related diseases. There is an increasing prevalence of type 2 diabetes worldwide which is causing significant morbidity, with statistics obtained from the World Health Organisation showing that about 422 million people worldwide have diabetes, particularly in low-and middle-income countries, and 1.6 million deaths per annum directly attributed to diabetes each year. The key gastrointestinal incretin hormones are known as glucose insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) stimulate insulin secretion from pancreatic b-cells. Dipeptidyl peptidase-4 (DPP-4) enzyme is responsible for splices in the N-terminal of GIP and GLP-1 resulting in inactivation of these enzymes. This results in decreased insulin secretion. Inhibition of DPP-4 allows the incretins to stimulate insulin secretion for longer and can result in lowering of the blood glucose level in patients with type 2 diabetes. This is the mechanism of the class of drugs known as gliptins. Complementary and alternative medicine-based therapies are gaining interest for a number of chronic diseases such as diabetes, however, there is a limited evidence base for efficacy. In fact, products listed with the medicines regulatory body in Australia (Therapeutic Goods Administration) are not allowed to be promoted for the treatment of diseases such as diabetes. There has been limited work performed on DPP-4 inhibition utilising naturally derived products. The potential benefit of such nutraceuticals for glycaemic control warrants further investigation and may provide data to support naturally derived DPP-4 inhibitors for anti-diabetic activity. Objective The overall objective of this study was to assess the potential for naturally-derived supplements (citrus bioflavonoids and phytosterols) to improve glycaemic control through the DPP-4 inhibitory pathway. Firstly, DPP-4 inhibitory activities of individual citrus bioflavonoids and phytosterols were assessed through in silico and in vitro techniques. The in vitro DPP-4 inhibitory activity of commercial citrus bioflavonoid and phytosterol supplements was then assessed. Ten commercially available citrus bioflavonoid supplements from different international markets were chemically characterised, with this data used to determine the DPP-4 inhibitory activity. A small scale pharmacokinetic pilot study was developed to determine the bioavailability from a single dose of a commercial citrus bioflavonoid supplement. A randomised placebo-controlled double-blinded study involving 50 patients with type 2 diabetes was developed to determine pharmacodynamics of a commercial citrus bioflavonoid supplement with respect to glycaemic control. Methods The Docking Server (https://www.dockingserver.com/web) online tool was used to perform in silico molecular docking studies. A commercial DPP-4 inhibitory assay kit was used for in vitro studies, with results compared to drugs from the gliptin drug class (sitagliptin, saxagliptin, vildagliptin). The citrus bioflavonoid supplements and standards (rutin, naringin, naringenin, eriocitrin, eriodictyol, hesperidin and hesperetin) were tested individually in this study for DPP-4 inhibitory activity. The three supplements used in the study were Thompson’s Super Bioflavonoid Complex®(SB); Ethical Nutrients Bioflavonoids Plus Vitamin C®(EN); and Citrus Bioflavonoids and Rutin®(CB). The DPP-4 inhibitory activity of phytosterol supplements and standards (stigmasterol, bsitosterol and campesterol) was tested. The three supplements used in the study were Thompson’s Cholesterol Manager® (CM); Blackmores Cholesterol Health® (CH); and Mega Strength Beta Sitosterol® (BS). The chemical constituents of the citrus bioflavonoid supplements were investigated by UPLCMS/MS (ultra-performance liquid chromatography-tandem mass spectrometry) using citrus bioflavonoids standards of rutin, naringin, naringenin, eriocitrin, eriodictyol, hesperidin and hesperetin. The constituents of the phytosterol supplements were investigated by GC-MS/MS (gas chromatography-tandem mass spectrometry) using phytosterol standards stigmasterol, β-sitosterol and campesterol. Convenience sampling was used to select 10 different citrus bioflavonoid supplements, labelled CBF-1 to CBF-10, from products available over-the-counter or online in Australia. Supplements were manufactured in Australia (AUS), the United States of America (USA) and Canada (CAN) and each had label claims regarding constituents. A protocol was developed to assess the pharmacokinetics of the citrus bioflavonoid supplement Thompson's Super Bioflavonoid Complex® in participants (n=6) through blood and urine samples at time points 0, 1, 2, 4, 8 hours with analysis for flavonoids and flavonoid catabolites by LC-MS/MS. Results In silico Molecular Docking The free binding energy (FBD) is a measure of the binding affinity of a ligand to a target protein. The FBD is presented in kcal/mol and the more negative the value the stronger the binding. The inhibition constant (IC\(_{50}\)) is the concentration of ligand at which enzyme activity is reduced by 50%. The IC50 can be predicted from the FBD and can also be determined experimentally by in vitro testing. The maximum in silico activity for citrus bioflavonoids was shown by rutin with FBD -9.08 kcal/mol and IC50 (in silico) 0.220 µM followed by naringin, hesperidin, naringenin, eriocitrin, hesperetin and eriodictyol. In the case of phytosterols, greatest in silico activity was shown by stigmasterol with FBD -8.78 kcal/mol and IC\(_{50}\) (in silico) 0.152 µg/mlfollowed by β-sitosterol and campesterol. The in silico activity of gliptins for comparison showed the maximum activity by sitagliptin FBD -10.33 kcal/mol and IC\(_{50}\) (in silico) 0.027 µM followed by saxagliptin and vildagliptin. In vitro DPP-4 inhibition In vitro testing of DPP-4 inhibition was performed for the citrus bioflavonoids and three commercially-available supplements. Rutin demonstrated the greatest inhibitory activity with IC\(_{50}\) 296 µg/ml, followed by naringenin 1500 µg/ml, eriodictyol 1640 µg/ml, hesperetin 1720 µg/ml, eriocitrin 2280 µg/ml, hesperidin 2390 µg/ml, and naringin 3160 µg/ml. The maximum tested concentration of the supplements was 100 mg/ml. The greatest in vitro DPP-4 inhibition was shown by CB followed by SB and EN with IC\(_{50}\) values of 2.7 mg/ml, 3.4 mg/ml and 16.9 mg/ml respectively. The total flavonoid content for the supplements CB, SB and EN were 12.0 %w/w, 14.5 %w/w and 5.3 %w/w respectively. For the phytosterols, no in vitro inhibition of DPP-4 activity was exhibited by stigmasterol or β-sitosterol at any tested concentration up to 50 mg/ml. Bioflavonoid chemical characterisation UPLC-MS/MS analytical testing performed on 10 commercially available citrus supplements showed a wide range of flavonoid content on a %w/w basis. The lowest content was shown by CBF-6 with only 0.8% followed by CBF-2 with 1.9%. The highest content was for CBF-2 with 33.3 %w/w followed by CBF-7 with 32.1 %w/w. The main bioflavonoid in 9 of 10 supplements was hesperidin with CBF-4, CBF-7 and CBF-8 also having high rutin content. The potential DPP4 inhibitory activity was also calculated based on their rutin equivalence. The two highest rutin equivalence activities were shown by CBF-7 and CBF-4 with 400 mg and 222 mg respectively. The two lowest rutin equivalence activities were shown by CBF-2 and CBF-6 with 11.2 mg and 1.9 mg respectively. Planned Clinical Studies The Human Research Ethics Committee of the University of Tasmania approved the randomised placebo-controlled double-blinded trial, incorporating a pilot PK study (approval number H001572). Analytical methods were developed for the citrus bioflavonoids and their respective catabolites from plasma and urine for the pharmacokinetic phase. The restrictions implemented due to the COVID-19 pandemic prevented the completion of the study in the available timeframe. Conclusions The in silico molecular docking results of phytosterols looked promising and showed DPP-4 inhibitory potential, however, the in vitro DPP-4 inhibitory assay demonstrated that phytosterols do not possess any useful DPP-4 inhibitory activity. The citrus bioflavonoids in silico molecular docking results also demonstrated potential DPP-4 inhibitory activity, and the in vitro DPP-4 inhibitory assay demonstrated some inhibition but with lower efficacy and potency than predicted in silico. The citrus bioflavonoid which showed the highest DPP-4 inhibition was rutin. Furthermore, the supplements containing phytosterols showed no DPP4 inhibitory activity and the citrus bioflavonoid supplements showed some modest DPP-4 inhibitory activity. The utility of in silico docking studies to predict the efficacy and potency of natural products must also be questioned. The chemical composition of the supplements was highly variable and the concentration of flavonoids within the supplements was found to be low compared with the label claim for several products. There are some quality assurance concerns related to flavonoid composition and content in the supplements, particularly for products manufactured outside of Australia. Citrus bioflavonoid supplements, but not phytosterol supplements have the potential to improve glycaemic control through the DPP-4 inhibitory pathway, but the effect is likely to be modest. A study to determine the pharmacokinetics and pharmacodynamics clinical effects for glycaemic control of citrus bioflavonoids was developed and approved.