Secondary metabolites, including essential oils, comprise a class of products
related to the physiological development of plants. They represent a chemical
interface between the plants and the environment and therefore, their synthesis
is often affected by environmental conditions (Kutchan,
2001), such as water and nutrient availability (Alsafar
and Al-Hassan, 2009; Azizi and Kahrizi, 2008; Pirzad
et al., 2006; Supanjani et al., 2005)
and growing habitat (Verma et al., 2011; Azizi
and Kahrizi, 2008; Mosayebi et al., 2008).
Additionally, phenology and harvesting age have been identified as important
effects on the essential oil yield (Okoh et al.,
2007; Ozguven et al., 2006; Ayanoglu
et al., 2005; Telci and Sahbaz, 2005).
According to Brant et al. (2008), plant species
belonging to the same botanical family do not present a similar behavior as
function of environmental conditions. Therefore, it is not possible to establish
a pattern and then various environmental factors can differentially alter the
quantitative and qualitative aspects of essential oils. As one of the many factors
that may influence the characteristics of essential oils, the climatic variations
that occur over the course of a year have been the focus of many researchers
attempting to identify the most appropriate time of the year for optimal extractions
in terms of yield and/or compound concentration. When the set of climatic factors
in seasonal climates with two well-defined seasons is modified, these variations
act on the plants and generally alter their metabolism (Scherer,
In research on essential oils, plants of the Myrtaceae family have proven to
be of great importance, largely because they naturally exhibit storage structures
for essential oils called translucent dots (Joly, 2002).
However, in Brazil, the effect of seasonal fluctuations on the chemical composition
and yield of essential oils is still unknown for the vast majority of the species
in this family in their native environments (Souza, 2009).
In general, knowledge about the forest species that produce these oils is still
lacking and is especially sparse for the Cerrado (Brazilian savannah), a biome
exclusive to Brazil that includes more than 11,000 species of phanerogamic plants.
This study analysed the essential oils of two species of plants from the Myrtaceae family native to the cerrado, the maria preta (Blepharocalyx salicifolius (Kunth.) O. Berg) and the araçá do cerrado (Psidium myrsinites DC). The study focused on describing the quantitative and qualitative behaviour of essential oil production as a function of the climatic variations that mark the typical season changes observed in Midwestern Brazil.
MATERIALS AND METHODS
Material collection: The samples were collected from a region of the
Stricto sensu Cerrado, an experimental plot of the University of Brasilia
(Brasília, Brazil) located on the Agua Limpa Farm. Leaves and thin branches
from 12 previously marked and identified individuals (six species each of B.
salicifolius and P. myrsinites) were collected. The species were
preliminarily identified and later compared with the material deposited in the
UNB Herbarium (UB) under the following catalogue numbers: Blepharocalyx salicifolius-88293,
To analyse the influence of seasonality on the essential oils of these species,
collections were scheduled to be conducted in four months, November, March,
July and September, between 2009 and 2010. However, it was not possible to complete
the collection during the last month (September 2010) because of sufficient
plant material to obtain samples from both species. P. myrsinites is
deciduous and its foliation period occurs only between August and September
(Silva-Junior, 2005). All materials collected in the field
were placed in plastic bags and the samples were marked. Then, the material
was stored in a cold chamber (10°C, 50% relative humidity) until essential
oil extraction, which occurred 1 day after collection for P. myrsinites and
2 days after collection for B. salicifolius.
Climatological data, including temperature, rainfall, sunlight and relative
humidity for each sampling month, were obtained from the National Institute
of Meteorology website (www.inmet.gov.br).
Essential oil extraction, yield and composition analysis: Extractions were performed by steam distillation using laboratory equipment to obtain small extractions of the essential oils (Linax, D1). Approximately 250 g of fresh leaves was used in each extraction. Each extraction was performed for 90 min to allow for maximum yield. By the end of each extraction, approximately 900 mL of the hydrosol was obtained and stored in a properly identified amber glass bottle. All bottles were conditioned in a cold chamber until the next step of separating the aqueous and organic portions of the hydrosol.
The separation of each sample was performed using a decanting funnel and four portions of ethyl acetate (100 mL each), with an electromagnetic stirrer used in 10 min cycles at each addition of solvent. During each cycle, the organic fraction of the hydrosol was reused and the aqueous fraction was discarded. Next, 50 mL of saturated sodium chloride solution (NaCl) was added to the organic fraction to dry the material and decrease any emulsification formed during the initial process, allowing for the removal of any aqueous component still present. To remove possible water residue, sodium sulphate (Na2SO4) was also added. Finally, the organic fraction was filtered through a funnel and the solvent was recovered using a rotary evaporator.
To calculate the essential oil yield, a relationship between the wet leaf mass used in distillation and the essential oil mass obtained was determined using a scale accurate to 1x10-4. To evaluate the effect of the collecting season on the essential oil yield, an analysis of variance (ANOVA) and a multiple comparison test, the Least Significant Difference (LSD) test, both at a significance level of 5%, were used.
After obtaining the pure oil samples, the samples were divided according to the time of year of collection. Initially, to identify the chemical profiles of the oils, spectroscopic Nuclear Magnetic Resonance (NMR) analyses, 1H (300 MHz) and 13C (75 MHz) were used to identify the functional groups. In all experiments, a 5 mm internal diameter ATB probe at room temperature with a 45° pulse was used. The 1H and 13C spectra, referenced to TMS and CDCl3 (77.0 ppm), respectively, with chemical shifts (δ) expressed in ppm, scalar couplings (J) in Hz and multiplicities defined for singlet (s), doublet (d), double doublet (dd), multiplet (m) and heptet-triplet (th), were obtained using a Varian Mercury Plus spectrometer. Infrared analyses were performed using a Bomen MB-100 spectrometer and vibrational frequencies were expressed in cm-1.
To identify the major components of the oils, the same previously used samples were injected into a gas chromatograph coupled to a mass selective detector (GC/MS) operated in the electron ionisation mode (70 eV). A Shimadzu 7890 A chromatograph with a 5%-phenyl/95%-methyl-silicone (HP5, 30 mmx0.32 mmx0.25 μm) capillary column and helium as the carrier gas (1.0 mL min-1) was employed to analyse the oils. The oven temperature was programmed from 60 to 240°C at a heating rate of 3°C min-1. The oil was diluted to 1% in ethyl acetate and 1 μL of the solution was injected into the injector at 250°C in split flow mode (1:20). The mass spectra obtained were compared to data from the Wiley library, sixth Edn.
RESULTS AND DISCUSSION
Quantitative evaluation: The B. salicifolius species did not
demonstrate a variation in yield between the first two analysed periods, November
and March; the average yield for both months was 0.16% but the yield increased
to 0.21% in July. However, this difference was not statically significant according
to LSD test. For P. myrsinites, there was a variation statistically significant
between the first two periods and the average yields for November and March
were 0.18 and 0.22%, respectively. During July, the average yield further increased
to 0.38%, the highest value observed.
For both species, the dry season, represented by July, produced a slight increase
in essential oil production. This result possibly occurred because of the more
severe weather conditions that generally influence plant physiology by causing
the plants to defend against adverse external conditions (Evans,
1996; Salisbury and Ross, 1991). The last day of rain
before the dry season occurred on 27 May 2009, followed by 46 days without rain
until the collection date in July. These conditions resulted in an adverse situation
for basic plant physiological functions in terms of the relative humidity present.
It is possible that essential oils in these species play an important role in the defence of the plants against adverse weather conditions because for both species, the period with the greatest yield had the worst relative humidity and rainfall conditions. Water supply can often be critical for essential oil production when associated with other climatic factors such as high temperatures. However, in this study, although July had the lowest relative humidity during the collection months (55%), the average variation in the maximum temperature for each period was low (25-27°C) (Table 1). This condition may have resulted in only an alteration in leaf stomatal activity, thereby reducing oil volatilisation.
As stated by Taiz and Zeiger (2004) under water-deficient
circumstances, in a slow and long-term process, plants generally develop resistance
strategies by diminishing leaf area and deepening root depth. When the onset
of stress is rapid or the plant has already established these adaptations, stomatal
activity can be drastically reduced. The stomata, which are cellular structures
responsible for controlling air intake and air/water output as mentioned by
Appezzato-da-Gloria and Carmello-Guerreiro (2003), a re
formed b y two guard cells that control the stomatal opening, which is the channel
between this organ and the environment. According to Appezzato-da-Gloria
and Carmello-Guerreiro (2003), the opening and closing of the stomata depend
on turgor variation and the stomata open in the presence of higher amounts of
potassium ions in the guard cells. Located on the leaf epidermis, the guard
cells lose turgor by releasing water to the atmosphere. This release is likely
triggered by low relative humidity, causing hydropassive closure (Taiz
and Zeiger, 2004). The absence of water or reduced quantities of water leads
to stomatal closure, which reduces the amount of water and air leaving the cells.
|| Average weather conditions for each collection month
Thus, the loss of volatile material may be less than what would be found during
periods of high relative humidity, which favour stomata opening and high-intensity
rainfall, which can leach volatile material from the plants.
The dry season in central Brazil can last for up to 7 months and each plant
adapts by developing survival strategies to avoid suffering from the lack of
rain. As stated by Eiten (1972) the diverse vegetative
types of the Cerrado are marked by differences in floristic composition as a
function of the soil characteristics, such as nutrient stock and concentration,
depth and drainage. For the development of arboreal species in the more closed-vegetation
types, deeper soils are required to provide a greater nutrient supply (Henriques,
2005) and a higher water content in the top soil is needed during the dry
season relative to that needed b y the more open-vegetation types (Franco
and Luttge, 2002). Along with these soil characteristics, analyses of the
arboreal vegetation in the Cerrado have revealed that most woody plants produce
deep root systems that can access the soil layers that store water, providing
these plants access to water for vegetation throughout the year in the restricted
formations of the Cerrado (Ferri, 1944). Even in the
dry season, these arboreal plants do not suffer from a lack of water and possibly
exhibit diminished leaf water content only due to the reduction in relative
humidity. Accordingly, the plants that have established the necessary adaptations
can continue to produce essential oils and only exhibit reduced stomatal activity,
reducing oil volatilisation but not production.
In addition to water stress, other aspects may be related to the physiology
in B. salicifolius and P. myrsinites species that make the dry
period the most favourable for essential oil production. Full development of
the glandular trichomes that store essential oils in some species may be light
dependent, as observed for basil (Ocimum basilicum L.) and thyme (Thymus
vulgaris L.) (Gobbo-Neto and Lopes, 2007). In July,
there was a higher incidence of sunlight, reaching an average of 8.5 h day-1,
versus 4.7 and 5.9 h in November and March, respectively Future studies of the
leaf anatomy of these plants as they relate to the production of essential oils
can help better identify which climatic factors actually interfere with the
production of these oils.
The results obtained here are different from those found by Pirzad
et al. (2006). They studied the effect of different irrigation regimes
on the yield of essential oil from Matricaria chamomilla and observed
that the highest oil yield was obtained for irrigation at 85% of field capacity,
while the lowest yield was at 55%. Surely, these studies cannot be directly
compared since they were conducted in very different environment. Nevertheless,
they highlight that water supply is an important factor affecting the essential
Analysing only the quantitative aspect of essential oil production, the role
of these essential oils in protection against predators and attraction of pollinators/dispersers
may not be as relevant. It can be inferred since that the period of increased
intake did not coincide with the flowering (November to December for P. myrsinites
and August to January for B. salicifolius) and fruiting (November to
February for P. myrsinites and January to March for B. salicifolius)
seasons (Fig. 1). Ozguven et al.
(2006) observed that the yield of essential oil form Origanum syriacum
var. bevanii was higher during the full blooming period. However, the
effect of phenological period can vary according to the site as observed by
Ayanoglu et al. (2005) for yield essential oil
from Melissa officinalis. They observed an opposite behaviour: depending
on the site, the yield can be higher before or after flowering.
Comparing data from the literature, it is noted that the average yield of 0.18%
for B. salicifolius was very close to the value of 0.17% described by
Marques (2007) but was higher than those found by Castelo
et al. (2010) (0.10%) and Limberger et al.
(2001) (0.09%). For the P. myrsinites species, the average yield
of 0.26% was twice that found by Castelo et al. (2010)
(0.13%) in a study in the cerrado but was lower than that found by Freitas
et al. (2002) (0.4%) in a study in the Caatinga.
Although, a trend toward a greater yield in the dry period was observed with the two species during the periods analysed, the LSD test revealed that this variation was statistically significant only for P. myrsinites. By comparing the three periods, a statistically significant difference was observed between the yield from the rainy season, November (0.18%) to March (0.22%) and the dry season in July (0.38%).
For B. salicifolius, the variation was not statistically significant
among the periods analysed: November (0.16%), March (0.16%) and July (0.21%).
One hypothesis for the non-significant yield variation in this species is that
unlike most plants used in essential oil studies, which have annual or biennial
life cycles and which are found in herbaceous or shrubby habitats, B. salicifolius
is an arboreal plant with a perennial life cycle.
||Yield of essential oil according to the month of collection,
means followed by same letter within each species are not statistically
significant, bars are standard deviation
The difference in the physiological development between plants with a perennial
life cycle and those that are annual or biennial (Arthur and
Wilson, 1967) may be a relevant factor that can cause plants to have shorter
life cycles during the peak years of essential oil production. The development
of these plants must be faster so that all stages of growth, development and
reproduction can be completed for the successful propagation of the species.
Annual plants, usually weeds and vegetables, have a cycle from seed to adult
plant in which they form new seeds within a single growing season that can last
for several weeks. Biennials have cycles of two growing seasons, the first near
the ground with root formation, a small stem and leaves, followed by the second
phase with flowering, fruiting and death; these plants rarely become woody (Raven
et al., 1996). Perennials can be woody or herbaceous and they have
a vegetative structure that survives year after year, with a longer life cycle
and the evolution of all of the developmental stages occurring over several
seasons (Raven et al., 1996). This attribute can
therefore be a factor that leads to more continuous production throughout the
year because there is no urgent need to complete all of the developmental stages
in only a few seasons.
GC/MS: In the GC/MS analysis, the oils of B. salicifolius
contained the following main components: p-cymene (aromatic hydrocarbon), monoterpenes
(α-pinene and α-terpineol), sesquiterpenes (aromadendrene, globulol
and caryophyllene oxide) and others, such as cis-3-hexane-1-ol and 2(1H)-naphthalene,
with variation occurring between periods.
In a study by Limberger et al. (2001) with the
same species, 1,8-cineole, linalool and β-caryophyllene were found to be
the major compounds. Moreira et al. (1999) studied
species in southern Brazil and reported 1,8-cineole, (-)-β-pinene and (-)-limonene
as the main components. In a study in Argentina, Tucker
et al. (1993) found 1,8-cineole, limonene and linalool. It is important
to note that although these studies examined the same species, many factors,
such as genetics, soil characteristics, extraction techniques and the climatic
characteristics of the region, affect the production quantity and quality of
the essential oils. Therefore, it is normal and even expected that there would
be variations between the results from different studies with varied characteristics.
Because there was no significant variation in yield between the periods examined, essential oil extraction from B. salicifolius in the region should be directed according to the desired chemical compounds, i.e., depending on the type of market that will use the oils or the type of markets the producers hope to reach.
GC/MS analysis of P. myrsinites revealed a predominance of principal
components that included sesquiterpenes (caryophyllene oxide, β-caryophyllene,
β-guaiane, α-humulene and viridiflorol) and monoterpenes (myrcene).
There was also variation in the principal components among the periods analysed.
Despite the predominance of sesquiterpenes in the species composition, the presence
of a monoterpene was detected among the major components. This monoterpene,
β-myrcene, is a natural olefin compound found in several plants that is
used in the production of fragrances. In the study by Freitas
et al. (2002) with the same species, major components, such as sesquiterpenes,
β-caryophyllene and caryophyllene oxide, were also identified. Additionally,
July was the period of highest yield, that is, with the greatest commercial
potential and the diversity of organic compounds among the oils major
components was demonstrated using chromatography. Analysing these two aspects
it is concluded that this dry period is the most suitable for the extraction
of essential oils from P. myrsinites.
1H and 13C NMR: Using 1H and 13C NMR spectroscopic techniques, it was observed that essential oils from these two species are complex mixtures of saturated and unsaturated hydrocarbons with oxygenated functional groups. In the 13C NMR spectra of oils from the B. salicifolius species, signs of a ketone carbonyl and traces of ester were observed in the three periods analysed. These compounds greatly affect the aromatic aspect of the essential oils. Regions of olefinic and aromatic carbons were also observed, structures that also influence the aroma by being very volatile. Aliphatic and oxygenated carbons were also found. In 1H NMR spectra, the presence of aliphatic, oxygenated and olefinic hydrogen was also confirmed (Table 2, 3).
||The functional groups identified by 13C NMR for
||The functional groups identified by 1H NMR for
According to Stewart (2006), ketone and ester carbonyls
have different physical and chemical properties than simple hydrocarbons. The
ketone functional group is responsible for some of the most powerful aromas
and flavours in natural products. This functional group can act therapeutically
as a sedative, analgesic and relaxant but can also be dangerous for humans.
An example is the oil from Salvia officinalis L. which is rich in camphor,
a neurotoxic product. In plants, the compounds act as pheromones and are excellent
natural insecticides when used in large quantities. According to the same author,
the presence of esters greatly influences the fragrance of the essential oils,
even in small quantities. A wide variety of fragrances is composed of esters
and esters form the most popular class of compounds in the perfume industry.
Esters are generally not toxic and produce relaxing effects. Some of the compounds
detected in GC/MS were found in the 13C NMR spectra using the compound
spectroscopic data, verifying the presence of these compounds in the essential
oils of the species (Table 4).
The qualitative analysis of the 13C NMR spectra of B. salicifolius
demonstrated that the profiles differ amongst themselves and an expansion of
the spectra made the difference more evident. Of the periods analysed, the March
extract was the richest in volatile compounds, with the greater signal in the
ketone carbonyl regions (Table 4). The July extract exhibited
lower signal from the differentiated carbons and the extract from November was
in the intermediate range. A possible explanation for the March extract being
the richest in compounds is that this month marks the fruiting period of the
species, which begins in January. Although, the individuals analysed did not
bear fruits, the presence of ketones in the oils may be related to the need
to attract dispersers or to protect the plant from predators.
|| The 13C NMR chemical shifts of compounds obtained
from Blepharocalyx salicifolius* and from Psidium myrsinites**
|*The Blepharocalyx species presented pinene, α-terpineol,
p-cymene and caryophyllene oxide compounds. **The Psidium species
presented only caryophyllene oxide and myrcene compounds, Source: Silverstein
et al. (2005), Note: Experimentally obtained chemical shift values
were obtained from the 13C spectra of the three periods analysed
||The functional groups identified by 13C NMR for
A possible explanation of the July results is that the essential oil production
was enhanced in the sexual organs and reduced in the leaves during the month
preceding the flowering period of the species to attract its natural pollinators,
bees and small insects. As previously noted, among the periods analysed, there
was no significant variation in essential oil production in the species, with
a trend toward increased production found only in the dry season or in the chemical
composition of its oil. It is evident that there was considerable variation
among the three periods independent of the rainy or dry period. It is possible
that the essential oil of B. salicifolius plays a major role in the interaction
between the species and other biological agents, as opposed to adverse weather
conditions. A great similarity between the species was observed in the 1H
NMR spectra of the three periods. Because it is related to a type of natural
product, 13C analysis reveals a larger number of compounds and its
chemical shift range is larger than that found with the 1H analysis.
In the 13C NMR spectra from P. myrsinites, the presence of
ester was observed in all periods, unlike the ketone carbonyl functional group,
which was found only in July. This result is important because the presence
of ketones greatly influences the aromatic aspect of the essential oils and
is greater in the oil extracted during July than in that extracted during the
other months. Aliphatic, oxygenated and olefinic carbons were also observed
and confirmed in the 1H NMR spectra (Table 5, 6).
Some of the major compounds detected with GC/MS were also found in 13C
NMR spectra, verifying their presence.
||The functional groups identified by 1H NMR for
Through, qualitative analysis of the 13C NMR spectra, it was evident
that the March extract had fewer compounds and lower intensity peaks than the
July extract, which exhibited the highest number of different carbons. The November
extract had an intermediate quantity.
One possible explanation for this result is that the flowering period occurs from November to December and the fruiting period occurs from November to February. One of the functions of essential oils is the attraction of pollinators/dispersers and it is possible that more intense production of these compounds may act as an attractant during that period. With the end of the fruiting period in March, production of these compounds becomes less intense until July, when the climatic conditions become less favourable for plant physiology and a new peak in compound production occurs. According to the records, quantitative and qualitative production is even more intense during the dry period than during the early flowering and fruiting periods.
In the 1H and 13C NMR spectra for P. myrsinites
in all periods, the presence of the monoterpene linalool was observed, as reported
by Castelo et al. (2010). Comparing the spectra
of the species with those from synthetic and pure linalool it was possible to
identify the characteristic peaks of the detected compounds, especially in the
olefinic regions (Table 7).
The 1H NMR spectroscopic analysis indicated the presence of linalool
but this compound was not detected by chromatography among the five major compounds.
Competition with other sources, particularly cultivated sources, may hamper
the incorporation of the product obtained from this species in the market. However,
compound extraction from these species can be enhanced by the use of extracts
from the natural environment that generate less damage to the environment, which
is highly valued by the market.
|| The 1H and 13C NMR chemical shifts
of compounds obtained from linalool and Psidium myrsinites
|D: Doublet, dd: double doublet, m: Multiplet, th: Heptet-triplet,
The Brazilian rosewood (Aniba rosaeodora Duck) is the most used and
known natural source of linalool but it is under great pressure due to unmanaged
extraction and new sources would be important to ensure the survival of the
species. Some native plants, such as Amazonian sacaca (Croton cajucara
Benth) have been analysed for the presence of linalool (Chaves
et al., 2006); however, P. myrsinites could become a new source
for the product in a new region, as the demand for new sources is currently
concentrated in northern Brazil. This creates the possibility that other communities
could compete within the market, stimulating diverse production sources and
placing value on the social and environmental aspects of production.
Infrared (IR): The functional groups observed in the 1H and 13C NMR spectra were confirmed by the IR technique, excluding the acyl ester groups, which were poorly defined despite an experimental period (128 transients) that was sufficient to detect them. Because the 13C NMR technique detects the best features of organic groups, the presence of acyl groups may still be considered. The presence of ketone carbonyl at 1708 cm-1, although less prominent in the IR spectrum, was confirmed in July for P. myrsinites.
Through, chemical analyses, it may conclude that the chemical profile of the essential oil produced by B. salicifolius is formed by a complex array of compounds and this oil could potentially be used in various industries, especially pharmaceuticals. As the quantitative variation was not significant, this oil may be extracted during different times of the year. The essential oil produced by P. myrsinites has potential applications, as observed by linalool. This species presents a chemical profile rich in volatile substances and by evaluating the qualitative and quantitative data together, the dry season proved the most optimal for extracting its oil.
The authors are grateful for the CAPES, for the Scholarship grant to the first author and for CNPq and FINEP which provided financial support to purchase equipments.