EXTRACTION OF ELIONURUS HENSII
K. SCHUM ESSENTIAL OIL IN A DOMESTIC SCALE IN THE “PLATEAU DES CATARACTES”
(CONGO-BRAZZAVILLE) Silou T 1, 2*, Nombault Nienzy
JP 1, 2, Nsikabaka S 1, 2, Loumouamou AN 3, 4,
Bikindou K 3, 4 1Faculté des Sciences ET Techniques,
BP 69 Brazzaville, Congo 2Ecole Supérieure de Technologie
des Cataractes, BP 389, Brazzaville, Congo 3Ecole Normale Supérieure B.P. 69
Brazzaville, Congo 4Institut National de Recherche en
Sciences Exactes ET Naturelles BP 2400 Brazzaville, Congo DOI: https://doi.org/10.29121/IJOEST.v2.i1.2017.05 ABSTRACT Elionurus hensii yields essential oils composed mainly of para-menthadienols in its stems and about 50 % aristo lone in its roots. These oils, which exhibit anti-microbial and anti-oxidant properties, were extracted by hydro distillation with a local extractor and was analyzed by gas chromatography (GC/FID and GC/MS). To optimize this domestic process, a 23 full factorial design is used to assess the effects and interactions of potentially influential factors. Stems yield 1-2% in essential oil. The extraction time and the degree of division of the plant material had negligible effects on the yield. Only the residual water content had a significant positive effect, together with the interaction between this content and the degree of division. The cumulated content of the four isomeric para-menthadienols present in the oil was insensitive to the effects and interactions of the factors. Roots yield 0.5 - 0.9 % in essential oil. The overall effects and interactions of factors on the yield and the aristo lone content were negligible. Principal component analysis of samples obtained with the experimental design, and their radar plots, indicated a very strong resemblance between oils obtained from the same plant part, and a marked difference between those obtained from different plant parts. Keywords: Experimental Design; Effects of Factor; Multivariate Analysis; P- Menthadienols; Aristo lone, Elionurus Hensii. Cite This Article: Silou T, Nombault Nienzy, Nsikabaka S, Loumouamou AN, and Bikindou K. (2017). EXTRACTION OF ELIONURUS HENSII K. SCHUM ESSENTIAL OIL IN A DOMESTIC SCALE IN THE “PLATEAU DES CATARACTES” (CONGO-BRAZZAVILLE). International Journal of Engineering Science Technologies, 2(1), 37-57. doi: 10.29121/IJOEST.v2.i1.2017.05 1. INTRODUCTION The
genus Elionurus (Poaceae family),
comprising some 20 species, is not well described in the literature. What work
is reported concerns Elyonurus muticus
[1], [2], [3], Elionurus elegans [4]
and Elyonurus viridulus [5]. The
essential oil derived from both the aerial parts (stems) and the below-ground parts
(roots) of Elionurus elegans possesses
antibacterial, antifungal and antioxidant properties [4]. The methanol extracts
of Elyonorus muticus, composed mainly
of phenolic compounds, exhibit a high antioxidant activity [6]. Elionurus hensii, which is found in tropical and subtropical
regions of South America (Brazil and Argentina), Africa (Republic of Congo, Gabon, DR Congo, Angola) and
Australia [8], has been studied only in the Republic of Congo, where this wild plant grows
abundantly on the “Plateau des Cataractes”. In the
first description we published in 2006 of the essential oils from different
parts of the plant (roots, stems, leaves, flowers), we reported that the oils
from the aerial parts were mainly composed of p-menthadienols, whereas the roots yielded an oil containing more
than 40 % aristolone [9]. In a
previous very detailed study of (i) the
volatile components from stems and roots obtained by hydrodistillation and
head-space SPME, and (ii) the phenolic compounds and flavonoids extracted with
methanol, ethyl acetate and dichloromethane, the antioxidant activities of the
extracts were demonstrated and compared [8]. Loumouamou
et al. assess the seasonal variation
in the composition of essential oils as a function of different harvest sites,
and the impact of this composition on the biological activities [10], [11],
[12]. However,
the massive presence of p-menthadienol
isomers points to possible antimicrobial properties, as in Cymbopogon gigantus from Burkina Faso [13], Cameroon [14], Benin
[15], Mali [16], and Côte d’Ivoire [17]. Further,
aristolone, a ketone that is very abundant in the roots, could either be
isolated or gainfully used in its native state, or after conversion into its
oxime: several studies have found this family of substances to have antitumor,
antimicrobial, antioxidant, and antidepressant, anticonvulsive and antiviral
properties [18]. We
describe here the essential oil extraction from stems and roots of Elionurus hensii, to assess the effects
of factors controllable at the artisanal level on the yield and quality of the
products obtained. The
results of this work will support controlled artisanal production of this oil,
which is of potential medicinal interest, by essential oil producers grouped in
the OIBT project as part of the Republic of Congo anti-poverty programme [19]. 2.
MATERIALS AND METHODS 2.1. Plant Material Elionurus hensii K. Schum is a perennial
grass composed of culms 60–100 cm long, with strongly developed side branches
forming blades 7–10 cm long, 2–3 cm wide that flower at maturity [20], [21]. In the
Republic of Congo, where it grows under the lower-Congo climate, on clay-sand
to sandy-moist soil, this plant, which does not exceed 1 m in height,
presents upwardly branched culms, thin sinuous roots 4–8 cm long, rolled leaves
8–15 cm long and 2 cm wide, and a glabrous base.
Figure 1: Fresh
Plant of Elyonurus Hensii (Stems, Leaves, Flowers, Roots,) The
samples studied were collected on the “Plateau des Cataractes” at Loufoulakari,
Loukoko and Sese (District of Louingui, Pool Department, R Congo). 2.2. Extraction In the Laboratory scale, the essential oils were obtained by steam distillation. Water and plant material (200 g of plant material for stems, or 135 g for roots.) were placed in a Clevenger apparatus for 4 h. The organic phase of the resulting condensate was separated from the aqueous phase by extraction with diethyl ether. The organic phase was dried over sodium sulphate and the essential oil was recovered after evaporation of the solvent. The artisanal extraction is led in a cylindrical distiller (60 L, 4kg). The vapor resulting from the hydrodistllation passes in a pipe (2 cm diameter) crossing a (100 cm length, 30 cm broad and 30 cm height) reserve, filled with cool water circulating in opposite direction (figure 2).
Figure 2: Local equipment for hydrodistillation of essential oils (60 L) 2.3. Gaz Chromatography GC analysis was performed on an Agilent GC 6890
instrument equiped with a split injector
(280°C), a flam ionization detector (FID) and a DB-5 column (20m x 0,18mm x
0,18µm).The temperature program was 50°C (3.2 min) rising to 330°C (10°C /min). Dihydrogene was used as
carrier at a flow rate 1ml/min. 2.4. Gaz Chromatography / Mass Spectrometry GC/SM analysis was performed on Agilent GC 7890 /Agilent MS 5975 operating in EI mode (70 eV), equiped with a DB-5 column (20m x 0,18mm x 0, 18µm). The temperature of injector was 280°C and helium was the carrier gas at 0.9 mL/min. The temperature program was 50°C (3.2 min) rising to 330°C (8°C /min). The identification was carried out by calculating retention indices (RI) and comparing mass spectra with those in data bancks [22], [23], [24], [25]. 2.5. Modelling of Essential Oil Extraction (Hydrodistillation) The
variables influencing extraction yield were:
time, temperature, condensation rate, the state of division of the plant
material, the mass ratio of plant material to water, and water loss from
plant material [26]. A model
with six variables, even in the case of a first degree model, would need 26
= 64 experiments [27]. For experimental convenience some variables were thus
kept constant. We considered three variables:
extraction duration, residual water content (X2) and state of division of the plant
material (X3). These three factors offered the advantage of being
easy to control, even in a small scale production unit. Extraction yield y and p- menthadiol content z depend on
factors X1, X2 and X3. Mathematically, this is
expressed as y or z = f(X1, X2,
X3) where y and z are
the responses, f(X) is the response function and X1, X2
and X3, are the factors taken
into account. The
experiment is designed to determine the effects of certain factors on each
response. The
two-level factorial design as developed by Davies [28] is well-suited to
addressing this type of question, and has the advantage of needing only very
elementary mathematical skills [29].The
general formula for a complete factorial plan with N experiments is N = 2k,
where k is the number of variables in the factorial. If k = 3, then N = 2k
= 23 = 8 experiments. To
construct the experiment matrix we define reduced variables xi as: xi = (Xi – X0)
/ΔX; X0
is the base value, at the centre of the experimental domain (level 0),
and ΔX is the
variation step, i.e., the unit of variation of the variables. Table 1 give the
two levels of the variables in steam extraction of Elionurus hensii stems. Table 1: Levels of the variables in steam extraction of Elionurus hensii stems
The combination of these 3 variables and the 2 levels by variable lead to the following experimental design (table 2) Table 2: Experimental design for essential oil extraction from Elionurus hensii stems
The domain of the study, with coded variables, becomes the domain (-1, +1) and the eight responses described by the experimental matrix are set up after randomisation (table 3). Table 3: Experimental matrix for essential oil
extraction from Elionurus hensii stems
For a
first degree model with interactions, the representative points of a
three-variable experimental design are located in three-dimensional space. The
corresponding response function is a first degree polynomial for each factor
taken separately. It is notated: y = a0 + a1x1
+ a2x2 + a3x3 + a12x1x2
+ a13x1x3 + a23x2x3
+ a123x1x2x3 If the
mathematical model associated with the factorial design is constructed with
centred, reduced variables, the coefficients of the polynomial thus have very
simple meanings: average a0,
main effects ai, and
interactions aij, and aijk [27]. 2.6. Statistical Treatment Means, standard deviations and the usual graphs were obtained with Excel software. Multivariate analysis was performed on XLSTAT software (Addinsoft an add up of Excel Microsoft). 3.
RESULTS AND DISCUSSION 3.1. Chemical Composition of Stem and Root Essential Oils of Elionurus Hensii Previously
works show that the essential oils from all the aerial
parts were similar, and were mostly composed of oxygenated monoterpenes, in
particular p-menthadienols, and
that those from the below-ground parts contained some 40 % aristolone [9],
[8].
In this study, all the above-ground parts were pooled to make a single sample,
hereafter called “stems”; likewise for the below-ground parts, called “roots”. Tables 4
and 5 show respectively chemical composition of stem and root essential oils
from Elionurus hensii, table 6
recapiulates the main constituents of
stem and root essential oils from Loufoulakari, Loukoko and Sese sites. Table 4: Chemical composition of the essential
oil from stems of Elionurus hensii (Loufoulakari site)
Table 5: Chemical composition on root essential oil (Loufoulakari
site)
Table 6: Major constituents (**)
of essential oils from stems (S) and roots (R) collected at the sites of
Loufoulakari (sample 1), Loukoko (sample 2), and Sese (sample 3)
* S1 means: stem of
sample 1; R2: root of sample 2) The
essential oil from stems was composed of a large number of menthadienol isomers
like in Cymbopogon giganteus [15], [16], [17], [30]. The
identification of these different isomers is difficult. Garneau et al. [31] determined Kovats indices on two columns and
recorded the mass spectra of six menthadienol isomers of known structures,
which they synthesised. From these data, after analysis by gas phase
chromatography coupled with mass spectrometry, we identified the isomers
present in the essential oils studied. They were in decreasing order of
content: cis-p-mentha-1(7),8-dien-2-ol,
trans-p-mentha-1(7),8-dien-2-ol, trans-p-mentha-2,8(9)-dien-1-ol, cis-p-mentha-2,8(9)-dien-1-ol, and trans-p-mentha-1,8-dien-6-ol (carveol).
Then came limonene, 2-undecanone, carvone, and 2-tridecanone. These
oils differed appreciably from those extracted from roots, which had lower
essential oil contents, more p-cymene
and intermedeol, and noteworthy levels of aristolone (more than 40 % against 3%
in the aerial parts). Aristolone
was isolated and identified spectrally by NMR spectroscopy and mass
spectrometry [10] This difference
can be visualised using a polar coordinate representation (radar plot). One
notes that (i) the radar plot of the essential oil from stems differs from that
of oil from roots, and (ii) those of oils from the same parts of the plant
(stems or roots) are similar. Figure 3 shows radar plots of stem and root
essential oils. Figure 3:
Radar- plots of Stem and root essential oilsextracted from Elionurus
hensii 3.2. Modelling of Essential Oil Extraction by Hydrodistillation from Elionurus Hensii in A Laboratory Scale Steam
distillation and water distillation are used to extract useful essential oils from
aromatic plant resources. Skaria et al. [32] distinguish
three techniques: (i) hydro-distillation (the
plant matter is partially or totally immersed in the distillation water), (ii)
steam distillation (steam
produced outside the extractor in a steam generator passes through the plant
material, which is not in water) et (iii) vapo-hydro-distillation (steam is produced in situ in the extractor, where a grille
separates the water from the plant material). Only hydro-distillation and vapo-hydro-distillation can be used at artisanal scale. 3.2.1.
Hydrodistillation of Elionurus Hensii Stems The
modelling of the extraction process is in principle complex, but can be
simplified by a judicious choice of the factors to be studied. We selected
three factors: extraction duration (h),
X2; residual water content, X2 ; state of division (cm),
X3 (tables 1, 2 and 3). Table 7
recapitulates the experimental matrix and the responses: essential oil content
and cumulated p-menthadienol
content. Table 7: Experimental matrix and responses (essential oil
content and cumulated p-menthadienol content).
The mathematical equation
representing the quantitative yield of the hydrodistillation of stems of Elionurus hensii generated by the model
is: y = 1.34 + 0.07x1
+ 0.61x2 +
0.02x3 + 0.04x1x2 – 0.001x1x3 – 0.17x2x3 +
0.10x1x2x3 With: a0 = 1.34, a1
= 0.07, a2 = 0.61, a3 = 0.02 a12 = 0.04, a13
= -0.001, a23 = -0.17 a123 = - 0.10 This
relation shows that the optimum should be located in the experimental domain or
very close to it, as the values of an
are not very high. The average yield of essential oil we can extract from the
experimental set-up is a0
= 1.3 %. The
influence of residual water content
(a2 = 0.61) is important
in the response, the combined effect of residual
water content and state of division of the plant material was favourable
(a23 = 0.17). However, we
note the very low a values for the other
interactions, which can therefore be ignored in practice, especially for
small-scale production units. The mathematical equation
representing the cumulated p-menthadienol
content in the essential oils from stems of Elionurus hensii
generated by the model is: z = 49.95 + 0.686x1 + 3.966x2
– 0.563x3 + 1.578x1x2 + 0.658x1x3
+ 0.128x2x3 + 0.366x1x2x3 The
average content of cumulated p-menthadienols was 49.95% and the factor, residual water content
in plant material, exhibits the most important effect in the response (+
3.966). Extraction duration and state of
division presents opposite effects: the first is positive (+0.686), the second,
negative (-0.563) with a very close magnitude. All interactions are positive
and x1x2 interaction was the most important (+1.578). 3.2.2. Hydrodistillation of the Elionurus
Hensii Roots Tables 8
and 9 give the levels of variation of the three variables, the experimental
matrix, and the responses (essential oil yield and aristolone content). Table 8. Levels of variation of variables
forthe extraction of essential oils from Elionurus hensii (roots)
Table 9: Experimental matrix and responses forthe
extraction of essential oils from Elionurus hensii (roots)
The mathematical equation
representing the quantitative yield of the hydrodistillation of roots of Elyonurus hensii generated by the model
is: y = 0.747 + 0.112x1
+ 0.0525x2 + 0.0575x3 – 0.0725x1x2 –
0.0375x2x3 + 0.0775x1x3 + 0.0975x1x2x3 Average yield
(0.747), effects of factors (0.0575 - 0.112) and effects of interactions (0.0375 –
0.0775) are less important in root essential oil extraction. The extraction duration
presents the most important positive effect. Interaction effects are negligeted. The mathematical equation
representing the aristolone content in essential oils of Elyonurus hensii generated by the model is: z = 41.181
– 0.456x1 + 3.466x2 + 0.568x3 – 0.828x1x2
– 0.413x2x3 + 3.516x13 + 4.133x1x2x3 Aristolone
extraction is more sensitive to the effects of factors and interactions. With an
average yield of 41.181 %, the extraction was impacted by the residual water
content (3.466) and the interaction of extraction duration/state of division
(3.516).The three- factor interaction lead to the most important effect on the
response (4.133). 3.2.3.
Characterisation of Oils During Extraction The analysis
of essential oils collected during the execution of the experimental
design can yield information on how the
different constituents of these oils are extracted in relation to the factors
studied. If light
fractions are collected first during a distillation, we can expect to obtain
oils rich in light fractions for short times and rich in heavy fractions for
long times. If by
contrast all the constituents are collected at the steam temperature, the parts
affected first by the steam will be collected together, and we can expect an
oil that from the very first drop will have the average composition of the
plant’s total essential oil. The
multivariate analysis of 16 samples from two experimental designs gave very
interesting results. Principal
component analysis (PCA) carried out on the data given in Tables 11 and 12
indicates a very close correlation of variables, mainly around the first
principal axis (F1) in the first principal plane (F1F2). Table 10: Yield (%) and major
component contents (Table 6 *) in
essential oils from stems (S**) and roots (R***) of Elionurus hensii obtained in
the execution of the experimental design
This means
that the analysis can be carried out with a much smaller number of variables
(constituents of essential oils). Ten of the 16 variables used lie on the
correlation circle. F1 contains
75,25 % of the information on the variability of the essential oils, and 83,14
% on F1F2. The study can therefore be limited to the first principal plane F1F2
(Figure 4). Figure 4: Correlation circle of variables in principal component
analysis (PCA) The
distribution of individuals in the first principal plane F1F2 shows a clear
separation between oils of the plant’s aerial parts and those of its
below-ground parts, suggesting a close similarity of oils from the same plant
part, and a marked difference in the oils from the two parts (Figure 5). Figure 5: Distribution of individuals (essential oil samples) in principal
component analysis (R: roots; S: stems) Ascending
Hierarchical Clustering (AHC) confirms the distribution into two groups each
totally separate and homogeneous (oils from stems and oils from roots)
(Figure 6, Table 11). Figure 6: Classes generated by
HAC of essential oils Table 11: Distribution of
individuals into classes by HAC
3.3. Modelling of the Artisanal Extraction (Hydrodistillation) A local extractor, used in a domestic scale in Elionurus hensii essential oil extractions, was tested in following conditions (figure 2, tables 12 and 13). Table 12: Levels of variation of variables in the artisanal extraction of essential oils from Elionurus hensii (aerial parts).
Table 13: Experimental matrix and responses for the extraction
of essential oils from Elionurus hensii (aerial
parts).
3.3.1. Response: Essential Oil Yield The average of yield of essential oil extraction is 0.75. Figure 7 gives the values and the relative importance of the principal and interaction effects of the first order factor. The principal effects b1, b2 and b3 are weak and of the same order of magnitude, the two first are positive and the third is negative. The effects of interactions 1-2 and 1-3 are nulls. On figure 8, the diagrams a, b, c and d the features in dotted strait lines has representing the principal effects, their slopes are identical (similar principal effects) and thus are parallel (absence of interaction effects). The diagrams e and f illustrate the existence of interaction effects. Then interaction x2-x3 produces the most important effect on the extraction yield contenu de p-menthadienol and it is positive. The mathematical expression of the first degree polynom associated to the full factorial design model selected here is: y = 0.75 + 0.03 x1
+ 0.03 x2 - 0.02 x3 + 0.05 x1x3 With a coefficient of regression R2= which validates a posteriori the model.
Figure 7: Graphic representation of the coefficient of factor effects (principal and interaction) on the yield of essential oil extraction. Figure
8: Graphic representation of factor interaction effectson the yield of essential oil extraction. 3.3.2. Response : P-Menthadienol Content The statistics of the coefficients of the model leading to the coefficient of regression R2=0.958 validate this last, which results mathematically in: z= 40.0 -0.055x1-0.88x2-0.70 x3- 0.15x1x2 -0.13x1x3+0.98x2x3-0.33x1x2x3. The average of the p-menthadienol content is of 40.0 %. The content of p-methadienols is more sensitive to the principal and interaction effects of the factors; these effects are overall negative. Only the interaction x2x3 is positive (figure 9).
Figure 9: Graphic representation of the coefficient of factor effects (principal
and interaction) on p-menthadienol
content. 3.3.3.
Composition of 8 Samples Extracted Via The
Artisanal Scale The composition of 8 samples extracted via the artisanal scale were closed similar according to the SD values of the major constituent contents and the representative radar plots of the essential oils (table 14, figure 10). Table 14: Essential oil composition of samples extracted via the artisanal experimental design (Elionurus hensii stems).
Figure 10: Representative radar plot of the essential oil
extracted via the artisanal hydrodistillation
from Elionurus hensii stems (similar
radar plots for the 8 samples). 4.
CONCLUSION Hydrodistillation
is used to extract useful essential oils from aromatic plant resources. Their efficiency depends on both the plant
material and the extraction process. It
is important to work with homogeneous plant material obtained in the most
favourable conditions (collection of plant material when the content and
composition of the essential oil is optimal) The
modelling of the extraction process is in principle complex, but can be
simplified by a judicious choice of the factors to be studied. We selected
three factors for the steam distillation and four factors for the water
distillation. These were used as variables in a two-level factorial design.
This design, involving a mathematical model in the form of a first degree
polynomial, was used to calculate average response, and effects of factors. The factor considered and the experimental domain selected have very weak influence on the extraction of the essential oil, which is on average 0, 75 %. For the aerial part (stems, leaves and flowers) of Elionurus hensii, the values of the factor effects was close to 0. This means which one is very close to the optimum of extraction. The artisan can thus keep, the current extraction conditions. A finer research of optimum, with the actuel extractor, will not produce any significant profit. The effects of these same factors have impact more important on the contents of p-menthadienol, but non-significant on an artisanal scale. The composition of the 8 samples resulting from the experimental design have a similar compositions; the factor studied do not have any impact on the quality of oils: nearly 50 % of p-menthadienols, which are responsible known biological properties of this plant. REFERENCES
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