A Pilot Screening of Agro-Food Waste Products as Sources of Nutraceutical Formulations to Improve Simulated Postprandial Glycaemia and Insulinaemia in Healthy Subjects

Abstract

The control of glucose homeostasis is the main goal for both the prevention and management of diabetes and pre-diabetes. Numerous drugs are available, despite their side effects. This is constantly leading people to be inclined to natural alternative treatments. Evidence indicates antioxidant-based nutraceuticals as an optimal tool for the glycaemic control. Currently, a great interest has been focused on the valorisation of agro-food by-products as sources of bioactive compounds including polyphenols. In this sense, we tested the efficacy of novel nutraceutical products based on polyphenolic extract from nectarines (NecP), tomato peels (TP), and olive leaves (EOL) on glycaemic and insulinemic responses. The three formulations contained, respectively, 0.007 mg abscisic acid (ABA)/g, 0.5 mg carotenoids/g, and 150 mg oleuropein/g. Twenty healthy subjects consumed a regular glucose solution (RG) or a treatment beverage (TB) obtained by mixing RG with the individual formulations (TB NecP, TB EOL, and TB TP), separately, and on different days. All three formulations significantly lowered the 30 min glucose plasma peak (p < 0.05 for all); similarly, NecP and TP also significantly lowered the 30 min insulin plasma peak (p < 0.05 for all). These results may lead to the hypothesis of a formulation of a multi-component nutraceutical with a synergistic efficacy for the glycaemic control.

Full text

nutrients
Article
A Pilot Screening of Agro-Food Waste Products as
Sources of Nutraceutical Formulations to Improve
Simulated Postprandial Glycaemia and Insulinaemia
in Healthy Subjects
Gian Carlo Tenore 1, Domenico Caruso 2, Maria D’Avino 2, Giuseppe Buonomo 3,
Giuseppe Caruso 4, Roberto Ciampaglia 1, Elisabetta Schiano 1, Maria Maisto 1,
Giuseppe Annunziata 1, * and Ettore Novellino 1
1
Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Naples, Italy;
giancarlo.tenore@unina.it (G.C.T.); roberto.ciampaglia@unina.it (R.C.); elisabetta.schiano@gmail.com (E.S.);
maria.maisto@unina.it (M.M.); ettore.novellino@unina.it (E.N.)
2Department of Internal Medicine, Hospital Cardarelli, Via Antonio Cardarelli, 80131 Naples, Italy;
dr.domenicocaruso@gmail.com (D.C.); dott.mariadavino@gmail.com (M.D.)
3Coop. Samnium Medica, Viale C. Colombo 18, 82037 Benevento, Italy; giuseppebuonomo@tin.it
4Department of Emergency, Hospital Cardarelli, Via Antonio Cardarelli, 80131 Naples, Italy;
giuseppe.caruso@gmail.com
*Correspondence: giuseppe.annunziata@unina.it; Tel.: +39-340-001-6504
Received: 26 March 2020; Accepted: 26 April 2020; Published: 1 May 2020


Abstract:
The control of glucose homeostasis is the main goal for both the prevention and management
of diabetes and pre-diabetes. Numerous drugs are available, despite their side effects. This is constantly
leading people to be inclined to natural alternative treatments. Evidence indicates antioxidant-based
nutraceuticals as an optimal tool for the glycaemic control. Currently, a great interest has been focused
on the valorisation of agro-food by-products as sources of bioactive compounds including polyphenols.
In this sense, we tested the efficacy of novel nutraceutical products based on polyphenolic extract from
nectarines (NecP), tomato peels (TP), and olive leaves (EOL) on glycaemic and insulinemic responses.
The three formulations contained, respectively, 0.007 mg abscisic acid (ABA)/g, 0.5 mg carotenoids/g,
and 150 mg oleuropein/g. Twenty healthy subjects consumed a regular glucose solution (RG) or a
treatment beverage (TB) obtained by mixing RG with the individual formulations (TB NecP, TB EOL,
and TB TP), separately, and on different days. All three formulations significantly lowered the 30 min
glucose plasma peak (p<0.05 for all); similarly, NecP and TP also significantly lowered the 30 min
insulin plasma peak (p<0.05 for all). These results may lead to the hypothesis of a formulation of a
multi-component nutraceutical with a synergistic efficacy for the glycaemic control.
Keywords: glucose homeostasis; OGTT; abscisic acid; carotenoids; oleuropein; nutraceutical
1. Introduction
A wide range of natural substances of plant origin, specifically, polyphenols, carotenoids,
and terpenoids [
1
–
5
] have been demonstrated to be active on glycaemia in humans. To this regard,
agro-food waste products are increasingly attracting a great interest from the nutraceutical industry,
since they represent still rich sources of bioactive compounds which can be conveniently recovered for
the formulation of food supplements indicated for the control of glycaemia.
Fruit thinning is a widespread agronomical practice that involves removing excess unripe fruits,
measuring 1–2 centimetres in diameter, to produce better-sized, ripe, and healthy fruits, albeit in
Nutrients 2020,12, 1292; doi:10.3390/nu12051292 www.mdpi.com/journal/nutrients

Nutrients 2020,12, 1292 2 of 12
smaller numbers. It is generally applied to a specific range of tree fruits, including apples, pears,
plums, peaches, and nectarines, and consists of leaving a minimum of one fruit every 5–8 cm (plums
and apricots) to a maximum of one fruit every 10–15 cm (apples and pears) and 20–25 cm (peaches
and nectarines) on tree branches [
6
]. Since this practice may interest up to 40% of the entire tree fruit
load, fruit thinning may lead to a massive agricultural waste product which is generally destined
to fertilising or feeding. Interestingly, these waste fruits are supposed to be a significant source of
abscisic acid (ABA). This phytohormone is majorly responsible for the regulation of plant growth and
differentiation [
7
–
9
]. Specifically, the influence of ABA on fruit ripening has been well documented,
although its mechanism of action is still unclear. Studies have revealed that there is a progressive
accumulation of ABA during fruit ripening, reaching its maximum concentration at a specific stage
after full bloom and then decreasing to its minimum level at the fruit fully ripe/harvest stage [
10
–
14
].
Abscisic acid is known as a suppressive of plant growth regulator, inducing expression of cell cycle
inhibitors effective on DNA [
15
] and protein synthesis [
16
] and thereby arresting cell divisions [
17
] and
blocking cell cycle progression at the initial stages [
18
]. Probably, this effect would be highly requested
by the fruit at an immature stage when cell cycle progression can be disturbed by several environmental
factors such as oxidative stress [
18
]. Later, the production of increasing levels of protective compounds,
such as antioxidants, would make possible the completion of fruit development, so that the action of
ABA is no longer required [
18
]. The plant hormone ABA is also produced by pancreatic
β
-cells [
19
],
adipocytes and myoblasts [
1
] in response to glucose and active in humans. The most recent studies
concerning the mechanism of interaction of ABA with the receptor lanthionine synthetase C-like 2
(LANCL2) in glycaemic control and their influence on insulin and glucagon-like peptide 1 (GLP-1)
release highlight a leading role of ABA in the physiological regulation of plasma glucose levels in
humans [
20
]. Plasma levels of ABA increases in healthy individuals administered with a concentrated
glucose solution thus indicating the capacity of incretins to stimulate the release of ABA, similar to their
effect on insulin secretion [
1
]. The mechanism at the base of the hypoglycaemic action of supplemented
ABA
in vivo
at low doses (few micrograms compared to hundreds milligrams/kg body weight) would
depend on its upregulating effects of glucose transport receptors, rather than its stimulatory capacity
of insulin release [
10
]. Therefore, the administration of ABA at low doses may be suggested as a useful
tool for the improvement of glucose tolerance in diabetic patients with deficiency of or resistance to
insulin. To this regard, the fruit thinning waste product may be considered as a more convenient
agro-food matrix for nutraceutical applications. Moreover, these formulations, as a source of ABA,
would favour the intake of this human endogenous hormone, contributing to its plasma levels and,
thus, to its physiological capacity to modulate glycaemia.
Olive leaves are a massive agricultural by-product of the harvesting or processing technology
of olive fruits. They contain high amounts of polyphenols of which the many beneficial properties
to human health of olive leaf extracts, used in traditional medicine, have always been ascribed.
Specifically, oleuropein, one of the most abundant constituents of olive leaf extract, has been referred
to as able to counteract oxidative stress correlated to plasma glucose levels thus indicating its ability
to improve postprandial glycaemia [
21
,
22
]. Oleuropein seems to improve postprandial glycaemia,
by counteracting Nox2-mediated oxidative stress, recognized as being majorly responsible for the
cellular production of reactive oxygen species (ROS) [
2
]. Reactive oxygen species are indicated as
being able to activate dipeptidyl peptidase-4 (DPP-4) which promptly disables incretin activity, thus
unbalancing insulin secretion [
23
,
24
]. However, a specific role of oleuropein on insulinaemia has
recently been elucidated. Carnevale et al. [
25
] demonstrated that 20 mg of pure oleuropein was able
to lower postprandial glycaemia in healthy subjects by enhancing DPP-4 activity, plasma GLP-1 and
insulin levels.
Tomato industrial processing originates a huge amount (up to 3% by fresh fruit weight) of
industrial by-product (tomato pomace), consisting mainly of peels, seeds and pulp. Tomato pomace
has no commercial value and is currently disposed in landfills and only partially recovered by drying
or composting for the production of animal feed. Nevertheless, the abundance of several bioactive

Nutrients 2020,12, 1292 3 of 12
compounds, especially carotenoids (mainly, lycopene), up to five times the concentration in the pulp,
suggests the possibility of employing the tomato pomace as a cheap and sustainable source, for the
extraction of these valuable natural substances. Recent studies including clinical evidence of the
bioactive properties of carotenoids have shown that these compounds may play a significant role in
the treatment of diabetes by improving insulin resistance which has been indicated as a major risk
factor for the development of type 2 diabetes mellitus (T2DM) [
26
].
In vivo
experimental data have
demonstrated the capacity of lycopene to improve glycaemia as well as other metabolic disorders in
mice given a high-fat diet after its twelve-week oral supplementation, either as a pure compound or as
tomato powder, at the same dosage [
27
]. Interestingly, previous human trials have indicated a linear
correlation between plasma lycopene and
β
-carotene concentration and insulin sensitivity in healthy
volunteers [
28
,
29
]. Specific carotenoids would act as peroxisome proliferator-activated receptor gamma
(PPAR
γ
) agonists, through a similar mechanism to thiazolidinediones, a class of oral antidiabetic
drugs, which are adopted in clinical therapy [
30
]. It has also been observed that carotenoid intake
has an inverse relation with glycosylated haemoglobin (HbA1c) levels [
31
]. To date, the molecular
mechanisms at the base of the effects of carotenoids on glycaemia and insulinaemia remain unclear.
Nevertheless, there is general agreement that the beneficial effects of carotenoids in diabetes cannot
simply be associated with their antioxidant properties.
The first aim of the present work was to formulate pilot nutraceutical products based on a water
extract of unripe fruits derived from fruit thinning (fruit thinning waste products, FTWPs); ethanol
extract from olive leaves (EOL); and dried tomato peel powder (TP). Their exact contents of ABA,
oleuropein, and carotenoids, respectively, were detected. Then, each formulation was tested on healthy
human subjects in order to evaluate its effects on glycaemic and insulinemic responses to a standard
glucose drink.
2. Materials and Methods
2.1. Reagents and Standards
All chemicals and reagents used were either analytical-reagent or high-performance liquid
chromatography (HPLC) grade. The water was treated in a Milli-Q water purification system (Millipore,
Bedford, MA) before use. (
±
)-2-Cis-4-trans-abscisic acid (ABA), cartridges Discovery SPE DSC-MCAX
(bed wt, 300 mg; volume, 6 mL; Supelco Analytical, Bellefonte PA, USA), Supelclean SPE LC-NH
2
(bed wt, 300 mg; volume, 6 mL; Supelco Analytical), acetone, ammonium acetate, hexanes, methanol
(MeOH), methyl tert-butyl ether (MtBE),
β
-Carotene (
≥
95%), and lycopene (
≥
90%), oleuropein (
≥
98%)
were all purchased from Sigma–Aldrich (Milano, Italy). Glucose syrup 75 g/150 mL was provided by
Sclavo Diagnostics International S.r.l. (Sovicille, Siena, Italy).
2.2. Sample Collection and Sample Preparation for HPLC Analyses
The FTWPs (apples, pears, plums, peaches, and nectarines) were collected in June 2018 at the
orchards of “Giaccio Frutta” society (Vitulazio, Caserta, Italy, 41
◦
10
0
N–14
◦
13
0
E), at 20–25 days after full
bloom, coinciding with the fruit thinning stage. Tomato peels (cultivar San Marzano) were provided
in September 2018 by La Torrente S.r.l. (Angri, Salerno, Italy, 40
◦
43
0
N–14
◦
33
0
E). Olive leaves (cultivar
Ravece) were provided in September 2018 by Agriturismo Petrilli (Flumeri, Avellino, Italy, 41
◦
4
0
N–15
◦
9
0
E).
All the procedures performed to obtain both the extract and the samples for the HPLC analyses are
described in detail in the Supplementary Materials.
2.3. HPLC-DAD Analyses of Samples
The chromatographic apparatus consisted of a Jasco Extrema LC-4000 system (Jasco Inc., Easton,
MD) provided with the following modular components: a vacuum degassing unit, a quaternary pump,
an autoinjector, a column oven, and a diode array detector photodiode array detector (DAD). The ABA
was determined according the method described by Bosco et al. [
32
] with slight modifications [
32
].

Nutrients 2020,12, 1292 4 of 12
Carotenoids were analysed as previously described by Cooperstone et al. [
33
]. Determination of
oleuropein in olive leave extracts was carried out as previously described by Cooperstone and
colleagues (2016) [
34
]. Information about the HPLC-DAD system used and method are detailed in the
Supplementary Materials.
2.4. Nutraceutical Product Preparation
Large-scale production of the nutraceutical products from agri-food waste matrixes was
accomplished by MBMed Company (Turin, Italy).
Among all of the FTWPs, as the objects of this study, nectarines were found to be the richest in
ABA content (Table 1). Thus, they were chosen as the ideal candidate to be used for the industrial
transformation. Nectarines were extracted with water at 50
◦
C. After centrifugation, the extract
underwent a spray-drying process with maltodextrins as support, obtaining a fine powder, containing
an extract:maltodextrins ratio 1:1 (w/w) (nectarine dry extract powder, NecP).
Table 1. Content of abscisic acid in fruit thinning waste products.
Peaches Nectarines Apples Plums Pears
µg/g FW 0.9 ±0.5 a4.5 ±1.5 c0.8 ±0.3 a0.4 ±0.2 b0.3 ±0.1 b
µg/g DW 9.5 ±1.6 a15.0 ±3.0 c8.1 ±1.1 a6.5 ±0.9 b5.5 ±0.8 b
Values are the means
±
SD (n=5; p<0.01).
abc
Mean values in rows with different superscript letters are significantly
different by the Tukey–Kramer multiple comparison test. Abbreviations: FW, fresh weight; DW, dry weight.
Tomato peels were dried at 42
◦
C in industrial ovens to obtain a fine powder (dried tomato peel
powder, TP).
Olive leaves were extracted with pure ethanol at room temperature. After centrifugation,
the extract underwent a spray-drying process with maltodextrins as support, obtaining a fine powder,
containing an extract:maltodextrins ratio 1:1 (w/w) (ethanol extract from olive leaves, EOL).
2.5. HPLC-DAD Analyses of Nutraceutical Products
Chromatographic analyses for the determination of ABA, carotenoids, and oleuropein levels in
the nutraceutical products based on NecP, TP, and EOL, respectively, were conducted as reported in
Section 2.3. The results are shown in Table 2.
Table 2. HPLD-DAD determination of the main bioactive components in the nutraceutical products.
Abscisic Acid (from NecP) Carotenoids (from TP) Oleuropein (from EOL)
mg/g 0.007 ±0.004 0.5 ±0.1 150.0 ±5.6
Values are the mean
±
SD (n=5; p<0.01); Abbreviations: NecP, nectarine dry extract powder; TP, dried tomato peel
powder; EOL, ethanol extract from olive leaves.
2.6. Study Population and Protocol
This was a randomised, single centre, double-blind trial. The study was conducted on 18–70 years,
normal-weight, and normal-glycaemic subjects recruited by the Samnium Medical Cooperative
(Benevento) in January 2019. Participants completed six test sessions, each on a different day with
consecutive sessions, separated by at least 1 week. Each participant tested the oral glucose solution on
sessions 1, 3, and 5, and one of the three treatment beverages during each of the remaining sessions
in a random, counterbalanced order. Participants consumed the reference glucose solution on three
separate occasions and each test beverage on one occasion only. Subjects were informed not to drink
alcohol or perform hard physical activity 48 h prior to blood sampling. Participants maintained their
usual dietary and lifestyle patterns throughout the study. The reference glucose solutions and the
treatment beverages all contained 75 g glucose. The three treatment beverages (TBs) were prepared by

Nutrients 2020,12, 1292 5 of 12
mixing the glucose solutions with the following samples: 2 g of NecP (14
µ
g ABA)
→
TB NecP; 2 g TP
(1.0 mg total carotenoids)
→
TB TP; 400 mg EOL (60.0 mg oleuropein)
→
TB EOL. Both TB and reference
glucose solutions were served into dark jars, in order to blind subjects and researchers of the study to
the different colours of the solutions mixed with the nutraceutical products. The nutraceutical products
required to prepare each treatment beverage were added to the glucose solutions immediately before
being served to the subjects. The study was conducted in accordance with the 1964 Helsinki Declaration
(revised in 2000) and approved by the Scientific Ethics Committee of AO Rummo Hospital (Benevento,
Italy) with protocol no. 28 of 15 May 2017. Additional information concerning the study protocol,
including study procedures and statistical analyses, are detailed in the Supplementary Materials.
3. Results
3.1. Enrolment
A total of 20 healthy subjects (11 women and 9 men) with a mean age of 45.1
±
15.8 years
and an average BMI of 23.3
±
3.4 kg/m
2
were assigned to the study (Table 3). The group was well
balanced for demographics and clinical factors. No subject prematurely terminated study participation.
All participants performed the six test sessions (dropout rate: 0%). The mean within-individual
coefficient of variation for the glycaemic responses to the three repeated glucose solutions was 11%
which was within the accepted level of ≤30% (ISO 26642:2010).
Table 3. Baseline characteristics of randomised subjects.
Characteristics Value
Demographics
Age (years) 45.1 ±15.8
Male sex (No (%)) 9 (45.0%)
White ethnicity (No (%)) 20 (100%)
Clinical parameters
BMI (Kg/m2)23.3 ±3.4
TC (mg/dL) 190.1 ±11.2
LDL-C (mg/dL) 98.0 ±10.1
HDL-C (mg/dL) 57.2 ±8.5
Triglycerides (mg/dL) 147.3 ±12.7
Glucose (mg/dL) 82.5 ±14.2
Values are means ±SD (n=5).
3.2. Tolerability of Treatment Beverages
The TB were palatable and well tolerated. No adverse events were reported.
3.3. Glycaemia and Insulinaemia Responses to Reference Glucose Solution and Treatment Beverages
All of the three TB revealed lower peak plasma glucose concentrations at 30 min compared to the
reference glucose solution (TB NecP, p=0.02; TB TP, p=0.02; TB EOL, p=0.02) (Figure 1). Particularly,
TB TP, and TB EOL demonstrated higher effects respect to TB NecP (p=0.02 and p=0.02, respectively),
showing no significant difference among each other (p=0.48).
As regards the postprandial insulin response curves (Figure 2), TB NecP and TB TP produced
lower peak plasma concentrations at 30 min respect to the reference glucose solution (p=0.03 and
P=0.04,
respectively), whereas TB NecP demonstrated the lowest effect compared to TB TP (p=0.03).
Conversely, TB EOL led to a higher peak insulin glucose concentration compared to the reference
glucose solution (p=0.02).

Nutrients 2020,12, 1292 6 of 12
Nutrients 2020, 12, x FOR PEER REVIEW 6 of 14
Figure 1. Change in postprandial plasma glucose concentration in healthy adults for the three
treatment beverages. TB NecP (2 g of NecP, equivalent to 14 µ g ABA); TB EOL (400 mg EOL,
equivalent to 60.0 mg oleuropein); TB TP (2 g TP, equivalent to 1.0 mg total carotenoids). Data are
mean ± SD. * Indicates a significant difference between peak 30 min glucose concentration for each
treatment test compared to the reference test (p < 0.05). Abbreviations: RG, regular glucose solution;
NecP, nectarine dry extract powder; TP, dried tomato peel powder; EOL, ethanol extract from olive
leaves.
As regards the postprandial insulin response curves (Figure 2), TB NecP and TB TP produced
lower peak plasma concentrations at 30 min respect to the reference glucose solution (p = 0.03 and P
= 0.04, respectively), whereas TB NecP demonstrated the lowest effect compared to TB TP (p = 0.03).
Conversely, TB EOL led to a higher peak insulin glucose concentration compared to the reference
glucose solution (p = 0.02).
Figure 1.
Change in postprandial plasma glucose concentration in healthy adults for the three treatment
beverages. TB NecP (2 g of NecP, equivalent to 14
µ
g ABA); TB EOL (400 mg EOL, equivalent to 60.0 mg
oleuropein); TB TP (2 g TP, equivalent to 1.0 mg total carotenoids). Data are mean
±
SD. * Indicates a
significant difference between peak 30 min glucose concentration for each treatment test compared to
the reference test (p<0.05). Abbreviations: RG, regular glucose solution; NecP, nectarine dry extract
powder; TP, dried tomato peel powder; EOL, ethanol extract from olive leaves.
Nutrients 2020, 12, x FOR PEER REVIEW 7 of 14
Figure 2. Change in postprandial plasma insulin concentration in healthy adults for the three
treatment beverages. TB NecP (2 g of NecP, equivalent to 14 µg ABA); TB EOL (400 mg EOL,
equivalent to 60.0 mg oleuropein); TB TP (2 g TP, equivalent to 1.0 mg total carotenoids). Data are
mean ± SD. * Indicates a significant difference between peak 30 min insulin concentration for each
treatment test compared to the reference test (p < 0.05). Abbreviations: RG, regular glucose solution;
NecP, nectarine dry extract powder; TP, dried tomato peel powder; EOL, ethanol extract from olive
leaves.
The total glucose response over 150 min was expressed as the postprandial glucose incremental
area under the curve (iAUC) ignoring the area under the baseline using the trapezoidal rule [35,36].
All of the three TB produced lower postprandial glucose iAUC compared to the reference glucose
solution (TB NecP versus RG, 8317 mg/dL.min versus 9378 mg/dL.min, p = 0.02; TB TP versus RG,
7558 mg/dL.min vs. 9378 mg/dL.min, p = 0.02; TB EOL vs. RG, 7611 mg/dL.min vs. 9378 mg/dL.min,
p = 0.02) (Figure 3). Particularly, TB TP and TB EOL demonstrated higher effect respect to TB NecP (p
= 0.02 and p = 0.02, respectively), showing no significant difference between each other (p = 0.48).
Figure 2.
Change in postprandial plasma insulin concentration in healthy adults for the three treatment
beverages. TB NecP (2 g of NecP, equivalent to 14
µ
g ABA); TB EOL (400 mg EOL, equivalent to 60.0 mg
oleuropein); TB TP (2 g TP, equivalent to 1.0 mg total carotenoids). Data are mean
±
SD. * Indicates a
significant difference between peak 30 min insulin concentration for each treatment test compared to
the reference test (p<0.05). Abbreviations: RG, regular glucose solution; NecP, nectarine dry extract
powder; TP, dried tomato peel powder; EOL, ethanol extract from olive leaves.

Nutrients 2020,12, 1292 7 of 12
The total glucose response over 150 min was expressed as the postprandial glucose incremental
area under the curve (iAUC) ignoring the area under the baseline using the trapezoidal rule [
35
,
36
].
All of the three TB produced lower postprandial glucose iAUC compared to the reference glucose
solution (TB NecP versus RG, 8317 mg/dL.min versus 9378 mg/dL.min, p=0.02; TB TP versus RG,
7558 mg/dL.min vs. 9378 mg/dL.min, p=0.02; TB EOL vs. RG, 7611 mg/dL.min vs. 9378 mg/dL.min,
p=0.02) (Figure 3). Particularly, TB TP and TB EOL demonstrated higher effect respect to TB NecP
(p=0.02 and p=0.02, respectively), showing no significant difference between each other (p=0.48).
Nutrients 2020, 12, x FOR PEER REVIEW 8 of 14
Figure 3. Incremental area under the curve (iAUC) postprandial glucose responses for the three
treatment beverages. TB NecP (2 g of NecP, equivalent to 14 µ g ABA), TB EOL (400 mg EOL,
equivalent to 60.0 mg oleuropein), and TB TP (2 g TP, equivalent to 1.0 mg total carotenoids)
compared to the reference glucose solution. Data are mean ± SD. abc Mean values with different
superscript letters are significantly different by the Tukey–Kramer multiple comparison test (p <
0.05). Abbreviations: RG, regular glucose solution; NecP, nectarine dry extract powder; TP, dried
tomato peel powder; EOL, ethanol extract from olive leaves.
The postprandial insulin iAUC was calculated in the same manner as for the postprandial
glucose iAUC, using the trapezoidal rule [37]. As shown in Figure 4, TB NecP and TB TP produced
lower effects respect to the reference glucose solution (TB NecP versus RG, 3572 µ IU/mL.min versus
5649 µ IU/mL.min, p = 0.03; TB TP versus RG, 4116 µ IU/mL.min versus 5649 µ IU/mL.min, p = 0.02),
whereas TB NecP demonstrated the lowest effect compared to TB TP (p = 0.03). Conversely, TB EOL
led to a higher postprandial insulin iAUC compared to the reference glucose solution (p = 0.02).
Figure 3.
Incremental area under the curve (iAUC) postprandial glucose responses for the three
treatment beverages. TB NecP (2 g of NecP, equivalent to 14
µ
g ABA), TB EOL (400 mg EOL, equivalent
to 60.0 mg oleuropein), and TB TP (2 g TP, equivalent to 1.0 mg total carotenoids) compared to the
reference glucose solution. Data are mean
±
SD.
abc
Mean values with different superscript letters are
significantly different by the Tukey–Kramer multiple comparison test (p<0.05). Abbreviations: RG,
regular glucose solution; NecP, nectarine dry extract powder; TP, dried tomato peel powder; EOL,
ethanol extract from olive leaves.
The postprandial insulin iAUC was calculated in the same manner as for the postprandial
glucose iAUC, using the trapezoidal rule [
37
]. As shown in Figure 4, TB NecP and TB TP produced
lower effects respect to the reference glucose solution (TB NecP versus RG, 3572
µ
IU/mL.min versus
5649
µ
IU/mL.min, p=0.03; TB TP versus RG, 4116
µ
IU/mL.min versus 5649
µ
IU/mL.min, p=0.02),
whereas TB NecP demonstrated the lowest effect compared to TB TP (p=0.03). Conversely, TB EOL
led to a higher postprandial insulin iAUC compared to the reference glucose solution (p=0.02).

Nutrients 2020,12, 1292 8 of 12
Nutrients 2020, 12, x FOR PEER REVIEW 9 of 14
Figure 4. Incremental area under the curve (iAUC) postprandial insulin responses for the three
treatment beverages. TB NecP (2 g of NecP, equivalent to 14 µg ABA), TB EOL (400 mg EOL,
equivalent to 60.0 mg oleuropein), and TB TP (2 g TP, equivalent to 1.0 mg total carotenoids)
compared to the reference glucose solution. Data are mean ± SD. abcd Mean values with different
superscript letters are significantly different by the Tukey–Kramer multiple comparison test (p <
0.05). Abbreviations: RG, regular glucose solution; NecP, nectarine dry extract powder; TP, dried
tomato peel powder; EOL, ethanol extract from olive leaves.
3.4. Glycaemic Index and Insulinemic Index of Treatment Beverages
For the calculation of the glycaemic index (GI), the absolute iAUC glucose value for each TB
was expressed as a percentage of the mean iAUC glucose values of the standard glucose solution,
and the resulting values were averaged to obtain the GI value for each TB [37]. All of the three TBs
led to significant reductions in GI compared to the reference glucose solution (TB NecP, p = 0.02; TB
TP, p = 0.02; TB EOL, p = 0.02) (Table 4). Specifically, TB TP and TB EOL demonstrated higher effects
respect to TB NecP (p = 0.02 and p = 0.02, respectively), showing no significant difference among each
other (p = 0.48).
The insulinemic index (II) was calculated in the same manner as the GI, using the absolute
iAUC insulin values [37]. As shown in Table 4, TB NecP and TB TP produced lower values in respect
to the reference glucose solution (p = 0.03 and p = 0.04, respectively), whereas TB NecP demonstrated
the lowest effect compared to TB TP (p = 0.03). Conversely, TB EOL led to a higher II compared to the
reference glucose solution (p = 0.02).
Figure 4.
Incremental area under the curve (iAUC) postprandial insulin responses for the three
treatment beverages. TB NecP (2 g of NecP, equivalent to 14
µ
g ABA), TB EOL (400 mg EOL, equivalent
to 60.0 mg oleuropein), and TB TP (2 g TP, equivalent to 1.0 mg total carotenoids) compared to the
reference glucose solution. Data are mean
±
SD.
abcd
Mean values with different superscript letters
are significantly different by the Tukey–Kramer multiple comparison test (p<0.05). Abbreviations:
RG, regular glucose solution; NecP, nectarine dry extract powder; TP, dried tomato peel powder; EOL,
ethanol extract from olive leaves.
3.4. Glycaemic Index and Insulinemic Index of Treatment Beverages
For the calculation of the glycaemic index (GI), the absolute iAUC glucose value for each TB was
expressed as a percentage of the mean iAUC glucose values of the standard glucose solution, and the
resulting values were averaged to obtain the GI value for each TB [
37
]. All of the three TBs led to
significant reductions in GI compared to the reference glucose solution (TB NecP, p=0.02; TB TP,
p=0.02; TB EOL, p=0.02) (Table 4). Specifically, TB TP and TB EOL demonstrated higher effects
respect to TB NecP (p=0.02 and p=0.02, respectively), showing no significant difference among each
other (p=0.48).
Table 4.
Glycaemic index (GI) and insulinemic index (II) for the three treatment beverages. TB NecP
(2 g of NecP, equivalent to 14
µ
g ABA), TB EOL (400 mg EOL, equivalent to 60.0 mg oleuropein), TB TP
(2 g TP, equivalent to 1.0 mg total carotenoids) in relation to the reference glucose solution (RG).
Test Solution GI Value II Value
RG 100 a100 a
NecP 90 ±5b63 ±4b
EOL 80 ±4c120 ±7c
TP 83 ±5c72 ±6d
Data are mean
±
SD.
abcd
Mean values in columns with different superscript letters are significantly different by the
Tukey–Kramer multiple comparison test (p<0.05). Abbreviations: NecP, nectarine dry extract powder; TP, dried
tomato peel powder; EOL, ethanol extract from olive leaves.
The insulinemic index (II) was calculated in the same manner as the GI, using the absolute iAUC
insulin values [
37
]. As shown in Table 4, TB NecP and TB TP produced lower values in respect to the

Nutrients 2020,12, 1292 9 of 12
reference glucose solution (p=0.03 and p=0.04, respectively), whereas TB NecP demonstrated the
lowest effect compared to TB TP (p =0.03). Conversely, TB EOL led to a higher II compared to the
reference glucose solution (p=0.02).
3.5. Insulin Sensitivity of Subjects in Response to Reference Glucose Solution and Treatment Beverages
Insulin sensitivity of subjects was evaluated in reference to data of glucose tolerance (Figure 1)
and insulin secretion (Figure 2) and expressed as values of Matsuda Indexes [
38
] as follows: RG, 5.99;
TB NecP, 8.31; TB TP, 7.61; TB EOL, 5.98. The samples TB NecP and TB TP revealed to improve insulin
sensitivity in the treatment subjects compared to the reference glucose solution.
3.6. Study Strength and Limitations
The major strengths of the clinical trial herein presented reside in the originality of the study
and in the evaluation of the treatment effects in real-world settings. The positive results, herein
reported, can inform physicians about a novel treatment/intervention which can represent a valuable
support/alternative in the clinical practice. Conversely, the main limitations of our study included
a small sample size of healthy participants with normal glucose tolerance and insulin sensitivity;
the short-term assessment for the treatment of a chronic condition which only allowed the investigation
of acute postprandial effects of the nutraceutical formulations; the lack of a dose assessment in order to
define the range of minimum effective–maximum non-toxic concentrations of therapeutic interest.
4. Discussion
Our results revealed that all of the three nutraceutical formulations were able to significantly
lower simulated postprandial glycaemia in healthy adults when added to the reference glucose
solution (average reduction of glucose peak at 30 min: NecP,
−
9%; EOL,
−
17%; TP,
−
20%) (Figure 1).
As regards the prediabetes management, previous authors reported that the occurrence of T2DM may
be significantly decreased by favouring a minimum lowering of 10 GI units through diet and/or the use
of food supplements [
39
]. The present work indicated that the acute consumption of NecP, TP, and EOL
may lead to an average decrease of 10, 17, and 20 GI units, respectively (Table 3). Interestingly, our
results indicated lowering effects by the treatment beverages on simulated postprandial insulinaemia,
in comparison with the reference glucose solution with the exception of TB EOL (average variation of
insulin peak at 30 min: NecP,
−
28%; TP,
−
36%; EOL, +20%; average variation of the II: NecP,
−
37 units;
TP,
−
28 units; EOL, +20 units) (Figure 2; Table 3). Thus, the present study would highlight that
two out of three of the nutraceutical formulations (mainly, NecP and TP) would be able to influence
postprandial glycaemia through an insulin-saving mechanism, while EOL would preferentially
modulate plasma glucose levels by stimulating insulin release. Recent scientific literature has identified
ABA, carotenoids, and oleuropein, occurring in NecP, TP, and EOL, respectively, as among the natural
bioactive components with the major effectiveness on animal glycemia and insulinemia [
20
–
22
,
28
,
29
].
Nevertheless, it must be pointed out that these nutraceutical formulations are characterised by very
heterogeneous phytocomplexes. Thus, our results regarding their influence on human glycemia and
insulinemia could be ascribed to a plethora of different phytochemicals rather than to an individual
constituent or a specific class of compounds. In the light of these data and concerning the many
different mechanisms of action of NecP, TP, and EOL, it may be hypothesised the formulation of a
multi-component synergistic product, potentially useful to modulate glycaemia and insulinaemia in
multifactorial patients such as diabetic subjects.
Overall, an aspect of major interest of the present study is represented by the possibility of
obtaining useful nutraceutical formulations from agricultural by-products. The management of solid
waste originating from agricultural processing is a serious emerging problem both in Western and
developing countries. Particularly, the costs of drying, storage and shipment of by-products for their
disposal are economically crucial factors. Therefore, agro-food waste is often employed as fertilizer
or feed. Many attempts have been made by researchers and industries in order to face agro-food

Nutrients 2020,12, 1292 10 of 12
by-products. Specifically, many by-products which were first underexploited and disregarded are
being increasingly converted into valuable ingredients. Some of these ingredients are commercialized
and widely used by the industries as food products or as nutraceutical ingredients in functional
foods [
40
]. Thus, our study is in line with the current worldwide trend to recover such agro-food
wastes for environmental, economic, and healthy purposes.
5. Conclusions
Our results indicated that all of the nutraceutical formulations obtained from agro-food by-products
were clinically able to significantly reduce simulated postprandial glucose levels. As regards the
postprandial insulin responses, NecP and TP produced lower peak plasma concentrations, while EOL
led to a higher peak insulin concentration, in respect to the reference glucose solution. Thus, the present
study highlighted that two of the three nutraceutical formulations were able to influence postprandial
glycaemia through an insulin-saving mechanism, while EOL would preferentially modulate plasma
glucose levels by stimulating insulin release. Overall, the difference in the mechanisms of action,
attributable to the main bioactive constituents of the three nutraceutical formulations, may lead to the
hypothesis of a multi-component synergistic preparation which may be regarded as an innovative
and promising nutritional intervention for the management of postprandial glucose homeostasis in
pre-diabetic and, possibly, diabetic subjects. Undoubtedly, the present work represents a preliminary
study for the evaluation of the effects of specific nutraceuticals on human glycaemia and insulinaemia
which must be supported by further
in vitro
,
in vivo
, and clinical investigations to confirm the
proposed results.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/12/5/1292/s1,
supplementary file: materials and methods.
Author Contributions:
Conceptualization, G.C.T., D.C., M.D., G.B. and E.N.; methodology, G.C.T., D.C., M.D.,
G.B. and E.N.; validation, G.C.T., D.C., M.D., G.B. and E.N.; investigation, G.C.T., D.C., M.D., G.B., R.C., E.S., M.M.,
G.A. and E.N.; data curation, G.C.T., D.C., M.D., G.B., R.C., E.S., M.M., G.A. and E.N.; writing—original draft
preparation, G.C.T., D.C., M.D., G.B., R.C., E.S., M.M., G.A. and E.N.; writing—review and editing, G.C.T., D.C.,
M.D., G.B., G.C., R.C., E.S., M.M., G.A. and E.N.; visualization, G.C.T., D.C., M.D., G.B., G.C., R.C., E.S., M.M., G.A.
and E.N.; supervision, E.N.; funding acquisition, E.N. All authors have read and agreed to the published version
of the manuscript.
Funding:
This work was supported by a grant: “Combattere la resistenza tumorale: piattaforma integrata
multidisciplinare per un approccio tecnologico innovativo alle oncoterapie—CAMPANIA ONCO TERAPIE”
Regione Campania and EiCURE-Regione Campania.
Acknowledgments: The assistance of the staffis gratefully appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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