Influence of Organic and Mineral Soil Fertilization on Essential Oil of Spilanthes oleracea cv. Jambuarana
Luciana da S. Borges,
Maria A.R. Vieira,
Marcia O.M. Marques,
Giuseppina P.P. Lima
In the present study, the composition of essential oil of leaves and inflorescences of jambu (Spilanthes oleracea . Jambuarana), under organic manuring and mineral fertilization, was studied. Jambu plants show important chemical properties and their production has been addressed for the extraction of the essential oils for cosmetics industries, due to their pharmacological properties. The experimental area of treatments contained urea as mineral fertilizer (120 g m2), applied twice and organic fertilizer (8 kg m2), applied at the planting. Jambu leaves and flowers were harvested twice: the first at 90 days after seedling transplantation and at the opening of the flower buds. Branches were cut at 7 cm from the soil, thus new branches can bud for the accomplishment of the second crop which happened 40 days after the re-budding. The essential oil was analyzed by gas chromatography coupled with mass-spectrometry. According to our results the most representative compounds were trans-caryophyllene, germacrene-D, 1-dodecene, spathulenol and spilanthol (a compound presenting anesthetic properties) occurring in inflorescences. Fertilization procedure does not affect the content and the quality of the essential oil in Jambu plants.
Received: October 30, 2011;
Accepted: March 09, 2012;
Published: May 17, 2012
Jambu plants (Spilanthes oleracea cv. Jambuarana) (Asteraceae), also
known as cresson du Pará, belong to the Asteraceae family,
native of Amazonian region, living in tropical climate and widely consumed in
the Northern area of Brazil, mainly in Para State. The plant presents important
chemical properties, waking up the interest of pharmaceutical industries, mainly
for the presence of its active compound, spilanthol. Others properties, such
as a larvicidal action, of the powder of Spilanthes mauritiana (Ohaga
et al., 2007a), on two mosquito species (Anopheles gambiae
and Culex quinquefasciatus) were described (Ohaga
et al., 2007b).
Early phytochemical studies on Spilanthes were carried out by Jacobson
(1957), reporting spilanthol as an amide (an N-isobutylamide), occurring
also in other species of Spilanthes genre (Ramsewak
et al., 1999). Studies showed that flowers and leaves of Spilanthes
contain amino acids (Mondal et al., 1998; Peiris
et al., 2001), alkaloids (Peiris et al.,
2001) and flavonoids (Makambila-Koubemba et al.,
Some reports considered the effects of soil conditions, determined by agricultural
history and crop management, on plants production of primary and secondary metabolites
(Upadhyay and Patra, 2011). The knowledge of how soil
conditions and essential oil production are related may contribute not only
to understand the way plants respond to environmental stresses but may also
provide useful information to design sustainable agricultural strategies, since
sustainable production could be achieved by decreasing the amount of inputs
needed to sustain high yields in poor environments (De la
Fuente et al., 2003).
The intensive use of chemical fertilizers has side effects in polluting underground
water, destroying microorganisms and insects, making plants more susceptible
to the attack of diseases and reducing soil fertility (Malik
et al., 2009). Soil fertilization, among several other factors, represents
the main aspect affecting plant chemical composition. External factors can affect
the content of active molecules and many researchers reported the influence
of agriculture procedure on plant content of many substances. Altitude, photoperiod,
temperature, incidence of solar light and conditions related to soil composition
are examples, beyond the effects of environmental cultivation conditions. For
instance, variations of these factors can influence the biomass and the quality
of essential oils in aromatic plants, as already verified, studying the effect
of nitrogen addition in the production of alkaloids. Ren
et al. (2001) studied antioxidant activity in vegetable species produced
following two different cultivation procedures (organic manuring and mineral
fertilization), observing significant differences between treatments, demonstrating
that plant nutrition can alter secondary metabolism. Concerning the production
of essential oil in lemongrass (Cymbopogon citratus), several papers
reported that the build-up of the essential oil was promoted by organic cultivation
and in relation to biomass, this kind of cultivation procedure increased the
production of the aerial and radicular plant parts (Costa
et al., 2008).
Companies using natural products, such as pharmaceutical and cosmetics industries,
have been opting for plants cultivated without mineral addition (organic production),
in particular because of consumers take increasing care about health and it
is clear the effect of fertilization procedure on environment conservation.
Thus, we asked if the influence of fertilization can increase the phytochemicals,
as the volatile substances of Spilanthes oleracea cv. Jambuarana. Therefore,
the objective of this work is to determine the chemical composition of essential
oil in jambu leaves and inflorescence, under organic manuring and mineral fertilization.
MATERIALS AND METHODS
Experimental: Jambu plants (Spilanthes oleracea cv. Jambuarana)
were cultivated during the spring (January/June) with an average temperature
of 21 °C, under organic manuring and mineral fertilization, within the approximate
area corresponding to Greenwich geographical coordinates latitude 22° 44'
50'' S and longitude 48° 34' 00'' W, at an altitude around 765 m. The mean
annual rainfall is 1534 mm, the mean rainfall during the wettest month (January)
is around 240 mm and 38 mm is observed during the driest period (July and August).
A sprinkler irrigation device was used for water supply. Irrigation was carried
out twice a day.
The seeding was performed in January 2008, in polystyrene trays of 128 cells
containing the commercial substrate Plantimax®. The seeds of
jambu jambuarana were obtained from producers in the state of Para (Brazil)
with good features phytosanitary field production. The germination occurred
at seven days after the seeding and transplanting to 40 days after seeding.
The planting was done manually in 6 m2 (for each experiment: organic
and mineral), placing 18 plants per row and each plot consisted of five lines.
The spacing used was 20x25 cm which allowed the cultivation of 90 plants. The
experiment presented as doses of mineral fertilizer treatment (120 g m-2
urea) applied in two times (15 days after planting) and the doses of organic
fertilizer (8 kg m-2 castor pomace) applied at planting.
The first crop of jambu was collected in the morning, 90 days after seedling
transplantation (seedling was cultivated on Plantimax® substrate),
at the opening of the flower buds. Branches were cut at 7 cm from the soil,
thus new branches can bud for the accomplishment of the second crop which happened
40 days after the re-budding.
Picked plants (leaves and flowers) were transferred in the laboratory and washed
for the elimination of sludge. The first wash was accomplished with clean water
in order to eliminate residues from the field. The second wash was carried out
with a solution containing 150 mg L-1 of sodium hypochlorite and
in order to reduce microbial contamination, plant material was further immersed
in this solution for 5 min. The third and the fourth washes were in distilled
water. Leaves and inflorescences were separated, weighted and transferred in
a greenhouse, under forced circulation at 40°C, until a constant weight
Isolation of essential oils: In order to obtain essential oils, leaves
and inflorescences were finely ground and separately subjected to distillation
in a Clevenger apparatus for 2 h. Obtained oils were separated from the aqueous
phase by liquid-liquid extraction with dichloromethane.
Separation and quantification: Separation and quantification (normalization
area method) of the substances were carried out by gas chromatography (Shimadzu,
GC-2010), equipped with a flame ionization detector, using a DB-5 (J and W Scientific;
30 mx0.25 mmx0.25 μm) capillary column. Substance identification was accomplished
by gas chromatography coupled to a mass spectrometer (Shimadzu, QP-5000), operating
by electron impact (70 eV), equipped with a capillary column OV-5 (Ohio Valley
Specialty Chemical, Inc., 30.0 m x 0.25 mm x 0.25 μm). CG analyses in were
carried out as follows: injector and detector temperatures were 220 and 230°C,
respectively. Helium was used as carrier gas at a flow rate of 1.0 mL min-1
with 1:20 split ratio. Samples were diluted (1 μL mL-1) in ethyl
acetate and the injection volume was 1 μL. Temperature program was: 60-120°C
at 6°C min-1, 120-195°C at 3°C min-1 and 195-240°C
at 8°C min-1.
Identification of volatile components: Compound identification was carried
out by comparison of the mass spectra with the system database CG-MS (Nist.
62 lib.) and retention times according to Kortvelyesi et
al., (1995). Substance retention times were obtained by co-injection
of the essential oil with a mixture of hydrocarbons (C9-C24),
using the equation of Van Den Dool and Kratz (1963).
Statistics: Data were subjected to statistical analysis using SAS program
package. The one-way analysis of variance (ANOVA) followed by Duncan multiple
range test were used and the differences between individual means were deemed
to be significant at p<0.05.
RESULTS AND DISCUSSION
The CG-MS analysis of essential oil of Spilanthes oleracea coming from
the first crop resulted in the identification of twelve compounds (Table
1, Fig. 1). Total identified substances were 89.05% in
inflorescences and 85.04% in leaves. As regards the second crop (cultivar),
in plants cultivated under mineral fertilization, 90.82% were identified in
inflorescences and 94.90% in leaves. Regarding plants from organic manuring
it was noticed that total identified substances in essential oil of the first
crop were 87.13 % in inflorescences and 99.56% in leaves while 86.30 and 86.33%
in inflorescences and in leaves of the second, respectively.
Although there was no significant difference between the methods of cultivation,
was observed higher levels of trans-caryophyllene in relation to others components
of the oil analyzed. In plants coming from mineral fertilizer cultivation, the
average content of trans-caryophyllene was 48.64 and 47.42% in inflorescences
of the first and the second crop, respectively. In leaves it was found at 45.39
and 33.61% in the first and in the second crop, respectively. Regarding organic
manure cultivation, the average content of trans-caryophyllene varied from 47.83%
in inflorescences of the first crop to 43.85% of the second. For leaves the
contents found was 59.29 and 42.13% in the first and in the second crop, respectively.
The analysis of essential oil in Micromeria biflora ssp. arabica also
showed that the major sesquiterpene component was trans-caryophyllene (43.7%)
Another compound found at high concentration Spilanthes oleracea was
germacrene-D. In inflorescences of plants grown under mineral fertilization
it was 20.75 and 19.57% of the total essential oil in the first and the second
crop, respectively. While in leaves it was found at a concentration of 20.06%
in the first and 17.46% on the second crop. On the other hand, organically cultivated
plants showed a content of germacrene-D of 19.87 and 19.86% in inflorescences
of the first and the second crop, respectively. In leaves, the contents were
of 22.36 and 23.40%, in the first and the second crop, respectively. These germacrene-D
contents were higher than those reported for Salvia spinosa (10.66%)
(Salehi Sourmaghi et al., 2006) and Melissa
officinalis L. cultivated in two sites of Turkey (2.04% and 1.89%) (Ayanoglu
et al., 2005) and lower than that found in Marrubium astracanicum
Jacq. (23.4%) (Teimori et al., 2008).
|| Chemical composition of essential oil of Jambu (S. oleracea
cv. Jambuarana), grown under organic and mineral manuring
|IK exp.: Experimental Kovats index, IK lit: Kovats Index from
bibliography, tr:≤ 0.2 and nd: Not detectable
|Fig. 1 (a-b):
||Chromatogram of total ions in essential oil of jambu (Spilanthes
oleracea cv. Jambuarana) inflorescence, cultivated under (a) Mineral
manuring and (b) Organic, Peaks 5, 7, 8 and 10 are trans-caryophyllene,
germacrene-D, 1-dodecene and spathulenol, respectively
We detected spilanthol between 3.70% and 4.09% in plants under organic manuring
and 2.53 and 3.84% in those under mineral fertilization, in the first and second
crop, respectively (Table 1). Other important compounds of
Spilanthes oleracea were also observed during the chemical analysis of
the oil, such as 1-dodecene and spathulenol, mainly in leaves, showing an average
content of 29.32% in the second crop, against 7.02% of the first crop of plants
grown under mineral fertilization. While in Spilanthes oleracea cultivated
under organic manuring, these compounds appeared in lower amount in leaves (5.28%
in the first crop and 8.07% in the second), when compared to plants coming from
mineral fertilized fields.
The quality and the yield of essential oils have been reported previously to
be influenced by fertilizers and soil pH (Alvarez-Castellanos
and Pascual-Villalobos, 2003; Upadhyay and Patra, 2011).
Fertilizers have been found to increase the yield of essential oil from established
crops, like Valeriana officinalis (Letchamo et
al., 2004), Artemisia annua (Malik et
al., 2009) and Matricaria recutita (Upadhyay
and Patra, 2011). The results reported in the present work, regarding the
analysis of essential oil, showed that fertilization procedure was not influenced
essential oil composition of Spilanthes oleracea (Table
1). From our analysis, trans-caryophyllene content is well above that reported
by Almeida et al. (2005), finding 33.43% trans-caryophyllene
in leaves of Leonurus sibiricus. The presence of trans-caryophyllene
was previously observed in essential oil of Piper duckei (Abraham,
2001) and this substances, trans-caryophyllene and germacrene-D, have been
studied for having an anti-fungal and anti-microbial activity (Duarte
et al., 2005). Thus, the use of essential oil of jambu (Spilanthes
oleracea), due to its content of trans-caryophyllene and germacrene-D, may
find an application in crop biological control.
According to our results, germacrene-D was not influenced by the fertilization
procedure and its content was generally lower than that reported by others authors.
As a comparison, Almeida et al. (2005) obtained
24.95% germacrene-D content in leaves of Leonurus sibiricus. This substance
(peak 7 of Fig. 1) was also found in Artemisia parviflora
(Rana et al., 2003), Piper friedrichsthalii
and P. pseudollindeni (Vila et al., 2003),
Juniperus turbinata (Cosentino et al., 2003)
and Lippia javanica, showing, in vitro, an antibacterial and
fungicidal activity (Ngassapa et al., 2003).
Literature reports showed that others plants containing high percentages of
germacrene D present eminent anti-microbial activity, as well (Azimi
et al., 2011). The increasing interest on plants containing high
amount of this genre of substances is certainly due to the biological properties
of their molecular structure (sesquiterpenes), showing a wide antibacterial
and anti-fungal activity and acting as enzymatic inhibitors (Abraham,
The content of spilanthol observed in the present study was lower than that
described in literature. The analysis of essential oil of jambu performed by
Vulpi et al. (2007) reported an average content
of spilanthol of 15.16% in inflorescences: value well above that obtained in
the present work. The same authors verified its presence at an average content
of 1.46% in essential oil from jambu leaves. However, the presence of this substance
was not detected in our study, in the essential oil of jambu leaves cultivated
under organic manuring and mineral fertilization, neither in the first nor in
the second crop (Table 1). Although, Vulpi
et al. (2007) reported in leaves a higher content of germacrene-D
(38.51%), β-farnesene (36.04%) and 3-tridecene (2.97%). While in inflorescences,
15.38% 3-7-dimethyl-1,3,6-octatriene, 15.16% spilanthol and 15.02% β-farnesene
were found. In the present study, the data reveal a higher average content of
trans-caryophyllene, germacrene-D, 1-dodecene and spathulenol was observed,
regardless of fertilization.
According to the literature, the component of jambu essential oil that causes
the sensation of anesthesia provided by spilanthol, which influences its cultivation
for medicinal or culinary purposes. However, we detected low spilanthol content
found in jambu plants, which can be attributed to the period in which the experimental
study was carried out, i.e., the temperature was quite unstable, showing temperatures
below the recommended values (16-18°C). Has been suggested that climatic
factors influence the concentration of active molecules (secondary metabolites)
in plants and, among climatic factors, temperature affects very deeply the survival
of vegetables, being linked to plant growth and development. Species showing
scarce adaptation to temperature variation in a specific area will present serious
problems in biomass and active molecule (secondary metabolites) production,
because it influences primary metabolism (breathing and photosynthesis) and,
as a consequence, secondary metabolism. According to Malik
et al. (2009) among those that can affect the chemical composition
of a plant, is nutrition. The deficiency, or the excess, of nutrients can promote
a lower or a higher secondary metabolite production in plants. For this reason,
organic manuring and mineral fertilization used in the present work could interfere
with the content and the quality of essential oil of jambu plants, but this
effect did not occur during this experiment. Several studies pointed out that
environmental, genetic and agronomic factor can cause changes in the chemical
composition of products from vegetable origin. For this reason periodic evaluations
are so important.
In the present study, results reported allow to give several answers about
the effect of organic and mineral manuring on jambu plant cultivation. From
the results neither of the cropping system (organic or conventional) changed
significantly the composition of essential oil of jambu in relation to spilanthol
and the others components of essential oil. In this study, the benefit from
organic agriculture is the absence of pesticides. Furthermore, these results
allow to conclude that the most abundant compounds found in this study were
trans-caryophyllene, germacrene-D, 1-dodecene and spathulenol and spilanthol,
mainly in inflorescences.
Authors would like to thank CAPES (Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior) and CNPq (National Council for Scientific
and Technological Development).
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