International Journal of Food Science and Technology 2020 1

Original article
Mango (Mangifera indica L.) leaf extracts as ingredient for the
formulation of functional beverages with biological activity

Guadalupe Yeraldin Medina-Saavedra,1† Jos
e Andr
es Herrera-Corredor,2† Yamill Vargas-Rivera,1

Oscar Valeriano S

anchez-Valera, Ad

1
an Cabal-Prieto, 3
Witoon Prinyawiwatkul,4
Emmanuel de Jes
us Ram
ırez-Rivera *
1
& Lorena Guadalupe Ram
on-Canul5*
1 Innovaci
on Agricola Sustentable, Tecnol
ogico Nacional de M
exico/Instituto Tecnol

ogico Superior de Zongolica, Veracruz, M
exico
2 Posgrado en Innovaci
on Agroalimentaria Sustentable, Colegio de Postgraduados, Campus C
ordoba, Veracruz, M
exico
3 Maestr
ıa en Ingenier
ıa, Tecnol

ogico Nacional de M
exico/Instituto Tecnol

ogico Superior de Huatusco, Veracruz, M
exico
4 School of Nutrition and Food Sciences, Louisiana State University Agricultural Center, Baton Rouge LA, 70803, USA
5 Instituto de Nutrici

on, Universidad de la Sierra Sur, Oaxaca, M
exico
(Received 4 August 2020; Accepted in revised form 16 November 2020)

Summary The biological activity of mango leaf extracts from different mango varieties was evaluated in terms of
total phenolic content, flavonoids, antioxidant activity and inhibition of a-amylase and a-glucosidase
enzymes. Leaf extract-based beverages were formulated and evaluated for some physicochemical, microbi-
ological and sensory properties. Results indicated that the extract from ’Tommy Atkins’ mango leaves
had the highest total phenols content (137.08 mg of GAE g
1) and antioxidant activity
(DDPH = 38.26 mg TEAC 100 g
1; ABTS = 59.13 mg TEAC 100 g
1). The beverage formulated with
20% leaf extract presented the highest percentage of antioxidant activity (38.63%) and inhibition of
enzymes a-amylase (41.9%) and a-glucosidase (37.53%). All beverages presented a yellow hue and con-
sumers rated it with a degree of liking between 4.8 and 7.3 according to a hedonic scale. Results showed
that the biological properties of beverages could be an alternative for the control of free radicals and glu-
cose levels.
Keywords Aqueous extract, beverage, biological activity, flavonoid content, mango leaf, phenolic content.

disease appear when hyperglycaemia is not controlled,

Introduction
Type 2 diabetes mellitus is a disease characterised by
chronic hyperglycaemia. This disease results from a
progressive deterioration in the function of pancreatic
cells. This causes a decrease in insulin synthesis, a
resistance of peripheral tissues to insulin and a
decrease in the metabolic response to insulin. The
development of type 2 diabetes mellitus can be
described as a series of cellular and metabolic alter-
ations that affect and impair glucose homeostasis
(ADA, 2010; Rojas et al., 2012). In addition to hyper-
glycaemia, metabolism of carbohydrates, proteins and
fats are also affected by this disease (Rojas et al.,
2012). The main symptoms are polyuria, polyphagia,
polydipsia and accelerated weight loss (Maldonado et
al., 2010; Rojas et al., 2012). Complications from this
*Correspondent: E-mails: l_g_r_c@hotmail.com (LGR-C),
ejramirezrivera@zongolica.tecnm.mx (EJR-R)
†
First authors.
causing organ dysfunction or failure and long-term
complications such as neuropathy, retinopathy,
nephropathy, diabetic foot, among others (Maldonado
et al., 2010). During the development and complica-
tions of this pathology, oxidative stress increases when
glucose auto-oxidation and protein glycation occurs,
reducing the antioxidant defence system and promot-
ing the generation of free radicals (Sigh et al., 2013).
A therapeutic practice in the management of type 2
diabetes mellitus is controlling the postprandial hyper-
glycaemia by inhibiting the a-amylase and a-glucosi-
dase enzymes present in the gastrointestinal tract
(Chen & Kang, 2013). Inhibitions of these enzymes
can significantly reduce the postprandial increase in
blood glucose, which is an important strategy for the
treatment of patients with type 2 diabetes mellitus
(Chen & Kang, 2013; Ru
ız-Ru
ız et al., 2015).
Unfortunately, therapeutic drugs designed as inhibi-
tors of these enzymes have several side effects such as
flatulence, bowel sounds and diarrhoea (Chen & Kang,

doi:10.1111/ijfs.14910
© 2020 Institute of Food Science and Technology
2 Mango leaf functional beverage G. Y. Medina-Saavedra et al.
2013). By-products and residues (e.g. stems, husks,
seeds, barks and leaves) from different plants are a
potential alternative for the study of various biological
activities such as antidiabetic, antioxidant, antibacte-
rial, among others. Roots and Mangifera indica L.
leaves have been used since ancestral times as tradi-
tional medicine (D
ıaz-Torres et al., 2017). This species
is native to the Indo-Burmanic region which include
tropical and subtropical areas (Infante et al., 2011). In
M
exico, the states with the largest mango planted area
are as follows: Chiapas, Sinaloa, Guerrero, Nayarit,
Oaxaca, Michoac
an and Veracruz (SIAP, 2019). In
2017, M
exico reported 202 000 ha of planted area of
Mangifera indica L., of ’Manila’, ’Ataulfo’, ’Tommy
Atkins’, ’Haden’, ’Kent’ and Creole varieties (SIAP,
2019). Despite fruit from these varieties are of great
economic importance in M
exico, leaves are not com-
monly used. Leaves represent a promising source of
bioactive compounds such as phenols, flavonoids,
triterpenes, phytosterols and polyphenols (Mohan et
al., 2013; Medina et al., 2016). Guan-Lin et al. (2014),
Lin & Lee (2014), Jibaja & S
anchez (2015) and Xiang
et al. (2016) have shown that mango leaves contain
different bioactive compounds that contribute to the
reduction of blood glucose and neutralisation of cellu-
lar damage by oxygen. Mangiferin (1,3,6,7-tetrahy-
droxyxanthone-C2-b-D-glucoside) is among these
bioactive compounds. This phenol compound has
strong free radical scavenging activity and has the abil-
ity to chelate metals. It also inhibits, in a non-competi-
tively way, the a-amylase enzyme and the substrate-
enzyme complex, causing a decrease in starch degrada-
tion. In addition, some studies have shown that mango
leaves do not have a genotoxic, cytotoxic or clasto-
genic effect making them suitable for human consump-
tion (Zhang et al., 2014; Reddeman et al., 2019; Villas
et al., 2019).
To date, there is limited information on the bio-
logical activities of the varieties ’Ataulfo’, ’Manila’
and ’Tommy Atkins’ neither on the use of their leaf
extracts for beverage formulation. For this reason, it
is necessary to study and test leaf extracts from dif-
ferent mango varieties in order to identify which
variety had the highest biological potential (antioxi-
dant activity and inhibition of the enzymes a-amy-
lase and a-glucosidase) for the development
functional beverages. Consumer interest in products
that promote health has led to an increase in the
formulation and marketing of infusion-based bever-
ages, which may be rich in phytochemicals and have
properties for reducing the risk of diseases such as
diabetes, cardiovascular diseases and cancer (P
erez-
Ram
ırez et al., 2015). Infusions based on fruit fla-
vours and aromas are well accepted by consumers
due to their sensory properties and antioxidant prop-
erties. The use of leaf extracts is an alternative for
International Journal of Food Science and Technology 2020
developing beverages with antioxidant and hypogly-
caemic potential.
The objectives of this study were as follows: (i) to
determine the biological activity of mango leaf extracts
in terms of phenols content, flavonoids content,
antioxidant activity and inhibition of the a-amylase
and a-glucosidase enzymes on leaves from different
varieties of Mangifera indica L. and (ii) to formulate
leaf extract-based beverages focusing on their physico-
chemical, microbiological and sensory properties.
Materials and methods
Plant material and chemicals
Mangifera indica L. leaves were collected from the
experimental field at Instituto Nacional de Investiga-
ciones Forestales, Agr
ıcolas y Pecuarias (INIFAP),
located in Cotaxtla, Veracruz, M
exico. Mango vari-
eties included for the study were as follows: Manila,
Ataulfo and Tommy Atkins. Mango leaves were col-
lected from this research centre because the agricul-
tural practices are controlled and environmental
factors were common for all varieties. Botanical plant
identification was carried out in the Department of
Botany of the INIFAP (Family: Anacardiaceae;
Genus: Mangifera; reference voucher: 2362842). Chem-
icals used were reagent-grade and purchased from
Sigma AldrichÒ of M
exico (Qu
ımica S.A. de C.V.,
Toluca, M
exico).
Chemical analysis proximal of Mangifera indica L. leaves
The proximal chemical composition was performed
according to the methods as described in the Official
Methods of analysis (AOAC International, 2005). The
moisture and dry matter content were carried out
according to method 926.08. Ash, crude protein, fat
and crude fibre contents were determined according to
methods 923.03, 920.23, 920.39 and 962.09 (AOAC
International, 2005).
Obtaining the aqueous extract of Mangifera indica L.
leaves
The mango leaves were dried by convection at 60 °C
for 24 h. They were then crushed in a Thomas Scien-
tificÒ Wiley Mill Model 4 mill (PARMEX, Mexico,
NJ, USA) to obtain a powder with particle size of
approximately 1 mm. Samples were stored at room
temperature in polyethylene bags until required for
extract preparation (Ru
ız-Ru
ız et al., 2015). Mango
leaf extracts were processed according to Ru
ız-Ru
ız et
al. (2015). A 500 mg sample of crushed leaves was
weighed and then added to 5 mL of distilled water at
100 °C for 30 min. The mixture was centrifuged at
© 2020 Institute of Food Science and Technology

2000 g (10 °C) for 10 min in a Clinical Centrifuge
device (Model Z100A; Labnet, Newark, NJ, USA).
The supernatant was decanted, and the solid residue
was again added to 5 mL of distilled water, and the
procedure was repeated two more times. The aqueous
phase obtained from three extractions was decanted
into a 25 mL volumetric flask and then filtered with
membrane 0.45 lm. Finally, the aqueous extract was
concentrated using a lyophiliser (LabconcoÒ freeze
dryer 5, Kansas, MO, USA).
Determination of total phenols of Mangifera indica L. leaf
extracts
Phenolic compounds were determined using the Folin
and Ciocalteu’s Phenol method (Singleton & Rossi,
1965). A total of 100 µL of aqueous extract was mixed
with 600 µL of distilled water and then 500 µL of
Folin-Ciocalteu 2 N reagent were added in a tube. The
mixture was allowed to stand for 8 min in dark condi-
tions, then 1.5 mL of sodium carbonate (Na2CO3)
(20%) solution was added and adjusted to 10 mL with
distilled water. The mixture was allowed to stand
again for 2 h in dark conditions. After this time, the
readings were performed at 760 nm in a Beckman
DUÒ 730 spectrophotometer (San Francisco, CA,
USA). The total amount of phenolic compounds was
determined in mg of gallic acid equivalent (GAE) g
1
sample and calculated by comparing the standard
curve (0–1 mg of gallic acid mL
1) against the absor-
bance of each sample.
Determination of flavonoids of Mangifera indica L. leaf
extracts
Flavonoid content was determined using the alu-
minium chloride technique (AlCl3) proposed by
Dewanto et al. (2002). A total of 250 µL of aqueous
extract was taken and 75 µL of sodium nitrite (NaNO2
prepared at 5%), 150 µL of aluminium chloride (AlCl3
prepared at 10%) and 500 µL of sodium hydroxide
(NaOH prepared at 1 M) were mixed in a tube. Then
the mixture was adjusted to a volume of 2.5 mL with
distilled water and allowed to stand for 5 min. The
readings were performed at 510 nm on a Beckman
DUÒ 730 spectrophotometer (CA). The total amount
of flavonoids was determined in mg of quercitin equiv-
alent (QE) g
1 sample and calculated by comparing
the standard curve (0–300 µg of quercitin mL
1) with
the absorbance of each sample.
Antioxidant activity of Mangifera indica L. leaf extracts
The antioxidant activity was determined by DDPH
(2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azinobis-3-
ethylbenzothiazoline-6-sulphonic acid) and ferric
© 2020 Institute of Food Science and Technology
Mango leaf functional beverage G. Y. Medina-Saavedra et al. 3
reduction-FRAP (Ferric Reduction Antioxidant
Power). DPPH (2,2-diphenyl-1-picrylhydrazyl) assay
was conducted according to the Brand-Williams et al.
(1995) technique. The stock solution was prepared by
mixing 2.5 mg of DPPH radical with 100 mL of
methanol. The solution absorbance was adjusted to
0.7  0.02 in 515 nm using an UV–Vis spectropho-
tometer Beckman DUÒ 730 (CA, USA). 3.9 mL of
DPPH radical were placed in a test tube, and 100 lL
of the sample extract or standard was added (metha-
nol was used as blank). The decrease in absorbance at
515 nm was measured at 1 min intervals for the first
10 min and then at 5 min intervals until stabilisation.
The time required to obtain DPPH readings of each
extract was 30 min. Trolox was used as standard for
the calibration curve, and the results were expressed as
mg of trolox equivalent antioxidant capacity (TEAC)
100 g
1 of sample.
For the ABTS method, 19.2 mg of ABTS was mix-
ing with 5 mL of distilled water and 88 lL of Potas-
sium Persulphate (K2S2O8) (0.0378 g mL
1) (Re et al.,
1999). This solution was incubated at room tempera-
ture for 16 h in dark conditions. Then, 1 mL of the
activated ABTS solution was taken and mixed with
100 mL of ethanol. The radical was adjusted to an
absorbance of 0.7  0.02 at 734 nm. After adjusting
the blank with methanol, a 30 lL aliquot of diluted
extract was added to 2970 lL of ABTS solution. The
decrease in absorbance at 734 nm was first measured
at intervals of 1 min for 10 min and then every 5 min
until stability was achieved using a Beckman DUÒ 730
spectrophotometer. Trolox was used as standard for
the calibration curve, and the results were expressed as
mg of trolox equivalent antioxidant capacity (TEAC)
100 g
1 of sample.
The antioxidant activity was carried out through fer-
ric reduction-FRAP (ferric reduction antioxidant
power) according to Arnous et al. (2002). A total of
100 µL of extract, 100 µL of ferric chloride (3 mM)
and 1800 µL of TPTZ (2.4.6-Tris (2-pyridyl)-s-triazine)
(1 mM) solution were mixed. The reaction was main-
tained in a water bath at 37 °C for 30 min. Absor-
bance was measured at 620 nm. Results were
expressed as mg of TEAC 100 g
1 sample using a cali-
bration curve with Trolox standards.
Inhibition of the a-amylase and a-glucosidase enzymes
from Mangifera indica L. leaf extracts
The assay was carried out following the standard pro-
tocol as described by Dineshkumar et al. (2010).
Starch (2 mg; substrate) was suspended in a test tube
containing 0.2 mL of Tris-HCl (0.5 M, pH 6.9) with
calcium chloride (0.01 M). The suspension was heated
at 100 °C for 5 min and then pre-incubated at 37 °C
for 5 min. The aqueous extract was dissolved with
International Journal of Food Science and Technology 2020
4 Mango leaf functional beverage G. Y. Medina-Saavedra et al.
1 mL of dimethyl sulphoxide (0.1%) to obtain concen-
trations of 50, 100, 200, 400, 600, 800 and
1000 lg mL
1. Then, 0.2 mL of aqueous extract was
placed in the tube containing the substrate solution.
Swine pancreatic amylase enzyme dissolved in Tris-
HCl buffer (2 units mL
1) was added to the tube con-
taining the extract and starch. The process was carried
out at 37 °C for 10 min. The reaction was stopped by
adding 0.5 mL of 50% acetic acid solution to each
tube. The mixture was centrifuged at 2000 g for 5 min
in a clinical centrifuge device (Model Z100A; Labnet).
Finally, the absorbance of the supernatant was mea-
sured at 595 nm in a spectrophotometer Beckman
DUÒ 730. The concentration of the extract which
reduced the amylase activity by 50% was defined as
the IC50 value. The in vitro a-glucosidase inhibitory
assay was performed according to Dineshkumar et al.
(2010). The a-glucosidase was mixed with 20 lL of
aqueous extract at various concentrations (50, 100,
200, 400, 600, 800 and 1000 lg mL
1) and incubated
for 5 min at 37 °C. Then, 20 lL of paranitrophenyl
glucopyranoside (pNPG) was added at 1 mM to start
the reaction. The mixture was incubated at 37 °C for
20 min. The reaction was terminated by the addition
of 50 lL of sodium carbonate (Na2CO3) 1 mM and the
final volume was completed to 150 lL. The a-glucosi-
dase activity was determined spectrophotometrically at
405 nm on a ChroMateÒ 4300 Microplate reader by
measuring the quantity of paranitrophenol released
from pNPG. The concentration of the aqueous extract
required to reduce the a-glucosidase activity in 50%
was defined as the IC50 value. Acarbose drug (Glu-
cobayÒ, Bayer, Mexico, NJ, USA) was used as a refer-
ence standard for both tests, since it is currently used
as an oral antidiabetic treatment.
Formulation of mango leaf extract-based beverages
The beverages were prepared using the leaf extracts of
the variety that presented a higher content of phenolic
compounds, antioxidant activity, and inhibition of the
a-amylase and a-glucosidase enzymes. Dehydrated
leaves were extracted with purified hot water at a 1:10
ratio (water at 100 °C for 30 min) with constant stir-
ring. The beverages with the leaf infusions were pre-
pared according to the methodology proposed by P
erez-
Ram
ırez et al. (2015). The ingredients used were: 0.7 g
L
1 sodium benzoate (Sigma AldrichÒ, M
exico) as a
preservative, 10 g L
1 SteviaÒ (Walmart de M
exico SA
de CV) as a sweetener, 10 g L
1 mandarin juice as
flavouring and 0.2 g L
1 citric acid (Sigma AldrichÒ,
M
exico) as a stabilising agent. The concentration of
additives used in this study was consistent with that
used in the beverage industry (Codex, 1995). Beverages
were formulated using different porportions of mango
leaf extract (10%, 15% and 20%). A beverage
International Journal of Food Science and Technology 2020
formulated at 0% mango leaf extract was used as the
control. Each beverage was pasteurised (95 °C for
10 min) and stored in a sterile glass amber bottle with a
screw cap at 7  2 °C until microbiological, physico-
chemical, biological activities and sensory evaluation.
Microbiological analysis of mango leaf extract-based
beverages
The Colony Forming Units (CFU mL
1) measurement
for aerobic mesophiles, total coliforms, fungi and yeast
were determined according to the guidelines of the
Official Mexican Standards NOM-092-SSA1, 1994,
NOM-111-SSA1, 1994 and NOM-113-SSA1, 1994.
Physicochemical analysis of mango leaf extract-based
beverages
Colour determination was carried out with a Konica
MinoltaÒ colorimeter (Model CR-400; USA) using a
tristimulus scale (L*, a*, b*), where the value of L* rep-
resents the relative luminosity and has values between 0
and 100, the value of a* goes from green to red, that of
b* from blue to yellow. Chroma (C) and Hue Angle
(°H) were also determined (G
ongora et al., 2018). In
addition, pH and acidity were determined according to
the methodologies of the AOAC International (2005).
Total Sugars (TS) content was determined according to
the methodology of phenol-sulphuric from Dubois et al.
(1956). Reducing sugars (RS) contents were measured
according to Miller (1959). Total soluble solids (TSS)
were performed using a digital refractometer (Sper Sci-
entific, Model 300034, Phoenix, AZ, USA). Results were
reported as °Brix (AOAC International, 2005005). Total
phenolic and flavonoid contents were also determined
by the Folin–Ciocalteu and aluminium chloride (AlCl3)
methods, respectively (Singleton & Rossi, 1965;
Dewanto et al., 2002).
Antioxidant activity and inhibition of the a-amylase and
a-glucosidase enzymes of mango leaf extract-based
beverages
The antioxidant activity was determined by scavenging
effect on DDPH (2,2-diphenyl-1-picrylhydrazyl) free
radical as a described by Brand-William et al. (1995).
The antioxidant activity was expressed as percentage
of DPPH radical scavenging. The enzyme inhibition
was carried out as described by Dineshkumar et al.
(2010), and the results were expressed in percentages
of enzyme inhibition.
Sensory evaluation of mango leaf extract-based beverages
Sensory evaluation was carried out with 100 con-
sumers (fifty women and fifty men between the ages of
© 2020 Institute of Food Science and Technology

20 and 35 years old) from the Universidad de la Sierra
Sur. Consumers were selected according to their level
of consumption of this type of products (ISO 8586-1,
1993). Consumers were informed about product ingre-
dients. Participation in the study was voluntary. Prin-
ciples established by the Declaration of Helsinki were
followed in order to protect subjects participating in
the study. Each consumer determined their level of lik-
ing for each beverage sample using a hedonic scale (1–
9, where 1 = dislike extremely and 9 = like extremely).
Samples were coded with a three-digit random code
and served sequentially monadic (MacFie et al., 1989).
Statistical analysis
The treatments were distributed in a completely ran-
domised design with five repetitions. Data were sub-
jected to a one-way analysis of variance (ANOVA) with
a = 5% level of significance. Mean comparison was
performed using the Tukey’s test. The Pearson correla-
tion coefficient (r) was determined between the bioac-
tive compounds and the antioxidant capacity by two
levels of significance (P < 0.05 and P < 0.01). The sen-
sory properties values were analysed with a one-way
factor ANOVA. Statistical analyses were performed using
the Statgraphic Plus version 5.1 program (Statistical
Graphics Corp., The Plains, VA, USA).
Results and discussion
Proximate analysis of the Mangifera indica L. leaves
Proximate analysis results of mango leaves are shown
in Table 1. Mango leaves of Ataulfo variety had the
highest (P < 0.0001) moisture and ash content (14.13
and 8.87 g 100 g of sample
1) compared to the Manila
and Tommy Atkins varieties, which had moisture con-
tents of 7.33 and 8.37 g 100 g
1 of sample, respectively
and ashes of 6.43 and 5.03 g 100 g
1 of sample,
respectively (Table 1). Moisture values from this study
were similar to those reported by Rakholiya & Chanda
(2012), who obtained 8.9 g 100 g
1 of wet-basis sam-
ple on Kesar mango leaves from India. Rambabu et
al. (2018) found 7.2 g 100 g
1 of moisture sample in
Indian Neelam mango leaves. Ash content values were
similar to those reported by Rakholiya & Chanda
(2012) who reported 9.6 g 100 g
1 of sample of ashes
on Kesar mango leaves. On the other hand, Rambabu
et al. (2018) reported 1.9 g 100 g
1 of sample in Nee-
lam mango leaves. The similarities of the moisture and
ash contents in the present study and those described
above could be due to the similarities in agricultural
practices (e.g. fertilisation, irrigation, pest management
and disease controls) used in M
exico and India. The
Tommy Atkins mango leaves obtained the highest
content (P < 0.001) of protein and crude fat (10.40
© 2020 Institute of Food Science and Technology
Mango leaf functional beverage G. Y. Medina-Saavedra et al. 5
and 5.50 g 100 g
1 of sample, respectively) compared
to Manila and Ataulfo leaves, in which, a crude pro-
tein content of 8.23 and 6.77, respectively, and fat of
2.90 and 4.33, respectively, was obtained. The crude
protein results in this research were higher than those
reported by Rambabu et al. (2018) who obtained a
value of 0.3 g 100 g
1 of sample in Neelam mango
leaves. The difference in the values obtained in this
research and the mentioned authors may be due to the
collection period of mango leaves. The Manila mango
leaves showed higher (P < 0.001) crude fibre content
(31.10 g 100 g
1 of sample) compared to Ataulfo and
Tommy Atkins who contained 26.60 and 24.97 g
100 g
1 of sample, respectively.
Total phenols and total flavonoids content of Mangifera
indica L. leaf extracts
Results of the total phenol and flavonoid content of
Mangifera indica L. leaves are shown in Table 2.
Tommy Atkins mango leaves showed higher
(P < 0.0001) total phenolic content (137.08 mg of
GAE g
1) compared to Manila and Ataulfo having
117.19 and 125.22 mg of GAE g
1, respectively.
Results of the total phenolic content in the aqueous
extracts of the Mangifera indica L. leaves in this study
were higher than those reported by Saleh-Gazwi &
Mahmoud (2019) (86.20 mg of GAE g
1). However,
they were lower than those values of 390.6, 376.7 and
292.7 mg of GAE g
1 in mango leaves of Tommy
Atkins, Haden and Edward from Ecuador, respectively
(D
ıaz-Torres et al., 2017). The difference in the total
phenolic content may be due to the fact that these
authors used 70% ethanol as the extraction solvent
which caused a higher interaction of the solvent with
the secondary metabolites, facilitating the release of
the total phenols. Regarding the flavonoid content,
Tommy Atkins mango leaves had a higher (P < 0.05)
content (135.28 mg of QE g
1) compared to Manila
and Ataulfo having between 109.99 and 120.04 mg of
QE g
1. Regarding the content of flavonoids, the val-
ues found in this study were higher than those
reported by Saleh-Gazwi & Mahmoud (2019), who
observed a value of 42.64 mg of QE g
1 in Mangifera
indica L. leaves using aqueous extract. According to
Ma et al. (2011) the differences in flavonoids may be
due to the type of mango leaf variety analysed. Addi-
tionally, flavonoid contents found in this study may be
affected by climate, soil, water, air quality, among
others (Ma et al., 2011).
Antioxidant activity of Mangifera indica L. leaves extracts
Results of DPPH, ABTS and ferric-FRAP free radical
scavenging activity for the samples evaluated ranged
between 22.27 to 38.26 mg of TEAC 100 g
1, 30.95 to
International Journal of Food Science and Technology 2020
6 Mango leaf functional beverage G. Y. Medina-Saavedra et al.

Table 1 Proximate analysis of Mangifera indica L. leaves

Moisture Ash Crude protein Crude fat Crude fibre N.F.E

g 100 g
1
Variety of sample

Manila 7.33  0.06c 6.43  0.15c 8.23  0.76b 2.90  0.20c 31.10  0.95a 44.03  1.78a
Ataulfo 14.13  0.31a 8.87  0.12a 6.77  0.15c 4.33  0.32b 26.60  1.81b 39.37  2.50b
Tommy Atkins 8.37  0.06b 5.03  0.06b 10.40  0.26a 5.50  0.10a 24.97  1.77b 45.70  1.65a

Different letters in the same column indicate significant differences according to the Tukey (n = 5; P < 0.05).
N.F.E, Nitrogen Free Extract.

Table 2 Total phenols and total flavonoids content of Mangi- Table 3 Antioxidant activity of Mangifera indica L. leaves
fera indica L. leaf extracts extracts

Total phenols (mg of Total flavonoids (mg of DPPH (TEAC ABTS (TEAC FRAP (TEAC
Variety GAE g
1) QE g
1) Variety 100 g
1) 100 g
1) 100 g
1)

Manila 117.19  0.82c 109.99  0.08c Manila 23.95  2.11b 30.95  3.94c 39.25  4.05c
Ataulfo 125.22  1.69b 120.04  1.27b Ataulfo 22.27  2.60b 37.09  2.78b 50.12  6.31b
Tommy 137.08  0.54a 135.28  0.23a Tommy 38.26  2.94a 59.13  3.70a 64.58  7.01a
Atkins Atkins

Different letters in the same column indicate significant differences
according to the Tukey (n = 5; P < 0.05).
GAE, gallic acid equivalents; QE, quercitin equivalents.
59.13 mg of TEAC 100 g
1 and 39.25 to 64.58 TEAC
mg 100 g
1, respectively (Table 3). The sample that
presented the highest antioxidant activity on the three
techniques evaluated was the Tommy Atkins variety.
Mohan et al. (2013) evaluated the antioxidant activity
in the aqueous extracts of mango leaves and found
that free radical scavenging activity with the DPPH
method was 31.42 µg mL
1. The antioxidant activity
in the aqueous extracts of mango leaves (80 mg mL
1)
using the FRAP method has also been reported by
some authors (Udem et al., 2018). This biological
activity is due to the antirradical activity and the inhi-
bitory effect on metal ions present in mango leaves
(G€ulcin et al., 2010). Results showed that antioxidant
activity values from the ABTS technique were higher
than those from the DDPH technique for all samples.
This is related to the low selectivity of the ABTS. This
fact can be attributed to (i) the ABTS radical reacts
with any hydroxylated aromatic compound regardless
of its real antioxidant potential and (ii) the DPPH rad-
ical does not react with flavonoids lacking hydroxyl
groups in ring B with aromatic acids containing a sin-
gle hydroxyl group.
In addition, if the antioxidant capacity of extracts
is caused by the presence of flavonoids and other
types of polyphenols, it should be taken into
account that DPPH is more selective than ABTS
and does not react with flavonoids that have no
hydroxyl groups in ring B or with aromatic acids
that contain a single hydroxyl group. This explains
Different letters in the same column indicate significant differences
according to the Tukey (n = 5; P < 0.05).
ABTS, 2.20 -Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); DPPH,
2,2-Diphenyl-1-picrylhydrazyl; FRAP, Ferric Reduction Antioxidant
Power; TEAC, mg of Trolox Equivalent Antioxidant Capacity.
that the DPPH values obtained are lower than those
of ABTS. Likewise, high values were presented in
the FRAP technique, which indicates that the chelat-
ing property of the samples with the highest reduc-
ing power can provide protection against oxidative
damage (Aseervatham et al., 2012; Mercado-Mercado
et al., 2013). Pearson’s correlation coefficients
(Table 4) show that all antioxidant analysis presented
significant correlation at P < 0.05 and P < 0.01. This
fact shows that the bioactive compound contents (to-
tal phenols and flavonoids) found in this study
showed a significant correlation with antioxidant
activity (DDPH, ABTS and FRAP), suggesting that
these compounds contributed significantly to antioxi-
dant activity.
Inhibitory activity of the a-amylase and a-glucosidase
enzymes in Mangifera indica L. leaf extracts
In this study, extracts of mango leaves showed an inhi-
bitory capacity of the a-amylase and a-glucosidase
enzymes. The varieties that presented high inhibitory
activity for the a-amylase enzyme were Ataulfo and
Tommy Atkins (263.65 and 236.41 µg mL
1)
(Table 5). In the case of a-glucosidase, the variety with
the higher inhibitory activity was Tommy Atkins
(270.96 µg mL
1). These values were lower than those
obtained by Ganogpichayagrai et al. (2017), who

International Journal of Food Science and Technology 2020 © 2020 Institute of Food Science and Technology
 Mango leaf functional beverage G. Y. Medina-Saavedra et al. 7

Table 4 Pearson’s correlation coefficient between antioxidant Table 5 Inhibitory activity of the a-amylase and a-glucosidase
activity and total phenols and total flavonoids content enzymes in Mangifera indica L. leaf extracts

Manila variety† a-amylase IC50 a-glucosidase IC50

Variety (µg mL
1) (µg mL
1)
Coeficiente de Total Total

n (r)
correlacio phenols flavonoids DDPH ABTS FRAP Manila 348.61  17.45a 314.90  31.62a
Ataulfo 263.65  4.75b 372.53  2.60a
Total phenols 0.84 0.80 0.83 0.85 Tommy 236.41  15.45b 270.96  23.2b
Total flavonoids 0.91 0.90 0.90 0.91 Atkins
DPPH 0.85 0.80 Acarbose 23.39  10.23c 49.38  9.37c
ABTS 0.80
Different letters in the same column indicate significant differences
Ataulfo variety†
according to the Tukey (n = 5; P < 0.05). IC50: concentration of the
Correlation Total Total aqueous extract required to inhibit 50% of the enzyme. Acarbose drug
coefficient (r) phenols flavonoids DDPH ABTS FRAP (Glucobayâ, Bayer) was used as a reference standard for both tests.

Total phenols 0.88 0.86 0.80 0.75

Total flavonoids 0.88 0.90 0.92 0.88 possibly attributed to various bioactive compounds,
DPPH 0.85 0.90 one of which was the phenolic compounds. Phenolic
ABTS 0.90 compounds have been shown to inhibit a-amylase
Tommy Atkins variety† and a-glucosidase enzymes by the action of OH
groups that tend to form hydrogen bonds with amino
Correlation Total Total acids present in the active sites of the enzymes. Man-
coefficient (r) phenols flavonoids DDPH ABTS FRAP
giferin (1,3,6,7-tetrahydroxyxanthone-C2-b-D-glu-
Total phenols 0.88 0.81 0.85 0.87 coside) is the main phenolic component in mango
Total flavonoids 0.89 0.88 0.9 0.92 leaves (Medina et al., 2016). Mangiferin acts as a
DPPH 0.87 0.79 non-competitive inhibitor, which binds to enzymes
ABTS 0.85 and the enzyme-substrate complex, causing a decrease
in starch degradation and controlling blood glucose
ABTS, 2.20 -Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); DPPH,
2,2-Diphenyl-1- picrylhydrazyl; FRAP, Ferric Reduction Antioxidant
peaks. Mangiferin reduces the postprandial glucose
Power. response through impaired secretion of the glucose-
†
All data show a significant correlation of P < 0.05 and P < 0.01. dependent insulinotropic polypeptide (GIP) of the
incretin hormones and glucagon-like peptide 1 (GLP-
1). In addition, it stimulates the secretion of insulin
found that extracts from the leaves of M. indica from pancreatic b cells and the modulation of glucose
Okrong had an IC50 of 1452.8 µg mL
1 for the a-glu- release from the liver, making it possible to control
cosidase enzyme and 2284 µg mL
1 for the a-amylase patients with type 2 diabetes mellitus (ADA, 2010;
enzyme. Kulkarni & Rathod (2018) evaluated the Zhang et al., 2014; Reddeman et al., 2019). Phenolic
aqueous extracts of mango leaves for inhibition of a- compounds also facilitated the reduction of glucose
amylase and a-glucosidase enzymes. They found that a absorbed in the gastrointestinal tract (Sigh et al.,
concentration of 100 lg mL
1 caused an inhibition of 2013; Ganogpichayagrai et al., 2017).
64.5% and 77.8% of amylase and a-glucosidase activi- The biological activities present in the extracts were
ties respectively. lower compared to the reference standards (IC50
The inhibition of amylase and a-glucosidase a-amylase = 23.39 lg mL
1 and IC50 a-glucosi-
enzymes caused by the mango leaf extracts was dase = 49.38 µg mL
1). However, as it was found that

Table 6 Physicochemical analysis of the beverages containing the Mangifera indica L. leaves extracts

Formulation Acidity (%) pH TSS (°Brix) TS (g of glucose L
1) RS (g of glucose L
1)

F1 0.026  0.00c 3.15  0.03d 5.47  0.06b 28.90  0.32a 4.70  0.04c
F2 0.028  0.00b 3.39  0.02c 5.43  0.21b 28.79  2.60a 4.84  0.02b
F3 0.029  0.00a 3.47  0.01b 5.53  0.06b 28.33  1.61a 4.87  0.01b
F4 0.027  0.00b 3.57  0.01a 5.80  0.10a 29.03  1.51a 4.96  0.02a

Different letters in the same column indicate significant differences according to the Tukey (n = 5; P < 0.05). F1: 0% leaf extract (control beverage);
F2: 10% leaf extract; F3: 15% leaf extract and F4: 20% leaf extract.
RS, reducing sugars; TS, total sugars; TSS, total soluble solids.

© 2020 Institute of Food Science and Technology International Journal of Food Science and Technology 2020
8 Mango leaf functional beverage G. Y. Medina-Saavedra et al.

there was inhibition of the amylase and glucosidase (14.79 mg of QE mL
1) compared to F1, F2 and F3
enzymes in the present study, the extracts can be used formulations having 1.77, 7.46 and 11.50 mg of QE
as natural anti-a-amylase and a-glucosidase agents and mL
1, respectively. Results of flavonoids in the sam-
can also be considered as an alternative for the formu- ples were superior to those obtained by Rubio-P
erez et
lation of a functional food aimed to help people with al. (2014) who prepared a beverage based on green tea
type 2 diabetes mellitus. and apple and found values of 5.2 mg of QE mL
1.
The flavonoid content in beverages evaluated in this
study may help decrease the onset and progression of
Microbiological and physicochemical analysis of the
some chronic degenerative diseases (Rubio-P
erez et al.,
beverage based on the Mangifera indica L. leaves extracts
2014).
Results from microbiological analysis of the beverage
based on leaves extracts evidenced the absence of aero-
Antioxidant activity and inhibition of a-amylase and a-
bic bacteria, moulds, yeasts and coliforms, which sug-
glucosidase enzymes of mango leaf extract-based
gested compliance with hygiene standards during
beverages
product preparation (NOM-218-SSA1, 2011). The
acidity content of beverages ranged from 0.026% to The results of antioxidant activity and enzyme inhibi-
0.029% (Table 6). F3 had the highest (P < 0.05) acid- tion are shown in Table 8. Formulation F4 showed
ity content (0.029%) and F4 showed the highest pH higher (P < 0.0001) antioxidant activity (38.63%) com-
value (3.57; P < 0.05). The beverage that obtained the pared to formulations F1, F2 and F3 (9.56%, 8.45%
highest (P < 0.05) TSS content was F4 (5.80 °Brix). and 19.59%, respectively). The efficiency of the bever-
However, there were no differences (P > 0.05) among ages containing mango leaf extracts to reduce diges-
formulations for TS content. tion of polysaccharides and glucose absorption was
reflected by the inhibitory activity of the a-amylase
and a-glucosidase enzymes. The percentage inhibition
Total phenols and flavonoids of beverages based on the
of the a-amylase and the a-glucosidase enzymes was
Mangifera indica L. leaves extracts
below 50% (10.7–41.9% and 12.90–37.53%, respec-
Total phenol and flavonoid content of beverages are tively). However, the results suggest a possible antidia-
shown in Table 7. Results indicated that formulation betic effect that could contribute to the production of
F4 showed a higher total phenolic content (83.83 mg short-chain fatty acids, which are considered beneficial
of GAE mL
1) than F1, F2 and F3 formulations with for digestive system. Future research should be focused
41.63, 51.27 and 72.28 mg of GAE mL
1, respectively. on how to improve the hypoglycaemic function in for-
Results of phenol content in the samples were lower mulated beverages by ensuring the enzymatic activity
than those reported by Medina et al. (2016) who eval- using nanoencapsulation of bioactive compounds from
uated an infusion of Mangifera indica L. leaves and the mango leaf. Nanoencapsulation can be prepared
found a content of 381 mg of GAE mL
1. However, using carbohydrates such as succinated starch, mal-
phenol contents found in this study can help increase todextrin and other compounds as wall materials. This
the intake of phenols by approximately 30% by method can increase the solubility and chemical stabil-
increasing the antioxidant capacity in the human diet ity of bioactive compounds during manufacture and
(Medina et al., 2016). Regarding flavonoid content,
the F4 formulation had a higher (P < 0.05) content
Table 8 Antioxidant activity and inhibition of the a-amylase
and a-glucosidase enzymes in beverages containing the Man-
Table 7 Total phenols and total flavonoids of beverages con- gifera indica L. leaf extracts
taining the Mangifera indica L. leaves extracts
Antioxidant activity a-amylase a-glucosidase
Total phenols (mg de Total flavonoids (mg de Formulation (%)† (%)‡ (%)‡
Formulation GAE mL
1) QE mL
1)
F1 9.56  1.25c 10.7  0.85d 12.90  2.50c
F1 41.63  3.43d 1.77  0.63d F2 8.45  3.13c 30.5  0.70c 26.73  1.60b
F2 51.27  5.97c 7.46  0.41c F3 19.59  2.53b 37.9  1.65b 34.63  1.69a
F3 72.28  3.78b 11.50  0.74b F4 38.63  0.43a 41.9  1.32a 37.53  1.15a
F4 83.83  4.37a 14.79  0.051a
Different letters in the same column indicate significant differences
Different letters in the same column indicate significant differences according to the Tukey (n = 5; P < 0.05). F1: 0% leaf extract (control
according to the Tukey (n = 5; P < 0.05). F1: 0% leaf extract (control beverage); F2: 10% leaf extract; F3: 15% leaf extract and F4: 20% leaf
beverage); F2: 10% leaf extract; F3: 15% leaf extract and F4: 20% leaf extract.
†
extract. Per cent Removal of DDPH (%).
‡
GAE, Gallic acid equivalents; QE, Quercitin equivalents. Per cent inhibition of enzyme (%).

International Journal of Food Science and Technology 2020 © 2020 Institute of Food Science and Technology
 Mango leaf functional beverage G. Y. Medina-Saavedra et al. 9

Table 9 Colour analysis in beverages containing the Mangifera indica L. leaf extracts

Formulations L* a* b* C °H

F1 90  0.56a
0.5  0.03b 27  0.43c 80  0.16a 80  0.15c

F2 83  0.56b 0.5  0.25b 43  2.63b 43  2.37b 89  0.32a
F3 84  0.56b 2  0.08a 50  0.26a 50  0.71c 88  0.11b
F4 75  0.56c 1  0.17a 48  1.96a 48  0.18c 89  0.18b

Different letters in the same column indicate significant differences according to the Tukey (n = 5; P < 0.05). F1: 0% leaf extract (control beverage);
F2: 10% leaf extract; F3: 15% leaf extract and F4: 20% leaf extract. L*: represents the relative luminosity and has values between 0 and 100; a*: goes
from green (
) to red (+); b*: from blue (
) to yellow (+); C: Chroma; °H: Hue Angle.

storage, and protect them against decomposition by
light. In addition, nanoencapsulation can help achieve
a controlled release of bioactive compounds and main-
tain their biological activity for during storage.
(Rashidinejad & Mahdi, 2017). The antioxidant effect
of beverages could contribute to preventing or delay-
ing the oxidation of various substances such as fatty
acids, which could cause important physiological alter-
ations with the triggering of multiple diseases (Ru
ız-
Ru
ız et al., 2015; Shuyuan et al., 2017).
Colour analysis and sensory evaluation of mango leaf
extract-based beverages
Colour results of beverage formulations are shown in
Table 9. Significant differences (P < 0.05) were
observed in all colour variables measured. Regarding
the luminosity (L*), the F2 (10% extract) and F3
(15% extract) formulations were similar (P > 0.05)
between them and presented values between 83 and 84
of L*. The F1 formulation exhibited a higher value of
L* with a slightly perceptible red (a*) and with a ten-
dency to a yellow colour (b*). However, F4 formula-
tion showed a low luminosity (P < 0.05) with an
almost imperceptible red tone but with a higher ten-
dency towards yellow. Therefore, the values of chroma
(C) and hue angle (°H) indicated that the formulations
tended to the yellow region of the colour space. The
yellow colour of the different formulations of this
study could be due to the concentration of polyphe-
nols, which are a group of yellow pigments produced
by oxidation reactions.
Results from the consumer study indicated that for-
mulations based on leaf extracts were located in the
region ‘Neither like nor dislike’ to ‘Like slightly’ of the
hedonic scale. F2 (10% mango leave extract Tommy
Atkins) and F3 (15% mango leave extract Tommy
Atkins) formulations were scored with 5.8 and 6.2,
respectively. The F4 formulation (20% Tommy Atkins
mango leave extract) obtained a value of 4.9 on the
hedonic scale while the control sample (0% Tommy
Atkins mango leave extract) had a score of 7.3.
Results from this study were similar (5.7) to those
reported by Medina et al. (2016) whom evaluated
beverage from mango Ub
a leaves. This could be
caused by the concentration of sweetener used to make
the beverage (Keast, 2016). Medina et al. (2016) men-
tioned that sweeteners can influence the consumer lik-
ing for this type of beverages.
Conclusion
Mango leaf extracts were found to have hypogly-
caemic and antioxidant activity. The beverage formu-
lated with 20% leaf extract presented the highest total
phenols content, antioxidant activity and inhibition of
enzymes a-amylase and a-glucosidase. In addition, the
microbiological quality indicates that beverages evalu-
ated were safe for human consumption. According to
the consumer study, the beverages were rated between
‘Neither like nor dislike’ to ‘Like slightly’. Results
showed that the biological properties of beverages
could be an alternative for the control of free radicals
and glucose levels. However, it is important to per-
form in vivo studies to determine their biological activ-
ity. Improving the flavour the beverage should be
done in the future to improve sensory liking of this
functional beverage.
Acknowledgments
The research and development of the thesis of the first
author was financed with the resources of the Convo-
catoria de Apoyo a la Investigaci
on Cient
ıfica y Tec-
nol

ogica 2019 with the code 6770.19-P (entitled:
Activiades biol
ogicas in vitro de diversas plantas
medicinales que ayudan a enfermedades incluidas den-
tro del s
ındrome metab
olico) of the Tecnol
ogico
Nacional de M
exico. The Centro de Investigaci
on en
Nutrici

on y Alimentaci
on de la Universidad de la
Sierra Sur is thanked for the facilities granted for the
completion of the first author’s research stay. Also,
thanks to Dr. Enrique No
e Becerra Leor of Instituto
Nacional de Investigaciones Forestales, Agr
ıcolas y
Pecuarias (INIFAP) Campo Experimental Cotaxtla,
Veracruz for the facilities granted for the collection of
mango leaves.

© 2020 Institute of Food Science and Technology International Journal of Food Science and Technology 2020
10 Mango leaf functional beverage G. Y. Medina-Saavedra et al.
Conflicts of interest
The authors report no conflicts of interest.
Author contribution
Guadalupe Yeraldin Medina-Saavedra: Data curation
(equal); Formal analysis (equal); Investigation (equal);
Methodology (equal); Writing-original draft (equal).
Jos
e Andr
es Herrera-Corredor: Data curation (lead);
Formal analysis (lead); Investigation (lead); Methodol-
ogy (lead); Writing-review & editing (lead). Yamil Var-
gas-Rivera: Investigation (equal); Methodology
(equal); Writing-review & editing (equal). Oscar Vale-
riano S
anchez-Valera: Conceptualization (equal);
Investigation (equal); Methodology (equal); Writing-
review & editing (equal). Ad
an Cabal-Prieto: Investiga-
tion (equal); Methodology (equal); Writing-review &
editing (equal). Witoon Prinyawiwatkul: Conceptualiza-
tion (equal); Data curation (equal); Formal analysis
(equal); Investigation (equal); Writing-review & editing
(equal). Emmanuel de Jes
us Ram
ırez-Rivera: Conceptu-
alization (lead); Data curation (lead); Formal analysis
(lead); Funding acquisition (lead); Investigation (lead);
Methodology (lead); Writing-original draft (lead);
Writing-review & editing (lead). Lorena Guadalupe
Ram
on-Canul: Conceptualization (lead); Data curation
(equal); Formal analysis (lead); Investigation (lead);
Methodology (lead); Supervision (lead); Writing-origi-
nal draft (lead); Writing-review & editing (lead).
Ethical approval
Consumers were informed about product ingredients.
Participation in the study was voluntary. Principles
established by the Declaration of Helsinki were fol-
lowed in order to protect subjects participating in the
study.
Peer Review
The peer review history for this article is available at
https://publons.com/publon/10.1111/ijfs.14910.
Data availability statement
The data that support the findings of this study are
available from the corresponding author upon reason-
able request.
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