Industrial Crops & Products 115 (2018) 52–61

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Industrial Crops & Products

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Application of mixture design for optimum antioxidant activity of mixtures T

of essential oils from Ocimum basilicum L., Origanum majorana L. and
Rosmarinus officinalis L.
⁎
Tomasz Baja, , Andrew Barylukb, Elwira Sieniawskaa
a
Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodzki Str., Lublin, Poland
b
Lakeridge Health Oshawa, University Heath Network, 1 Hospital Court, Oshawa, ON, L1G 2B9, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Compounds with potent antioxidant properties are gaining interest among food manufacturers and consumers
Mixture design alike. With rising concerns about the safety of synthetic substances being added to food products, companies are
Antioxidant activity beginning to search for natural sources of antioxidants. Because essential oils have well studied antioxidant
Essential oils properties they can be used as food preservatives. What is more, mixtures of essential oils prepared by combining
individual essential oils may have a more potent antioxidant profile compared to any single essential oil, without
compromising the overall antioxidant potential of the mixture. This may be related to increased biological
diversity of a mixture. Hence, the purpose of this work was to demonstrate the usefulness of the simplex-lattice
mixture design method for the design of essential oil mixtures with potent antioxidant properties, as well as
generate a discussion of potential interactions existing between essential oils. Statistical modeling was utilized to
design a mixture of three essential oils: marjoram, basil and rosemary. The highest antioxidant activity was
obtained for a 75:8:17 percentage composition mixture respectively. However, the proposed composition of the
individual ingredients in the design mixture showed a higher percentage of inhibition (90%) compared to the
activity of the single component (ol. marjoram 88%) which was the most active. Hence, statistical planning and
the development of utility profiles for mixtures of essential oils can be used to predict the optimal composition as
well as determine their antioxidant profile.

1. Introduction
The antioxidant activity of natural products has been widely studied
in recent years in both in vitro and in vivo models, which prove their
significant radical scavenging properties. This particular pharmacolo-
gical action is confirmed to influence a variety of biochemical processes
and slow down the progression of serious civilization diseases, such as
cardiovascular diseases, cancer, inflammatory diseases or infections
(Kukula-Koch et al., 2015, 2016). Also Central Nervous System can be
affected by the action of free radical scavengers, which slow down the
progression of dementia or depression (Kukula-Koch and Mroczek,
2015; Poleszak et al., 2016).
Essential oils (EO) – a group of plant derived secondary metabolites
− have well known antioxidant properties, which can be characterized
directly via interaction with peroxyl radicals or indirectly (in vivo)
through transcriptional induction of antioxidant enzymes, through
Keap1-Nrf2-antioxidant responsive element signalling pathway or with
modulation of cellular glutathione levels (Aggarwal, 2002). The direct
antioxidant activity of essential oils via free radical scavenging and
inhibition of linoleic acid oxidation, may be useful to the food industry
prolonging the storage stability of food (Ebrahimi and Khosravi-Darani,
2013). However, EO aromas should not interfere with the organoleptic
properties of food. The investigation of EO antioxidant activity in real
systems showed that whole EO, containing several active compounds,
or mixtures of isolated EO compounds can extend the shelf life of food
products. What is more, vapors of EO were described as components of
active packaging (Valero et al., 2006) and introduction of antioxidant
EO directly in the polymer films of packaging was proposed to reduce
microbial decay and preserve the antioxidant properties of food
(Amorati et al., 2013; Ribeiro-Santos et al., 2017a).
Basil (Ocimum basilicum L.), marjoram (Origanum majorana L.) and
rosemary (Rosmarinus officinalis L.) are herbs widely used for culinary
purposes. Their EOs have confirmed antioxidant activity, are non-toxic
(Elgndi et al., 2017; Hajlaoui et al., 2016) and have already been stu-
died as potential food preservatives (Sirocchi et al., 2017). However,
the scientific evidence regarding the antioxidant properties of EO

⁎
Corresponding author.
E-mail address: tbaj@pharmacognosy.org (T. Baj).

https://doi.org/10.1016/j.indcrop.2018.02.006
Received 31 October 2017; Received in revised form 1 February 2018; Accepted 2 February 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
T. Baj et al.
Fig. 1. Arrangement of mixture points using the simplex lattice design (S-LD) method.
mixtures is still limited to our knowledge. Hence, the aim of this study
was to develop a highly active mixture of three EO and to create a
general concept about interactions between their components utilizing
mixture design methodology. In this approach, the antioxidant response
depends solely on the ratio of mixture components. Theoretically, the
antioxidant properties of the mixture can be modified by optimizing
this ratio. To our knowledge, this is the first application of the Simplex-
Lattice Design (S-LD) to optimize the antioxidant profile of a basil,
marjoram and rosemary essential oil mixture.
2. Materials and methods
2.1. Materials
Commercially available EO; basil (Ocimum basilicum EO, producer
Aromatika Ukraine LLC, Kiev, Ukrain), marjoram (Origanum majorana
EO, producer Aromatika Ukraine LLC, Kiev, Ukrain) and rosemary
(Rosmarinus officinalis EO, producer Etja, Elblag, Poland) were pur-
chased from a local Polish herbal store.
2.2. GC/MS analysis
Gas chromatography − mass spectrometry analysis was performed
on a Shimadzu GC-2010 Plus coupled to a Shimadzu QP 2010 Ultra
mass spectrometer according to the method described previously (Baj
et al., 2017). Briefly, the separation of compounds was performed in a
fused-silica capillary column ZB-5 MS (30 m, 0.25 mm i.d.) with a film
thickness of 0.25 μm (Phenomenex) and a calibrated temperature gra-
dient (50 °C for 3 min, then steadily increase up to 250 °C over 25 min,
holding at 250 °C for 2 min). A temperature of 250 °C, 250 °C, and
220 °C was maintained for the injector, interface, and ion source, re-
spectively. Helium was the carrier gas with a flow rate of 1 ml/min
while the split ratio was 1:20. Ions were produced using the electron
impact method at 70 eV. A scan mode in the range of 40–500m/z, with
a scan rate of 0.20 s/scan was applied for data acquisition. A homo-
logous series of n-alkanes (C8–C24) were used to determine retention
indices under the same operating conditions. Identification was per-
formed on the basis of MS resources from literature (http://webbook.
nist.gov) and from a computer-supported spectral library (http://www.
massfinder.com).
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Industrial Crops & Products 115 (2018) 52–61
2.3. Composition of experimental points
2.3.1. Determination of the composition of reference mixtures
The Simplex-Lattice Design (Cornell, 2011; Goupy and Creighton,
2006) was used to determine the measurement points. This system has
symmetrically distributed experimental points and a well chosen
polynomial equation to represent the surface response over the entire S-
LD region. For the S-LD {q m} (q − number of factors, m − degree of
polynomial), the composition of each component ratio can be written
using Eq. (1) (Aggarwal, 2002):
xi = 0,1/m, 2/m,…,1; (i = 1,2,3…,q) (1)
Ranging between 0 and 1, all possible mixtures with these compo-
nent proportions are used. The final result of the S-LD is formation of
mixtures consisting of at least 2 solutions occurring in different re-
ciprocal proportions. The ratio of mixtures xi has restrictions that fulfil
the expression of Eqs. (2) and (3):
0 ≤ xi ≤ 1 (i = 1,2,… q) (2)
and
∑ xi = 1 (3)
where q is the number of components. In general, the number of
measurement points can be written as Eq. (4). (Aggarwal, 2002):
q+m−1
Cm. (4)
In the presented experiment, the S-LD for 3 components and a 3rd
degree polynomial {3,3} will consist of 5C4 = 9 points + one central
point. Each xi can take m + 1 = 4 possible values xi = 0,1/3, 2/3,1
where the possible design points are: (1 + 0 + 0), (2/3 + 0 + 1/3), (1/
3 + 0 + 2/3), (0 + 0 + 1), (0 + 1/3 + 2/3), (0 + 2/3 + 1/3),
(0 + 1 + 0), (1/3 + 2/3 + 0), (2/3 + 1/3 + 0), the central point is
located inside the triangle and consists of equal parts of all components
of the mixture (1/3:1/3:1/3). Consequently, we have 3 points of pure
EO (vertices of the triangle), 6 designed mixtures of EO (arranged at the
edges of the triangle) and one central point (inside the triangle) are
obtained (Fig. 1). The experiment deign is shown in Table 2.
T. Baj et al. Industrial Crops & Products 115 (2018) 52–61

Table 1 the S-LD, were combined with 180 μl of DPPH (Sigma-Aldrich, USA) in
Chemical composition and percentage of essential oils of basil, marjoram and rosemary. methanol (78 μg/mL) on a 96-well plate, in a strictly timed fashion. The
plate was isolated from all sources of light for 30 min at room tem-
No. RI Compounds basil marjoram rosemary
perature. After this incubation period, the absorbance was measured at
1. 927 α-thujene 0.1 515 nm in a BioTek ELx808 plate-reader (BioTek, USA). The positive
2. 934 α- pinene 0.3 0.7 16.6 control was a DPPH solution where methanol was added instead of the
3. 952 camphene 0.1 tr 5.3
test solution. The free radical scavenging activity of each mixture was
4. 974 sabinene 0.1 1.8 1.0
5. 979 β-pinene 0.2 0.1 11.0 then calculated as percent inhibition according to Eq. (5):
6. 989 β-myrcene 0.1 0.5 0.6
7. 1008 α-phellandrene 0.1 0.4
Inhibition (%) = (Acontol − Asample)/Acontrol * 100 (5)
8. 1112 α −ocimene 5.6
All experiments were repeated triplicate.
9. 1117 1.4-cineole 0.1
10. 1019 α-terpinene 0.2 1.9 2.8
11. 1027 p-cymene 1.5 3.8 2.1 2.5. Statistical model match
12. 1032 limonene 0.7 11.5 1.5
13. 1034 1.8-cineole 4.6 8.1 15.2
The effect of S-LD on the antioxidant activity (percent inhibition)
14. 1037 trans-β-ocimene tr
15. 1047 cis-β-ocimene 0.3 was analyzed using the last square multiple regression method. For this
16. 1060 γ-terpinene 1.7 1.9 2.2 experiment, typical multiple regression equations were used as follows:
17. 1074 trans-sabinene hydrate 0.1 1.6 Linear model:
18. 1087 α-terpinolene 0.2 0.8 0.3
19. 1102 linalool 4.5 14.7 1.5 y = b1 * x1 + b2 * x2 + b3 * x3 (6)
20. 1105 cis-sabinene hydrate 3.9
21. 1123 fenchol 0.1 tr Quadratic model:
22. 1130 trans-p-2 menthen 1- ol 0.5
23. 1153 camphor 0.2 0.2 15.1 y = b1 * x1 + b2 * x2 + b3 * x3 + b12*x1 * x2 + b13 * x1 * x3
24. 1180 borneol 0.1 0.1 1.9 + b23 * x2 * x3 (7)
25. 1182 camphol 2.8
26. 1186 terpinen-4-ol 2.9 8.2 0.2 Special cubic model:
27. 1194 p-cymen-8-ol 0.2
28. 1201 α-terpineol 13.8 5.0 y = b1 * x1 + b2 * x2 + b3 * x3 + b12 * x1 * x2 + b13 * x1 * x3
29. 1207 γ-terpineol 1.8 + b23 * x2 * x3 + b123 * x1 * x2 * x3 (8)
30. 1208 metylchavicol 75.1
31. 1215 trans-piperitol 0.2 Cubic model:
32. 1223 fenchyl acetate 0.1
33. 1229 nerol 0.1 y = b1 * x1 + b2 * x2 + b3 * x3 + b12 * x1*x2 + b13 * x1 * x3
34. 1232 tymolmethylether 0.1 + b23 * x2 * x3 + d12 * x1 * x2 * (x1 − x2) + d13 * x1 * x3 * (x1 − x3)
35. 1249 linalyl acetate 16.0
+ d23 * x2 * x3 * (x2 − x3) + b123 * x1 * x2 * x3 (9)
36. 1253 geraniol tr
37. 1287 bornyl acetate 0.1 3.1
where, y–is the starting value, xi- represents the mixture ingredients
38. 1349 α-terpineol acetate 0.1
39. 1363 eugenol 1.3 5.3 (independent, input points), and bi, bij, bijk, dij- are the coefficients of
40. 1376 geranyl acetate 0.3 the regression function (Yolmeh et al., 2017).
41. 1382 α-copaene tr The models described above, utilizing a 3rd degree polynomial
42. 1395 β-elemene 0.1 function, were used in surface matching. The following parameters:
43. 1406 metyleugenol 0.2
Sum of Square (SS), Mean of Square (MS), df (degree of freedom), test
44. 1423 longifolene 0.4
45. 1429 (E)-β-caryophyllene 0.8 1.3 1.0 F, p-values, R-square (R2), R-square adjusted (R2adj.) and eta-squared
46. 1443 (E)-β-bergamotene 1.1 (η2) were considered when selecting the appropriate statistical model.
47. 1465 α-humulene 0.1 0.1 0.3 When analyzing the statistical model, a general model match, evalua-
48. 1490 germacrene D 0.2 0.1
tion of the effect, Pareto graph as well as residual rating were per-
49. 1504 bicyclogermacrene 0.1
50. 1511 β-bisabolene 0.1 formed. The last part of the experiment was to determine the utility
51. 1525 δ-cadinene 0.5 profile of response for the mixture of EO. All statistical calculations
52. 1590 spathulenol 0.1 were carried out using Statistica 12 (StatSoft, USA).
53. 1595 caryophyllene oxide 0.3 0.2 0.1
54. 1619 1,10-di-epi-cubenol 0.1
55. 1656 β-eudesmol 0.1 0.1 3. Results and discussion
56. 1657 α-cadinol 0.3
Total identified 98.5 98.2 98.2 3.1. Chemical composition of EO

tr < 0.05.
2.3.2. Preparation of mixtures of essential oils
Pure EO were placed in eppendorf tubes in aliquoted proportions
(Table 2) and were vortexed (Grant-bio PV-1, Grant Instruments Ltd.,
England) for 3 min. The obtained solutions were then diluted 1:100 v/v
with methanol (J.T. Baker, USA), vortexed again and stored at 4 °C until
needed. The final volume of the mixtures was 1000 μl.
2.4. Antioxidant activity assay
The antioxidant activities were determined using a free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH%) based on Brand-Williams et al.
(1995) with modifications. 20 μl mixtures of EO, obtained according to
Commercially available EO were subjected to GC–MS analysis to
determine their chemical composition (Table 1). Thirty-seven active
compounds were identified in basil EO, with methyl chavicol as the
major constituent (75.1%). Other quite abundant compounds were 1,8-
cineole (4.6%) and linalool (4.5%). This basil-chemotype is frequently
described in literature (Padalia et al., 2017; Tiwari et al., 2017) al-
though other chemotypes with predominating linalool (Govindarajan
et al., 2013; Koroch et al., 2017), geranial (Koroch et al., 2017), methyl
cinnamate (Koroch et al., 2017) or methyl eugenol (Koroch et al., 2017)
have also been reported.
In EO extracted from marjoram, thirty-four compounds were iden-
tified. Limonene (11.5%), linalool (14.7%), α-terpineol (13.8%) and
linalyl acetate (16.0%) were predominant. This chemotype was de-
scribed previously in Finland, Morocco and Iran (Lis et al., 2007).

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Table 2
S-LD and the mean results of percent inhibition assays obtained experimentally and approximated from the regression equation for tested mixtures.

Exp. no.a Coded level Measured inhibition (%) Approx. inhibition (%) Residues

Basil (x1) Marjoram (x2) Rosemary (x3)

1 1 0 0 48.6 49.8 −1.2

2 0 1 0 87.9 84.5 3.4
3 0 0 1 1.2 2.5 −1.3
4 1/3 2/3 0 83.7 86.5 −2.8
5 1/3 0 2/3 18.2 20.8 −2.6
6 0 1/3 2/3 70.4 64.0 6.4
7 2/3 1/3 0 76.8 74.9 1.9
8 2/3 0 1/3 38.3 36.6 1.7
9 0 2/3 1/3 83.9 91.3 −7.4
10 1/3 1/3 1/3 69.1 70.7 −1.6
11 1/3 1/3 1/3 74.3 70.7 3.6

a
–non-randomized sequence.

However, the predominating marjoram chemotype is rich in terpinen-4-
ol (Sellamia et al., 2009).
Rosemary EO yielded twenty-nine compounds with α-pinene
(16.6%), 1,8-cineole (15.5%), camphor (15.1%) and β-pinene (11.0%)
predominating. These results are in agreement with Bajalan et al.
(2017), who also described α-pinene, 1,8-cineole and camphor as the
main compounds.
TIC chromatograms of pure essential oils from basil, marjoram and
rosemary are presented in Fig. 2A–C.
3.2. Antioxidant activity
The percent inhibition of DPPH was determined for each designed
mixture, tested in a concentration of 1 mg/mL (Table 2). When pure EO
were studied (exp. no 1–3), the best activity was observed for marjoram
EO (87.9%). Intermediate activity was observed for basil (48.6%),
while rosemary exhibited the lowest antioxidant activity (1.2%). The
antioxidant activity of EO chemotypes used in this study correlates with
existing literature. Basil, rich in methyl chavicol, was quite a weak
antioxidant (Beatovic et al., 2015), rosemary was described previously
as having low free-radical scavenging activity (Raskovic et al., 2014),
whereas O. majorana showed significantly high free-radical scavenging
activity (Olfa et al., 2016). The results obtained for marjoram can be
attributed to the presence of oxygenated monoterpenes (linalool, α-
terpineol, linalyl acetate), which possess higher antioxidant activity
than monoterpene hydrocarbons (pinenes), sesquiterpene hydrocarbons
or their oxygenated derivatives (Hajlaoui et al., 2016).
When the antioxidant potential was measured in each experimental
mixture, all had a greater antioxidant response than basil alone. The
percent inhibition of exp. no 4–11 ranged between 18.2% and 83.9%
(Table 2). The addition of marjoram EO to the mixture, in a ratio of 2/3,
resulted in a percent inhibition of above 80%. When marjoram EO
constituted 1/3 of the mixture, the obtained antioxidant activity was
above 70%. Interestingly, similar activity was calculated in a central
point of mixture design, where all components were in equal ratios (1/
3 + 1/3 + 1/3), suggesting that the antioxidant activity is dependent
on the marjoram EO content. The predicted percentage of antioxidant
activity calculated from the quadratic equation ranged from 2.5% to
91.3%.
3.3. Essential oil mixture optimization and evaluation of the statistical
model match
EO compounds are particularly complex, so attempting to char-
acterize their biological properties can be cumbersome. These com-
pounds have multidirectional activity, which is influenced by their rich
chemical composition. This composition is, however, dependent on
various external factors that hinder standardization. Furthermore, there
are numerous chemotypes occurring within the same species that may
affect their measured activity. These variables are additionally multi-
plied when developing mixtures of essential oils, when it is difficult to
predict the interactions between their constituents. Therefore, every
effort should be made to optimize both; the process of essential oil
extraction and characterization of their antioxidant profile. Further
work should focus on identifying optimal processing conditions within
the food, chemical and pharmaceutical industries to maximize perfor-
mance and improvement of qualitative or quantitative parameters of
EOs. Performing accurate measurements at every point during the
mixture design is expensive, time consuming or simply impractical. One
possible method to address these issues is Design of Experiments (DOE)
(Goupy and Creighton, 2006). This approach may be used to optimize
experiment plans. The goal is performing the lowest number of ex-
periments while gaining the maximum amount of data for the devel-
opment of an efficient and reproducible model with the desired prop-
erties.
Variance analysis results using a hierarchical set of models with
increasing complexity is presented in Table 3. The first analysis, using
the ANOVA simple linear model [Eq. (6)] showed statistical sig-
nificance at the level of P < .05. The test value F0.05(2.8) calculated for
this equation was higher than that obtained from the F0.05(v1,v2) dis-
tribution = 4.458, level of significance P < .0019. The linear model
explained the variance at the level of R2 with a frequency of 79.16%
and 73.96% for R2adj. The value of eta2 showed that the linear model
explained the variance of the dependent variable at a frequency of 79%.
Expanding from the linear to a quadratic model [Eq. (7)] improved the
fit for the regression analysis. The coefficients of determination were
also improved and the quadratic model explained 98.24% and 96.49%
of the variance and adjusted variance respectively. The eta2 coefficient
increased to 92%, indicating a better fit than the linear model pre-
viously analyzed. This evaluation may indicate a statistically significant
interaction between two-component systems. Introducing a ternary
system to the Special cubic [Eq. (8)] and Cubic [Eq. (9)] models re-
sulted in the absence of statistical significance at P < .05. This could
be explained by the higher complexity of the model and interactions
that may occur between ingredients of the three selected EOs. Surface
fitting for all tested models is shown in Fig. 3.
Due to a higher coefficient of determination and the strength of the
eta2 effect, a square model was chosen to further evaluate the statistical
effect of the composition of the EO mixture based on the percent in-
hibition. The regression model for the experiment is shown in Eq. (10):
Inhibition
(%) = 49.78 * x1 + 84.50 * x2 + 2.47 * x3 + 61.14 * x1 * x2 + 11.70 * -
x1 * x3 + 153.65 * x2 * x3. (10)
where, x1, x2, x3 – individual components of the EO mixture
The general analysis of the square model is presented in Table 4,

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Fig. 2. Chromatograms of essential oils: A – basil; B – marjoram; C – rosemary.

confirming its statistical significance and demonstrating no relevance to were approximated, and residual values were calculated as the differ-
the “lack of fit”. ence between the measured value and the approximated value
Based on the calculations using the selected model [Eq. (8)], values (Table 1). A comparison of empirical data with approximated data

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Table 3
Analysis of variance results for different statistical models.

Model SS df MS F p-values R2 R2adj. η2(%)

Linear 8389.29 10 3539.148 15.198 0.0019 0.7916 0.7396 79

Quadratic 1747.98 8 563.015 18.102 0.0041 0.9824 0.9649 92
Special cubic 147.38 5 39.454 0.097 0.7715 0.9828 0.9571 2
Cubic 143.9 4 56.98 3.214 0.3841 0.9984 0.9839 91
Total corr. 8389.29 10 838.93

Abbreviations: SS–sum of square, df–degrees of freedom, MS–mean of square, F–F-values for p < .05, R2–coefficient of determination, R2 adj.–coefficient of determination
adjusted,η2–eta-squared.

showed a high correlation of R2 = 0.9824 (Fig. 4A). To analyze the Table 4

validity of the regression model, a raw residue analysis was performed. Coefficients of the overall fit for the regression model (p < .05).
A normal distribution was observed by comparing raw residue results to
SS df MS F p–values
the expected normal values (Fig. 4B). A comparison of raw residues
with approximated values and experiment number is shown in Fig. 5 Quadratic model 8241.92 5 1648.383 55.925 0.0002
respectively. The raw residue analysis supports the use of this regres- Total error 147.37 5 29.475
sion model, where no random distribution trends were observed. Lack to fit 133.85 4 33.463 2.475 0.4404
Pure error 13.52 1 13.520
In order to evaluate the antioxidant activity of essential oils and Total corr 8389.29 10 838.929
their mixtures, 3 were chosen with a variable percent of inhibition (%I),
where rosemary < basil < marjoram. From the analyzed mixtures,
the most potent EO, with the greatest antioxidant activity, was mar- the second ingredient in the mixture 83.7–83.9%I (exp. 4 and 9). The
joram (Table 1). In the mixtures where the marjoram concentration was obtained percent inhibitions were higher for the studied mixtures than
67% the antioxidant activity was almost exactly the same regardless of for any pure marjoram EO as calculated using the regression equation

Fig. 3. Models of surface matching: A – linear, B – square, C special cubic, D – full cubic.

57
T. Baj et al.
Fig. 4. The correlation between the experimental and approximated values (A) and plot of raw residues vs. expected normal value (B).
Fig. 5. Plots of raw residues vs. experiment number (A) and approximated values (B).
Fig. 6. Pareto effect for quadratic model (significance p < .05).
for the square model. This may indicate the additive properties of the
EO in a mixture. Also, the Pareto analysis (Fig. 6) indicates the statis-
tical significance at p < .05 for a mixture of marjoram EO and mar-
joram EO alone.
The purpose of the experiment was to obtain clear information on
the proportions of individual essential oils in a mixture characterized by
the highest percent inhibition. The above can be obtained by de-
termining a profile of approximate antioxidant response and surface
usefulness. Utilization models are often used in industry to optimize
production process parameters (Candioti et al., 2014), extraction
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Industrial Crops & Products 115 (2018) 52–61
methods (Sammani et al., 2017) and to obtain the optimal antioxidant
mixture of various components (Maia et al., 2011).
During the experimental phase, a desirability profile was generated
with values of 1.2% – low (0.00); 44.55% – intermediate (0.50) and
87.9% – high (1.00). Based on the fit to the model of the square
equation adopted for this experiment, the usability profile for ap-
proximated values was generated and subsequently the optimal com-
position of the EO mixture was determined. A graphic representation of
the predicted values and desirability profile are shown in Fig. 7. A
predicted composition of the EO reference mixture and experimental
data are presented in Table 5.
Table 5 demonstrates a failure to obtain an experimentally ap-
proximated percent inhibition of 91% over 30 min. However, the ex-
perimentally measured percent inhibition significantly exceeded the
lower confidence interval for the theoretically determined values, and
was greater than the single most active EO. The above results may
further indicate a synergistic effect of the components in the mixture. A
mixture of EO with a higher percent inhibition was developed as a
result of the experiment, compared to each EO individually.
Previously reported in the literature optimization procedures for
essential oils using the Response Surface Methodology (RSM) focused
mainly on the physical parameters of raw plant material processing. In
order to obtain the highest yield of EO, the most frequently optimized
parameters of a hydrodistillation process were; the distillation time, the
solute/solvent ratio and plant part (Timung et al., 2016), as well as
temperature and pressure in supercritical fluid extraction (Haloui and
Meniai 2017; Zermane et al., 2016).
Furthermore, at present more and more research is being carried out
T. Baj et al.
Fig. 7. Desirability profile for optimization of references mixture of essential oils.
attempting to optimize EO mixtures to generate the highest anti-
microbial effect (Fadil et al., 2017; Ouedrhiri et al., 2016; Ribeiro-
Santos et al., 2017b).
To date, the most commonly used method to optimize the compo-
sition of EO is the Simplex-Centroid Design (S-CD). Ouedrhiri et al.
(2016) analyzed the synergistic effects of EO from Origanum compactum
L., Origanum majorana L., Thymus serpyllum L. on: Escherichia coli, Sta-
phylococcus areus, Bacillus subtilis and Pseudomonas aeruginosa. The au-
thors utilized S-CD and special cubic regression to determine the
composition of an EO mixture with an optimal antibacterial response
profile to Gram positive bacteria. The mixture consisted of 28% oregano
EO, 30% marjoram EO and 42% wild thyme EO. However, the optimal
mixture against E. coli turned out to be a composition of 75% oregano
EO and 25% marjoram EO. Subsequent authors (Ribeiro-Santos et al.,
2017b) also used S-CDs to study the antioxidant properties of EO
mixtures and their effects on E. coli, S. aureus and Penicillium spp. EO
from the following plant sources were used; Cinnamomum cassia (L.) J.
Presl, Cinamomum zeylanicum Blume, Rosmarinus officinalis L., Ocimum
basilicum L. The optimal EOs mixture had a composition of 51%, 34%,
Table 5
Approximate and experimentally determined percentage of inhibition (n = 8) for values obtained from desirability profile evaluation.
Data Contribution of components in the mixture (%)
Basil Marjoram
Predicted values 8 75
Values measured experimentally
CI – confidence interval.
59
Industrial Crops & Products 115 (2018) 52–61
15%, 0% respectively. The authors reported that this composition had
the potential to inhibit the growth of microorganisms at a concentration
of 5% (w/w). Fadil et al. (2017) evaluated EO antimicrobial activity
against Salmonella typhimurium. A mixture of Thymus vulgaris L., Myrtus
communis L. and Rosmarinus officinalis L. was proposed. The strongest
antimicrobial response was achieved using a mixture of 55% thyme EO
and 45% myrtle EO.
As shown in the examples above, the highest antimicrobial activity
may be obtained using a combination of two out of three or three out of
four tested EO. This may be due to an antagonistic effect of one of them.
The S-CD method predicts interactions between EO mixed in pairs,
in equal proportions (1: 1), but does not convey clear information as to
whether one of the EO in the mixture influences the other. This re-
presents the risk of introducing confounding variables to the measured
parameters within the EO mixture. Additionally, authors utilizing this
S-CD approach were forced to attempt these calculations with a mixture
consisting of 3 or more EO. Therefore, in this work, a S-LD was used
which, unlike S-CD, provides valuable data concerning EO properties
and their mutual influence while designing basic triangular mixtures.
Inhibition (%) −95%CI +95%CI
Rosemary
17 91.6 83.9 98.6
89.97 +/− 0.26 89.33 90.60
T. Baj et al.
For a ternary mixture, the use of a 3° polynomial will produce a ratio of
1/3 and 2/3. As in the S-CD, the mixture of all EO is central. So, we get
a more complex system for evaluating the interactions between EO,
while providing a greater freedom of data interpretation using S-LD.
The mixture optimization method has also found application in the
development of optimal extraction systems for isolation of bioactive
ingredients (Ozturk et al., 2014) within the food (Meinhart et al., 2017)
and pharmaceutical industries (Rao et al., 2016). It enabled the opti-
mization of isolation of lycopene form tomato and tomato products
(Periago et al., 2004), soy isoflavones (Yoshiara et al., 2012), or frac-
tions rich in total polyphenols form Schinus terebinthifolius Raddi
(DiCiaula et al., 2014). The application of statistical methods during the
optimization of the composition of mixtures of essential oils as well as
extraction solvents in order to isolate the biological compounds from
the plant matrix seems to be a useful tool for obtaining the best bio-
logical parameters.
4. Conclusion
As presented in this work, the use of a simplex-lattice design can be
helpful to study the relationships between EO. This model also provides
a reasonable template for the preparation of EO mixtures, by appre-
ciating their synergistic or cumulative antioxidant potential for an op-
timal response to oxygen free radicals in industrial applications. The
simplex-lattice plan proved to be a useful modeling tool for quantifying
antioxidant activity. Statistical planning along with the development of
a desirability profile can therefore serve as a predictor of EO mixture
quality as expressed by its antioxidant potential.
Conflicts of interest
The authors declare that there is no conflict of interest regarding the
publication of this article.
Acknowledgments
The authors would like to thank MSc Paulina Zdunek for her tech-
nical assistance during the experiments.
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