Antityrosinase Activity of Combretum micranthum, Euphorbia hirta and Anacardium occidentale Plants: Ultrasound Assisted Extraction Optimization and Profiling of Associated Predominant Metabolites

Abstract

Tyrosinase is an important component of the enzyme polyphenol oxidase, which upon contact with the phenolic substrates forms the pigment melanin and induces undesirable food browning. The phenolic and triterpenoid compounds that naturally occur in plants are well known as tyrosinase inhibitors. Combretum micranthum (CM) leaves, Euphorbia hirta (EH) plant, and Anacardium occidentale (AO) fruits are traditionally known to have potential anti-tyrosinase activities. The aim of this study was to optimize the ultrasound-assisted extraction of secondary metabolites from these matrices, and to evaluate in tubo the antityrosinase activity of these extracts. Efforts were also taken to profile the secondary metabolites, mainly the phenolic and triterpenoid compounds, in order to understand their probable association with tyrosinase inhibition. The optimal ultrasound-assisted extraction conditions for simultaneous extraction of phenolic, and triterpenoid compounds were determined. The aqueous fraction of these extracts showed significant antityrosinase activity, with the CM leaves exhibiting the strongest inhibitory effect (IC50 of 0.58 g·L−1). The predominant metabolic compounds from these natural extracts were putatively identified by using a high-resolution quadrupole-time of flight (QToF) LC-MS instrument. The high-resolution accurate mass-based screening resulted in identification of 88 predominant metabolites, which included dihydrodaidzein-7-O-glucuronide, micromeric acid, syringic acid, morin, quercetin-3-O-(6″-malonyl-glucoside), 4-hydroxycoumarin, dihydrocaffeic acid-3-O-glucuronide, to name some, with less than 5 ppm of mass error.

Full text

molecules
Article
Antityrosinase Activity of Combretum micranthum,
Euphorbia hirta and Anacardium occidentale Plants:
Ultrasound Assisted Extraction Optimization and
Profiling of Associated Predominant Metabolites
Hussein Zeitoun 1, *, Zareen Khan 2, Kaushik Banerjee 2, Dominique Salameh 1and
Roger Lteif 1
1
Unit
é
de Technologie et Valorisation Alimentaire, Centre d’Analyses et de Recherche, Universit
é
Saint-Joseph,
Facultédes sciences, Campus des Sciences et Technologies, Mar Roukos, Mkallès, P.O. Box 11- 514,
Riad El Solh,
Beirut 1107 2050, Lebanon; Dominique.salameh@usj.edu.lb (D.S.); Roger.lteif@usj.edu.lb (R.L.)
2National Reference Laboratory, ICAR-National Research Centre for Grapes, Pune 412307, India;
Zareenk19@gmail.com (Z.K.); kbgrape@yahoo.com (K.B.)
*Correspondence: Hussein.zeitoun@net.usj.edu.lb; Tel.: +221-77-644-21-21
Received: 29 April 2020; Accepted: 15 May 2020; Published: 9 June 2020


Abstract:
Tyrosinase is an important component of the enzyme polyphenol oxidase, which upon
contact with the phenolic substrates forms the pigment melanin and induces undesirable food
browning. The phenolic and triterpenoid compounds that naturally occur in plants are well
known as tyrosinase inhibitors. Combretum micranthum (CM) leaves, Euphorbia hirta (EH) plant,
and Anacardium occidentale (AO) fruits are traditionally known to have potential anti-tyrosinase
activities. The aim of this study was to optimize the ultrasound-assisted extraction of secondary
metabolites from these matrices, and to evaluate in tubo the antityrosinase activity of these
extracts. Efforts were also taken to profile the secondary metabolites, mainly the phenolic and
triterpenoid compounds, in order to understand their probable association with tyrosinase inhibition.
The optimal ultrasound-assisted extraction conditions for simultaneous extraction of phenolic,
and triterpenoid compounds were determined. The aqueous fraction of these extracts showed
significant antityrosinase activity, with the CM leaves exhibiting the strongest inhibitory effect
(IC
50
of 0.58 g
·
L
−1
). The predominant metabolic compounds from these natural extracts were
putatively identified by using a high-resolution quadrupole-time of flight (QToF) LC-MS instrument.
The high-resolution accurate mass-based screening resulted in identification of 88 predominant
metabolites, which included dihydrodaidzein-7-O-glucuronide, micromeric acid, syringic acid, morin,
quercetin-3-O-(6”-malonyl-glucoside), 4-hydroxycoumarin, dihydrocaffeic acid-3-O-glucuronide,
to name some, with less than 5 ppm of mass error.
Keywords:
Combretum micranthum;Euphorbia hirta;Anacardium occidentale; ultrasound
assisted-extraction; metabolic profiling; antityrosinase activity
1. Introduction
The browning effect can degrade the appearance, nutritional value, shelf life and marketability [
1
]
of fruits and vegetables. In the food industry the use of tyrosinase enzyme inhibitors as anti-browning
agents is not common due to the concerns related to their food safety, off-flavors, and lack of
economic feasibility. The most used anti-browning compounds include ascorbic acid, sodium chloride,
L-cysteine, and sodium metabisulfite. Although the sulfite-containing compounds are also known
for their anti-browning effects, their use might cause allergic reactions to consumers, and therefore,
Molecules 2020,25, 2684; doi:10.3390/molecules25112684 www.mdpi.com/journal/molecules

Molecules 2020,25, 2684 2 of 24
have been banned by the U.S. Food and Drug Administration since 1986. The high importance of food
safety in the industry leads researchers across the world to a quest for natural plant-based browning
inhibitors as they are anticipated to be free of unkind side effects [2,3].
The anti-browning agents that are commonly used in food industry include butylated
hydroxytoluene, butylated hydroxyanisole, and propyl gallate, however, these compounds are
suspected to cause liver damage, and carcinogenesis [
4
,
5
]. Therefore, it became necessary to find
alternatives to these chemical agents. In literature, the secondary metabolites in plant extracts have
been reported to provide a safer alternative to food industries in preventing browning of foods
and beverages [
4
]. It has been demonstrated that the tyrosinase inhibitors in plant extracts work
synergistically, and provide browning inhibitory activities [
6
]. Since plant extracts contain numerous
phenolic and triterpenoid compounds with potential antityrosinase activities, they are expected to
provide a high inhibitory effect on browning reactions. Lim et al. examined the inhibitory effects
of chemical agents vis-
à
-vis natural products on the polyphenol oxidase activity in sweet potatoes,
and reported a slightly lower inhibition in comparison to the chemical agents [3].
The distribution of phenolic compounds in the plant kingdom is widespread and includes a wide
range of molecules with diverse chemical structures and functions. These molecules have in their
structure at least one aromatic ring grafted with one or more hydroxyl groups, which allow them to
act as inhibitors of melanogenesis that prohibits the expression of tyrosinase enzyme [
7
,
8
]. A recent
review on phenolic and terpenoid compounds has also shown the importance of these compounds as
food additives [
9
]. In fact, these compounds may be regarded as food preservatives since they have
antimicrobial, antioxidant, and anti-browning properties [
10
]. In addition, extracts containing phenolic
and terpenoid compounds may be useful for the development of products with enhanced nutritional
value, potential health benefits, longer shelf-life, and good sensory profile [
11
]. The triterpene group
of compounds (include triterpenes and sterols) might accumulate in plants as glycosides (saponins)
in extensive amounts. The terpenoid compounds have diverse industrial applications because of
their wide variety, which range from simple (flavor and fragrance) to complex (tetraterpene and
polyterpene) compounds [
12
]. Triterpenoids are also reported to inhibit the production of melanin
because of their antityrosinase properties [
8
,
13
]. During food processing, wounding stimulates
oxidation of phenolic compounds and enzymatic activity, which might lead to change in color attributes
(browning) that damages the appearance of foods. This reaction is mediated by the activity of
polyphenol oxidase, which upon contact with the phenolic substrates, form melanin pigment [
14
].
Since phenolic and triterpenoid compounds inhibit the enzymatic activity of tyrosinase (an important
class of polyphenol oxidase), these plant metabolites may be useful for preventing enzymatic browning
in fruits and vegetables.
Generally, chemical agents are used for the prevention of browning in plant-derived foods.
Plant extracts (and/or compounds from natural sources) that have health benefits for consumers,
if provide safe and effective control of browning in food products, are much appreciated.
So, plant extracts have recently become a subject of high interest for their antityrosinase activity
because of their richness in bioactive compounds. Researchers around the world take attempts to
identify such inhibitors from plant sources considering their less toxicity and better bioavailability for
food applications [
6
,
14
]. Most researchers focus on identifying plant extracts with antityrosinase activity
without understanding which compounds are associated to this effect [
15
,
16
]. However, only few papers
report their chemical composition [
16
]. It is well known that phenolic and triterpenoid compounds are
the most bioactive compounds that are able to inhibit the tyrosinase enzymatic activity [6].
In this study, three plant materials from Senegal were selected as raw materials based on their
bioavailability, popularity, and low cost. These include Combretum micranthum leaves, Euphorbia hirta
plant and Anacardium occidentale fruits. The leaf of Combretum micranthum (CM) is widely known
for its medicinal properties in traditional African medicine. However, the metabolite profile of the
leaves of this plant has largely remained under-explored [
17
,
18
]. Euphorbia hirta (EH) is another plant,
the extract of which is known for the treatment of gastrointestinal diseases, and disorders [
19
]. It is

Molecules 2020,25, 2684 3 of 24
also used as an antidote and pain reliever for scorpion stings or snakebites [
20
]. However, information
on the compounds that might be responsible for such bio-efficacies is scarce. Similarly, fruits of
Anacardium occidentale (AO) [
21
] are becoming more and more popular as new evidences on the
biological properties of its extract are being reported that include antimicrobial, anti-mutagenic,
and anti-inflammatory activities. It also serves as a urease inhibitor, and exerts lipoxygenasic activity,
to name some. The major classes of bioactive compounds in this fruit that have been reported so
far include carotenoids, vitamin C, and polyphenols [
22
]. In the literature, only few investigations
have been reported so far on the phenolic and triterpenoid profiling of CM leaves, EH plant, and AO
fruit extracts. Some studies have reported HPLC-based identification of select phenolic compounds,
which includes isolation and identification of 13 phenolic compounds in CM leaves [
17
], 14 flavonoids
in AO fruits [
22
], and 17 phenolic compounds in EH plant extracts [
20
,
23
]. In this study, these three
extracts were screened for the predominant phenolic compounds and other phytochemicals with a
non-target approach using a high-resolution quadrupole-time of flight (QToF) LC-MS. All phenolic
compounds and other phytochemicals were identified based on high-resolution accurate mass analysis
with the data processing through UNIFI
®
, which is a unique compound identification software solution.
The aims of this study were to establish the optimal conditions of ultrasound-assisted extraction
of phenolic and triterpenoid compounds from CM leaves, EH plant, and AO fruits, measure their
antityrosinase activity, and establish the profile of the predominant bioactive metabolites that might be
responsible for their antityrosinase activity.
2. Results and Discussion
2.1. Fitting the Models
The complete design consisted of twenty experiments. The average values of two responses
(total phenolic and total triterpenoid contents) and variances expressed by standard variation (n=3)
for each plant are presented in Table 1. To measure how well our model fitted to the experimental
data, the parameters such as p-value, F-value, and coefficient of determination (R
2
) were evaluated.
The ANOVA analysis evaluated the significance of the quadratic polynomial models. The R
2
value
was always between 0 and 1. The closer the R
2
value to 1, the stronger was the model to predict
the response. For a good fit of a model, an R
2
value of at least 80% was considered [
24
]. From the
ANOVA (Table 2), it was found that R
2
for CM, EH and AO for the two responses were higher than 80%.
The form of the models chosen to explain the relationship between the three factors (A, B and C) and
the response (TPC and TTC) were well correlated and attested so that the developed models described
the true behaviour of the process. The significance of each coefficient was determined using F-value
and the corresponding p-value. Therefore, factors A, B, AA, AC and BC showed significant effects
(p<0.05)
on the extraction recovery of total phenolic compounds for CM. Factors A, B, AA, AC, and
CC also showed significant effects (p<0.05) on the extraction of total triterpenoid compounds for CM.
The only two significant effects for EH on the extraction of TPC were the factors A and AA, while A,
B, C, and AA had significant effects on the extraction of TTC for the same plant. Statistical analysis
revealed that the significant effects concerning TPC included A, B, C, AA, and CC for AO, while the
significant effects concerning TTC comprised B, C, AA, AB, AC, BB, and BC. The larger the value of F
and the smaller the value of p, the more significant was the corresponding coefficient term. At 95%
confidence level, the model was significant when p-value was lower than 0.05 [
25
]. The lack-of-fit
test was used to investigate the fitness of a model. A p-value above 0.05 indicated suitability of the
model to accurately predict the variations [
24
]. All models showed statistically insignificant lack of fit
(p-value greater than 0.05) except for models fitted for CM which were very close to 0.05 but not greater
(0.0497 for TPC and TTC) than 0.05. In general, a model was found to be well fitted to the experimental
data if it presented a significant regression, and a non-significant lack of fit [26].

Molecules 2020,25, 2684 4 of 24
Table 1. Experimental data for the responses obtained from C. micrathum leaves, E. hirta plant and A. occidentale fruits.
Run
Combretum micranthum Leaves Euphorbia hirta Plant Anacardium occidentale
TPC (mg GAE/g DW) TTC (mg UAE/g DW) TPC (mg GAE/g DW) TTC (mg UAE/g DW) TPC (mg GAE/g DW) TTC (mg UAE/g DW)
Average
Value
Standard
Deviation
Average
Value
Standard
Deviation
Average
Value
Standard
Deviation
Average
Value
Standard
Deviation
Average
Value
Standard
Deviation
Average
Value
Standard
Deviation
1 73.01 2.69 70.30 4.48 36.89 1.94 13.40 0.61 9.91 0.28 16.22 0.14
2 67.44 0.51 73.06 2.94 26.32 1.68 11.43 0.68 8.38 0.17 9.15 0.18
3 80.29 1.1 68.36 6.36 43.33 4.61 14.46 0.32 10.83 0.33 15.94 0.73
4 74.37 2.04 85.60 3.60 32.49 2.38 12.44 1.78 8.93 0.17 14.46 1.33
5 70.97 0.74 60.70 0.75 41.11 4.22 14.52 0.84 9.82 0.12 16.34 1.10
6 78.09 1.02 89.82 8.39 27.55 2.40 12.29 0.09 8.54 0.10 15.85 0.21
7 69.37 1.95 69.35 3.33 41.37 2.24 14.92 0.61 10.65 0.26 14.67 0.50
8 78.73 1.1 88.57 2.19 31.39 1.26 13.76 0.52 10.00 0.14 15.58 0.68
9 78.61 0.17 81.44 2.53 37.13 3.71 13.10 0.83 10.19 0.17 18.46 1.55
10 78.09 0.74 79.03 3.40 40.27 1.68 13.15 0.92 10.29 0.37 18.18 1.17
11 80.98 1.87 80.56 1.94 43.15 3.89 13.15 0.40 10.74 0.45 20.15 1.67
12 77.93 1.9 80.95 2.59 38.64 2.72 12.49 0.35 10.26 0.08 19.98 2.90
13 80.17 0.2 83.10 1.87 38.70 3.07 12.70 0.61 10.08 0.16 18.40 0.21
14 77.97 0.34 76.83 2.36 37.73 0.68 12.49 0.17 10.43 0.08 18.06 1.37
15 83.78 0.99 88.43 3.78 40.56 2.80 12.70 0.15 10.41 0.18 18.03 0.14
16 77.81 0.85 81.88 1.56 35.06 0.85 12.55 0.52 11.71 0.11 19.55 1.51
17 78.89 0.85 77.80 2.56 37.78 1.63 12.34 0.38 10.25 0.02 11.61 1.76
18 84.46 0.23 81.51 2.50 37.99 1.40 13.86 0.53 11.43 0.08 19.25 0.88
19 62.08 1.59 49.93 3.04 38.96 2.71 14.36 0.76 9.14 0.15 14.49 0.76
20 71.01 0.76 75.84 2.13 20.93 0.85 12.60 0.35 7.35 0.13 14.15 0.60

Molecules 2020,25, 2684 5 of 24
Table 2.
Analysis of variance (ANOVA) for the second order polynomial models of Combretum micranthum leaves, Euphorbia hirta plant and Anacardium occidentale fruits.
Total Phenolic Content Total Triterpenoid Content
Source SS 1Df 2MS 3F-Ratio p-Value SS Df MS F-Ratio p-Value
Combretum micranthum
A429.314 1 29.314 17.32 0.0088 917.124 1 917.124 196.96 <0.0001
B537.4578 1 37.4578 22.13 0.0053 43.0225 1 43.0225 9.24 0.0288
C64.67492 1 4.67492 2.76 0.1575 0.000796 1 0.000796 0 0.9901
AA 337.811 1 337.811 199.54 <0.0001 544.127 1 544.127 116.86 0.0001
AB 0.446512 1 0.446512 0.26 0.6294 2.62205 1 2.62205 0.56 0.4868
AC 97.7901 1 97.7901 57.76 0.0006 100.394 1 100.394 21.56 0.0056
BB 3.71458 1 3.71458 2.19 0.1986 0.66977 1 0.66977 0.14 0.7201
BC 28.7661 1 28.7661 16.99 0.0092 1.28 1 1.28 0.27 0.6225
CC 0.556849 1 0.556849 0.33 0.5911 43.0799 1 43.0799 9.25 0.0287
Lack-of-fit 42.884 5 8.57679 5.07 0.0497 117.926 5 23.5851 5.07 0.0497
Pure error 8.46488 5 1.69298 23.2815 5 4.6563
Total (corr.) 606.903 19 1829.9 19
R291.5392 92.2834
R2adjusted 83.9245 85.3384
Euphorbia hirta
A 414.882 1 414.882 87.43 0.0002 7.82864 1 7.82864 74.68 0.0003
B 21.3192 1 21.3192 4.49 0.0876 3.09019 1 3.09019 29.48 0.0029
C 3.44571 1 3.44571 0.73 0.4331 0.900954 1 0.900954 8.59 0.0326
AA 154.863 1 154.863 32.63 0.0023 1.22537 1 1.22537 11.69 0.0189
AB 1.36951 1 1.36951 0.29 0.6142 0.13005 1 0.13005 1.24 0.316
AC 0.567113 1 0.567113 0.12 0.7436 0.045 1 0.045 0.43 0.5413
BB 3.19556 1 3.19556 0.67 0.4492 0.356344 1 0.356344 3.4 0.1245
BC 9.05251 1 9.05251 1.91 0.2258 0.005 1 0.005 0.05 0.8358
CC 3.56558 1 3.56558 0.75 0.4257 0.00164 1 0.00164 0.02 0.9051
Lack-of-fit 28.0986 5 5.61971 1.18 0.4287 1.89648 5 0.379295 3.62 0.0922
Pure error 23.7274 5 4.74548 0.524133 5 0.104827
Total (corr.) 658.003 19 15.9239 19
R292.1237 84.7989
R2adjusted 85.0351 71.1179

Molecules 2020,25, 2684 6 of 24
Table 2. Cont.
Total Phenolic Content Total Triterpenoid Content
Source SS 1Df 2MS 3F-Ratio p-Value SS Df MS F-Ratio p-Value
Anacardium occidentale
A 5.1303 1 5.1303 96.19 0.0002 5.54457 1 5.54457 6.31 0.0537
B 2.41633 1 2.41633 45.3 0.0011 18.6022 1 18.6022 21.18 0.0058
C 0.724866 1 0.724866 13.59 0.0142 6.23314 1 6.23314 7.1 0.0447
AA 9.73445 1 9.73445 182.51 <0.0001 50.4501 1 50.4501 57.44 0.0006
AB 0.00845 1 0.00845 0.16 0.707 6.10751 1 6.10751 6.95 0.0461
AC 0.28125 1 0.28125 5.27 0.0701 10.0576 1 10.0576 11.45 0.0196
BB 0.131704 1 0.131704 2.47 0.1769 31.5062 1 31.5062 35.87 0.0019
BC 0.08405 1 0.08405 1.58 0.2648 6.07261 1 6.07261 6.91 0.0466
CC 0.433212 1 0.433212 8.12 0.0358 1.21737 1 1.21737 1.39 0.2921
Lack-of-fit 0.977908 5 0.195582 3.67 0.0901 22.1435 5 4.4287 5.04 0.0502
Pure error 0.266683 5 0.0533367 4.39168 5 0.878337
Total (corr.) 20.9194 19 153.931 19
R294.0505 82.7616
R2adjusted 88.696 67.2471
1Sum of squares; 2Degrees of freedom; 3Mean square; 4Ethanol concentration; 5Temperature; 6Processing time

Molecules 2020,25, 2684 7 of 24
Multiple regression equations were obtained in terms of coded factors, which described the effect
of three independent parameters: ethanol concentration, extraction temperature, and extraction time.
The equations generated allowed prediction of TPC and TTC extraction efficiency by empirical models
(Equations (1) to (6)):
TPC (CM) =44.825 +0.752 A +0.280 B −0.064 C −0.012 AA +0.0016 AB+0.0175 AC
+0.0090 BB −0.025 BC +0.0020 CC (1)
TTC (CM) =51.428 +1.363 A +0.585 B −2.192 C −0.015 AA +0.0038 AB+0.018 AC
−0.0038 BB −0.0053 BC +0.017 CC (2)
TPC (EH) =−28.733 +0.630 A +1.364 B +1.101 C −0.0082 AA +0.0028 AB −0.0013 AC
−0.0084 BB −0.014 BC −0.0050 CC (3)
TTC (EH) =22.518 −0.181 A −0.240 B +0.028 C +0.00073 AA +0.00085 AB+0.00038 AC
+0.0028 BB −0.00033 BC −0.00011 CC (4)
TPC (AO) =13.273 +0.168 A −0.173 B −0.237 C −0.0021 AA +0.00022 AB+0.00094 AC
+0.0017 BB +0.0014 BC +0.0017 CC (5)
TTC (AO) =−62.084 +0.029 A +2.768 B +0.515 C −0.0047 AA +0.0058 AB +0.0056 AC
−0.026 BB −0.012 BC −0.0029 CC (6)
2.2. Effect of Process Variables
The results presented in contour plots in Figures 1–3show the effect of the ultrasound-assisted
extraction parameters on the responses (TPC and TTC). These graphs were drawn by maintaining one
factor constant and varying the two other factors.
2.2.1. Effect of Ethanol Concentration and Extraction Time on TPC and TTC
The effects of ethanol concentration (A) and extraction time (C) on TPC and TTC corresponding to
the extraction temperature of 47.5
◦
C are reflected in Figure 1a–c, which show that TPC increased as
the ethanol concentration increased.
However, beyond a certain ethanol concentration, TPC decreased significantly. In fact, extraction
of phenolic compounds from plant material and their solubility depended on the nature of the solvent
used and its polarity [
27
]. At the optimized level of ethanol concentration, TPC increased with
increasing extraction time for CM and AO. A larger contact time between the solvent and the solids
improved the diffusion of the compounds to be extracted [
28
]. For EH, TPC decreased with an increase
in extraction time. This was probably due to the degradation of certain compounds after a long time
of exposure to ultrasonic irradiation [
29
]. Figure 1d–f showed that TTC increased significantly with
increasing level of ethanol concentration up to a certain value, after which it diminished progressively
except for EH for which TTC always decreased with a higher ethanol concentration. The extraction
yield was affected in response to variations in solvent polarity from water to ethanol. The extraction
yield was also decreased with a lower water percentage due to the change in polarity and decrease in
effective swelling of plant matrix [
30
]. At the optimum ethanol concentration, TTC always increased
when ultrasonic extraction time was longer. Exposure to ultrasound irradiation for 60 min did not
degrade triterpenoids, and the equilibrium of desorption was also not attainable.

Molecules 2020,25, 2684 8 of 24
Molecules 2020, 25, x 8 of 24
2.2. Effect of Process Variables
The results presented in contour plots in Figures 1−3 show the effect of the ultrasound-assisted
extraction parameters on the responses (TPC and TTC). These graphs were drawn by maintaining
one factor constant and varying the two other factors.
2.2.1. Effect of Ethanol Concentration and Extraction Time on TPC and TTC
The effects of ethanol concentration (A) and extraction time (C) on TPC and TTC corresponding
to the extraction temperature of 47.5 °C are reflected in Figure 1a–c, which show that TPC increased
as the ethanol concentration increased.
Figure 1. Response surface plot showing the effect of ethanol concentration and extraction time on
total phenolic and total triterpenoid compounds from Combretum micranthum (a,d), Euphorbia hirta
(b,e) and Anacardium occidentale (c,f) corresponding to extraction temperature of 47.5 °C.
However, beyond a certain ethanol concentration, TPC decreased significantly. In fact,
extraction of phenolic compounds from plant material and their solubility depended on the nature
of the solvent used and its polarity [27]. At the optimized level of ethanol concentration, TPC
increased with increasing extraction time for CM and AO. A larger contact time between the solvent
and the solids improved the diffusion of the compounds to be extracted [28]. For EH, TPC decreased
Figure 1.
Response surface plot showing the effect of ethanol concentration and extraction time on
total phenolic and total triterpenoid compounds from Combretum micranthum (
a
,
d
), Euphorbia hirta (
b
,
e
)
and Anacardium occidentale (c,f) corresponding to extraction temperature of 47.5 ◦C.
2.2.2. Effect of Extraction Temperature and Ethanol Concentration on TPC and TTC
The effects of temperature (B) and ethanol concentration (A) on TPC and TTC corresponding to
an extraction time of 40 min are presented in Figure 2a–c.
These show that TPC increased when the extraction temperature increased.
Indeed, an augmentation of extraction temperature increased the solvent diffusivity into the
plant matrix and enhanced the desorption and solubility of the targeted compounds [
31
]. Until the
optimum temperature was reached, TPC increased with increasing ethanol concentration, but beyond
a certain concentration, TPC decreased significantly. These results are explained in Figure 1a–c.
Figure 2d,e show that TTC always increased when the extraction temperature increased for CM and
EH. Figure 2f shows that for AO, TTC increased with a rise in temperature until it reached a certain
value, after which, TTC diminished progressively. These results prove that the triterpenoids content in
AO degraded at a high temperature. TTC had an increasing trend with a rise in ethanol concentration.
However, beyond certain level of ethanol in CM and AO, TTC started decreasing. However, the results
for EH were opposite. In this case, with a rise in ethanol concentration, TTC recovery initially
decreased, which subsequently increased with a further rise in ethanol concentration. For CM and AO,
the results are in agreement with Figure 1d,f. For EH, the nature of triterpenoids extracted through
sonication were different according to the polarity of the solvent used in the extraction. At a lower

Molecules 2020,25, 2684 9 of 24
ethanol concentration, the polar triterpenoids were extracted, but at a higher ethanol concentration,
less polar triterpenoids were extracted.
Molecules 2020, 25, x 9 of 24
with an increase in extraction time. This was probably due to the degradation of certain compounds
after a long time of exposure to ultrasonic irradiation [29]. Figure 1d–f showed that TTC increased
significantly with increasing level of ethanol concentration up to a certain value, after which it
diminished progressively except for EH for which TTC always decreased with a higher ethanol
concentration. The extraction yield was affected in response to variations in solvent polarity from
water to ethanol. The extraction yield was also decreased with a lower water percentage due to the
change in polarity and decrease in effective swelling of plant matrix [30]. At the optimum ethanol
concentration, TTC always increased when ultrasonic extraction time was longer. Exposure to
ultrasound irradiation for 60 min did not degrade triterpenoids, and the equilibrium of desorption
was also not attainable.
2.2.2. Effect of Extraction Temperature and Ethanol Concentration on TPC and TTC
The effects of temperature (B) and ethanol concentration (A) on TPC and TTC corresponding to
an extraction time of 40 min are presented in Figure 2a,b, and c.
Figure 2. Response surface plot showing the effect of ethanol concentration and extraction
temperature on total phenolic and total triterpenoid compounds from Combretum micranthum (a,d),
Euphorbia hirta (b,e) and Anacardium occidentale (c,f) corresponding to extraction time of 40 min.
These show that TPC increased when the extraction temperature increased. Indeed, an
augmentation of extraction temperature increased the solvent diffusivity into the plant matrix and
enhanced the desorption and solubility of the targeted compounds [31]. Until the optimum
temperature was reached, TPC increased with increasing ethanol concentration, but beyond a certain
Figure 2.
Response surface plot showing the effect of ethanol concentration and extraction
temperature on total phenolic and total triterpenoid compounds from Combretum micranthum (
a
,
d
),
Euphorbia hirta (b,e) and Anacardium occidentale (c,f) corresponding to extraction time of 40 min.
2.2.3. Effect of Extraction Time and Extraction Temperature on TPC and TTC
The effects of extraction temperature (B) and extraction time (C) on TPC and TTC corresponding
to an ethanol concentration of 60% are presented in Figure 3a,b.
For CM and EH, the results showed that TPC decreased when the extraction time increased at the
higher temperature. On the other hand, at a lower temperature, TPC increased when extraction time
increased. For AO (Figure 3c), regardless of temperature, TPC increased with increasing extraction
time. In fact, ultrasound treatment disrupted the plant matrix rapidly and augmented the contact area
with the solvent. Nevertheless, on a prolonged exposure to ultrasonic wave and high temperature,
the targeted compounds had a chance to get oxidized or decomposed [
32
]. Moreover, at a lower
temperature, when the vapor pressure was lower, there were few cavitation bubbles, but those
collapsed with relatively high intensity and thereby enhanced the cell disruption. However, at the
higher temperature, more bubbles were created, which collapsed with relatively less intensity due
to a smaller pressure difference between inside and outside of the bubbles [
33
,
34
]. Figure 3e,f show
that TTC increased with an increase in extraction time for EH and AO. Figure 3d showed that TTC
diminished when the extraction time was increased until it reached to a minimum level. Subsequently,
it increased progressively with extraction time. In addition, when the optimum extraction time was

Molecules 2020,25, 2684 10 of 24
maintained, TTC always increased when extraction temperature increased. These results demonstrated
the synergic effect of time and temperature in ultrasound-assisted extraction.
Molecules 2020, 25, x 10 of 24
concentration, TPC decreased significantly. These results are explained in Figure 1a,b and c. Figure
2d,e show that TTC always increased when the extraction temperature increased for CM and EH.
Figure 2f shows that for AO, TTC increased with a rise in temperature until it reached a certain value,
after which, TTC diminished progressively. These results prove that the triterpenoids content in AO
degraded at a high temperature. TTC had an increasing trend with a rise in ethanol concentration.
However, beyond certain level of ethanol in CM and AO, TTC started decreasing. However, the
results for EH were opposite. In this case, with a rise in ethanol concentration, TTC recovery initially
decreased, which subsequently increased with a further rise in ethanol concentration. For CM and
AO, the results are in agreement with Figure 1d,f. For EH, the nature of triterpenoids extracted
through sonication were different according to the polarity of the solvent used in the extraction. At a
lower ethanol concentration, the polar triterpenoids were extracted, but at a higher ethanol
concentration, less polar triterpenoids were extracted.
2.2.3. Effect of Extraction Time and Extraction Temperature on TPC and TTC
The effects of extraction temperature (B) and extraction time (C) on TPC and TTC corresponding
to an ethanol concentration of 60% are presented in Figure 3a,b.
Figure 3. Response surface plot showing the effect of extraction temperature and extraction time on
total phenolic and total triterpenoid compounds from Combretum micranthum (a,d), Euphorbia hirta
(b,e) and Anacardium occidentale (c,f) corresponding to ethanol concentration of 60%.
Figure 3.
Response surface plot showing the effect of extraction temperature and extraction time on
total phenolic and total triterpenoid compounds from Combretum micranthum (
a
,
d
), Euphorbia hirta (
b
,
e
)
and Anacardium occidentale (c,f) corresponding to ethanol concentration of 60%.
2.3. Determination of Optimum Conditions
The optimum ultrasonic-assisted extraction conditions from CM leaves, EH plant and AO fruits
were determined to maximize TPC and TTC. Based on the experimental results and the desirability
function, the optimum ultrasound-assisted extraction conditions could be decided. The experiments
were conducted in triplicate at the optimum conditions, and the average values were recorded.
The desirability values were 0.991, 0.988 and 0.917 for CM, EH and AO, respectively. The optimal
conditions were obtained with the ethanol concentrations of 65.25, 35.77 and 66.66%, at the temperatures
of 60.08, 59.65, and 47.48
◦
C, and extraction time of 23.18, 26.12, and 56.82 min for CM, EH, and AO,
respectively. At these conditions, the optimum TPC (87.04
±
0.60; 40.43
±
0.45 and 11.31
±
0.28
(mg GAE/g DW)) and TTC (90.08
±
2.57; 12.50
±
0.58 and 18.10
±
1.07 (mg UAE/g DW)) were obtained
for CM, EH and AO, respectively. The predicted values were 88.73, 42.80, and 11.48 mg GAE/g DW for
TPCs, and 89.11, 14.92, and 18.91 mg UAE/g DW for CM, EH, and AO, respectively. The experimental
values were found to be in agreement with the predicted values obtained from the quadratic models
developed in this study.

Molecules 2020,25, 2684 11 of 24
2.4. In-Tubo Tyrosinase Activity Assay
The effects of the aqueous extracts of CM leaves, EH plant, and AO fruits on mushroom tyrosinase
activity (using L-tyrosinase as a substrate), are reported in Figure 4. The in-tubo antityrosinase activity
assays were carried out on the aqueous extracts considering the fact that ethanol might alter the
tyrosinase activity, and the results would be inaccurate and unreproducible. Moreover, the use of
a hydroethanolic extract as an anti-browning agent for food processing was not feasible since the
consumption of ethanol in relatively higher level is reported to increase the risk of developing certain
diseases [
35
]. The results show that all aqueous extracts have a significant and dose-dependent
inhibitory activity of tyrosinase enzyme. The aqueous extract from CM leaves exhibited the strongest
inhibitory effect. In fact, CM aqueous extract showed an IC
50
of 0.58 g
·
L
−1
. However, the IC
50
of
EH and AO was not reached even at a 6-fold higher concentration. Kojic acid, the molecule used as
a standard reference, showed an IC
50
of only 0.22 g
·
L
−1
. In the plant extracts, the concentration of
bioactive metabolites was lower than the IC
50
value of the extracts. It is obvious since the plant extracts
comprised a mixture of many compounds, whereas kojic acid is a single molecule [
36
]. These results
were in good accordance with the previous findings and stipulated that phenolic and triterpenoid
compounds could be responsible for the tyrosinase enzymatic inhibition. In fact, CM extract showed a
higher concentration of both phenolic and triterpenoid compounds. However, EH extract showed a
higher concentration of metabolites than AO extract but a less percentage of tyrosinase inhibition. It is
not surprising as the percentage of tyrosinase inhibition of an extract is not only related to the quantity
of phenolic and triterpenoid compounds, but also to its chemical composition. The influence of their
chemical composition will be discussed later in this article.
Molecules 2020, 25, x 12 of 24
Figure 4. In tubo tyrosinase activity assays results for Combretum micranthum leaves, Euphorbia hirta
plant and Anacardium occidentale fruits aqueous extract with kojic acid solution used as reference. * p
< 0.05 indicates a significant difference between aqueous extract test and negative control.
In addition to having many biological properties, these plant extracts show significant
antityrosinase activity. These plants could be used as a food additive because of their safety for the
consumers. Ping et al. have demonstrated that EH plant extract shows no signs of toxicity or
symptoms related to oral toxicity [37]. However, it is possible that a middle-level toxicity appears at
very high doses, and therefore, it is necessary to know the doses when the extract is used as an
antibacterial, antioxidant or antibrowning agent in food [38]. The toxicity of AO fruits associated with
dairy products has long been discussed. However, a recent study concluded that the juice and cow
milk mixture are not toxic to animal cells, but the juice-yogurt mixture has some toxic effects on the
liver cells [39]. CM leaf extract, which has shown the highest antityrosinase activity appeared to be
the most promising one, without any symptoms of toxicity. In fact, an acute and subchronic oral
toxicity assessments of CM leaves extract in Wistar rats recently showed that the oral dose up to 5000
mg/kg of CM leaves extract has no evidence of toxicity or treatment-related mortality in animals. In
addition, repeated doses of the hydroalcoholic extract (1000 mg/kg) of CM leaves for 28 days showed
no significant change in food and water intake. It was concluded that the risk/benefit ratio is in favour
of usage of CM leaves extract [40].
2.5. Identification of Metabolic Compounds
A total of 88 metabolic compounds was tentatively identified in the ethanol-water extract of the
plant matrices using high-resolution LC-MS (Table 3), out of which 75 were phenolic compounds (26
phenolic acids and 49 flavonoids). A total of 22, 29 and 24 phenolic compounds were detected in CM
leaves, EH plant and AO fruit, respectively. All the detected compounds were tentatively identified
based on the accurate mass of their precursor and one or more diagnostic product ions, each with <5
ppm of mass errors. The m/z of the observed precursor ions (from the in-house developed database
of natural compounds) and their characteristic fragment ions (through in-silico fragmentation of the
chemical structure of the compound, feature of UNIFI software) were matched either with the spectra
of the reference standards, or with spectral library from literature (previous research articles or public
databases including ChemSpider (http://www.chemspider.com), SciFinder Scholar
(https://scifinder.cas.org), FooDB (http://foodb.ca/) and Phenol-Explorer (www.phenol-explorer.eu).
Figure 4.
In tubo tyrosinase activity assays results for Combretum micranthum leaves, Euphorbia hirta
plant and Anacardium occidentale fruits aqueous extract with kojic acid solution used as reference.
*p<0.05 indicates a significant difference between aqueous extract test and negative control.
In addition to having many biological properties, these plant extracts show significant
antityrosinase activity. These plants could be used as a food additive because of their safety for
the consumers. Ping et al. have demonstrated that EH plant extract shows no signs of toxicity or
symptoms related to oral toxicity [
37
]. However, it is possible that a middle-level toxicity appears
at very high doses, and therefore, it is necessary to know the doses when the extract is used as an
antibacterial, antioxidant or antibrowning agent in food [
38
]. The toxicity of AO fruits associated
with dairy products has long been discussed. However, a recent study concluded that the juice and
cow milk mixture are not toxic to animal cells, but the juice-yogurt mixture has some toxic effects on

Molecules 2020,25, 2684 12 of 24
the liver cells [
39
]. CM leaf extract, which has shown the highest antityrosinase activity appeared
to be the most promising one, without any symptoms of toxicity. In fact, an acute and subchronic
oral toxicity assessments of CM leaves extract in Wistar rats recently showed that the oral dose up to
5000 mg/kg of CM leaves extract has no evidence of toxicity or treatment-related mortality in animals.
In addition, repeated doses of the hydroalcoholic extract (1000 mg/kg) of CM leaves for 28 days showed
no significant change in food and water intake. It was concluded that the risk/benefit ratio is in favour
of usage of CM leaves extract [40].
2.5. Identification of Metabolic Compounds
A total of 88 metabolic compounds was tentatively identified in the ethanol-water extract of the
plant matrices using high-resolution LC-MS (Table 3), out of which 75 were phenolic compounds
(26 phenolic acids and 49 flavonoids). A total of 22, 29 and 24 phenolic compounds were detected
in CM leaves, EH plant and AO fruit, respectively. All the detected compounds were tentatively
identified based on the accurate mass of their precursor and one or more diagnostic product ions,
each with <5 ppm of mass errors. The m/zof the observed precursor ions (from the in-house developed
database of natural compounds) and their characteristic fragment ions (through in-silico fragmentation
of the chemical structure of the compound, feature of UNIFI software) were matched either with
the spectra of the reference standards, or with spectral library from literature (previous research
articles or public databases including ChemSpider (http://www.chemspider.com), SciFinder Scholar
(https://scifinder.cas.org), FooDB (http://foodb.ca/) and Phenol-Explorer (www.phenol-explorer.eu).
The identifications of all metabolic compounds were based on certain criteria as mentioned in
Table 3. For instance, luteolin-7-O-malonylglucoside (Table 3, No. 53) was identified based on its
protonated molecular ion m/z 534.10096 (mass error, 3.86 ppm with elemental composition, C
24
H
22
O
14
)
and its two characteristic fragment ions (m/z287.0559 and m/z163.0394). Naringenin-4
0
-O-glucuronide
(Table 3, No. 18 and 49) appeared at two different retention times which indicated that this compound
probably appeared in two isomeric forms. Myricetin-3-O-glucoside (Table 3, No. 21) was identified
based on its protonated molecular ion m/z480.09039 (mass error 2.46 ppm), elemental composition
(C
21
H
20
O
13
) and characteristic fragment ions with m/z153.0183 and m/z319.0456. In a similar manner,
the other compounds (Table 3) were identified.
The results in this study were consistent when compared to previous studies. Welch et al.
had also reported (
−
)-epigallocatechin and myricetin-3-O-glucoside in the leaf extract of CM [
17
].
According to [
23
,
41
], EH extract was earlier reported to contain chlorogenic acid and kaempferol.
Besides, myricetin, (
−
)-epigallocatechin, myricetin-3-O-glucoside, and quercetin-3-O-galactoside were
reported in previous studies in the fruit extract of AO [
21
,
22
,
42
]. All other compounds reported
in Table 3are reported for the first time in these plant extracts. Hence, the current results appear
much more comprehensive as compared to the previous studies. There were few compounds which
differed from the results of previous studies. This difference could be due to variation in methods of
extraction and analysis. Moreover, the possibility of compounds being identified largely depend on
the size of the database used. The UNIFI software allowed us to screen the samples against a database
comprising thousands of compound entries. This offered a comprehensive screening of the compounds.
Furthermore, variations in metabolite profile might also depend upon the cultivar, geographical and
climatic conditions, etc. [43].

Molecules 2020,25, 2684 13 of 24
Table 3. Identification of phenolic compounds and other phytochemicals by high-resolution LC-MS (R. T.: Retention time; R. P.: Relative percentage).
No. Compound Name Formula R. T.
(min)
Expected
Mass (m/z)Adducts Observed
Mass (m/z)
Mass Error
(ppm) Fragments (relative%) Detector
Counts R. P. (%)
C. micranthum leaves extract
Phenolic acid
1. Syringic acid
C
9
H
10
O
50.59 198.05282 −e 198.05279 2.60 182.0572 (100%) 133170.27 11.38
2. p-Coumaric acid ethyl ester
C
11
H
12
O
31.56 192.07864 +H 193.08632 2.06 175.0755 (100%) 35086.45 3.00
3. Sesamol C7H6O32.02 138.03169 +H 139.03933 2.61 121.0287 (100%) 24158.38 2.06
4. Dihydrocaffeic acid-3-O-glucuronide
C
15
H
18
O
10 0.62 358.09000 +Na 381.07991 1.81 198.0526 (100%) 19538.90 1.67
5. Prodelphinidin trimer GC-C-C
C
45
H
38
O
20 2.28 898.19564 +H, +Na 899.20573 3.13
729.1436 (100%), 605.1285 (80%)
14474.68 1.24
6. p-Coumaric acid C9H8O33.01 164.04734 +H 165.05504 2.56
147.0443 (100%), 119.0493 (60%)
9977.19 0.85
7. Vanillic acid C8H8O42.03 168.04226 +H 169.04982 1.69 139.0393 (100%), 151.0393 (80% 3730.41 0.32
8. Eugenol 1
C
10
H
12
O
21.56 164.08373 +H 165.09132 1.92 147.0807 (100%), 2514.33 0.21
Isoflavonoid
9. Dihydrodaidzein-7-O-glucuronide
C
21
H
20
O
10 4.70 432.10565 +H, +Na 433.11431 3.20 415.1037 (60%), 313.0714
(100%), 283.0608 (100%) 390762.65 33.38
Anthocyanins
10. Cyanidin-3-O-(6”-p-coumaroyl-glucoside)
C
30
H
27
O
13 2.21 595.14517 −e 595.14572 1.86
287.0544 (100%), 425.0877 (50%)
74911.14 6.40
11. Pelargonidin-3-O-coumarylglucoside
C
30
H
27
O
12 3.18 579.15025 −e 579.15211 4.15
272.0663 (100%), 563.1574 (20%)
8481.75 0.72
12. Delphinidin-3-O-(6”-p-coumaroyl-glucoside)
C
30
H
27
O
14 0.78 611.14008 −e 611.14093 2.29
303.0505 (100%), 287.0553 (80%)
5366.72 0.46
Flavonol
13. Dihydroquercetin
C
15
H
12
O
73.44 304.05830 +H 305.06674 3.82
163.0395 (100%) 153.0185 (80%)
3683.11 0.31
Flavans
14. Leucocyanidin 4
C
15
H
14
O
72.02 306.07395 +H, +Na 307.08202 2.56
291.0877 (100%), 181.0501 (30%)
52544.44 4.49
15. Leucopelargonidin
C
15
H
14
O
63.00 290.07904 +H 291.08711 2.75
229.0504 (20%), 165.0551 (100%)
43785.59 3.74
16. (-)−epigallocatechin
C
15
H
14
O
71.82 306.07395 +H, +Na 307.08240 3.81
263.0532 (100%), 153.0546 (80%)
29619.58 2.53
17. 6-Geranylnaringenin
C
25
H
28
O
54.59 408.19367 +Na 431.18347 1.34 273.0768 (100%), 250.820 (80%) 10237.19 0.87
18. Naringenin-40-O-glucuronide
C
21
H
20
O
11 4.08 448.10056 +H 449.10878 2.09
271.0604 (100%), 257.0824 (80%)
10048.21 0.86
19. Naringenin
C
15
H
12
O
56.21 272.06847 +H 273.07601 0.97 153.0183 (100%) 3875.93 0.33
20. (+)-Catechin-3-O-gallate
C
22
H
18
O
10 2.21 442.09000 +H 443.09772 1.00
287.0554 (100%), 291.0875 (80%)
3315.07 0.28
Flavone

Molecules 2020,25, 2684 14 of 24
Table 3. Cont.
No. Compound Name Formula R. T.
(min)
Expected
Mass (m/z)Adducts Observed
Mass (m/z)
Mass Error
(ppm) Fragments (relative%) Detector
Counts R. P. (%)
21. Myricetin-3-O-glucoside
C
21
H
20
O
13 4.48 480.09039 +H, +Na 481.09885 2.46
153.0183 (100%), 319.0456 (90%)
18064.01 1.54
22. Baicalin hydrate
C
21
H
20
O
12 3.51 464.09548 +H 465.10369 2.02
285.0766 (100%), 325.0667 (30%)
1838.75 0.16
Triterpenoid
23. Micromeric acid
C
30
H
46
O
311.22 454.34470 +H 455.35284 1.90 383.3285 (100%), 393.3519
(100%), 437.3421 (50%) 194168.06 16.59
24. Cucurbitacin P
C
30
H
48
O
79.32 520.34000 −e 520.34081 2.60
455.3519 (100%), 337.2736 (80%)
53383.81 4.56
25. Cucurbitacin F2
C
30
H
46
O
79.70 518.32435 −e 518.32157 −4.32
471.3466 (100%), 355.2645 (30%)
15060.36 1.29
Amino acid
26. Tryptophan
C
11
H
12
N
2
O
21.80 204.08988 +H 205.09785 3.42
188.0714 (100%), 144.0808 (50%)
8815.60 0.75
E. hirta plant extract
Phenolic acids
27. 4-Hydroxycoumarin C9H6O31.76 162.03169 +H 163.03911 0.85 145.0286 (100%) 279416.51 11.14
28. Caffeoylquinic acid
C
16
H
18
O
92.58 354.09508 +Na, +H 377.08446 0.43 163.0391 (100%), 145.0287
(40%), 177.0548 (30%) 53371.32 2.13
29. Chlorogenic acid
C
16
H
18
O
91.76 354.09508 +Na, +H 377.08440 0.25
163.0391 (100%), 215.0530 (10%)
40229.57 1.60
30. 3,4-Dihydro-1-benzopyran-2-one C9H8O21.65 148.05243 +H 149.05981 0.73
105.0336 (100%), 123.0441 (30%)
29274.70 1.17
31. 2-S-Glutathionyl caftaric acid
C
23
H
27
N
3
O
15
S
4.58 617.11629 −e 617.11476 −1.59
153.0184 (100%), 307.0608
(60%), 529.1350 (10%), 409.0923
(10%),
25104.15 1.00
32. Punicalin
C
34
H
22
O
22 2.42 782.06027 +H 783.06859 1.33
277.0346 (100%), 303.0141 (80%)
19495.48 0.78
33. 4,5-Dicaffeoylquinic acid
C
25
H
24
O
12 4.67 516.12678 +Na, +H 539.11657 1.07 163.0392 (100%), 499.1239
(50%), 287.0555 (30%) 16282.13 0.65
34. Feruloyl tartaric acid
C
14
H
14
O
92.25 326.06378 +H 327.07182 2.31
153.0185 (100%), 309.0611 (10%)
13817.19 0.55
35. Feruloyl malic acid
C
14
H
14
O
80.84 310.06887 −e 310.06978 4.71
200.0449 (100%), 135.0294 (70%)
11092.96 0.44
Isoflavonoids
36. Genistin
C
21
H
20
O
10 4.81 432.10565 +H, +Na 433.11317 0.56 415.1020 (100%), 397.0920
(80%), 379.0814 (90%) 91534.53 3.65
37. Dihydrodaidzein-7-O-glucuronide
C
21
H
20
O
10 4.48 432.10565 +H 433.11318 0.59 415.1021 (100%), 255.0646
(80%), 367.0811 (80%) 15379.65 0.61
Anthocyanins

Molecules 2020,25, 2684 15 of 24
Table 3. Cont.
No. Compound Name Formula R. T.
(min)
Expected
Mass (m/z)Adducts Observed
Mass (m/z)
Mass Error
(ppm) Fragments (relative%) Detector
Counts R. P. (%)
38. Pelargonidin-3-O-sambubioside
C
26
H
29
O
14 4.14 565.15573 −e 565.15593 1.32
547.1449 (100%), 379.0816 (80%)
87443.37 3.49
39. Pelargonidin-3-O-sophoroside
C
27
H
31
O
15 3.89 595.16630 −e 595.16623 0.81
577.1547 (100%), 271.0596
(90%), 529.1332 (70%), 559.1457
(30%)
58442.79 2.33
40. Peonidin-3-O-arabinoside
C
21
H
21
O
11 5.58 449.10839 −e 449.10960 3.93 303.0508 (100%), 287.0557
(20%), 413.08617 (10%), 34687.76 1.38
41. Pelargonidin-3-O-coumarylglucoside
C
30
H
27
O
12 1.67 579.15025 −e 579.15016 0.79
149.0598 (100%), 275.0559 (80%)
15007.82 0.60
Flavonols
42. Quercetin-3-O-(6”-malonylglucoside)
C
24
H
22
O
15 5.24 550.09587 +H, +Na 551.10419 1.90
303.0502 (100%), 345.0609 (20%)
282214.49 11.25
43. Quercetin-7-O-glucoside
C
21
H
20
O
12 5.02 464.09548 +H, +Na 465.10365 1.93
303.0502 (100%), 433.1132 (30%)
107974.95 4.30
44. Quercetin-3-O-glucuronide
C
21
H
18
O
13 2.25 478.07474 +H 479.08307 2.19 309.0611 (100%), 303.0521 (805) 58211.34 2.32
45. Quercetin-3-O-rhamnosyl-galactoside
C
27
H
30
O
16 3.44 610.15338 +H, +Na 611.16197 2.14
153.0184 (100%), 303.0550 (40%)
24565.15 0.98
46. Quercetin-3-O-xylosylglucuronide
C
26
H
26
O
17 3.85 610.11700 +Na 633.10908 4.52
315.0512 (100%), 319.0457 (90%)
10086.09 0.40
47. Methylgalangin
C
15
H
10
O
65.81 286.04774 +H 287.05573 2.50
213.0547 (100%), 163.0394 (80%)
9976.99 0.40
Flavans
48. Naringenin-7-O-glucuronide
C
21
H
20
O
11 2.85 448.10056 +H 449.10811 0.60 287.0552 (100%) 206146.03 8.22
49. Naringenin-40-O-glucuronide
C
21
H
20
O
11 5.58 448.10056 +Na, +H 471.09085 2.26 303.0508 (100%), 274.0477
(30%), 287.0557 (10%) 59330.32 2.36
Flavones
50. Morin
C
15
H
10
O
75.57 302.04265 +H 303.05090 3.21 153.0187 (100%), 285.0402
(40%), 737073.25 29.38
51. Kaempferol
C
15
H
10
O
66.09 286.04774 +H 287.05597 3.32
231.0640 (100%), 229.0497 (80%)
68887.11 2.75
52. Apigenin-7-O-apiosyl-glucoside
C
26
H
28
O
14 4.60 564.14791 +H, +Na 565.15639 2.14
547.1458 (100%), 529.1350
(60%), 303.0503 (40%), 337.0486
(30%)
43427.69 1.73
53. Luteolin-7-O-malonyl-glucoside
C
24
H
22
O
14 5.82 534.10096 +H, +Na 535.11030 3.86
287.0559 (100%), 163.0394 (40%)
27471.22 1.09
54. 6-Hydroxyluteolin-7-glucoside
C
20
H
18
O
13 2.86 466.07474 +H 467.08270 1.46
287.0552 (100%), 321.0242 (90%)
9781.46 0.39
55. Kaempferol-3-O-rhamnoside
C
21
H
20
O
10 6.09 432.10565 +Na, +H 455.09458 −0.64
287.0559 (100%), 153.0186 (60%)
9303.30 0.37
Triterpenoids
56. Cucurbitacin E
C
32
H
44
O
88.37 556.30362 +Na 579.29556 4.71 301.1419 (100%), 277.2167
(50%), 317.2063 (30%) 30532.85 1.22

Molecules 2020,25, 2684 16 of 24
Table 3. Cont.
No. Compound Name Formula R. T.
(min)
Expected
Mass (m/z)Adducts Observed
Mass (m/z)
Mass Error
(ppm) Fragments (relative%) Detector
Counts R. P. (%)
57. Cucurbitacin R6
C
30
H
46
O
78.89 518.32435 −e 518.32630 4.82
453.2625 (100%), 184.0736
(80%), 442.2353 (60%), 335.2584
(30%)
9589.35 0.38
Amino acid
58. Tryptophan
C
11
H
12
N
2
O
21.90 204.08988 +H 205.09736 1.00
188.0707 (100%), 170.0601 (90%)
23930.23 0.95
A. occidentale fruits extract
Phenolic acid
59. Dihydrocaffeic acid-3-O-glucuronide
C
15
H
18
O
10 0.62 358.09000 +Na 381.08111 4.97 198.0526 (100%) 114811.09 33.61
60. o-Coumaric acid C9H8O30.76 164.04734 +H 165.05492 1.81
147.0442 (100%), 109.0657 (70%)
17841.73 5.22
61. Cinnamoyl glucose
C
15
H
18
O
74.89 310.10525 +Na 333.09472 0.74
204.1017 (100%), 275.0926 (50%)
12415.40 3.63
62. p-Coumaroyl glucose
C
15
H
18
O
82.49 326.10017 +Na 349.08884 −1.57
147.0437 (100%), 119.0492 (70%)
7877.05 2.31
63. 2-Hydroxyphenylacetic acid C8H8O30.80 152.04734 +H 153.05465 0.22
119.0490 (100%), 107.1491 (50%)
6340.16 1.86
64. 4-Hydroxybenzaldehyde C7H6O20.76 122.03678 +H 123.04446 3.25 95.0494 (100%), 107.0491 (70%) 3808.08 1.11
65. 3,4-Dihydro-1-benzopyran-2-one C9H8O24.35 148.05243 +H 149.05992 1.45
131.0494 (100%), 103.0545 (80%)
3467.34 1.01
66. 3,4-Dihydroxyphenylglycol
C
8
H
10
O
40.80 170.05791 +H 171.06566 2.79
139.0388 (100%), 153.0546 (80%)
1729.20 0.51
67. Salvianolic acid C
C
26
H
20
O
10 0.73 492.10565 +H 493.11517 4.56
207.0288 (100%), 225.0389 (90%)
1399.04 0.41
Anthocyanin
68. Delphinidin-3-O-galactoside
C
21
H
21
O
12 4.89 465.10330 −e 465.10344 1.49 303.0502 (100%) 2014.41 0.59
Flavonols
69. Quercetin-3-O-galactoside
C
21
H
20
O
12 4.88 464.09548 +Na, +H 487.08535 1.34
153.0184 (100%), 303.0502 (20%)
8556.56 2.50
70. Dihydroquercetin-3-O-rhamnoside
C
21
H
22
O
11 3.00 450.11621 +Na 473.10555 0.25 303.0517 (100%) 1800.84 0.53
Flavans
71.
3
0
-O-Methyl-(-)
−
epicatechin-7-O-glucuronide
C
22
H
24
O
12 4.35 480.12678 +H 481.13465 1.23
313.0710 (100%), 245.0470 (20%)
8248.27 2.41
72. Naringenin-5-O-glucuronide
C
21
H
20
O
11 5.49 448.10056 +Na, +H 471.09023 0.96
303.0501 (100%), 287.0552 (30%)
2980.31 0.87
73. (-)−epigallocatechin
C
15
H
14
O
70.82 306.07395 +H 307.08107 −0.52
263.0532 (100%), 153.0546 (80%)
2464.54 0.72
74. 6-Prenylnaringenin
C
20
H
20
O
54.00 340.13107 +H 341.13985 4.39
323.1282 (100%), 193.0859 (80%)
2278.05 0.67
75. Leucopelargonidin-3-O
-alpha-l-rhamno-beta-d-glucopyranoside
C
27
H
34
O
15 4.67 598.18977 +H, +Na 599.19741 0.60 495.1482 (100%), 374.1589
(30%), 290.0399 (20%) 2086.70 0.61
Flavones

Molecules 2020,25, 2684 17 of 24
Table 3. Cont.
No. Compound Name Formula R. T.
(min)
Expected
Mass (m/z)Adducts Observed
Mass (m/z)
Mass Error
(ppm) Fragments (relative%) Detector
Counts R. P. (%)
76. Myricetin
C
15
H
10
O
84.67 318.03757 +H 319.04544 1.88
153.0182 (100%), 165.0183 (30%)
9795.79 2.87
77. Morin
C
15
H
10
O
75.49 302.04265 +H 303.05049 1.86
287.0552 (90%), 153.0437 (100%)
8258.29 2.42
78. Isovitexin
C
21
H
20
O
10 4.71 432.10565 +H, +Na 433.11372 1.85 337.0715 (100%), 415.1022
(90%), 283.0605 (80%) 5879.40 1.72
79. Myricetin-3-O-glucoside
C
21
H
20
O
13 4.23 480.09039 +Na, +H 503.08111 2.97 319.0453 (100%) 4580.01 1.34
Flavanone
80. Pinocembrin
C
15
H
12
O
46.09 256.07356 +H 257.08090 0.24 153.0185 (100%) 2037.62 0.60
Lactone
81. Coumarin C9H6O20.76 146.03678 +H 147.04406 0.04 123.0442 (100%), 95.0494 (50%) 3826.69 1.12
Chalcon
82. Phloretin
C
15
H
14
O
54.59 274.08412 +H 275.09162 0.79 131.0491 (100%), 151.0390
(70%), 133.0649 (60%) 3254.60 0.95
Triterpenoids
83. Micromeric acid
C
30
H
46
O
38.55 454.34470 +H 455.35167 -0.66
437.3409 (100%), 423.3296 (20%)
54817.17 16.05
84. Cucurbitacin E
C
32
H
44
O
88.37 556.30362 +Na 579.29310 0.45 301.1407 (100%) 24481.80 7.17
85. Cucurbitacin F2
C
30
H
46
O
79.72 518.32435 −e 518.32215 −3.19
471.3475 (100%), 454.2935 (50%)
11253.75 3.29
86. Cucurbitacin R6
C
30
H
46
O
78.72 518.32435 −e 518.32544 3.16 335.2584 (100%), 184.0736
(80%), 361.2357 (40%) 6412.54 1.88
87. Cucurbitacin P
C
30
H
48
O
79.12 520.34000 −e 520.34005 1.14
337.2739 (100%), 398.2676 (30%)
5347.24 1.57
Fatty acid
88. 3-Hydroxyphenylvaleric acid
C
11
H
14
O
37.66 194.09429 +H 195.10218 3.10 95.0493 (100%) 1556.22 0.46

Molecules 2020,25, 2684 18 of 24
The most dominant compounds in CM leaves included dihydrodaidzein-7-O-glucuronide
(isoflavonoid), micromeric acid (triterpenoid) and syringic acid (phenolic acid) with relative percentages
of 33.38, 16.59, and 11.38%, respectively. For EH plant, the most dominant compounds included morin
(flavone), quercetin-3-O-(6—malonylglucoside) (flavonol), and 4-hydroxycoumarin (phenolic acid)
with relative proportions of 29.38, 11.25, and 11.14%, respectively. AO fruits contained dihydrocaffeic
acid-3-O-glucuronide (phenolic acid), micromeric acid (triterpenoid), and cucurbitacin E (triterpenoid)
as the major compounds with relative percentages of 33.61, 16.05, and 7.17%, respectively. Micromeric
acid was a major and common compound for both CM and AO. All three plant extracts showed to
have common compounds, although in different proportional amounts. For example, naringenin
4
0
-O-glucuronide, and pelargonidin-3-O-coumarylglucoside were the common compounds between
CM and EH. (
−
)-epigallocatechin and myricetin-3-O-glucoside were present in both CM and AO. Some
of the compounds that were common between EH and AO included morin and cucurbitacin E.
A recent review article reports the tyrosinase inhibitors discovered from natural, semisynthetic,
and synthetic sources in the last four decades [
6
]. Most of the compounds identified in
this study, and many of their derivatives have been found to have great antityrosinase
activity. For example, quercetin, a flavonol, is well known for its antityrosinase activity,
and most of its derivatives show a similar effect
[6,13].
The extracts of EH plants
and AO fruits have shown significant amounts of quercetin derivatives that could be
responsible for their antityrosinase activity, especially quercetin-3-O-(6”-malonyl-glucoside),
which is the most predominant compound in EH plant extract, and have never been
tested for its antityrosinase activity earlier. Quercetin-3-O-(6-O-malonyl)-
β
-d-glucopyranoside,
and kaempferol-3-O-(6-O-malonyl)-
β
-d-glucopyranoside from mulberry leaves were identified as
tyrosinase inhibitors [
44
]. The compound dihydrodaidzein-7-O-glucuronide have never been
reported to have an antityrosinase activity. However, daidzein inhibited tyrosinase activity by
55.8 ±1.4%
at 100
µ
g
·
mL
−1
. We can expect to find a similar effect with daidzein derivatives, such as
dihydrodaidzein-7-O-glucuronide, which have been found to be the most prominent compound in
CM leaves [
45
]. Micromeric acid, an important predominant compound found in both CM leaves and
AO fruits, have never been tested for its antityrosinase activity. This triterpenoid should be explored in
future studies to confirm or refute its implication in the inhibition percentage of tyrosinase enzyme.
Enzymatic kinetics studies have shown that morin, the most predominant compound in EH plant extract,
reversibly inhibited tyrosinase in a competitive manner, and bound to tyrosinase at a single binding
site by van der Waals interactions and hydrogen bonds, inducing rearrangement and conformational
changes in the enzyme [
46
]. Asthana et al. showed that 4-hydroxycoumarin, a predominant compound
in EH plant extract, was not an inhibitor of tyrosinase enzyme [
47
]. Dihydrocaffeic acid-3-O-glucuronide
has never been reported to have antityrosinase activity. However, caffeic acid and its derivatives have
been largely reported to have significant antityrosinase activity [
48
]. These results were consistent with
all previous findings and allowed to explain where from the antityrosinase effect of these plant extracts
came from. These findings could definitely lead to the discovery of new active compounds to inhibit
the tyrosinase enzymatic activity. Now, experiments to isolate bioactive compounds responsible for the
inhibition of tyrosinase enzyme are underway. These compounds could be useful in food processing
industries as anti-browning agents.
3. Materials and Methods
3.1. Materials
Twenty kg of EH plant (leaves, stems, roots) were harvested from Tyre (Tyre, Lebanon) in April
2017. Twenty Kg of both CM leaves and AO fruits were harvested from Dakar (Dakar, Senegal) in July
2017. The plants were taxonomically authenticated by a botanist to confirm their genus and species.
The plants were air-dried in shade for three weeks and ground until fine and homogenous particles

Molecules 2020,25, 2684 19 of 24
were obtained. The same plant samples were preserved and used during the study. All chemical
reagents and standards mentioned were purchased from Sigma-Aldrich (Steinheim, Germany).
3.2. Ultrasound-Assisted Extraction
Ultrasound-assisted extraction was performed for screening of phenolic and triterpenoid contents
from leaves of CM, whole plant of EH and fruits of AO. An ultrasonic bath, Elmasonic S type S 15/H type
S 15 (manufactured by Elma Hans Schmidbauer GmbH & Co. KG, Singen am Hohentwiel, Germany,
bath frequency 37 KHz, power 280 W) was used. The set-up allowed regulation of temperature.
The grinded plants (30, 15 and 20 g of CM, EH and AO, respectively) were placed directly into the
ultrasonic bath with 300 mL of ethanol: water mixture to decide the optimal ratio utilizing the central
composite design. The reaction mass was filtered under vacuum and the filtrate was collected in a
volumetric flask for the determination of total phenolic and total triterpenoid contents.
3.3. Preliminary Study
An increase in the ratio of solvent/matrix lead to an enhancement of the gradient concentration
and improved the extent of diffusion of analytes in the medium. The high gradient concentration was
considered as the driving force during extraction until equilibrium was reached. Ultrasound-assisted
extraction required mechanical agitation to improve mass transfer and to avoid ultrasonic intensity
attenuation due to heterogeneity of the medium [
49
]. In order to optimize the liquid/solid ratio (mL
·
g
−1
)
and the agitation speed (rpm or
×
g), the maximum yield of extraction was considered. It was observed
that the optimum liquid/solid ratio were 10, 20 and 15 mL
·
g
−1
for CM, EH and AO, respectively. On the
other hand, the optimum agitation speeds were 80, 130 and 80 rpm for CM, EH and AO, respectively.
3.4. Total Phenolic Content
The total phenolic content (TPC) was estimated as gallic acid equivalents (GAE), expressed
as mg gallic acid equivalent/g dry weight (DW) according to the standard method [
50
] with slight
modifications. To 0.1 mL of plant extract, 0.4 mL of distilled water and 2.5 mL of Folin-Ciocalteu
solution (0.2 N) were added. After shaking, 2 mL of 7.5% (w/v) Na
2
CO
3
was added. The solution
was then incubated for 10 min at 60
◦
C, followed by and 10 min of incubation at
−
20
◦
C to stop
the reaction. The absorbance of the samples was measured at 760 nm and compared to gallic acid
calibration curve. Data are representative of three independent experiments. The linear range of gallic
acid was 0.1–1 mg·mL−1(R2=0.9985).
3.5. Total Triterpenoid Content
The total triterpenoid content (TTC) was determined by the method of Ming et al. with a slight
modification and expressed as mg ursolic acid equivalent (UAE)/g dry weight (DW) [
51
]. The sample
solution (200
µ
L) was heated to evaporation in a water-bath, and to it, 0.3 mL of freshly mixed
5% (w/v) vanillin-acetic solution and 1 mL sulfuric acid were added, mixed and incubated at 60
◦
C
for 30 min. After incubation, the mixed solution was cooled and diluted to 9.3 mL with acetic acid.
The absorbance was measured at 546 nm and compared to standard ursolic acid calibration curve.
Data are representative of three independent experiments. The linear range of ursolic acid was
1–10 mg·mL−1(R2=0.9979).
3.6. Antityrosinase Activity
Solution of L-tyrosine at 0.5 mg
·
mL
−1
, mushroom tyrosinase at 142 U
·
mL
−1
, as well as aqueous
fraction extracts of CM leaves, EH plant and AO fruits at different concentration were prepared in
phosphate buffer solution (PBS) at pH 6.6. The pure kojic acid served as the reference standard inhibitor
for comparison. Briefly, 50
µ
L of L-tyrosine solution and 50
µ
L of tyrosinase solution were mixed
with 50
µ
L of the aqueous fraction extracts or kojic acid solution. The mixture was incubated at 37
◦
C

Molecules 2020,25, 2684 20 of 24
for 60 min, and the dopachrome was measured by UV-Vis spectroscopy at 475 nm [
52
]. Data are
representative of three independent experiments. Data are analyzed using one-way ANOVA test.
Values of * p<0.05 are considered significant.
3.7. LC-MS [UPLC-(ESI)-QToFMS] Analysis of Metabolic Compounds
The metabolic compounds from natural extracts were identified by the method of Kumar et al.
2017 [
53
]. An Acquity Ultra Performance Liquid Chromatograph (UPLC) (Synapt G2 HDMS, Waters
Corporations, Manchester, UK) coupled to a quadrupole time of flight mass spectrometer (QToF-MS,
Synapt G2 HDMS, Waters Corporation, Manchester, UK) was used for analysis. The QToF-MS
was controlled by MassLynx 4.1 software (Waters, Manchester, UK) and operated with electrospray
ionization (ESI) in the positive mode at the mass resolution of 20,000, and acquisition in the MS
E
mode provided quick switching from low energy scan at 4 V (full scan MS) to high energy scan
(10–60 V ramping) during a single LC run. The low-collision energy (CE) experiments provided data
about the intact molecular ion (e.g. M
+
, [M +H]
+
) and the high-CE scan generated data on the fragment
ions. The source parameters included: capillary 3 kV, sampling cone 30 V, extraction cone 5 V, source
temperature 120
◦
C, desolvation temperature 500
◦
C, desolvation gas flow 1000 L
·
h
−1
, and cone gas flow
50 L
·
h
−1
. Nitrogen was used both as cone gas and drying gas. The calibration of the mass spectometer
was done with 0.5 mM of sodium formate. The mass correction was done using the lock spray and
the reference mass leucine enkephaline (m/z556.2771 in positive and 554.2670 in negative polarity)
at 2
µ
g
·
mL
−1
with 10
µ
L
·
min
−1
of flow rate at an interval of 20 s. The chromatographic separation
was performed on an Acquity UPLC BEH C18 column (2.1
×
100 mm, 1.8
µ
m, Waters Corporation,
Manchester, UK) at 35
◦
C. The mobile phase was composed of solvent A (methanol: water (10:90, v/v)
and solvent B (methanol: water (90:10, v/v) with 0.1 % formic acid in both phases. The following
gradient was applied: 90% A (0–0.5 min), 50% A (0.5–4.5 min), 50–2% A (4.5–8 min), 2% A (8–11 min),
2–90% A (11–12 min), 90% A (12–15 min). The flow rate was 0.4 mL·min−1.
3.8. Data Analysis
The acquired data (n=6 biological replicates) were processed in UNIFI software (version 1.7,
Waters Corporation) with a screening solution workflow which helped in automated data processing
to reporting the positive identifications by comparison with a database of phenolic compounds and
other phytochemicals. The phenolic compounds were identified with mass errors below 5 ppm for the
precursor and one or more product ion(s) having a similar mass accuracy. The product ions generated
through collision induced dissociation were matched against the theoretical fragmentation pattern.
Any new compounds could be added to the UNIFI software in order to create a customized library of
compounds. The relative percentages of the most predominant compounds were calculated.
3.9. Experimental Design
The extraction of phenolic compounds and triterpenoids as a function of ethanol concentration
(A), extraction temperature (B) and extraction time (C) were studied using a rotatable second order
design with six replicates in the center of the experimental domain. The conditions of the independent
variables studied were: A in the range of 40–80%, B in the range of 40–55
◦
C and C in the range
30–50 min. The two response variables to be optimized included total phenolic and total triterpenoid
contents. The total number of experiments (N) in a central composite design was calculated using the
following equation (Equation (7)):
N=2n+2n+x0(7)
Here, nrepresents the number of variables and x0 is the number of experimental central points [
54
].
Twenty experiments (consisting of eight factorial points, six star point and six replicates at the center)
were performed to optimize the parameters. In this study, three-level-three-factor central composite
design was employed, requiring 20 (n=3; x
0
=6) experiments. A second order polynomial equation

Molecules 2020,25, 2684 21 of 24
was used in order to develop an empirical model, which correlated the responses to the independent
variables. The general form of second order polynomial equation (Equation (8)) was:
R=b0+
n
X
i=1
biXi+
n−1
X
i=1
j>1
n
X
j=2
bijXiXj+
n
X
i=1
biiX2
i(8)
Here, R represents the predicted response, b
0
is a constant coefficient, B
i
, b
ij
, b
ii
are the coefficients
of linear, interaction effect and squared effects respectively, nis the number of variables, while X
i
and X
j
define the independent variables [
55
]. Analysis of variance (ANOVA), regression analysis and
response surface plots were performed in order to establish optimum conditions for total phenolic and
total triterpenoid contents.
In order to reach the maximal yield, we sought to determine the optimum extraction conditions of
total phenolic and total triterpenoid compounds using the desirability functions. The same weight was
used for both responses (w =1). The desirability function ranged from 0 (minimum desirability or
non-desirable situations) to 1 (maximum desirability). The importance of a goal ranged between 1
to 5 (1 for the least important and 5 for the most important). In this study, all the goals were equally
important and set at 3. All statistical analysis were performed using the software STATGRAPHICS
®
Centurion XVI (Statgraphics 18, The Plains, Virginia). Triplicate experiments were carried out in the
optimal condition. The experimental and predicted mean values were compared in order to determine
the validity of the models.
4. Conclusions
This work marks the first extensive study of the most predominant phenolic compounds and other
phytochemicals in the extracts of CM leaves, EH plant and AO fruits that have shown to be effective
in inhibiting the enzymatic activity of tyrosinase. A total of 88 predominant metabolic compounds
have been identified, including 75 polyphenols and 10 triterpenoids. On the one hand, 22, 29 and
24 polyphenols were identified in CM, EH and AO extracts, respectively. On the other hand, three,
two and five triterpenoids were identified in CM, EH and AO extracts, respectively. In this paper,
the extraction conditions to obtain the same plant extracts with consistent yield and quality were
very well detailed. The optimal ultrasound assisted-extraction conditions recommended an ethanol
concentration of 65.25, 35.77 and 66.66%, temperature of 60.08, 59.65 and 47.48
◦
C and extraction time
of 23.18, 26.12 and 56.82 min for CM, EH and AO, respectively. All plant extracts showed significant
anti-tyrosinase activity with the higher IC
50
of 0.58 g
·
L
−1
for CM extract. Moreover, the results
indicated that CM extract could be considered as a good source of phenolic (87.04
±
0.60 mg GAE/g
DW) and triterpenoid
(90.08 ±2.57 mg UAE/g DW)
compounds with potential utilization as bioactive
ingredients in food products. Thus, researchers and industrialists who will be interested in pursuing
studies on the same plant extracts will be able to do it. Recently, several benefits have been addressed
to phenolic and triterpenoid compounds. The food industry that are in search of multifunctional
ingredients from natural sources could be interested in using these extracts as anti-browning agents.
However, we recommend evaluating the safety of these extracts by
in vitro
and
in vivo
models before
their use in food products. Further studies should also focus on the isolation of these bioactive
compounds and develop them as products for utilization in food industries.
Author Contributions:
Conceptualization, H.Z., K.B. and R.L.; methodology, H.Z., Z.K., K.B., D.S. and R.L.;
software, H.Z., Z.K. and D.S.; validation, H.Z., K.B. and R.L.; formal analysis, H.Z., Z.K., K.B., D.S. and
R.L.; investigation, H.Z., Z.K., K.B., D.S. and R.L.; writing—original draft preparation, H.Z., Z.K. and D.S.;
writing—review and editing, K.B. and R.L.; supervision, K.B. and R.L.; funding acquisition, R.L. All authors have
read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Lebanese National Council for Scientific Research (CNRS-FS114),
the Research Council of Saint-Joseph University of Beirut (Project FS111) and Uniparco industry (Senegal).

Molecules 2020,25, 2684 22 of 24
Acknowledgments:
We would like to thank Ankita Lakade from ICAR-National Research Centre for Grapes for
her help in analyzing the LC-MS data.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Lim, W.Y.; Wong, C.W. Inhibitory effect of chemical and natural anti-browning agents on polyphenol oxidase
from ginger (Zingiber officinale Roscoe). J. Food Sci. Technol. 2018,55, 3001–3007. [CrossRef] [PubMed]
2.
Loizzo, M.R.; Tundis, R.; Menichini, F. Natural and Synthetic Tyrosinase Inhibitors as Antibrowning Agents:
An Update. Compr. Rev. Food Sci. Food Saf. 2012,11, 378–398. [CrossRef]
3.
Lim, W.Y.; Cheun, C.F.; Wong, C.W. Inhibition of enzymatic browning in sweet potato (Ipomoea batatas (L.))
with chemical and natural anti-browning agents. J. Food Process. Preserv. 2019,43, 1–8. [CrossRef]
4.
Paudel, P.; Seong, S.H.; Wagle, A.; Min, B.S.; Jung, H.A.; Choi, J.S. Antioxidant and anti-browning property
of 2-arylbenzofuran derivatives from Morus alba Linn root bark. Food Chem. 2020,309, 125739. [CrossRef]
5.
Botterweck, A.A.M.; Verhagen, H.; Goldbohm, R.A.; Kleinjans, J.; Van Den Brandt, P.A. Intake of butylated
hydroxyanisole and butylated hydroxytoluene and stomach cancer risk: Results from analyses in the
Netherlands Cohort Study. Food Chem. Toxicol. 2000,38, 599–605. [CrossRef]
6.
Zolghadri, S.; Bahrami, A.; Tareq, M.; Khan, H.; Munoz-munoz, J.; Garcia-molina, F. A comprehensive review
on tyrosinase inhibitors. J. Enzyme Inhib. Med. Chem. 2019,34, 279–309. [CrossRef]
7.
Afaq, F.; Katiyar, S.K. Polyphenols: Skin Photoprotection and Inhibition of Photocarcinogenesis. Mini. Rev.
Med. Chem. 2011,11, 1200–1215.
8. Chang, T. An Updated Review of Tyrosinase Inhibitors. Int. J. Mol. Sci. 2009,10, 2440–2475. [CrossRef]
9.
Lyu, X.; Lee, J.; Chen, W.N. Potential natural food preservatives and their sustainable production in yeast:
Terpenoids and polyphenols. J. Agric. Food Chem. 2019,67, 4397–4417. [CrossRef]
10.
Carocho, M.; Barreiro, M.F.; Morales, P.; Ferreira, I.C.F.R. Adding molecules to food, pros and cons: A review
on synthetic and natural food additives. Compr. Rev. Food Sci. Food Saf. 2014,13, 377–399. [CrossRef]
11.
Faustino, M.; Veiga, M.; Sousa, P.; Costa, E.M.; Silva, S.; Pintado, M. Agro-food byproducts as a new source
of natural food additives. Molecules 2019,24, 1056. [CrossRef]
12.
Boutanaev, A.M.; Moses, T.; Zi, J.; Nelson, D.R.; Mugford, S.T.; Peters, R.J.; Osbourn, A. Investigation of
terpene diversification across multiple sequenced plant genomes. Proc. Natl. Acad. Sci. USA
2014
,112, 81–88.
[CrossRef]
13.
Chang, T.S. Natural melanogenesis inhibitors acting through the down-regulation of tyrosinase activity.
Materials 2012,5, 1661–1685. [CrossRef]
14.
Rouf, A.; Naik, H.R.; Beigh, M.A.; Kanojia, V.; Mir, S.A.; Aafia, S.; Ayaz, Q.; Altaf, U. Enzymatic browning of
apple and its control by chemical treatment: A review. Int. J. Food Sci. Nutr. 2018,3, 81–88.
15.
Jdey, A.; Falleh, H.; Ben Jannet, S.; Mkadmini Hammi, K.; Dauvergne, X.; Ksouri, R.; Magn
é
, C. Phytochemical
investigation and antioxidant, antibacterial and anti-tyrosinase performances of six medicinal halophytes.
South African J. Bot. 2017,112, 508–514. [CrossRef]
16.
Haliloglu, Y.; Ozek, T.; Tekin, M.; Goger, F.; Baser, K.H.C.; Ozek, G. Phytochemicals, antioxidant,
and antityrosinase activities of Achillea sivasica Çelik and Akpulat. Int. J. Food Prop.
2017
,20, S693–S706.
[CrossRef]
17.
Welch, C.; Zhen, J.; Bass
è
ne, E.; Raskin, I.; Simon, J.E.; Wu, Q. Bioactive polyphenols in kink
é
liba tea
(Combretum micranthum) and their glucose-lowering activities. J. Food Drug Anal.
2018
,26, 487–496.
[CrossRef]
18.
De Morais Lima, G.R.; De Sales, I.R.P.; Filho, M.R.D.C.; De Jesus, N.Z.T.; De Sousa Falc
ã
o, H.;
Barbosa-Filho, J.M.; Cabral, A.G.S.; Souto, A.L.; Tavares, J.F.; Batista, L.M. Bioactivities of the genus
Combretum (Combretaceae): A review. Molecules 2012,17, 9142–9206. [CrossRef]
19.
Kausar, J.; Muthumani, D.; Hedina, A.; Anand, V. Review of the phytochemical and pharmacological activities
of Euphorbia hirta Linn. Pharmacogn. J. 2016,8, 310–313. [CrossRef]
20.
Al-Snafi, A.E. Pharmacology and therapeutic potential of Euphorbia hirta (Syn: Euphorbia
pilulifera)—A review. J. Pharm. 2017,7, 7–20. [CrossRef]

Molecules 2020,25, 2684 23 of 24
21.
Dedehou, E.; Dossou, J.; Anihouvi, V.; Soumanou, M.M. A review of cashew (Anacardiumoccidentale
L.) apple: Effects of processing techniques, properties and quality of juice. African J. Biotechnol.
2016
,15,
2637–2648.
22.
Cunha, A.G.; Brito, E.S.; Moura, C.F.H.; Ribeiro, P.R.V.; Miranda, M.R.A. UPLC–qTOF-MS/MS-based phenolic
profile and their biosynthetic enzyme activity used to discriminate between cashew apple (Anacardium
occidentale L.) maturation stages. J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
2017
,1051, 24–32.
[CrossRef]
23.
Yi, W.; Wei, Q.; Di, G.; Jing-yu, L.; Yang-li, L. Phenols and flavonoids from the aerial part of Euphorbia hirta.
Chin. J. Nat. Med. 2012,10, 40–42.
24.
Radojkovi, M.; Zekovi, Z.; Joki, S.; Vidovi, S.; Milo, S. Optimization of Solid-Liquid Extraction of Antioxidants
from Black Mulberry Leaves by Response Surface Methodology. Food Technol. Biotechnol. 2012,50, 167–176.
25.
Liu, Y.; Zheng, Y.; Wang, A. Response Surface Methodology for Optimizing Adsorption Process Parameters
for Methylene Blue Removal by a Hydrogel Composite. J. Adsorpt. Sci. Technol.
2011
,28, 913–922. [CrossRef]
26.
Bezerra, M.A.; Santelli, R.E.; Oliveira, E.P.; Villar, L.S.; Escaleira, L.A. Response surface methodology (RSM)
as a tool for optimization in analytical chemistry. Talanta 2008,76, 965–977. [CrossRef] [PubMed]
27.
Taha, F.S.; Mohamed, G.F.; Mohamed, S.H.; Mohamed, S.S.; Kamil, M.M. Optimisation of the Extraction of
Total Phenolic Compounds from Sunflower Meal and Evaluation of the Bioactivities of Chosen Extracts. Am.
J. Food Technol. 2011,6, 1002–1020. [CrossRef]
28.
Ghafoor, K.; Choi, Y.H.; Jeon, J.Y.; Jo, I.H. Optimization of Ultrasound-Assisted Extraction of Phenolic
Compounds, Antioxidants, and Anthocyanins from Grape (Vitis vinifera) Seeds. J. Agric. Food Chem.
2014
,
57, 4988–4994. [CrossRef]
29.
Xu, D.; Zhou, Y.; Zheng, J.; Li, S.; Li, A.; Li, H. Optimization of Ultrasound-Assisted Extraction of Natural
Antioxidants from the Flower of Jatropha integerrima by Response Surface Methodology. Molecules
2016
,21,
18. [CrossRef]
30.
Amirah, D.M.; Khan, M.R. Comparison of Extraction Techniques on Extraction of Gallic Acid from Stem
Bark of Jatropha curcas. J. Appl. Sci. 2012,12, 1106–1111. [CrossRef]
31.
Altemimi, A.; Watson, D.G.; Choudhary, R.; Dasari, M.R. Ultrasound Assisted Extraction of Phenolic
Compounds from Peaches and Pumpkins. PLoS ONE 2016,11, e0148758. [CrossRef]
32.
Liao, J.; Qu, B.; Zheng, N. Effects of Process Parameters on the Extraction of Quercetin and Rutin from the
Stalks of Euonymus Alatus (Thumb.) Sieb and Predictive Model Based on Least Squares Support Vector
Machine Optimized by an Improved Fruit Fly Optimization Algorithm. Appl. Sci. 2016,6, 340. [CrossRef]
33.
Charpe, T.W.; Rathod, V.K. Effect of Ethanol Concentration in Ultrasound Assisted Extraction of Glycyrrhizic
Acid from Licorice Root. Iran. J. Chem. Eng. 2014,11, 21–30.
34.
Gutte, K.B.; Sahoo, A.K.; Ranveer, R.C. Effect of ultrasonic treatment on extraction and fatty acid profile of
flaxseed oil. OCL 2015,22, 2–7. [CrossRef]
35.
Alzeer, J.; Abou Hadeed, K. Ethanol and its Halal status in food industries. Trends Food Sci. Technol.
2016
,58,
14–20. [CrossRef]
36.
Pintus, F.; Span, D.; Corona, A.; Medda, R. Antityrosinase activity of Euphorbia characias extracts. PeerJ
2015,3, e1305. [CrossRef]
37.
Yuet Ping, K.; Darah, I.; Chen, Y.; Sreeramanan, S.; Sasidharan, S. Acute and subchronic toxicity study of
euphorbia hirta L. methanol extract in rats. Biomed Res. Int. 2013,2013, 1–14. [CrossRef]
38.
Rajeh, M.A.B.; Kwan, Y.P.; Zakaria, Z.; Latha, L.Y.; Jothy, S.L.; Sasidharan, S. Acute toxicity impacts of
Euphorbia hirta L extract on behavior, organs body weight index and histopathology of organs of the mice
and Artemia salina. Pharmacogn. Res. 2012,4, 170–177.
39.
Innocent, B.Y.; Daniel, A.; Mikpons
è
, D.A.A.; Fr
é
d
é
ric, L.; Raphaël, D.; Evelyne, L.; C
é
lestin, T.C.K.; Sachi, P.;
Roseline, B.; Apollinaire, M.G. Histopathological study of toxicity mixture of cashew apple juice (Anacardium
occidentale), cow milk and yogurt on the wistar rat. J. Exp. Biol. Agric. Sci. 2016,4, 435–439.
40.
Kpemissi, M.; Metowogo, K.; Melila, M.; Veerapur, V.P.; Negru, M.; Taulescu, M.; Pot
â
rniche, A.V.; Suhas, D.S.;
Puneeth, T.A.; Vijayakumar, S.; et al. Acute and subchronic oral toxicity assessments of Combretum
micranthum (Combretaceae) in Wistar rats. Toxicol. Rep. 2020,7, 162–168. [CrossRef] [PubMed]
41.
Pi
ó
ro-Jabrucka, E.; Pawełczak, A.; Przybył, J.L.; W˛eglarz, Z. Accumulation of phenolic and sterol compounds
in Euphorbia hirta (L.). Herba Pol. 2011,57, 30–37.

Molecules 2020,25, 2684 24 of 24
42.
Su
ê
nia, M.; Vilar, D.A.; De Souza, G.L.; Vilar, D.D.A.; Leite, J.A.; Raffin, F.N.; Barbosa-Filho, J.M.; Henrique, F.;
Nogueira, A.; Rodrigues-Mascarenhas, S.; et al. Assessment of Phenolic Compounds and Anti-Inflammatory
Activity of Ethyl Acetate Phase of Anacardium occidentale L. Bark. Molecules 2016,21, 1–17.
43.
Sampaio, B.L.; Edrada-Ebel, R.; Batista, F.; Costa, D. Effect of the environment on the secondary metabolic
profile of Tithonia diversifolia: A model for environmental metabolomics of plants. Sci. Rep.
2016
,6, 1–11.
[CrossRef] [PubMed]
44.
Yang, Z.; Zhang, Y.; Sun, L.; Wang, Y.; Gao, X.; Cheng, Y. Analytica Chimica Acta An ultrafiltration
high-performance liquid chromatography coupled with diode array detector and mass spectrometry
approach for screening and characterising tyrosinase inhibitors from mulberry leaves. Anal. Chim. Acta
2012
,
719, 87–95. [CrossRef]
45.
Wang, B.; Juang, L.; Yang, J.; Chen, L.; Tai, H.; Huang, M. Antioxidant and Antityrosinase Activity of
Flemingia macrophylla and Glycine tomentella Roots. Evid.-Based Complement. Altern. Med.
2012
,2012, 1–7.
46.
Wang, Y.; Zhang, G.; Yan, J.; Gong, D. Inhibitory effect of morin on tyrosinase: Insights from spectroscopic
and molecular docking studies. Food Chem. 2014,163, 226–233. [CrossRef] [PubMed]
47.
Asthana, S.; Zucca, P.; Vargiu, A.V.; Sanjust, E.; Ruggerone, P.; Rescigno, A. Structure—Activity Relationship
Study of Hydroxycoumarins and Mushroom Tyrosinase. J. Agric. Food Chem.
2015
,63, 7236–7244. [CrossRef]
[PubMed]
48.
Taofiq, O.; Gonz
á
lez-Param
á
s, A.M.; Barreiro, M.F.; Ferreira, I.C.F.R.; McPhee, D.J. Hydroxycinnamic acids
and their derivatives: Cosmeceutical significance, challenges and future perspectives, a review. Molecules
2017,22, 281. [CrossRef] [PubMed]
49.
Vardanega, R.; Santos, D.T.; Meireles, M.A.A. Intensification of bioactive compounds extraction from
medicinal plants using ultrasonic irradiation. Pharmacogn. Rev. 2014,8, 88–95. [PubMed]
50.
Li, H.; Zhang, Z.; Xue, J.; Cui, L.; Hou, T.; Li, X.; Chen, T. Optimization of ultrasound-assisted extraction
of phenolic compounds, antioxidants and rosmarinic acid from perilla leaves using response surface
methodology. Food Sci. Technol. 2016,36, 686–693. [CrossRef]
51.
Zhen-Ming, L.; Zhe, G.J.; Hong-yu, X.; Wen-fang, D.; Zheng-hong, S.J. Optimization of Extraction of Total
Triterpenoids from Submergedly Cultured Antrodia camphorata using Response Surface Methodology.
Nat. Prod. Res. Dev. 2011,23, 946–951.
52.
El Khoury, R.; Michael, R.; Marc, J.; Beyrouthy, E.; Baillet, A.; Toufic, G.; Ali, R.; Roger, T. Phytochemical
screening and antityrosinase activity of carvacrol, thymoquinone, and four essential oils of Lebanese plants.
J. Cosmet Dermatol. 2018,18, 1–9. [CrossRef] [PubMed]
53.
Kumar, T.; Khan, Z.; Oulkar, D.; Singh, B.K.; Maurya, A.; Singh, B.; Banerjee, K. High resolution LC-MS
characterization of phenolic compounds and the evaluation of antioxidant properties of a tropical purple
radish genotype. Arab. J. Chem. 2017,13, 1355–1366. [CrossRef]
54.
Sahin, S.; Aybastıer, Ö.; Isik, E. Optimisation of ultrasonic-assisted extraction of antioxidant compounds from
Artemisia absinthium using response surface methodology. Food Chem.
2013
,141, 1361–1368. [CrossRef]
[PubMed]
55.
Amado, I.R.; Franco, D.; Sanchez, M.; Zapata, C.; Vazquez, J.A. Optimisation of antioxidant extraction from
Solanum tuberosum potato peel waste by surface response methodology. Food Chem.
2014
,165, 290–299.
[CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).