Industrial Crops and Products 89 (2016) 514521

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Industrial Crops and Products

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Antioxidant and anti-lipases activities in vitro of Mentha viridis and

Eucalyptus globulus extracts
Hamdi Belfeki a,c, , Mondher Mejri b , Mnasser Hassouna c
a
Process Engineering Department, Higher Institute of Technological Studies, Zaghouan, Tunisia
b
Molecules-Materials and Applications Laboratory, IPEST, La Marsa, Tunisia
c
Food Preservation Laboratory, High School of Food Industries, Elkhadra, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: Plants are a source of bioactive compounds such as antioxidants and enzyme inhibitors. In this work,
Received 21 January 2016 effect of the Eucalyptus globulus and Mentha viridis extraction by different solvents on phenolics content,
Received in revised form 30 May 2016 avonoids content, condensed tannins content, antioxidant activity and inhibitory effect against two
Accepted 1 June 2016
lipases from Aspergillus niger and olive was studied. Methanolic (MeOH) extract showed the strongest
antioxidant and anti-lipase activities. High correlation between total phenolics content, total avonoids
Keywords:
content, antioxidant activity and lipases inhibitory action was observed. Kinetic analysis of lipases inhi-
Lipase inhibition
bition was performed with Linweaver-Burk, Dixon and Cornish-Bowden plots. These results revealed
Polyphenols
Antioxidant activity
competitive inhibition mode by E. globulus and non competitive mixed inhibition mode by M. viridis
Eucalyptus globulus against the two lipases. Inhibition constant (KI ) values calculated with E. globulus MeOH extract were
Mentha viridis 0.271 and 0.188 mg/mL for Aspergillus niger and olive lipases respectively. With M. viridis MeOH extract
Kinetic analysis KI values were 0.303 and 0.421 mg/mL for the fungal and the plant lipases respectively. The inhibitory con-
centration 50% (IC50 ) values determined with E. globulus MeOH extract were 0.69 mg/mL for Aspergillus
niger lipase and 1.29 mg/mL for olive lipase. With M. viridis MeOH extract IC50 were 0.43 and 1.44 mg/mL
for Aspergillus niger and olive lipases respectively.
2016 Elsevier B.V. All rights reserved.

1. Introduction chemical and organoleptic characteristics of the extra virgin olive

Lipases (EC 3.1.1.3) are glycerol ester hydrolases that cat-
alyze the hydrolysis of triglycerides to free fatty acids and
glycerol (Joseph et al., 2008). Lipases are produced by most bio-
logical systems including animals, plants and microorganisms
(Mohanasrinivasan et al., 2009). Lipases are widespread in yeasts
and in fungi (Fickers et al., 2008). Lipase has been extensively stud-
ied because of its strong negative impact on oil quality. Hydrolysis
of triacylglycerol (TAG) is caused by endogenous and exogenous
lipases secreted by fungi (Ngando Ebongue et al., 2006). Olive
TAGs can be hydrolyzed during oil extraction process by an active
lipase present in olive seeds as well as by exogenous lipases pro-
duced by fungi which often contaminate the fruits. Recent research
has shown that freshly produced olive oil is contaminated with
lipase positive microorganisms capable of altering the physico-
Corresponding author at: Process Engineering Department, Higher Institute of
Technological Studies, Zaghouan, Tunisia.
E-mail address: hamdibelfeki@yahoo.fr (H. Belfeki).
oil (Panzanaro et al., 2010).
Lipase inhibitors can be used to prevent undesirable changes in
foods may be caused by lipase (Shimura et al., 1992). Some have
been isolated from the roots of Glycyrrhiza uralensis, from the fresh
roots of Platycodon grandioru, from Dioscorea nipponica, from the
leaves of Nelumbo nucifera, and from other kinds of plants (Zheng
et al., 2010). The presence of lipase inhibitors have been reported
in some natural sources like marine algae, soybean, wheat, citrus,
oolong tea and aqueous extract of some medicinal herbs (Sharma
et al., 2005).
There have been reports that naturally-occurring polyphenols
can inhibit lipase. Many studies have focused on polyphenols from
teas, herbal and fruit sources (McDougall et al., 2009). Inhibition
studies on lipases could contribute to better understand the mech-
anism of action of these enzymes in order to design novel substrate
specicities for catalyzing specic reactions useful for industrial
applications and new inhibitory compounds of either pharmaco-
logical or biotechnological interest (Ruiz et al., 2004). This is why,
it remains necessary to search for more efcacious lipase inhibitors
from natural sources.

http://dx.doi.org/10.1016/j.indcrop.2016.06.002
0926-6690/ 2016 Elsevier B.V. All rights reserved.
 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521 515

In this work, the potential of Eucalyptus globulus and Mentha
viridis as antioxidants and as two new sources of lipase inhibitors
has been shown. Aspergillus niger lipase and lipase extracted from
olive mesocarp were used as models. Kinetic study allowed the
authors of this study to calculate the parameters KM , Vmax , IC50 , KI
and also determine the mechanism of inhibition.
2. Materials and methods
2.1. Plant material
Mentha viridis (Lamiaceae) and Eucalyptus globulus (Myrtaceae)
were bought from Kamy SA company (Nabeul, Tunisia). Leaves
were cut into small pieces and air-dried in the shade. The dried sam-
ples were then ground to a ne powder using a grinder (moulinex,
LM240, France). Olives (Olea europea) were collected in Zaghouan
in north of Tunisia.
2.2. Lipases
for 5 min before 1 mL of a 7.5% sodium carbonate aqueous solution
was added. Samples were allowed to stand for 120 min at room
temperature before measuring their absorbance at 760 nm versus
the blank using a HACH UVvis spectrophotometer.
2.5. Determination of total avonoids content
Total avonoids content (TFC) was determined by spectropho-
tometric assay using the method of Dewanto et al. (2002). A
calibration curve of catechin (ranging from 0.01 to 0.225 g/L) was
prepared, and the results, determined by the regression equation of
the calibration curve (y = 0.05075 x, r2 = 0.99), were expressed as mg
catechin equivalents per gram of extract (mg CE/g extract). In this
method, 50 L of sample were mixed with 75 L of distilled water,
35.5 L of NaNO2 (5%) and 75 L of AlCl3 -6H2 O (10%) freshly pre-
pared. The mixture was allowed to stand for 5 min before 1250 L
of distilled water were added. The absorbance of all samples was
measured at 510 nm.

Lipase from Aspergillus niger was purchased from Sigma-Aldrich

2.6. Determination of condensed tannins
(St. Louis, MO, USA). Lipase from olive mesocarp was extracted
according to method of Panzanaro et al. (2010). The exocarp of
Condensed tannins content (CTC) are determined by the
the olive fruits at ripe stage was removed using a scalpel blade.
method described in Julkunen-Tiitto (1985) in the acidic medium
Mesocarp slices (approximately 5 g each) were homogenized with
vanillin with some modications. A calibration curve of catechin
mortar and pestle on ice upon the addiction of cold grinding
(0.151.5 g/L) was prepared, and the results, determined by the
medium (0.6 M sucrose, 0.1 M HEPES/NaOH, 10 mM KCl, 1 mM
regression equation of the calibration curve (y = 0.116 x, r2 = 0.996),
MgCl2 , 1 mM EDTA, 2 mM DTT, pH 7.5). The homogenate was cen-
were expressed as mg catechin equivalents per gram of extract (mg
trifuged at 10,000g at 4 C for 30 min. The oating fatty layer,
CE/g extract). An aliquot (500 L) of extract, 1.5 mL of vanillin (4%)
corresponding to the crude lipid body fraction, was removed from
diluted in methanol and 500 L of concentrated HCl (8%) diluted
the surface of the supernatant with a spatula and resuspended in
in methanol were mixed and kept for 15 min at room temperature.
buffer medium containing a non-ionic detergent (1 mM Tris, pH 7.5,
The same protocol was carried out for the control by replacing sam-
2 mM DTT, 0.5% Triton X-100, approximately 4 mL g1 fatty layer)
ple by the HCl solution. The absorbance was measured at 500 nm.
for 30 min at 4 C. The suspension was mixed vigorously with a vor-
tex and centrifuged again as above. The lipids, the top layer after
the second centrifugation, were removed and the remaining super-
2.7. -Carotene bleaching test
natant (soluble protein fraction) were ltered through two layers
of cheesecloth. The resulting ltrate is the crude lipase extract. Pro-
The antioxidant activity of extracts was evaluated according to
tein was quantied by method of Lowry et al. (1951) using bovine
a slightly modied version of the -carotene bleaching method
serum albumin as standard.
(Pratt, 1980). -Carotene (0.5 mg), 25 mg of linoleic acid and
200 mg of Tween 40 (polyoxyethylene sorbitan monopalmitate)
2.3. Preparation of plant extracts
were mixed with 1 mL of chloroform. Chloroform was removed
at 45 C, under vacuum, using a rotary evaporator (Heidolph,
Maceration of crude powder was carried out in a one-step
Germany). The resulting mixture was immediately diluted with
extraction (batch mode). A dried sample (20 g) was extracted with
100 mL of oxygenated distilled water and was mixed well for
200 mL of solvents with different polarity and mixed vigorously
12 min. Aliquots (2.5 mL) of this emulsion were transferred into
for 1 h. The solvents used were selected by increasing polarity:
different test tubes containing 0.35 mL of test samples. Butylated
dichloromethane DCM (3.1), ethyl acetate EtOAc (4.4), methanol
hydroxytoluene (BHT) was used for comparative purposes. A con-
MeOH (5.1), acetonitrile ACN (5.8) and distilled water DW (9). The
trol, containing 0.35 mL of corresponding solvent and 2.5 mL of the
resulting mixture was kept for 24 h at room temperature in dark-
above emulsion, was prepared. The tubes were placed, at 50 C, in
ness and ltered through a No. 1 Whatman paper. Solvents were
a water bath.
removed with rotary evaporator. Extracts obtained were diluted in
Absorbance of all the samples at 470 nm was measured at time
distilled water to give a concentration of 100 mg/mL and kept at
zero (t = 0). Measurement of absorbance was continued, until -
20 C for various analyses.
carotene color disappeared in control reaction (t = 140 min), at
20 min intervals. A mixture prepared as above, without -carotene,
2.4. Determination of total phenolics content
served as blank. The antioxidant activity (AA) of the extracts was
evaluated in terms of bleaching of the -carotene using the follow-
The total phenolics content (TPC) was determined with the
ing formula:
Folin-Ciocalteu reagent using the method of Lister and Wilson
(2001). A calibration curve of gallic acid (ranging from 0.01 to   
AA (%) = 100 1 A0 At )/(Ac0 Act
0.25 g/L) was prepared, and the results, determined by the regres-
sion equation of the calibration curve (y = 0.00971 x, r2 = 0.99), were
expressed as mg gallic acid equivalents per gram of extract (mg where A0 and Ac0 are the absorbance values measured at zero
GAE/g extract). In this method, 250 L of sample were dissolved time of the incubation for test sample and control, respectively.
in 1.125 mL of distilled water and 125 L of the Folin-Ciocalteu Absorbance values At and Act are measured in the test sample and
reagent. The samples were mixed well and then allowed to stand control, respectively at (t) time of incubation.
516 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521

2.8. Lipases inhibition assay Table 1

Extraction yields, total phenolics content, total avonoids content and condensed
tannins content of Eucalyptus globulus and Mentha viridis extracts.
Lipase inhibition assay was performed using olive oil emulsion
as a substrate (Zhang et al., 2012). The emulsion was prepared Samples EY TPC TFC CTC
by suspending 10 g of olive oil into 100 mL of 2% gum Arabic in Eucalyptus globulus
0.1 M citric acid-sodium citrate buffer solution (pH 5.0). The mix- DCM 6.10 0.18a 50.25 1.24a 21.35 0.44a 4.88 0.06a
ture was homogenized for 3 min in a blender. After the addition EtOAc 9.40 0.26b 30.52 1.33b 13.60 0.72b 3.91 0.07b
MeOH 13.08 0.22c 128.34 1.63c 30.74 0.68c 25.58 0.15c
of 2 mL of 1 M CaCl2 , the reaction mixture was incubated at 35 C
ACN 8.80 0.25b 60.31 0.8d 18.45 0.32d 13.50 0.2d
for 1 min. One mL of enzyme solution was preincubated 30 min DW 14.40 0.38c 102.52 2.12e 35.50 0.81e 34.19 0.12e
with 1 mL of plant extract at room temperature and added to 18 mL
Mentha viridis
of the emulsion. The mixture was incubated for 30 min at 35 C DCM 4.72 0.14d 42.15 0.66f 14.29 0.42b 16.11 0.26f
with agitation at 200 rpm. The enzymatic reaction was halted by EtOAc 19.72 0.36e 50.25 1.42a 17.40 0.27d 5.30 0.07g
the addition of 20 mL of acetone/ethanol mixture (1:1, v/v) and MeOH 22.22 0.52f 94.01 1.13eg 32.64 0.63c 36.39 0.23h
the liberated free fatty acids (FA) were titrated with a solution of ACN 5.83 0.15a 52.01 1.38a 20.36 0.34a 17.50 0.2i
DW 18.05 0.42e 85.70 2.5g 25.75 0.5f 6.03 0.13j
0.05 N NaOH. Phenolphthalein was used as indicator. Final enzyme
concentration was 25 g/mL. Negative control was used by adding Values are mean standard deviation of triplicate analyses. In the same column,
values followed by the same letter are not signicantly different (p 0.05). EY:
ethanol/acetone mixture at the beginning of the procedure. Posi-
extraction yields expressed as%; TPC: total phenolics content expressed as mg gal-
tive control representing 100% of the activity was prepared under lic acid equivalents per gram of extract; TFC: total avonoids content expressed
the same conditions by replacing plant extract with appropriate as mg catechin equivalents per gram of extract; CTC: condensed tannins content
solvent. The velocity of product formation was expressed as mol expressed as mg catechin equivalents per gram of extract. DCM: dichloromethane;
FA/min. Lipase inhibition (LI) was calculated according the follow- EtOAc: ethyl acetate; MeOH: methanol; ACN: acetonitrile; DW: distilled water.

ing formula:
  obtained with E. globulus. Variation in the yields of various extracts
LI (%) = 100 (A a) (B b) / (A a)
is attributed to polarities of different compounds present in the
where A is the activity without inhibitor, a: the negative control leaves. Yields extraction of E. globulus leaves are higher than E. glob-
without inhibitor, B: the activity with inhibitor, and b is the negative ulus wood (Fernndez-Agull et al., 2015). Yield MeOH extract from
control with inhibitor. M. viridis is higher than petroleum ether and ethanolic extracts from
Mentha spicata, Mentha pulegium and Mentha rotundifolia (Bimakr
2.9. Kinetic analysis of enzyme inhibition et al., 2011; Brahmi et al., 2015).

In order to measure the inhibition mode by MeOH extracts
of E. globulus and M. viridis, lipase activity was assayed with dif-
ferent concentrations of olive oil as substrate (4.5, 9, 22.5 and
45 mg/mL) in the absence and presence of three concentrations
of the extracts (0.625, 1.25 and 2.5 mg/mL). The inhibitory con-
centration 50% (IC50 ) was calculated from the regression equation
obtained by plotting the percentage of inhibition versus the con-
centrations of E. globulus and M. viridis extracts. The Michaelis
constant (KM ), maximal velocity (Vmax ) and the inhibition mode
was determined by double-reciprocal Lineweaver-Burk plot (1/V
versus 1/[S]) and Dixon plot (1/V versus [I]). In order to estimate the
inhibition constant (KI ), slopes of double-reciprocal lines were used
to construct a secondary Dixon plot and from the line equation of
the plot KI was calculated. The dissociation constant KI was deter-
mined by Cornish-Bowden plot ([S]/V versus [I]) (Bowden, 1974;
Dixon, 1953).
2.10. Statistical analysis
All determinations were made in triplicate and data is reported
as mean SD. Data were analyzed using ANOVA variance analysis,
Minitab 2012 Version 16.2.3 statistical software (Minitab Inc., Pen-
nysalvania, USA). Correlation between TPC, TFC, CTC, AA and LI was
carried out using the correlation and regression in the EXEL pro-
gram. A probability value at p 0.05 was considered statistically
signicant.
3. Results and discussion
3.1. Extraction yields
Yields of E. globulus and M. viridis extracts obtained by differ-
ent solvents were examined and are presented in Table 1. Highest
yields were recorded when extractions were performed with MeOH
and DW. The yields obtained with M. viridis were higher than those
3.2. Amount of total phenolics
The highest total phenolic levels were detected when MeOH was
used for extraction as shown in Table 1. The total phenolics content
in MeOH extract of E. globulus leaves was higher than in E. globulus
bark ethanol extract, Eucalyptus camaldulensis leaves chloroform
and hexane extracts. But it was less than in E. camaldulensis leaves
MeOH extract, E. globulus bark MeOH and DW extracts (Vzquez
et al., 2008; Ashraf et al., 2015). The total phenolics content in
M. viridis MeOH extract was higher than in M. longifolia and M.
pulegium MeOH extracts (Gulluce et al., 2007; Khaled-Khodja et al.,
2014). It was relatively similar to Mentha arvensis acetone extract
found by Sulaiman et al. (2011) with 95.7 mg GAE/g.
3.3. Amount of total avonoids
The highest TFC levels were detected when DW and MeOH were
used for E. globulus and M. viridis extraction respectively as shown
in Table 1. The total avonoids content in DW and MeOH extracts of
E. globulus was higher than MeOH, chloroform and hexane E. camal-
dulensis extracts (Ashraf et al., 2015). The amount of total avonoids
in M. viridis MeOH extract was higher than in M. arvensis in acetone
and ethanol extracts (Sulaiman et al., 2011).
3.4. Amount of condensed tannins
The highest CTC levels were detected when DW and MeOH were
used for E. globulus and M. viridis extraction respectively as shown
in Table 1. The amount of condensed tannins in DW and MeOH
extracts of E. globulus leaves was higher than some Eucalyptus
species such as Eucalyptus cladocalyx, Eucalyptus platypus, Eucalyp-
tus stowardii, Eucalyptus gomphocephala and Eucalyptus ocktoniae
(Martius et al., 2012). The condensed tannins content in M. viridis
MeOH extract was higher than in M. pulegium (Khaled-Khodja et al.,
2014) and in Mentha piperata MeOH extracts (Sujana et al., 2013).
 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521 517

Table 2 Table 3
Relationship between antioxidant activity and phytochemical composition of E. Inhibition of Aspergillus niger and olive lipases by E. globulus, M. viridis extracts and
globulus and M. viridis extracts expressed as coefcient of determination (r2 ). standards.

Composition Antioxidant activity (%) Samples Inhibition (%)

E. globulus M. viridis Aspergillus niger lipase Olive lipase

Total phenolics content 0.795 0.845 Eucalyptus globulus

Total avonoids content 0.590 0.880 DCM 20.85 0.25a 17.23 0.36a
Condensed tannins content 0.803 0.169 EtOAc 10.42 1.12b 25.33 0.66b
MeOH 78.92 1.33c 1 82.65 2.35c 1
ACN 35.5 0.85d 2 36.4 1.5d 2
3.5. Antioxidant activity DW 62.75 0.75e 68.63 2.2ef

Mentha viridis
Antioxidant activity of E. globulus and M. viridis leaves extracts DCM 35.42 1.48df 24.66 1.28b
EtOAc 40.42 0.9f 37.1 0.55d
was evaluated, in comparison with synthetic antioxidant BHT, by
MeOH 72.75 0.45g 3 75.75 1.33bf 3
-carotene/linoleic acid bleaching method as shown in Fig. 1. In ACN 46.35 2.25f 52.72 0.78g
this study, the extracts of E. globulus and M. viridis were found to DW 64.75 1.81e 4 61.39 1.05e 4
limit -carotene bleaching by neutralizing the linoleate-free radi-
Standards
cal. Its known that the type of solvent is the most important factors EDTA (1 mM) 66.25 0.66e 55.13 0.33g
in order to obtain a high quality natural antioxidant. Methanol and SDS (10 mg/mL) 99 0.1h 5 99 0.1h 5
DW were found to be more efcient than ACN, DCM or EtOAc in Values in the same line with same subscript numbers are not signicantly different
extracting the antioxidants present in E. globulus and M. viridis (p 0.05). In the same column, values followed by the same letter are not signif-
leaves. A large variation in the antioxidant activities was observed icantly different (p 0.05). DCM: dichloromethane; EtOAc: ethyl acetate; MeOH:
in E. globulus and in M. viridis extracts. Antioxidant activity in MeOH methanol; ACN: acetonitrile; DW: distilled water; EDTA: ethylene diamine tetra
acetic acid; SDS: sodium dodecyl sulfate.
and DW E. globulus leaves extracts were higher than acetone-water
(70:30, v/v) E. globulus fruits extract (Boulekbache-Makhlouf et al.,
2013). Antioxidant effect observed in E. globulus leaves was due to Table 4
Relationship between lipases inhibition and the others characteristics of E. globulus
its high TPC and CTC as shown in Table 2. The same results were
and M. viridis extracts expressed as coefcient of determination (r2 ).
found by Amakura et al. (2009).
The results of Table 2, conrming that phenolics and avonoids Characteristics Inhibition (%)
coumpounds are responsible for a signicant part of M. viridis Aspergillus niger lipase Olive lipase
antioxidant capacity. Similar relationships have also been observed
E. globulus
by Nickavar et al. (2008). The effectiveness of phenolics and Total phenolics content 0.990 0.922
avonoids as antioxidants is not only attributed to their compo- Total avonoids content 0.776 0.700
sition or relative amount but also to the degree of polymerization, Condensed tannins content 0.814 0.823
concentration and interaction of their diverse chemical structures Antioxidant activity 0.836 0.965

to the colorimetric assays (Sulaiman et al., 2011). M. viridis

3.6. Lipases inhibition and kinetic properties
Inhibition of Aspergillus niger and olive lipases by different
extracts from E. globulus leaves varied from 10.42 to 82.65% and
from 24.66 to 75.75% in M. viridis extracts as shown in Table 3.
The results of anti-lipases assay support that E. globulus and M.
viridis extracts were effective to inhibit the lipases studied. In fact,
MeOH E. globulus and M. viridis extracts presented greatest inhi-
bition against lipase of Aspergillus niger with 78.92 and 72.75%
respectively, and against lipase of olive with 82.65 and 75.75%
respectively. This anti-lipase activity is higher than that obtained
with EDTA at 1 mM but lower than that obtained with SDS at
10 mg/mL. Lipases inhibitory effect by MeOH extract of E. globu-
lus and M. viridis leaves was stronger than MeOH extracts from
36 traditional Chinese medicinal herbs (Zheng et al., 2010), many
ethanol/water (1:1) extracts of Korean medicinal plants (Kim and
Kang, 2005) and many water/methanol (1:1) extracts of Slovenian
medicinal plants (Slanc et al., 2004).
Correlation between TPC, TFC, CTC, antioxidant activity and
lipases inhibition was recapitulated in Table 4. It was suggested that
phenolic compounds might be a major contributor for the lipases
inhibition. In fact, for TPC and the fungal lipase inhibition, r2 calcu-
lated were 0.990 and 0.982 respectively in E. globulus and M. viridis
extracts. For TFC and olive lipase inhibition, r2 calculated were
0.922 and 0.948 respectively in E. globulus and M. viridis extracts.
For CTC in E. globulus extracts and LI, r2 calculated were 0.814 and
0.823 respectively for the fungal and olive lipases. Many polyphe-
nolics such as avones, avonols, tannins and chalcones are active
against lipase (Birari and Bhutani, 2007). Role of phenolic coum-
Total phenolics content 0.982 0.841
Total avonoids content 0.969 0.948
Condensed tannins content 0.249 0.298
Antioxidant activity 0.860 0.848
pounds against pancreatic lipase activity was demonstrated by You
et al. (2012) in muscadine, by McDougall et al. (2009) in berry and by
Gondoin et al. (2010) in tea. It is well-known that polyphenols from
plants have an afnity for proteins, primarily through hydrophobic,
as well as hydrogen bondings.
Much research has focused on the inhibition of human or
porcine pancreatic lipases but very few have focused on micro-
bial and plant lipases. However, this lipases are also involved in
several food alterations. Yet, most of the previous researches only
reported the inhibitive activity of the tested samples against the
lipase within the range of concentrations instead of the inhibitive
mode. In this study, the inhibitory mode was determined from the
Lineweaver-Burk and Dixon plots of samples compared with the
control as shown in Figs. 2 and 3.
In the LB plots for lipases inhibition by E. globulus extracts (Fig. 2a
and b), all lines nearly gave the same intercepts in the y axis in the
plot. Although the mathematical equations for them differ in slopes,
which indicated that, no matter of the lipase source (Aspergillus
niger or olive), the enzymatic inhibition modes were the same
belonged to the competitive type. Otherwise, LB plots for lipases
inhibition by M. viridis extracts (Fig. 2c and d) show an intersec-
tion above the x axis, which reected a mixed non competitive
inhibition mode.
Dixon plots (Fig. 3) shows an intersection above the x axis,
conrming the results obtained by LB plots for competitive inhi-
518 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521

a b
100 100

Percentage inhibion (%) 80 80

Percentage inhibion (%)

60 60

40 40
BHT BHT
DCM DCM
EtOAc EtOAc
20 20
DW DW
MeOH MeOH
ACN ACN
0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
Time (min) Time (min)

Fig. 1. Antioxidant activity of E. globulus (a) and M. viridis (b) extracts. Each point represents the mean of three experiments and the vertical bars represent the standard
error of measurement.

a 1.8 b
1.4
I = 0.625 mg/mL I = 0.625 mg/mL
1.6 I = 1.25 mg/mL
1.2 I = 1.25 mg/mL
1.4 I = 2.5 mg/mL I = 2.5 mg/mL
1/V (mol FA/min)-1

1/V (mol FA/min)-1

I = 0 mg/mL 1 I = 0 mg/mL
1.2
1 0.8
0.8
0.6
0.6
0.4
0.4
0.2 0.2

0 0
-0.1 0 0.1 0.2 0.3 0.4 0.5 -0.1 0 0.1 0.2 0.3 0.4 0.5
1/[S] (mg/mL)-1 1/[S] (mg/mL)-1

c d
1.4 1.4
I = 0.625 mg/mL I = 0.625 mg/mL
1.2 I = 1.25 mg/mL 1.2 I = 1.25 mg/mL
1/V (mol FA/min)-1

1/V (mol FA/min)-1

I = 2.5 mg/mL I = 2.5 mg/mL

1 I = 0 mg/mL 1 I = 0 mg/mL

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0 0
-0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5
1/[S] (mg/mL)-1 1/[S] (mg/mL)-1

Fig. 2. LB plots of E. globulus extracts with Aspergillus niger lipase (a), olive lipase (b) and of M. viridis extracts with Aspergillus niger lipase (c) and olive lipase (d). Each point
represents the mean of three experiments and the vertical bars represent the standard error of measurement.
 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521 519

a b 1.4
1.8 S = 2.25 mg/mL
S = 2.25 mg/mL
S = 9 mg/mL
1.6 S = 9 mg/mL 1.2 S = 22.5 mg/mL

1/V (mol FA/min)-1

S = 22.5 mg/mL
1.4 S = 45 mg/mL

1/V (mol FA/min)-1

S = 45 mg/mL 1
1.2
0.8
1
0.8 0.6
0.6
0.4
0.4
0.2
0.2
0 0
-1 0 1 2 3 -1 0 1 2 3
[I] (mg/mL) [I] (mg/mL)

c d 1.4
1.4 S = 2.25 mg/mL
S = 2.25 mg/mL
S = 9 mg/mL 1.2 S = 9 mg/mL
1.2 S = 22.5 mg/mL
S = 22.5 mg/mL
1/V (mol FA/min)-1

1/V (mol FA/min)-1

S = 45 mg/mL 1 S = 45 mg/mL
1

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0 0
-1 0 1 2 3 -1 0 1 2 3
[I] (mg/mL) [I] (mg/mL)

Fig. 3. Dixon plots of E. globulus extracts with Aspergillus niger lipase (a), olive lipase (b) and of M. viridis extracts with Aspergillus niger lipase (c) and olive lipase (d). Each
point represents the mean of three experiments and the vertical bars represent the standard error of measurement.

bition with E. globulus and mixed non competitive inhibition with Table 5
Kinetic parameters and inhibition modes.
M. viridis. The ndings, therefore, revealed that a crude extract of
E. globulus exerted a dose dependent competition with TAG for a Parameters Control E. globulus M. viridis
common binding site in Aspergillus niger or olive lipase. Similar
AnL OL AnL OL AnL OL
results for competitive inhibition mode were reported by Zhang
Vmax (mol FA/min) 5.58 7.24 5.64 7.35 2.84 4.21
et al. (2011) in grape skin and by Han et al. (2001) in tea. On the
KM (mg/mL) 0.82 1.01 19.58 19.66 6.27 9.13
other hand, crude extract of M. viridis has a higher afnity than KI (mg/mL) 0.271 0.188 0.303 0.421
substrate for binding the free or bound enzyme. This mixed non KI (mg/mL) 2.79 3.46
competitive inhibition was also observed in Levisticum ofcinale IC50 (mg/mL) 0.69 1.29 0.43 1.44
(Gholamhoseinian et al., 2010) and in cocoa extract (Gu et al., 2011) Inhibition mode competitive non competitive mixed

against pancreatic lipase.
Dixon plot using slope of double-reciprocal lines (Fig. 4) was
used for determination of inhibition constant (KI ) by regression
equation. In presence of MeOH extract of E. globulus leaves KI was
0.271 mg/mL for Aspergillus niger lipase and 0.188 mg/mL for olive
lipase. In presence of MeOH extract of M. viridis leaves KI was
0.303 mg/mL for Aspergillus niger lipase and 0.421 mg/mL for olive
lipase. In comparison with the present study, the KI value of tea
saponin was determined to be 0.25 mg/mL (Birari and Bhutani,
2007), and the KI values of EtoAc extracts of muscadine fruit and
seed were 46.22 mg/mL and 7.52 mg/mL (You et al., 2012).
Kinetic properties were calculated and are listed in Table 5.
Methanol extract of E. globulus does not affect maximal velocity of
Aspergillus niger or olive lipase. But Michaeilis constant was modi-
ed by increasing its value from 0.82 to 19.58 mg/mL for Aspergillus
niger lipase and from 1.01 to 19.66 mg/mL for olive lipase. Hence,
inhibitors from E. globulus act by reducing afnity of enzyme for
its substrate. The results indicate that 0.69 mg/mL and 1.29 mg/mL
of E. globulus MeOH extract were able to inhibit 50% of the lipase
activity of Aspergillus niger and olive respectively.
AnL: Aspergillus niger lipase; OL: olive lipase; Vmax : maximal velocity; FA: fatty acids;
KM : Michaelis constant; KI : dissociation constant of enzyme-inhibitor complex;
KI : dissociation constant of enzyme-substrate-inhibitor complex; IC50 : inhibitory
concentration 50%.
Methanol extract of M. viridis decreased Vmax of Aspergillus niger
and olive lipase from 5.58 to 2.84 and from 7.24 to 4.21 mol
FA/min.mg of enzyme, respectively. Michaeilis constant was mod-
ied by increasing its value from 0.82 to 6.27 mg/mL for Aspergillus
niger lipase and from 1.01 to 9.13 mg/mL for olive lipase. So, they
can bind to either enzyme or enzyme-substrate (ES) with the dis-
sociation constants KI or KI , respectively. Dissociation constant
KI values calculated from Cornish-Bowden plot (Fig. 5) were 2.79
and 3.46 mg/mL for Aspergillus niger and olive lipases respectively.
Values of KI exceed KI values, indicating a higher afnity to bind
free enzyme than the ES complex. Concentrations of 0.43 mg/mL
and 1.44 mg/mL of M. viridis MeOH extract were able to inhibit 50%
of the lipase activity of Aspergillus niger and olive respectively.
The results indicate that inhibitory effects of E. globulus and M.
viridis were strong in comparison with Cyclocarea paliurus extract
520 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521

a 4 b 3
3.5
2.5
3

LB slope
2

LB slope
2.5
2 1.5
y = 0.350 + 1.291x y = 0.191 + 1.012x
1.5
r = 0.982 1 r = 0.996
1
0.5 0.5

0 0
-1 0 1 2 3 -1 0 1 2 3
[I] (mg/mL) [I] (mg/mL)

c 2.5 d 2.5

2 2
LB slope

LB slope
1.5 1.5
y = 0.246 + 0.811x y = 0.320 + 0.760x
1 r = 0.987 1
r = 0.980
0.5 0.5

0 0
-1 0 1 2 3 -1 0 1 2 3
[I] (mg/mL) [I] (mg/mL)

Fig. 4. Dixon plot using slope of double-reciprocal lines for determination of inhibition constant (KI ), in presence of E. globulus with Aspergillus niger lipase (a), olive lipase
(b) and in presence of M. viridis with Aspergillus niger lipase (c) and olive lipase (d).

a 20 b 13
18
S = 2.25 mg/mL S = 2.25 mg/mL 11
16
S = 9 mg/mL S = 9 mg/mL
S = 22.5 mg/mL 14 S = 22.5 mg/mL 9
S = 45 mg/mL 12 S = 45 mg/mL
7
10
[S]/V

[S]/V

8 5
6
4 3
2 1
0
-4 -2 -2 0 2 4 -4 -2 -1 0 2 4
-4
-3
[I] (mg/mL) [I] (mg/mL)
 
Fig. 5. Cornish-Bowden plot for determination of inhibition constant KI in presence of M. viridis with Aspergillus niger lipase (a) and olive lipase (b). Each point represents
the mean of three experiments and the vertical bars represent the standard error of measurement.

which showed pancreatic lipase inhibition in a dose-dependent
manner at an IC50 value of 9.1 mg/mL (Birari and Bhutani, 2007).
Values of IC50 obtained in the present study were higher than IC50 of
21 aqueous-ethanol extracts of wild Mediterranean dietary plants
between 5.48 and more than 10 mg/mL (Conforti et al., 2012).
4. Conclusion
The study indicates that among the E. globulus and M. viridis
leaves extracts, MeOH extract showed the most important TPC,
TFC, CTC and antioxidant activity. Low values of KI and IC50 proved
strong inhibitory action against Aspergillus niger and olive lipases.
This anti-lipase effects were on coincidence with the highest TPC,
TFC and antioxidant activity in the MeOH extract. Moreover, the
results of kinetic analysis demonstrated that the MeOH extracts of
E. globulus obeyed the competitive inhibition modality against both
enzymes. Hence, the two ligands, substrate and inhibitor, compete
for the same form of the enzyme i.e., the free enzyme. Methanol
extract of M. viridis has exercised a non competitive mixed inhibi-
tion to both enzymes. It was capable of binding to the free enzyme
or the ES complex. Overall, the strong antioxidant activity of E. glob-
ulus and M. viridis leaves suggest that they could be advantageously
used as a functional or nutraceutical food, to prevent or moderate
oxidative stress-related diseases. On the other hand, their anti-
lipase effects suggest also that they could be used as food additive
to prevent lipids and oils lipolysis. A further study in vivo conditions
is also necessary to conrm antioxidant and anti-lipase capacities
 H. Belfeki et al. / Industrial Crops and Products 89 (2016) 514521
of E. globulus and M. viridis, which may be used for preservation
and/or extension the shelf-life of raw and processed foods.
Acknowledgement
This study received nancial support from Ministry of Higher
Education, Scientic Research and Information and Communica-
tion Technologies, Tunisia.
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