Effect of sulphur on yield and chemical composition of essential oil of Ocimum basilicum

1 Departamento de Farmácia, Universidade Paranaense, Praça Mascarenhas de Moraes, 4282, Umuarama-PR, Brazil. 2 Programa de Pós-graduação em Biotecnologia Aplicada à Agricultura, Universidade Paranaense, Praça Mascarenhas de Moraes, 4282, Umuarama-PR, Brazil. 3 Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowski s/n, Rio de Janeiro-RJ, Brazil. 4 Departamento de Farmácia, Centro Universitário de Maringá, Av. Guedner, 1.610, Maringá-PR, Brazil. 5 Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Maringá-PR Brazil. 6 Programa de Pós-graduação em Ciência Animal, Universidade Paranaense, Praça Mascarenhas de Moraes, 4282, Umuarama-PR, Brazil.


INTRODUCTION
The term "ocimum" comes from the Greek "ókimom" which means fragrant, due to the smell scent exhaled by the leaves and inflorescences.The genus Ocimum comprises about 30 species rich in essential oils.Ocimum basilicum (L.), commonly known as Basil, is used worldwide and has an estimated global production of 42.5 tons year -1 (Morais and Barbosa, 2012).In Brazil, O. basilicum is cultivated mainly by small farmers to be marketed as a condiment (Teixeira et al., 2002).In addition to be consumed fresh, basil is employed to obtain an essential oil, whose high concentration of linalool is highly valued in the *Corresponding author.E-mail: cristianigazim@unipar.br.
international market and widely used in the industries of condiments and cosmetics (Marotti et al., 1996).An estimated 3% of the world's oil is used by the pharmaceutical industry, 34% by the beverage industry and 63% for food and perfumery industries.The production of essential oils is commonly operated in developing regions due to a primary agriculture oriented to low-demanding crops.Brazil, Guatemala, India, Egypt, Indonesia, Sri Lanka and Turkey are the main traditional exporters of essential oils (Craveiro and Queiroz, 1993).
Brazil is prominent in world production of essential oil, but the lack of quality standards is still a chronic issue.ABRAPOE (Brazilian Association of Producers of Essential Oils) was founded recently to help to integrate producers and research centers in order to improve the quality of essential oils (Bizzo et al., 2009).
The industries of medicinal, aromatic and seasoning plants have great interest in purchasing a product with a high concentration of active ingredients.Consumers of these markets are increasingly less interested in conventional farming using pesticides and more oriented to organic products (Bastos, 2007).
Organic fertilization has been recommended to grow medicinal, aromatic and seasoning crops as a part of organic farming practices provided by the technical standards of the Brazilian Organic Agriculture Association.It is recommended to use 3 to 5 kg m -2 of organic compost or cattle manure, and avoid the use of urban-waste composts due to risk of contamination with heavy metals (Martins, 1998).
According to Corrêa Junior and Scheffer (2009) medicinal plants under organic farming are more resistant to pest and diseases, being the need for phytosanitary control reduced.The use of pesticides in medicinal crops can cause alteration in the composition of active ingredients, and in some instances, the use of the produce for medicinal purpose become impracticable.In recent years, the increased resistance to insecticides and high concerns on pesticide residues in agriculture produces has encouraged the adoption of new strategies of control by farmers (Wyss et al., 2010).
In this context, the Ministério da Agricultura e do Abastecimento (Ministry of Agriculture and Supply -Brazil), through the Normative Instruction n°7 of May 1999, recommends the use of high-dilution substances in organic food production (BRASIL, 1999).Such substances have improved the plant health in general, promoted vigor seeds, changed production and yield of active ingredients (phytochemicals), favored the adaptation to adverse conditions and productivity; thereby reducing the use of fertilizers and pesticides (Andrade et al., 2001;Shah-Rossi et al., 2009), assuring a safer produce and reducing risks to the farmer´s health (Bonato, 2006).
Sulphur has a broad spectrum of action that improves the general appearance of the plant, strengthens its natural defense, increases resistance to nutritional and climatic stresses and pest attack, and also influences positively other important aspects for essential oil production and development of shoots (Rezende, 2003).Within this context, the objective of this study was assessing the effect of different Suphur dilutions on the development of O. basilicum and the yield and chemical composition of its essential oil.

Botanical identification
The

Planting crop
The soil used in the experiment was collected from experimental plots at 20 cm depth.The soil was homogenized and then its chemical and physical properties were determined (Table 1).The soil was put up into 30 polypropylene pots with 10 L capacity (27 cm high and 25.5 cm diameter), with no nutrient correction, addition of fertilizers or pesticides.During the test period, data of temperature, humidity and rainfall were obtained from the Secretariat of Agriculture and Supply of Paraná -Brazil (SEAB) (Table 2).To grow seedlings, 180 cell seedling trays (3.5 cm²) were used and four seeds per cell sown.After 30 days, three cells, each with four seedlings, were transplanted equidistantly to each pot.After 40 days, each pot was left with only one seedling, the others being removed.

Preparing treatments and statistical analysis
Each treatment consisted of diluting Sulphur in a centesimal scale.In the total, five dilution rates were tested: 0c (control), 6c, 12c, 24c and 30c (Bonato et al., 2009).Distilled water was used to make the control.Dilutions were obtained in centesimal scale according to the pharmacotechnique for insoluble drugs, described in the Brazilian Homeopathic Pharmacopoeia (Farmacopéia Homeopática Brasileira, 2001).Each treatment was diluted to 0.1% with distilled water as solvent.Diluted solutions were applied to the seedlings in trays, and to the plants in pots.In the seedling trays, 2 ml of dilutions were weekly applied in each cell during four weeks before transplanting seedlings to pots.In the pots, 250 ml of dilutions were weekly provided to the plants up to the end of the vegetative cycle (Silva et al., 2003).The experiment was conducted at open atmosphere and for each treatment five replications (five pots with one plant in each) were used.The parameters assessed were: Plant height (cm), shoot and root fresh matter (g), yield and chemical composition of essential oil.Plant height was measured from the distance between the base and the shoot apex with a tape-measure.
The experimental design was completely randomized.Prior analysis of variance (ANOVA) excluded outliers on plant fresh mass using the box plot method.From then on, data was subjected to one-way ANOVA using general linear model with mixed-effects and balanced design, considering each Sulphur dilution as one treatment, and compared with the Duncan's test (P ≤ 0.05) by using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL, USA).To comply with ANOVA assumptions, data was previously checked with the Levene´s test.

Collection and yield of basil essential oil
The basil leaves were collected at the end of the vegetative cycle (about 5 months) and subjected to essential oil extraction by hydrodistillation for 2 h at room temperature, using a modified Clevenger equipment (Silva et al., 2003).The oil was removed from equipment with n-Hexane and filtered on anhydrous sodium sulfate, there after stored and cooled to 3°C.The yield of essential oil was measured in wet weight (g/g).

Chemical identification of basil essential oil by GC-MS
The GC-MS analyses were performed using an Agilent 5973N GC-MS System operating in EI mode, equipped with a DB-5 capillary column (30 m × 0.25 mm × 0.25 µm, Agilent, PA, USA) used to inject 1 µl of a solution sample.The initial temperature of the column was 40°C.The column was heated gradually to 300°C with a 6 °Cmin -1 rate.The injector (split 2:1; 2.1 mlmin -1 ) and the heated transfer line temperature were held at 250 and 320°C, respectively.The carrier gas (He) flow rate was 4.8 ml min -1 .The same temperature program was used for CG-FID.The identification of chemical components was based on comparisons among their retention indexes (RI) obtained using various n-alkanes (C7 -C26).Also, their EI-mass spectra were compared with the Wiley library spectra and with the literature data (Adams, 2007).

RESULTS
The physical composition of the soil consisted of 30.0%clay, 33.6% silt and 36.4% sand.The soil chemical analysis basing the basil cultivation for this study is shown in the Table 1.
The climatic conditions (air temperature, relative humidity and rainfall data) during the basil cultivation from October 2012 to February 2013 are presented on Table 2.In the period in which the basil crop was planted, no significant changes in the parameters of temperature, humidity and rainfall were observed (Table 2).
Results on effect of Sulphur treatments (different dilutions) in the plant height and in the shoot and root fresh mass are shown in the Table 3. Data in Table 3 indicate that Sulphur is not affecting plant height.The 24c Sulphur dilution significantly inhibited the production of shoot fresh mass (267.70 g) in comparison with the control (372.75g) and the 6c dilution rate (375.75g).The 30c Sulphur dilution led to greater root mass (46.10 g) than that of the 6c Sulphur dilution (33.35 g).The yields of basil essential oil under different Sulphur dilutions are showing in Figure 1.
With respect to the effect of different Sulphur dilution rates on the yield of basil essential oil, both the 6c and the 12c rates significantly resulted in the highest yields (0.152 and 0.149% respectively) in relation to the control (0.078%) and in comparison with other dilutions.
Chemical composition of the basil essential oil was assessed as the plants were subjected to different Sulphur treatments (Table 4).
Results in Table 4 demonstrate that the basil oil contains the linalol and eugenol as the major components.Expressive concentrations were also observed for other components identified in this study: αpinene, α-cadinol, α-bergamotene, cineole and epibicyclosesquiphelandrene.

Soil analysis results (Table
The basil crop was grown from October 2012 to February 2013 at temperatures varying from 25.8 to 27.7°C, relative humidity from 63.2 to 75.0%, and rainfall from 73.2 to 290 mm (Table 2).These are proper conditions to grow basil, a crop which originates in the Middle East and North Africa and was introduced in Brazil by the Italians (Carvalho-Filho et al., 2006).According to the literature, this crop adapts well to subtropical and temperate climates, warm and wet weather; and can be grown throughout the year.The plant tolerates low temperatures, but its development is slower in such conditions (Favorito et al., 2011).
The effects of Sulphur dilutions on the basil development were not significant at all, with exception to the 30c treatment that increased the production of root mass in comparison with other treatments, and the 24c  treatment that led to a decrease of production of shoot fresh mass (Table 3).
The 6c and 12c Sulphur dilution rates increased the yield of basil essential oil.Analysis of chemical components revealed intense synthesis of linalol and eugenolby dilution at 6c, 12c, 24c and 30c rates (Figure 1 and Table 4).Sulphur has been used in various studies, among them, an evaluation of its effect on the development of Mentha arvensis (L.) at 6c, 12c, 24c and 30c dilutions by Bonato et al. (2009).The authors observed an increased production of shoot fresh mass at 24c and 30c rates of dilution and an increased plant height from all treatments used.Also in this study, Sulphur decreased dry mass production, with exception to the 6c treatment, and substantially increased the oil content in peppermint.Bonato and Silva (2003), in another study, assessed the effect of Sulphur at 5c, 12c, 30c, 200c and 1 Mc dilutions on the growth and productivity of radish.Sulphur applications resulted in an improvement of general aspects of radish plants, which was demonstrated for all parameters studied, in relation to the control treatment.Dilutions at 5c, 12c, 30c and 1000c rates resulted in the best plant responses.The control treatment and the 200c rate caused the most negative plant responses.Castro (2002) evaluated the effect of Sulphur dilutions at rates 3c, 12c, 30c, 200c and 1000c on the yield of essential oil of Cymbopogon citratus, and observed that the 12c dilution increased the oil yield at 2.1% while the rate 200c had the lowest yield (1.32%).In another study done by Armond et al. (2005) reported the effect of the China substance at dilutions 2c, 4c, 6c, 8c, 10c, 12c, 14c, 16c, 18c, 20c, 22c and 24c on the yield of essential oil of Bidens pilosa (L.).The authors observed the influence of the dilution rates on the oil yield by decreased production at 2c and 6c rates followed by an increase in production at 8c and 10c, decrease at 12c and 14 c, increase at 16c and 18c, incrementing increase up to 22c followed by a decrease.These results confirm an oscillation similar to that observed by Andrade et al. (2001), Carvalho et al. (2003) and Castro (2002), who by experiments, found secondary metabolites in medicinal plants while using diluted substances.
The composition chemical analysis of the essential oil demonstrated an increased synthesis of eugenol and linalol, the major oil constituents, for all dilutions tested, as shown in Table 4.These compounds were also found by Silva et al. (2003) when assessing the yield and chemical composition of essential oil of O. basilicum.The authors reported a 32.61% share of eugenol and 21.24% of linalol in the basil oil.In another study, Fernandes et al. (2004) found linalol as the most abundant substance in the species O. minimum (44.3 to 59.8%) and O. basilicum (22.7 to 37.4%).Such alteration in the concentration of chemical components is explained by Davenas et al. (1988) and Bonato and Silva (2003).The authors say that in homeopathy, the same substance commonly influence differently the chemical contents, depending on the rates of dilution applied.For certain dilutions, increased concentrations may occur whereas inhibitions may be observed within a specific physiology (Hormesis phenomenon).Interpretations on this behavior are not satisfactory yet.One of the hypotheses, based on biodynamic agricultural data, is that such behavior may be related to the existent rhythmic movements in nature.Another hypothesis, based on experimental data, is that such behavior is due to similarity between the applied homeopathic drug and the organism (Bonato, 2007).
The increase of linalol and engenol concentrations by the application of Sulphur treatments is considered important as these substances are widely used by agrifood, pharmaceutical and cosmetic industries (Marotti et al., 1996).
Linalol is a monoterpene alcohol of the tertiary openchain group that been widely used as starting material for several importantly in theses as the linalyl acetate, which has acaricide, bactericide and fungicide action.In medicine, this substance has been successfully applied as a sedative, and currently, its anticonvulsant properties are being studied.It is also a substance widely used in perfumery as a component in the production of fragrances.Thus, linalol has a wide application in various areas of human knowledge, being its production required in ever-increasing amounts (Silva et al., 2003).
Eugenol is an aromatic compound with numerous biological activities such as anti-inflammatory, antinociceptive, healing, anesthetic and antifungal properties commonly used in the food industry (Daniel et al., 2009).Its main use is in dentistry, and it is applied associated with zinc oxide as temporary and intermediate cement in some restorations; for being a good thermal insulator, eugenol replaces the lost tooth dentin.In addition, eugenol is also used to produce other phenol compounds such as vanillin (Priefert et al., 2001).

Conclusion
The Sulphur different dilutions used in this study affected the yield and composition of the basil essential oil, resulting in an increased production of linalol and eugenol, the major chemical components found in the basil oil.This increased production is considered economically important as the essential oil of basil with high concentration of linalool is highly prized in the international market and widely used by the agri-food and cosmetic industries.Eugenol is widely used in dentistry and as a precursor in the synthesis of various phenol compounds.The present study also suggests that homeopathy can be an important tool in agro-ecological systems.

Table 1 .
Chemical properties of soil.

Table 2 .
Average of month air temperature, pluviosity and relative humidity in the period of study.

Table 3 .
Mean (± standard error) of plant height (cm), shoot fresh mass (g) and root fresh mass (g) of basil under different Sulphur dilutions.