processes

Article
Purification Process of Mangiferin from Mangifera indica L.
Leaves and Evaluation of Its Bioactivities
Nguyen Thi Truc Loan 1 , Dang Thanh Long 2 , Pham Nguyen Dong Yen 3 , Truong Thi Minh Hanh 1 ,
Tri Nhut Pham 4,5 and Dung Thuy Nguyen Pham 4,5, *

1 Department of Food Technology, The University of Danang, University of Science and Technology,
Danang 550000, Vietnam; nttloan@dut.udn.vn (N.T.T.L.); ttmhanh@dut.udn.vn (T.T.M.H.)
2 Institute of Biotechnology, Hue University, Hue 530000, Vietnam; dtlong@hueuni.edu.vn
3 Institute of Applied Materials Science, Vietnam Academy of Science and Technology, Ha Noi 100803, Vietnam;
phamngdongyen@gmail.com
4 Center of Excellence for Biochemistry and Natural Products, Nguyen Tat Thanh University,
Ho Chi Minh City 70000, Vietnam; ptnhut@ntt.edu.vn
5 NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City 70000, Vietnam
* Correspondence: pntdung@ntt.edu.vn

Abstract: Mangiferin (C19 H18 O11 ) is a C-glucoside xanthone that is mainly found in the leaves
of mango (Mangifera indica L). The compound has been recognized for several pharmacological
values, including antioxidant and antibacterial activities. Therefore, the present study aims to
utilize the old leaves of mango as a potential source for mangiferin extraction and purification,
and evaluate its antimicrobial and antioxidant properties. In the present study, mangiferin was



extracted from a local variety of mango leaves using ultrasonic-assisted extraction methods and


ethanol as the extraction solvent. The crude ethanolic extract of mangiferin was then purified by the
Citation: Loan, N.T.T.; Long, D.T.; liquid–liquid fractionation method with dichloromethane and ethyl acetate, then further separated
Yen, P.N.D.; Hanh, T.T.M.; Pham, T.N.; by HPLC. The crude extract, ethyl acetate fractions and purified mangiferin were investigated for
Pham, D.T.N. Purification Process of anti-microorganism activity against Escherichia coli, Salmonella spp. and Aspergillus flavus by using the
Mangiferin from Mangifera indica L. paper disc diffusion assay. The results have shown that the extraction efficiency was 14.17%. The
Leaves and Evaluation of Its
obtained mangiferin was 1.97 g of content and 94.2% of purity. The oxidation resistance of purified
Bioactivities. Processes 2021, 9, 852.
mangiferin was 1.77 times higher than the crude extract and 1.05 times higher than the standard
https://doi.org/10.3390/pr9050852
mangiferin (IC50 = 13.841 µg/mL). However, the purified mangiferin has shown no inhibitory action
Academic Editors: Luigi Menghini
against the experimental strains of microorganisms. The findings from the present study suggest an
and Claudio Ferrante effective scheme of extraction and purification to obtain mangiferin from the local variety of mango
with high purity and antioxidant potential.
Received: 29 April 2021
Accepted: 10 May 2021 Keywords: mangiferin; Mangifera indica L.; ultrasonic-assisted extraction; liquid–liquid fractionation;
Published: 12 May 2021 column chromatography; antioxidant activity; antimicrobial activity

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
Mangifera indica L. or mango is one of the common species of the genus Mangifera
(family Anacardiaceae) [1]. It is a medium-to-large evergreen tree with various parts (roots,
barks, leaves, ripe and unripe pulps, seeds, and flowers) that have long been used in eth-
nomedicines [2]. More than 1000 varieties of mango have been identified and distributed
Copyright: © 2021 by the authors. mainly in the tropical areas of South and Southeast Asia [3,4]. During the last decade, the
Licensee MDPI, Basel, Switzerland. global production and export volume of mango took the leading position in the tropical
This article is an open access article fruit section, reaching approximately 47.1 and 1.69 million tons, respectively [5,6]. Nowa-
distributed under the terms and
days, in the situation where the consumers are actively searching for health-promoting
conditions of the Creative Commons
alternatives for traditional crops, mango is one of the tropical fruits that has been in high
Attribution (CC BY) license (https://
demand [7,8]. The pharmaceutical activities of mango are tremendously diverse, which
creativecommons.org/licenses/by/
is considered to derive from the bioactive compounds present in different mango plant
4.0/).

Processes 2021, 9, 852. https://doi.org/10.3390/pr9050852 https://www.mdpi.com/journal/processes

Processes 2021, 9, 852 2 of 14

parts, including protein, vitamin A, vitamin C, carotenoids, phenolic compounds (e.g.,

mangiferin, catechin, quercetin, gallotannins, and iriflophenones), gallic acid, benzoic
acid, fiber, carbohydrates, and minerals [9–11]. The phytochemical profile of mango, and
consequently its biological effects, can be greatly varied according to the variety, maturity
stage and cultivation areas [12].
One of the major constituents of mango leaves, barks, peel and flesh is C-2-β-d-
glucopyranosyl-1,3,6,7-tetrahydroxyxanthone or mangiferin (C19 H18 O11 ) [13]. Numerous
studies have reported a broad range of biological activities displayed by mangiferin, lead-
ing to its role as a potential therapeutic agent for pharmaceutical and cosmetic fields [14].
The abundant presence of mangiferin in mango urges for an effective and sustainable
extraction process that can utilize all plant parts, especially old leaves, in order to carry the
economic implications in minimizing agricultural wastes [15–17]. Previously, mangiferin
was extracted by conventional methods such as Sohlex, heat reflux and maceration extrac-
tion [18]. However, these methods consumed a significant amount of time and solvents,
and the obtained compound was highly sensitive to the operation conditions [19]. With the
recent development of novel extraction methods, such as microwave-assisted extraction,
ultrasonic extraction, and subcritical fluid, several studies have reported the effectiveness
of mangiferin extraction from various sources [20–23]. For instance, ultrasonic extraction
alone and coupled with three-phase partitioning have produced a significantly high extrac-
tion efficiency of mangiferin, which were 58.46 ± 1.27 mg/g and 41 mg/g, respectively,
under optimal conditions [24,25]. These studies confirmed that the use of ultrasonic extrac-
tion improved the mangiferin extraction and shortened the extraction time period, thus this
method is economically feasible and highly applicable on an industrial scale. However, the
extraction efficiency can be significantly interfered with by the presence of impurities in the
crude extract. Furthermore, the biological activities of the desired compound, as well as the
safety and production cost, would also be affected, especially in large-scale production [26].
For these reasons, it is highly demanding to develop an effective purification scheme to
associate with the extraction process. In this regard, column chromatography is favorably
used due to cost effectiveness and applicability of a wide range of stationary phases [27,28].
To our updated knowledge, ultrasonic extraction using the leaves of mango trees grown
in Vietnam has not been performed, and a compatible process for mangiferin purification
from this source has not been established.
In addition, mangiferin has been well-recognized for its antioxidant and anti-microorganism
potentials [29,30]. The antioxidant activity of the compound lies on the formation of
mangiferin–iron chelating complexes and the generation of reactive oxygen species, whereas
the mechanism of action of its anti-microorganism activity has remained unexplored [31].
Mangiferin exhibited antibacterial activity against a wide range of Gram-positive and
Gram-negative bacteria and has also been employed in combinatory therapy to potentiate
the anti-staphylococcal activity of several antibiotics [32,33]. Besides, mangiferin and its
derivatives at a high concentration (30%) effectively prevented Thermoascus aurantiacus and
Aspergillus flavus [34]. However, the effect of mangiferin purity on its anti-microorganism
and antioxidant activities was not highlighted. Although it is known that both the antioxi-
dant and anti-microorganism activities of a compound are closely linked to its purity, to our
best knowledge, there are little to no studies on evaluating these activities of mangiferin
extracted from a local variety of mango leaves.
Considering all the aforementioned gaps, the present study aims to purify mangiferin
from extracts obtained from the ultrasonic extraction of local old mango leaves using liquid–
liquid fractionation and the high-performance liquid chromatography (HPLC) technique,
then evaluate its antimicrobial and antioxidant capabilities.
Processes 2021, 9, 852 3 of 14

2. Materials and Methods

2.1. Plant Materials
Approximately 300 g of fresh and healthy dark-green colored old leaves were har-
vested from a local orchard located in Da Nang (Vietnam). The harvested leaves were
washed thoroughly with tap water for three times, allowed to dry at 45–50 ◦ C for 24 h,
ground into fine powder of 1–2 mm in size and stored in zip bags at room temperature to
avoid material degradation.

2.2. Chemical and Reagents

The reference mangiferin was purchased from Central Institute of Drug Quality
Control of Vietnam (Hanoi, Vietnam) in the form of light yellow powder with 98.6% of
purity. Ethanol (EtOH) (≥96%), dichloromethane, ethyl acetate, chloroform, NaOH, FeCl3 ,
HCl, 2.2-diphenyl-1-picrylhydrazyl (DPPH), and ascorbic acid (vitamin C) were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Deionized water used in all experiments was
prepared by using a Milli-Q water purification system (EMD Millipore, Billerica, MA,
USA). Luria–Bertani (LB) medium, Hansen liquid medium, potato dextrose agar (PDA)
and ampicillin (50 µg/mL) were purchased from Thermo Fisher Scientific (Waltham, MA,
USA). All reagents and chemicals used in the experiments were of analytical grade.

2.3. Ultrasonic Extraction

Ultrasonic extraction was performed in an ultrasonic bath (Elmasonic S100H model,
37 kHz, 600 W) as previously described by Zou et al. [24]. The mango leaves powder (3 g)
was dissolved in ethanol 60% in a glass vessel, which was then immersed into water and
sonication was proceeded under the following conditions: the liquid-to-solid ratios of 5/1,
10/1, 20/1, 30/1, 40/1 and 50/1 v/w, ethanol concentration of 20◦ , 40◦ , 60◦ , 80◦ and 96◦ ,
extraction temperatures of 15 ◦ C, 30 ◦ C, 45 ◦ C, 60 ◦ C and 75 ◦ C and extraction time of 1,
2, 3, 4, 5, 7, 10 and 15 min. After the extraction process had completed, the extract was
centrifuged at 13,000 rpm for 15 min and then subjected to an absorbance measurement at
the wavelength of 318 nm. The experiment was repeated three times and results were the
average values presented as mean ± standard deviation (S.D.). The yield of crude extracts
was calculated using the following formula:

total mass o f crude extract ( g)

Extraction yield = × 100. (1)
total mass o f mango leaves powder ( g)

2.4. Fractionation and Purification of Crude Extract

The crude mango leaves extract was subjected to fractionation and purification by
using liquid–liquid fractionation combined with column chromatography as previously
described by Singh et al. (2012) [31]. Briefly, the crude extract was dissolved in 60% ethanol
and extracted with dichloromethane (1:1 v/v) three times for 24 h, forming upper and
bottom layers. The upper layer was then continuously extracted with ethyl acetate (1:1 v/v)
three times for 24 h, forming upper and bottom layers. Both layers were collected for HPLC
analysis and the layer that had a high mangiferin content was selected for normal-phase
column chromatography, using a glass column filled with silica gel 60 (0.04–0.063 mm) as
stationary phase and chloroform:ethanol with increasing polarity (90:10 to 50:50 v/v) as
mobile phase. Then, mangiferin crystals were collected from vacuum evaporation and
impurities were washed with methanol and acetone [35]. The purified mangiferin was
analyzed by HPLC.

2.5. High-Performance Liquid Chromatography (HPLC) Analysis

Identification of mangiferin content in the crude extracts, as well as its dicholoromethane
and ethyl acetate fractions was performed by using HPLC, following the previous protocol
with some modifications [33]. Briefly, 0.01 g of crude mango leaves extract, 0.03 g of upper
and 0.095 g of bottom layers of ethyl acetate fraction, as well as 1 mg of standard mangiferin
Processes 2021, 9, 852 4 of 14

were accurately weighed. Each sample was then dissolved in 60% ethanol and filtered
through a 0.22-µm nylon filter (Sigma-Aldrich, St. Louis, MO, USA).
The presence of mangiferin was detected by using Agilent 1260 Infinity II HPLC
coupled with a UV/Vis detector and C-18 column (4.6 × 50 mm, 5 µm). Acetonitrile and
acetic acid 0.5% (1:1 v/v) were used as the mobile phase. The flow rate and temperature
were 0.6 mL/min and 25 ◦ C, respectively. A total of 5 µL of each prepared sample was
injected onto the column. The detection wavelength was 318 nm. The chromatogram
processing involved the use of Agilent ChemStation software (Agilent Technologies, Santa
Clara, CA, USA). The mangiferin content and retention time (R.T.) were calculated using
a standard curve obtained from reference mangiferin and results were represented as
milligrams mangiferin per milliliter of extracts (mg·mL−1 ).

2.6. DPPH Scavenging Activity Assay

The antioxidant activity of mango leaves crude extract, ethyl acetate fraction, purified
mangiferin and standard mangiferin was tested by DPPH scavenging assay following
the procedure by Alam et al. (2019) [36]. In brief, the sample was firstly diluted with
ethanol 60 to 1/1700, 1/1500, 1/1300, 1/1100, 1/900, 1/700, 1/300 and 1/100 of the
initial concentrations. Then, 2 mg of DPPH was dissolved in 20 mL of 99.7% ethanol.
Afterwards, 1 mL of sample and 1 mL of DPPH solution were mixed thoroughly with 99.7%
ethanol, vortexed, incubated in the dark at room temperature for 30 min and measured for
absorbance (Abs) at 517 nm by using UV–Vis spectrometer (Hitachi, Tokyo, Japan). The
negative and positive controls were pure 60% ethanol and vitamin C, respectively. The
DPPH scavenging activity (in %) was calculated using Formula (2) as follows. The results
were presented as half maximal inhibitory concentration (IC50 value) [37].

Abs control − Abs sample

% inhibition o f the DPPH = × 100 (2)
Abs control
where Abs control was the Abs of the blank and DPPH mixture and Abs sample was the Abs
of the fraction and DPPH mixture.

2.7. Antimicrobial Assay

The antimicrobial activities of purified mangiferin, reference mangiferin, crude mango
leaves extract, and its ethyl acetate fraction were investigated by using disc diffusion
method as previously described by Mostafa et al. (2018) [38]. E. coli (ATCC 25922),
Salmonella spp. (ATCC 700623), and A. flavus (NBRC 33021) were kindly provided by
the Institute of Biotechnology, Hue University, Vietnam. The bacterial strains and the
fungal strain were maintained in LB broth and liquid Hansen medium (pH = 5.6) at −80 ◦ C,
respectively. Before the experiment, a seed culture of each strain was prepared by inocu-
lating a single colony in fresh media and incubating for 12 h at 37 ◦ C under continuous
agitation (100 rpm). The suspensions were then diluted with the same types of media
to yield a final cell density of approximately 106 colony-forming units (CFU) per mL at
600 nm wavelength.
The prepared cultures of E. coli, Salmonella spp. and A. flavus (10 µL) were uniformly
spread onto the surface of LB agar plates (for E. coli and Salmonella spp.) and PDA plates
(for A. flavus) using a spreader and allowed to dry for 5 min. Meanwhile, the purified
mangiferin, reference mangiferin (which had been dissolved in distilled water), crude
extract, and ethyl acetate fraction at concentrations of 12.5, 25, 50, 100 and 200 mg/mL
were pipetted onto sterilized Whatman paper discs (6 mm in diameter) which were placed
on an empty sterile Petri dish and allowed to dry for about 20 min. Ampicillin antibiotic
(50 µg/mL) which acted as positive control for the assay was also applied following the
same procedure. Subsequently, these sample- and control-loaded paper discs were carefully
and aseptically transferred onto the inoculated agar surface. The plates were incubated
at 37 ◦ C for 24 h. After 24 h of incubation, the diameter of the inhibition zone (mm)
Processes 2021, 9, 852 5 of 14

surrounding the paper discs was measured. The experiment was performed in triplicates
and the results of inhibition zone measurements were presented as mean ± S.D.

2.8. Statistical Analysis

Statistical analysis in the present study was performed using Excel 2010 and Minitab
software version 16 (Minitab, Pennsylvania State University, PA, USA). All results from the
present study were subjected to one-way ANOVA and were expressed as mean ± S.D. The
difference between means was calculated by using Tukey’s multiple comparison test and
was considered to be significant at p-value < 0.05.

3. Results and Discussion

3.1. Effects of Extraction Methods on the Mangiferin Content
3.1.1. Effect of Liquid-to-Solid Ratio
The effect of different liquid-to-solid ratios on the mangiferin content was examined
while the other conditions were set as follows: extraction temperature of 60 ◦ C and ex-
traction time 10 min. The results demonstrated in Figure 1A have shown that the content
of mangiferin decreased significantly from 0.814 to 0.372 Abs as the liquid-to-solid ratio
decreased from 5/1 to 50/1. The mangiferin content at 10/1 of liquid-to-solid ratio was
not significantly different to that of 5/1 (p < 0.05). Furthermore, this ratio allowed the
compound to dissolve in a sufficient quantity of solvents, thus facilitating the subsequent
collection of the extract. Therefore, the liquid-to-solid ratio of 10/1 was selected for the
consecutive experiment.

Figure 1. Effects of different parameters of extraction method on the mangiferin content, as follows: (A) effect of liquid-
to-solid ratio (v/w) on the mangiferin content; (B) effect of extraction temperature (◦ C) on the mangiferin content; and (C)
effect of extraction time (min) on the mangiferin content. Values showing different letters in the same column indicated
significant difference (p < 0.05).
Processes 2021, 9, 852 6 of 14

3.1.2. Effect of Extraction Temperature

The effect of different extraction temperatures on the mangiferin content was examined
while the other conditions were set as follows: liquid-to-solid ratio of 10/1 and extraction
time 10 min. Overall, as shown in Figure 1B, the mangiferin content increased significantly
from 0.778 to 0.842 Abs when the extraction temperature increased from 15 ◦ C to 60 ◦ C.
However, as the temperature continued to rise to 75 ◦ C, the content started to decline to
0.79533 Abs. Previous studies have also reported that the temperature of 60 ◦ C was suitable
to promote the solubility and desorption of mangiferin [24]. In the present study, increasing
the extraction temperature level to higher than 60 ◦ C has resulted in solvent evaporation
and mangiferin decomposition. For this reason, the extraction temperature of 60 ◦ C was
selected for consecutive experiments.

3.1.3. Effect of Extraction Time

The effect of different extraction times on the mangiferin content was examined while
the other conditions were set as follows: liquid-to-solid ratio of 10/1, ethanol concentration
60◦ and extraction temperature of 60 ◦ C. The results are demonstrated in Figure 1C. It can
be observed that the mangiferin content reached the maximum level (0.87 Abs) after 4 min
of extraction. However, as the extraction time prolonged from 5 to 15 min, the mangiferin
content slowly declined. This observation could be explained by the fact that as mangiferin
has achieved the equilibrium state of dissolution at 4 min, extending the extraction time
to more than 4 min would cause evaporation of the solvent as well as exposure to the
environmental degradation of the compound [39]. Therefore, 4 min was selected as a
suitable extraction time for consecutive experiments.

3.2. Mangiferin Quantification by HPLC Analysis

In the present study, the crude extract of local mango leaves has been produced by
using the ultrasonic extraction method and ethanol as the extraction solvent. The extraction
efficiency as calculated from Equation (1) was 14.17% (w/w), which was marginally higher
than the results of a previous work by Akinpelu and Onakoya (2006) (13.93% w/w) [40].
However, our procedure showed superiorities in terms of the extraction time and eco-
nomic efficiency. To be specific, Akinpelu and Onakoya (2006) selected methanol as the
solvent and theextraction process was prolonged for 4 days, while the current process
with the combination of ethanol solvent and ultrasonic extraction yielded a similar ef-
ficiency (14.17%) and only required 4 min of extraction time [40]. For this reason, the
current process can be considered as an improvement in terms of the extraction efficiency
and time, thereby providing an essential insight for future studies on optimization and
industrial-scaled experiments.
The results of the HPLC analysis of mangiferin in the crude extract of mango leaves
obtained from UE are presented in Table 1 and Figure 2. Briefly, when analyzing the
crude extract, there are seven peaks shown with different R.T. and peak areas (Figure 2A).
Two out of these seven peaks were shown at 2.132 and 14.296 min, which were relatively
close to the peaks of the standard mangiferin, shown at 2.086 and 14.164 min (Figure 2B).
Similar results have been obtained by Fernández et al. (2015) [41], confirming the presence
of mangiferin the crude mango leaves extract. However, the peaks shown at 2.132 min
and at 2.086 min of crude extract and standard mangiferin, respectively, were most likely
corresponding to mangiferin isomers such as isomangiferin and homomangiferin, which
are present in mango leaves extract in a minor quantity [2,42]. Therefore, based on the
concentration and the peak area of the crude mango leaves extract shown at 14.296 min,
the presence of mangiferin was identified at a content of 9.510 mg/g.
Processes 2021, 9, 852 7 of 14

Table 1. HPLC analysis of mangiferin content (mg/g) in standard mangiferin, crude mango leaves extract, upper layer of
dichloromethane fraction, bottom and upper layers of ethyl acetate fraction and purified mangiferin.

Sample Conc. (g/mL) Retention Time (min) Peak Area (LU*s) Mangiferin Content (mg/g)
Standard mangiferin 0.001 14.164 1506.542 1000
Crude leaves extract 0.01 14.296 143.269 9.51
Dichloromethane
0.01 14.286 637.637 42.325
fraction (upper layer)
Ethyl acetate fraction
0.03 14.286 4846.101 107.223
(upper layer)
Ethyl acetate fraction
0.095 14.271 125.46 0.877
(bottom layer)
Purified mangiferin 0.8 14.103 1135.188 941.882

Figure 2. Cont.
Processes 2021, 9, 852 8 of 14

Figure 2. Cont.
Processes 2021, 9, 852 9 of 14

Figure 2. Results of HPLC analysis of (A) standard mangiferin, (B) crude mango leaves extract, (C) upper layer of
dichloromethane fraction, (D) bottom layer of ethyl acetate fraction, (E) upper layer of ethyl acetate fraction, and (F)
purified mangiferin.

In the present study, purification was performed to purify mangiferin from the crude
extract of old mango leaves. This step aims to remove the impurities (e.g., colorants and
compounds with weak polarity), which interfere with the efficiency of the extraction process
as well as the biological activities of mangiferin [27,43]. Previously, mangiferin has been suc-
cessfully purified from Chinese mango cultivars by using macroporous HPD100 resin chro-
matography combined with high-speed counter-current chromatography (HSCCC) [43].
However, studies on mangiferin purification from old leaves of mango trees grown in
Vietnam have not been established. Therefore, in this study, mangiferin was purified from
Vietnamese mango leaves extract by liquid–liquid fractionation (e.g., dichloromethane and
ethyl acetate) and column chromatography.
The results from the HPLC analysis revealed the presence of mangiferin in the upper
layer of the dichloromethane fraction, as well as both layers of the ethyl acetate fraction
(Table 1). Firstly, for the dichloromethane fraction, the bottom layer mostly contained
impurities while the upper layer showed eight peaks, and the peak shown at 14.286 min of
R.T. corresponded to mangiferin presence (Figure 2C). The mangiferin content of the upper
layer of the dichloromethane fraction was calculated as 42.325 mg/g and was taken for sub-
sequent fractionation with ethyl acetate. Secondly, for the ethyl acetate fraction, mangiferin
presence was detected in both the upper and bottom layers, as shown by the peaks at
14.271 and 14.286 min, respectively (Figure 2D,E). However, as the mangiferin content was
more concentrated in the upper layer than the bottom layer (Table 1), the upper layer of
the ethyl acetate fraction was selected for column chromatography. After the process had
finished, washing with acetone and methanol helped remove the remaining solvent and
impurities without dissolving the mangiferin crystals. As evidenced by the HPLC results,
the mangiferin obtained after the two-step purification process showed two peaks at 2.088
and 14.103 min, which approached closely to the peaks of standard mangiferin at 2.086
and 14.164 min (Figure 2F). Therefore, similar to the standard mangiferin, the purified
mangiferin may also contain a small quantity of mangiferin isomers, which corresponded
to the peak shown at 2.088 min. Therefore, based on the peak shown at 14.103 min, the
content of the purified mangiferin was identified at a content of 1.97 g (Table 1).
In the present study, a total amount of 1.97 g of mangiferin was obtained from the
extraction and purification of a local variety of mango leaves. The compound recovery was
0.82% and the purity was 94.2%. This result was compared to previous studies, which were
also performed on the leaves of mango trees grown in Vietnam yet employed macroporous
D101 and solvents of a different polarity [44,45]. In these studies, it can be seen that the
recovery and purity of the obtained mangiferin were ranged from 0.37–0.49% and 68–72%,
Processes 2021, 9, 852 10 of 14

respectively, which were lower than the present study. The purity of the mangiferin in
the present study can be comparable to Nian et al. (2016), in which mangiferin of 92.15%
purity was extracted from Anemarrhenae Rhizome by using polyamide and macroporous
HPD400 adsorption resins chromatography [46], as well as Luo et al. (2012), in which
mangiferin (99.13% purity) was purified from Chinese mango cultivars using macroporous
HPD100 resin chromatography associated with HSCCC [43]. Based on the obtained results,
the present study proposed that liquid–liquid fractionation can be combined with column
chromatography to form a cost-effective purification scheme for mangiferin from mango
leaves extraction. Furthermore, HPLC analysis has confirmed that with well-developed
extraction and purification techniques, the local mango leaves appeared to be a highly
available source of mangiferin, thus adding to the value of this by-product and reducing
agricultural waste.

3.3. Antioxidant Activity of Purified Mangiferin

The antioxidant activity of crude ethanol extract, ethyl acetate fractions, standard
mangiferin, and purified mangiferin obtained from UE assisted with the purification, as
shown in Table 2.
Table 2. Antioxidant activity of crude extract, ethyl acetate fractions, standard mangiferin and
purified mangiferin from mango leaves.

Sample IC50 (µg/mL) Regression Equation r2

Crude extract 27.522 y = 2.3945 x0.9167 0.9918
Ethyl acetate fraction
68.769 y = 8.0215 x0.6964 0.9915
(bottom layer)
Ethyl acetate fraction
15.548 y = 4.7598 x0.8571 0.997
(upper layer)
Standard mangiferin 16.383 y = 4.1919 x0.8865 0.9925
Purified mangiferin 13.841 y = 1.7396 x0.7938 0.9978
Vitamin C 2.551 y = 33.427 x0.43 0.9915

As shown in Table 2, the purified mangiferin displayed a significantly low IC50 value
(IC50 = 15.548 µg/mL), as compared to the standard mangiferin (IC50 = 16.383 µg/mL),
upper and bottom layers of the ethyl acetate fraction (IC50 = 15.548 and 68.769 µg/mL) and
the crude extract (IC50 = 27.522 µg/mL) (p < 0.05). In comparison with previous studies, the
free radical scavenging activity exhibited by mangiferin obtained from the present study
was 2.48 and 1.43 times higher than the mangiferin purified by macroporous D101 resin
(IC50 = 38.5 µg/mL) and by solvents with varied polarization (IC50 = 22.2 µg/mL) [45,46]. It
is widely accepted that the evaluation of the biological activities of plant-based extracts such
as antioxidant and anti-microorganism could provide essential insights into the efficiency
of the extraction and purification methods. Therefore, from the results of the present study,
it can be concluded that the combination of ultrasonic extraction, liquid–liquid fractionation
and column chromatography have effectively produced mangiferin with a high antioxidant
potential. Along with the benefits of lowered energy and extraction time consumption, this
combinatory method is therefore highly recommended for mangiferin extraction.

3.4. Antibacterial Activity of Purified Mangiferin

The crude extracts, ethyl acetate fraction (upper layer), purified and standard mangiferin
at various concentrations (12.5–200 mg/mL) were evaluated for anti-microorganism activ-
ity against E. coli, Samonella spp. and A. flavus by measuring the diameter of the inhibition
zone which appeared after 24-h incubation at 37 ◦ C.
As shown in Table 3, the crude extract inhibited the growth of E. coli in a concentration-
dependent manner, with the inhibition zones ranging from 7.7 ± 0.3 to 15.6 ± 0.5 (mm).
In particular, at the concentration of 25 mg/mL, this activity of mango leaves extract
was lower than the methanol extract from mango stem bark, whose inhibition zone was
measured as 12 mm at the concentration of 20 mg/mL [40]. In contrast, the upper layer of
Processes 2021, 9, 852 11 of 14

the ethyl acetate fraction of the mango leaves extract showed a concentration-dependent
inhibition against both E. coli and Samonella spp., indicated by the range of inhibition zones
from 11.3 ± 0.4 to 21.6 ± 0.5 (mm) and from 11.5 ± 0.5 to 23.5 ± 0.5 (mm), respectively.
As compared to Sija (2009), the antibacterial activity of the ethyl acetate fraction of mango
leaves extract in the present study was improved against Samonella spp., while being
comparably effective against E. coli [47]. The inhibitory activity by the ethyl acetate fraction
of the mango leaves extract has been proposed to derive from the presence of mangiferin
as well as several flavonoids and alkaloids that were previously shown to exhibit a high
antibacterial effect [2]. No inhibition against E. coli and Samonella spp. was observed in the
presence of both purified and standard mangiferin. All of the tested samples were unable
to inhibit the growth of A. flavus. The inability of mangiferin to inhibit the fungal species in
this study was contradictory to previous reports by Singh et al. (2012) [31], Stoilova et al.
(2008) [34] and Raju et al. [48], where a high concentration of mangiferin was able to exert a
killing effect on A. flavus and Thermoascus aurantiacus. Overall, these results have indicated
that (1) the ethyl acetate fraction of mango leaves extract exhibited strong antibacterial
activity against E. coli and Samonella spp. at 12.5–200 mg/mL, and (2) this action was not
attributed to the presence of a mangiferin component.

Table 3. Anti-microorganism activity of purified mangiferin against E. coli, Samonella spp. and A.
flavus by using disc diffusion assay.

Inhibition Zone (mm)

Extract Conc. (mg/mL)
E. coli Samonella spp. A. flavus
200 15.6a ± 0.5 - -
100 11.4b ± 0.4 - -
Crude extract 50 10.4c ± 0.2 - -
25 9.5d ± 0.3 - -
12.5 7.7e ± 0.3 - -
200 21.6a ± 0.5 23.5a ± 0.5 -
100 17.5b ± 0.5 17.9b ± 0.3 -
Ethyl acetate
fraction 50 14.6c ± 0.5 15c ± 0.4 -
(upper layer)
25 13.2d ± 0.3 13.9d ± 0.4 -
12.5 11.3e ± 0.4 11.5e ± 0.5 -
200 - - -
100 - - -
Purified
mangiferin 50 - - -
25 - - -
12.5 - - -
200 - - -
100 - - -
Standard
mangiferin 50 - - -
25 - - -
12.5 - - -
200 12 19 -
100 12 19 -
Ampicilin
(positive control) 50 12 19 -
25 12 19 -
12.5 12 19 -
The experiment was performed in triplicates. Values showing different letters in the same column indicated
significant different (p < 0.05). “-“ indicated no inhibition zone in case of bacteria or sparse distribution in case of
fungal spores.
Processes 2021, 9, 852 12 of 14

4. Conclusions
Mangiferin is one of the major constituents of mango that contributes to several
valuable biological activities of the plant. The old leaves from a local variety which
are often considered as wastes were utilized as the raw materials for the present study.
By carrying out UE and developing an effective purification method that consisted of
liquid–liquid fractionation and column chromatography, a total amount of 1.97 g of pure
mangiferin with 0.82% of recovery and 94.2% of purity was obtained from the crude extract
of the old mango leaves. The purified mangiferin showed improved scavenging activity
against the DPPH free radicals, with IC50 = 13.841 ± 0.81 µg/mL, which was higher
than the crude extracts and its ethyl acetate fractions. However, the purified compound
alone exhibited no inhibition against E. coli, Salmonella spp. and A. flavus. The findings
from the present study provide a helpful insight into the utilization of the old leaves of
mango as a promising source of mangiferin extraction to reduce agricultural wastes, and
they show that ultrasound combined with fractionation and column chromatography are
effective extraction and purification methods to obtain mangiferin with a high purity and
antioxidant potential.

Author Contributions: Investigation, N.T.T.L., D.T.L., P.N.D.Y., T.T.M.H. and D.T.N.P.; supervi-
sion, N.T.T.L., D.T.L., P.N.D.Y. and T.N.P.; writing—original draft, N.T.T.L., D.T.L. and D.T.N.P.;
writing—review & editing, D.T.N.P. All authors have read and agreed to the published version of
the manuscript.
Funding: This research is funded by the Funds for Science and Technology Development of the
University of Danang under grant number B2018-ÐN02-45.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ediriweera, M.K.; Tennekoon, K.H.; Samarakoon, S.R. A Review on Ethnopharmacological Applications, Pharmacological
Activities, and Bioactive Compounds of Mangifera indica (Mango). Evid. Based Complementary Altern. Med. 2017, 2017, 1–24.
[CrossRef] [PubMed]
2. Shah, K.; Patel, M.; Patel, R.; Parmar, P. Mangifera indica (Mango). Phcog Rev. 2010, 4, 42. [CrossRef] [PubMed]
3. Solís-Fuentes, J.A.; del Carmen Durán-de-Bazúa, M. Mango (Mangifera indica L.) Seed and Its Fats. In Nuts and Seeds in Health and
Disease Prevention; Elsevier: Amsterdam, The Netherlands, 2011; pp. 741–748. ISBN 9780123756886.
4. Tharanathan, R.N.; Yashoda, H.M.; Prabha, T.N. Mango (Mangifera indica L.), “The King of Fruits”—An Overview. Food Rev. Int.
2006, 22, 95–123. [CrossRef]
5. Food and Agriculture Organization (FAO). Global Prospects for Major Tropical Fruits-Short-Term Outlook, Challenges and
Opportunities in a Vibrant Global Marketplace. 2017. Available online: http://www.fao.org/fileadmin/templates/est/COMM_
MARKETS_Monitoring/Tropical_Fruits/Documents/Tropical_Fruits_Special_Feature.pdf (accessed on 10 January 2021).
6. Evans, E.A.; Ballen, F.H.; Siddiq, M. Mango Production, Global Trade, Consumption Trends, and Postharvest Processing and
Nutrition. In Handbook of Mango Fruit; Siddiq, M., Brecht, J.K., Sidhu, J.S., Eds.; John Wiley & Sons, Ltd.: Chichester, UK, 2017;
pp. 1–16. ISBN 9781119014362.
7. Testa, R.; Tudisca, S.; Schifani, G.; Di Trapani, A.; Migliore, G. Tropical Fruits as an Opportunity for Sustainable Development in
Rural Areas: The Case of Mango in Small-Sized Sicilian Farms. Sustainability 2018, 10, 1436. [CrossRef]
8. Lauricella, M.; Emanuele, S.; Calvaruso, G.; Giuliano, M.; D’Anneo, A. Multifaceted Health Benefits of Mangifera indica L. (Mango):
The Inestimable Value of Orchards Recently Planted in Sicilian Rural Areas. Nutrients 2017, 9, 525. [CrossRef] [PubMed]
9. Coelho, E.M.; de Souza, M.E.A.O.; Corrêa, L.C.; Viana, A.C.; de Azevêdo, L.C.; dos Santos Lima, M. Bioactive Compounds and
Antioxidant Activity of Mango Peel Liqueurs (Mangifera indica L.) Produced by Different Methods of Maceration. Antioxidants
2019, 8, 102. [CrossRef] [PubMed]
10. Núñez Sellés, A.J.; Vélez Castro, H.T.; Agüero-Agüero, J.; González-González, J.; Naddeo, F.; De Simone, F.; Rastrelli, L. Isolation
and Quantitative Analysis of Phenolic Antioxidants, Free Sugars, and Polyols from Mango (Mangifera indica L.) Stem Bark
Aqueous Decoction Used in Cuba as a Nutritional Supplement. J. Agric. Food Chem. 2002, 50, 762–766. [CrossRef]
Processes 2021, 9, 852 13 of 14

11. Fitmawati, F.; Resida, E.; Kholifah, S.N.; Roza, R.M.; Almurdani, M.; Emrizal, E. Phytochemical Screening and Antioxidant
Profiling of Sumatran Wild Mangoes (Mangifera Spp.): A Potential Source for Medicine Antidegenerative Effects. F1000Res 2020,
9, 220. [CrossRef]
12. Maldonado-Celis, M.E.; Yahia, E.M.; Bedoya, R.; Landázuri, P.; Loango, N.; Aguillón, J.; Restrepo, B.; Guerrero Ospina, J.C.
Chemical Composition of Mango (Mangifera indica L.) Fruit: Nutritional and Phytochemical Compounds. Front. Plant. Sci. 2019,
10, 1073. [CrossRef] [PubMed]
13. Iseda, S. On Mangiferin, the Coloring Matter of Mango (Mangifera indica Linn.). V. Identification of Sugar Component and the
Structure of Mangiferin. BCSJ 1957, 30, 629–633. [CrossRef]
14. Telang, M.; Dhulap, S.; Mandhare, A.; Hirwani, R. Therapeutic and Cosmetic Applications of Mangiferin: A Patent Review. Expert
Opin. Ther. Pat. 2013, 23, 1561–1580. [CrossRef]
15. Sagar, N.A.; Pareek, S.; Sharma, S.; Yahia, E.M.; Lobo, M.G. Fruit and Vegetable Waste: Bioactive Compounds, Their Extraction,
and Possible Utilization. Compr. Rev. Food Sci. Food Saf. 2018, 17, 512–531. [CrossRef] [PubMed]
16. Jahurul, M.H.A.; Zaidul, I.S.M.; Ghafoor, K.; Al-Juhaimi, F.Y.; Nyam, K.-L.; Norulaini, N.A.N.; Sahena, F.; Mohd Omar, A.K.
Mango (Mangifera indica L.) by-Products and Their Valuable Components: A Review. Food Chem. 2015, 183, 173–180. [CrossRef]
[PubMed]
17. Alañón, M.E.; Palomo, I.; Rodríguez, L.; Fuentes, E.; Arráez-Román, D.; Segura-Carretero, A. Antiplatelet Activity of Natural
Bioactive Extracts from Mango (Mangifera indica L.) and Its By-Products. Antioxidants 2019, 8, 517. [CrossRef] [PubMed]
18. Lerma-Torres, J.M.; Navarro-Ocaña, A.; Calderón-Santoyo, M.; Hernández-Vázquez, L.; Ruiz-Montañez, G.; Ragazzo-Sánchez,
J.A. Preparative Scale Extraction of Mangiferin and Lupeol from Mango (Mangifera indica L.) Leaves and Bark by Different
Extraction Methods. J. Food Sci. Technol. 2019, 56, 4625–4631. [CrossRef]
19. Zhang, Q.-W.; Lin, L.-G.; Ye, W.-C. Techniques for Extraction and Isolation of Natural Products: A Comprehensive Review.
Chin. Med. 2018, 13, 20. [CrossRef]
20. Salomon, S.; Sevilla, I.; Betancourt, R.; Romero, A.; NuevasPaz, L.; AcostaEsquijarosa, J. Extraction of Mangiferin from Mangifera
indica L. Leaves Using Microwaveassistedtechnique. Emir. J. Food Agric. 2014, 26, 616. [CrossRef]
21. Kulkarni, V.M.; Rathod, V.K. Mapping of an Ultrasonic Bath for Ultrasound Assisted Extraction of Mangiferin from Mangifera
indica Leaves. Ultrason. Sonochemistry 2014, 21, 606–611. [CrossRef]
22. Fernández-Ponce, M.T.; Casas, L.; Mantell, C.; Rodríguez, M.; Martínez de la Ossa, E. Extraction of Antioxidant Compounds from
Different Varieties of Mangifera indica Leaves Using Green Technologies. J. Supercrit. Fluids 2012, 72, 168–175. [CrossRef]
23. Kulkarni, V.M.; Rathod, V.K. A Novel Method to Augment Extraction of Mangiferin by Application of Microwave on Three Phase
Partitioning. Biotechnol. Rep. 2015, 6, 8–12. [CrossRef]
24. Zou, T.-B.; Xia, E.-Q.; He, T.-P.; Huang, M.-Y.; Jia, Q.; Li, H.-W. Ultrasound-Assisted Extraction of Mangiferin from Mango
(Mangifera indica L.) Leaves Using Response Surface Methodology. Molecules 2014, 19, 1411–1421. [CrossRef]
25. Kulkarni, V.M.; Rathod, V.K. Extraction of Mangiferin from Mangifera indica Leaves Using Three Phase Partitioning Coupled with
Ultrasound. Ind. Crop. Prod. 2014, 52, 292–297. [CrossRef]
26. Tandon, S. Distillation technology for essential oils. In Extraction Technologies for Medicinal and Aromatic Plant; Handa, S.S., Khanuja,
S.P.S., Longo, G., Rakesh, D.D., Eds.; International Centre for Science and High Technology: Trieste, Italy, 2008; pp. 115–127.
27. Zhang, Z.; Pang, X.; Xuewu, D.; Ji, Z.; Jiang, Y. Role of Peroxidase in Anthocyanin Degradation in Litchi Fruit Pericarp. Food Chem.
2005, 90, 47–52. [CrossRef]
28. Altemimi, A.; Lakhssassi, N.; Baharlouei, A.; Watson, D.; Lightfoot, D. Phytochemicals: Extraction, Isolation, and Identification of
Bioactive Compounds from Plant Extracts. Plants 2017, 6, 42. [CrossRef] [PubMed]
29. Matkowski, A.; Kus, P.; Goralska, E.; Wozniak, D. Mangiferin—A Bioactive Xanthonoid, Not Only from Mango and Not Just
Antioxidant. Mini Rev. Med. Chem. 2013, 13, 439–455. [CrossRef] [PubMed]
30. Pardo-Andreu, G.L.; Sánchez-Baldoquín, C.; Avila-González, R.; Delgado, R.; Naal, Z.; Curti, C. Fe(III) Improves Antioxidant and
Cytoprotecting Activities of Mangiferin. Eur. J. Pharm. 2006, 547, 31–36. [CrossRef]
31. Singh, S.K.; Tiwari, R.M.; Sinha, S.K.; Danta, C.C.; Prasad, S.K. Antimicrobial Evaluation of Mangiferin and Its Synthesized
Analogues. Asian Pac. J. Trop. Biomed. 2012, 2, S884–S887. [CrossRef]
32. Mazlan, N.A.; Azman, S.; Ghazali, N.F.; Yusri, P.Z.S.; Idi, H.M.; Ismail, M.; Sekar, M. Synergistic antibacterial activity of mangiferin
with antibiotics against Staphylococcus aureus. Drug Invent Today 2019, 12, 14–17.
33. Schieber, A.; Berardini, N.; Carle, R. Identification of Flavonol and Xanthone Glycosides from Mango (Mangifera indica L. Cv.
“Tommy Atkins”) Peels by High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry. J. Agric. Food
Chem. 2003, 51, 5006–5011. [CrossRef]
34. Stoilova, I.; Gargova, S.; Stoyanova, A.; Krastanov, A.I.; Gargova, S.; Ho, L. Antimicrobial and antioxidant activity of the
polyphenol mangiferin. Electron. J. Environ. Agric. Food Chem. 2008, 7, 2706–2716.
35. Jutiviboonsuk, A.; Sardsaengjun, C. Mangiferin in leaves of three thai mango (Mangifera indica L.) varieties. Indian J. Pharm. Sci.
2010, 6, 122–129.
36. Alam, M.K.; Rana, Z.H.; Islam, S.N.; Akhtaruzzaman, M. Total Phenolic Content and Antioxidant Activity of Methanolic Extract
of Selected Wild Leafy Vegetables Grown in Bangladesh: A Cheapest Source of Antioxidants. Potravin. Slovak J. Food Sci. 2019, 13,
287–293. [CrossRef]
Processes 2021, 9, 852 14 of 14

37. González, A.F.; Balmaseda, I.H.; Gaitén, Y.I.G.; Lizama, R.S.; Carmenate García, L.M.; Pieters, L.; Rodeiro, I.; Hernandez, R.D.;
Garrido, G. Phytochemical study and antioxidant capacity of three fractions from the stem of Caesalpinia bahamensis Lam.
J Pharm. Pharmacogn. Res. 2018, 7, 12–20.
38. Mostafa, A.A.; Al-Askar, A.A.; Almaary, K.S.; Dawoud, T.M.; Sholkamy, E.N.; Bakri, M.M. Antimicrobial Activity of Some Plant
Extracts against Bacterial Strains Causing Food Poisoning Diseases. Saudi J. Biol. Sci. 2018, 25, 361–366. [CrossRef] [PubMed]
39. Falleh, H.; Ksouri, R.; Lucchessi, M.-E.; Abdelly, C.; Magné, C. Ultrasound-Assisted Extraction: Effect of Extraction Time and
Solvent Power on the Levels of Polyphenols and Antioxidant Activity of Mesembryanthemum Edule L. Aizoaceae Shoots. Trop. J.
Pharm Res. 2012, 11, 243–249. [CrossRef]
40. Akinpelu, D.; Onakoya, T. Antimicrobial Activities of Medicinal Plants Used in Folklore Remedies in South-Western. Afr. J.
Biotechnol. 2006. [CrossRef]
41. Fernández-Ponce, M.T.; Casas, L.; Mantell, C.; Martínez de la Ossa, E. Use of High Pressure Techniques to Produce Mangifera
indica L. Leaf Extracts Enriched in Potent Antioxidant Phenolic Compounds. Innov. Food Sci. Emerg. Technol. 2015, 29, 94–106.
[CrossRef]
42. Wei, X.; Liang, D.; Wang, Q.; Meng, X.; Li, Z. Total Synthesis of Mangiferin, Homomangiferin, and Neomangiferin. Org. Biomol.
Chem. 2016, 14, 8821–8831. [CrossRef]
43. Luo, F.; Lv, Q.; Zhao, Y.; Hu, G.; Huang, G.; Zhang, J.; Sun, C.; Li, X.; Chen, K. Quantification and Purification of Mangiferin
from Chinese Mango (Mangifera indica L.) Cultivars and Its Protective Effect on Human Umbilical Vein Endothelial Cells under
H2O2-Induced Stress. IJMS 2012, 13, 11260–11274. [CrossRef]
44. Nguyen, T.M.P. Purification of Mangiferin from Mango (Mangifera indica L.) Leaves by Using D101 Absorbent Chromatography
and Evaluation of Antioxidant and Antibacterial Activitites. Master’s Thesis, The University of Danang (University of Science
and Technology), Da Nang, Vietnam, 2019.
45. Tran, T.D. Purification of Mangiferin from Mango Leaves by Traditional Method and Investigation of Biological Activitites.
Master’s Thesis, The University of Danang (University of Science and Technology), Da Nang, Vietnam, 2019.
46. Nian, S.; Liu, E.; Fan, Y.; Alolga, R.N.; Li, H.; Li, P. Orthogonal Separation Protocol for the Simultaneous Preparation of Four
Medically Active Compounds from Anemarrhenae Rhizoma by Sequential Polyamide and Macroporous Resin Adsorbent
Chromatography: Sample Preparation. J. Sep. Sci. 2016, 39, 3195–3204. [CrossRef]
47. Sija, S.L. In Vitro Antioxidant Activity and Antibacterial Activity of Ethyl Acetate Extract of Medicinal Tree Species, Anacardium
occidentale L. and Mangifera indica L. IJAEB 2019, 12. [CrossRef]
48. Raju, N.V.; Sukumar, K.; Reddy, G.B.; Pankaj, P.K.; Muralitharan, G.; Annapareddy, S. In-Vitro Studies on Antitumour and
Antimicrobial Activities of Methanolic Kernel Extract of Mangifera indica L. Cultivar Banganapalli. Biomed. Pharm. J. 2019, 12,
357–362. [CrossRef]