INFLUENCE OF STORAGE TEMPERATURES AND STORAGE TIME OF DRY LEAVES ON PATCHOULI [Pogostemon cablin (Blanco) Benth.] ESSENTIAL OIL INFLUÊNCIA DO TEMPO E TEMPERATURAS DE ARMAZENAMENTO DAS FOLHAS SECAS NO ÓLEO ESSENCIAL DE PATCHOULI [Pogostemon cablin (Blanco) Benth.]

Patchouli [Pogostemon cablin (Blanco) Benth.] is a plant of the family Lamiaceae, widely used as an essential oil in the cosmetics and perfumery industry. This study aimed to evaluate the influence of storage time and temperature of dry leaves on the patchouli essential oil content and chemical composition. The experiment was performed in a completely randomized design in a 6x2x2 factorial scheme, testing storage time (0, 1, 2, 4, 8, and 16 weeks) and temperature (28°C and 33°C) of dry leaves of two patchouli genotypes (POG-015 and POG-021). The variables essential oil content and chemical composition, and the identification of fungus during storage were evaluated. Results showed that the storage significantly influenced the essential oil content. Patchoulol was identified as the major compound in both genotypes, ranging from 55.05% to 68.77% (POG-15) and from 52.83% to 64.06% (POG-021). Based on the results of patchoulol, dry leaves of both genotypes (POG-015 and POG-021) can be stored for up to eight weeks at 2833°C without altering the essential oil quality.

Patchouli essential oil stands out in the perfumery industry (SINGH; SHARMA; RAMESH, 2002) for providinga base and lasting character to a fragrance, and is among the 18 most important essential oils worldwide (BIZZO; HOVELL; REZENDE, 2009).
The chemical composition of patchouli essential oil varies according to the genotype, production organs, environmental and production conditions, and storage conditions. However, patchoulol and α-patchoulene are considered the most important compounds and aroma regulators (SINGH; SHARMA; RAMESH, 2002). Patchoulol is a sesquiterpene officially used as a chemical marker for the quality assessment of Pogostemonis Herba in the Chinese Pharmacopoeia. Tsai et al. (2007) reported that leaf material quality, dry leaf, essential oil aging, and plant age could also influence the quality of patchouli essential oil. A review study has shown that when considering 100 analyses of patchouli essential oils, the authors listed the main compounds and their observed average concentrations, such as patchoulol (39.0%), α-bulnesene (14.0%), α-guaiene (11.0%), seyclellene (6.6%), α-patchoulene (4.5%), (E) -βcaryophyllene (3.1%), β-patchoulene and pogostol (2.4%) (BEEK; JOULAIN 2018). Nizio et al. (2018) reported that, in general, the stages that follow the plant material harvest, such as the drying process, the storage of plant material, and the extraction process could influence the essential oil content and chemical composition. Leaf drying and the appropriate storage allow better plant material conservation. Thus, the drying process is widely used, but it must be performed in a way that does not compromise the product quality at the end of the production process (MARCHESE;FIGUEIRA, 2005). In a study with Myrcia lundiana, Alves et al. (2018) reported that one-day drying at 40 ° C was enough to dry the leaves without affecting the essential oil content.
In the case of aromatic species, the drying process must consider the storage structure and the volatile characteristics of the essential oil compounds. Drying is used to prevent microorganisms reproduction, consequently preserving the quality of the harvested plant. It also reduces the weight and volume of plants for transportation and storage (CORREA et al., 2004). Sant'ana et al. (2010) observed that the dry leaves of two patchouli genotypes stored for 28 days had an increase in the essential oil content. The novelty of the present study is the storage time and the comparison of the percentages of the main compounds at those storage times.
Due to the importance of patchouli essential oil, this study aimed to evaluate the effect of the storage time and temperature of dry leaves on the essential oil content and chemical composition.

Plant material and experimental design
This study used two patchouli genotypes (POG-015 and POG-021) from the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe. The exsiccates are deposited in the Herbarium of the Department of Biology, under the numbers 13173 and 13177.
Seedlings of the two genotypes were generated by cutting, in black plastic bags. Vermiculite and coconut coir dust (1: 1) were used as the substrate and were supplemented with 1g.L -1 of lime and 2g.L -1 of N-P-K (6 -24-12). Seedlings were transferred to a greenhouse (50% shaded), with an intermittent misting, at the Department of Agronomic Engineering of UFS, where they were kept until transplanting. At 40 days after planting, seedlings were transplanted to the field (January 2011).
The soil pH was corrected to 6.5, using dolomitic lime. Fertilization consisted of applying 6L.m -2 of bovine manure and 160g.m -2 of N-P-K (6-24-12) + micronutrients. The spacing was 0.50m between plants and 0.60m between rows. Drip irrigation was used, and plants were cultivated in double-colored plastic mulch (black on the bottom and white on the top).
In May 2011, shoots were harvested at 25 cm from the soil, and leaves were dried in a forcedair-circulation oven at 38 °C, for five days. Then, the shoot dry matter per plant was determined. Dry leaves were randomly separated into 60-gram lots and packed in black plastic bags. Leaves were stored in a BOD chamber, in the darkness, for 0, 1, 2, 4, 8, and 16 weeks, at 28 and 33 °C.

Essential oil extraction
Three 50g dry leaves samples were collected in the storage period for the essential oil extraction. The metabolite was extracted by hydrodistillation, in a Clevenger apparatus coupled to a 3,000mL round-bottomed glass flask, for 160 minutes. The flask was added with 2,000mL of distilled water and 50g of dry leaves (EHLERT et al., 2006). Contents were estimated based on the dry mass (mL.100g -1 ) and expressed in percentage. Essential oil samples were kept in capped amber glass flasks, in a freezer, until chemical analysis.

Essential oil chemical analysis
The qualitative analysis of the essential oil chemical composition was performed in a gas chromatograph coupled to a GC-MS mass spectrometer (Shimadzu, model QP 5050A) with an autosampler (AOC-20i, Shimadzu), and a J & W Scientific fused silica capillary column (5%diphenyl-95%-dimethylpolysiloxane) of 30 m x 0.25 mm i.d., 0.25 μm film thickness, at a constant helium flow rate of 1.2 mL/min. The temperature was set to 50•C for 1.5 min, then programmed to increase to 200•C at 4•C/min, followed by an increase of 15•C/min up to 250•C, then remaining constant for 5 min. The sampler temperature was 250° C, and the detector temperature was 280 °C. The injection volume was 0.5μL (ethyl acetate), with a split ratio of 1: 100 and a column pressure of 64.20kPa. The MS conditions consisted of an ionic capture detector with an electron energy of 70eV. The scanning speed was 1,000, at a scan rate of 0.3 scan/s, and the system was programmed to scan fragments/ions with m/z in the order of 40 to 500 Da.
The essential oil components were identified by matching their mass spectra to the spectra available in the database of the device (NIST05, NIST21, and WILEY8). These libraries allowed for the comparison of spectral data with a similarity index above 80%. The measured retention indices were also compared with indices from the literature (ADAMS, 2007). The Kovats retention indices (KI) were determined by a homologous series of n-alkanes (C 8 -C 18 ) injected under the same chromatographic conditions of the samples, using the equation of Van Den Dool and Kratz (1963).
The quantitative analysis of the essential oil compounds was performed by Gas Chromatography/Mass Spectrometry and Flame Ionization Detector -GC/MS/FID (Shimadzu GC-17A), coupled with a ZB-5MS fused silica capillary column (5% dimethylpolysiloxane), with 30m x 0.25mm i.d., 0.25μm film thickness, under the same GC-MS conditions. Compounds were quantified by area normalization (%). Compounds concentrations were calculated by the area and placed in order of GC elution.

Statistical analyses
Variables means were subject to analysis of variance and compared by the Scott-Knott test at the 5% probability level and polynomial regression, using the Sisvar software (FERREIRA, 2011).

RESULTS AND DISCUSSION
A triple interaction (genotype x storage time x storage temperature of leaves) was detected in the essential oil content. The essential oil content of genotype POG-021 varied when the leaves were stored at 28 °C. This variation is represented by a quadratic equation, in which the highest levels were obtained between the second and eighth weeks of storage. At this same temperature (28°C), no significant differences were observed for genotype POG-015. At 33°C results for both genotypes can be represented by quadratic equations, and contents ranged from 2.13% to 3.0% (POG-015) and from 2.43% to 3.0 % (POG-021) ( Table 1).  Sant'ana et al. (2010) reported the beneficial effect on the essential oil content provided by the storage of patchouli leaves for 28 days. The authors verified an increase from 1.60 to 2.7% in the essential oil content, depending on the genotype. They also detected 14 chemical compounds in two patchouli genotypes.
Considering β-patchoulene, the highest content (1.46%) reported for genotype POG-015 was obtained around the fourth week of leaves storage at 28°C. Conversely, for genotype POG-021, at the same temperature, the highest content (1.85%) was recorded in the sixteenth week of storage. The analysis of genotype POG-021 at 33°C showed no significant differences throughout the storage period. The same was observed for βelemene and cycloseychellene (Table 2). E-caryophyllene contents ranged from 1.07% in the eighth week to 2.10% in the second week (POG-015, at 28°C). For POG-021, the contents varied between 1.30% (eighth week) and 2.06% (first week) at 33°C. At 28°C, results for both genotypes are represented by cubic equations (Table 3).
In relation to α-guanene, contents were slightly higher when compared with those of E-caryophyllene, ranging from 4.14 to 7.20% (POG-015). For seychellene, the lowest contents were recorded in the eighth week for POG-015, at the two temperatures tested; conversely, the highest contents were registered for POG-021, in the sixteenth week, with no significant difference between the two temperatures (Table 3).    The contents of α-humulene, alloaromadendrene (Table 4), and 9-epi-(E) caryophyllene (Table 6) were below 1.0%. The content of α-patchoulene decreased from the eighth week of storage for POG-015 at both temperatures. POG-021 had no significant difference for storage time at 28 and 33 °C (Table 4). α-bulnesene contents ranged from 6.5 to 10% (POG-015) and from 5.7 to 9.6% (POG-021) ( Table 5). The contents of α-bulnesene in POG-015 increased until the fourth week of storage at 28°C.
The highest patchoulol content in POG-015 was reported in the eighth week of storage (68.77%) at 28 °C. Results are represented by cubic equations ARRIGONI-BLANK, M. F. et al. Biosci for the two genotypes (Table 6). Sant'ana et al. (2010) also observed that patchoulol concentrations of the essential oil of stored leaves were significantly higher than those of non-stored leaves.
The compounds found in this study agree with what is commonly observed for patchouli essential oil. According to a review published exclusively with this aromatic species, the three compounds observed at the highest concentrations for most published studies are patchoulol, αbulnesene, and α-guaiene (BEEK; JOULAIN 2018).
The storage time and the storage temperature influence the patchouli essential oil content.
The highest essential oil content of genotype POG-021 leaves was detected between the second and eighth weeks of storage at 28 °C.