Effect of Ginger Extract on Postprandial Glycaemia of Healthy Adults and Its Antioxidant Properties

This clinical trial was approved by Ethical Committee (process number 519 at 23 November 2016). All participants signed a written informed consent after aim and experimental risk procedures explanation and its protected confidentiality was guaranteed. The experimental procedure involving human was care out according Declaration of Helsinki.

This blind (to participants) randomized controlled clinical trial was conducted at Egas Moniz higher education school in 30 nondiabetic adults. Participants with ages between 18 and 40 were selected and randomly allocated in intervention group (IG) (n=15) and control group (CG) (n=15), in which participants were alternated include in the groups. The IG performed an oral glucose tolerance test (OGTT) followed by aqueous ginger extract administration and the CG performed an OGTT administration alone.

For ginger extract preparation, powder ginger (Zingibre officinalle Roscoe) was obtained from Portugal Company with India origin and stored in a dried environmental locally until needed. The product has a batch number of LI1GIGRNT150012. Powder ginger was individually weight (0.2g each dose) and added to 100mL of boiled water obtaining the ginger extract, infusing 10 minutes. Ginger extract solution was after cooled at room temperature and distributed to each participant. This method was adapted by Wilkinson, J. M. (2000). For chemical analysis, a aqueous ginger extract previously obtained was used.

Regarding to blood glucose level assessment, the blood sample were collected for each participant using a capillary drop blood before the intervention (fasting) and after 30, 60, 90 and 120 minutes. The blood glucose level analysis was performed using a strips for glucose meter (Onetouch Select Plus Flex), a sterilized lancet and a glucose meter equipment.

General characteristics data of the participants were collected, namely, anthropometrics data, pharmacologic treatment and medical condition using a questionnaire. In addition, a 24-hour dietary recall at the day before the intervention was employing to sample participants. The nutritional analyzed of diet ingested was performed by Food Processor SQL (version 10.5.0).

The total phenolic compounds determination of the aqueous ginger extract was determined according to Folin-Ciocalteu method. The total phenolic results were expressed as mg gallic acid equivalent (GAE)/L of ginger extract. A volume of 125 μL of ginger extract and 2 mL of sodium carbonate were added to 2.5 mL of Folin-Ciocalteu reagent. After 15 min the absorbance was measured at 765 nm. The flavonoids compounds determination of the aqueous ginger extract was determined according to Prabha method. The flavonoids results were expressed as mg quercetin equivalent (GAE)/L of ginger extract. A volume of 2 mL of ginger extract were added to 0.1 mL of aluminum chloride anhydrous solution (10%), 0.1 mL of potassium acetate (1M) and 2.8 mL of distilled water. After 30 min the absorbance was measured at 415 nm.

The antioxidant activity was measured through different assays:

The superoxide anion radicals scavenging activity was determined based on Morais et al method. Superoxide anion was generated by reacting phenazine methosulfate (PMS), nicotinamide adenine dinucleotide hydride (NADH), and oxygen causing reduced NBT in Formazan. A volume of 0.5mL of sample was added to 0.5mL of a solution containing NADH (189 microM) and NBT (120 microM) with Tris-HCl (40mM, pH = 8). The reaction started after the addition of 0.5mL of PMS (60microM). Control sample was measured using only distilled water. After 5min of incubation, control absorbance was measured at 560 nm at room temperature.

The nitric oxide inhibitory activity was determined according Khayami et al method. A volume of 1mL of sodium nitroprusside 10nm was added to 250microL de phosphate buffered saline (PBS) and 250microL of test solution and it was shaken. The previous solution was incubated for 150 min at 25ºC and following add 3mL of sulfanilic acid and 0.33% of acetic acid glacial. After 5 min at room temperature, it was add 3mL of n-(1-naphthyl)ethylenediamine dihydrochloride (NED, 0.1% m/v) and incubated for 30 min at 25ºC. The absorbance was measured at 533nm. The previous procedure was employed to control obtained using water.

The free radical 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was obtained by ABTS oxidation with potassium persulfate 140mM for 12h in dark, according Zulueta et al method. It was prepared a solution with 10mL ABTS 7nm and 176microL persulfate and storage at room temperature by 12h in dark. The previous solution was diluted with ethanol until 0.7 absorbance at 734nm. A volume of 2850microL of ABTS radical was added to 150microL of sample and to 150microL of water. After 30min in dark the absorbance was determined at 734nm. The Trolox concentration was using as standard (mM Trolox/L). This test was performed for several extract concentrations in order to calculate IC50.

Data statistical analysis was performed using SPSS Statistics (Statistical Package for Social Sciences) (version 22) software. Mean and standard error of the mean were used. Shapiro-Wilk and Repeated Measures ANOVA of mixed type were used. Independent samples T-test was used to assess the difference between the 2 groups for total caloric value, carbohydrates, protein and lipid ingested, Cmax (maximum concentration), ΔCmax (variation of maximum concentration), and AUC (area under the curve) Incremental values. The AUC was calculated by Software GraphPad Prim (version 7.03). All statistical tests were performed at the 5% level of significance.