Pharmacognosy Chemical composition and antinociceptive activity of volatile fractions of the aerial parts of Solidago chilensis (Compositae)

Thirty-six compounds were identified from aerial parts of Solidago chilensis cultivated at PAF/FIOCRUZ campus in Rio de Janeiro city (RJ) using solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) analysis. These compounds are mainly non-oxygenated mono and sesquiterpenes. Germacrene D is the major compound of two the essential oil analyzed samples (12 to 23%) and of two of the volatiles samples analyzed by SPME (central stems and dry inflorescences samples). Limonene is the major compound in the fresh inflorescences sample (about 21%). The bornil acetate is present in both volatile fractions (16%) and essential oils (7–8%). All the essential oils samples evaluated shown a great antinociceptive activity, considering the used dose of the samples (1 mg/kg) and the standard substance (50 mg/kg). Solidago chilensis is one of Brazil arnicas and it is a medicinal plant widely used by the brazilian population. Other plants of the genus Solidago are also used in traditional medicine in North America and Europe. This is the first report of SPME analysis from Solidago genus and of the antinociceptive effect from Solidago chilensis essential oil.


Pharmacognosy Chemical composition and antinociceptive activity of volatile fractions of the aerial parts of Solidago chilensis (Compositae) Introduction
The Solidago genus is the biggest of the Compositae family, and includes about 120 species, most of them occurring in North America. Many members of the Solidago genus and of all Compositae family are important as cut flowers and ornamental crops, as well as being medicinal and aromatic plants, many of which produce essential oils used in folk and modern medicine and in the cosmetics, pharmaceutical industries and as beverages (Abad et al. 2013). Species of Solidago genus have been reported to possess antibacterial, anti-inflammatory, spasmolytic, and carminative properties (Sung et al. 1999). Solidago chilensis Meyen (also called as S. microglossa, S. odora (Oliveira et al. 1998) is a natural species in Chile, with distribution in South America, including the Northeast, Midwest, Southeast and Southern Brazil. Its use increases based on folk tradition in which its inflorescences and roots are used as anticephalgic in the treatment of injuries, such as anti-inflammatory since the the end of the 19 th century, after the arrival of the European immigrants, principally the Italians, although its medicinal properties were known since the 13 th century. These new settlers called the Solidago plants they encountered in Brazil "arnica" due to the similar aromas and medicinal properties to that of A. montana L. from Europe (Vieira 1999;Miguel 2007). So, this species has been used by population in place to the hepatotoxic exogenous species Arnica montana L. (Digest 2001;Mercandeli et al. 2012). Despite of the great use by population, mainly in South America, little is known about the species called "Brazilian arnicas" because very few studies have examined their effectiveness in human health (Silva et al. 2010).
The hydroalcoholic extract of Solidago chilensis Meyen has been evaluated and suggested that in oral therapeutic doses (till 100 mg/kg) there is no health risk in the acute toxicity test (Paula-Freire et al. 2006). Flavonoids, labdane and clerodane diterpenes, saponins, carotenoids, tannins, essential oil and other substances are chemically and biologically described for the Solidago species (Torres 1985;Valverde et al. 2013).
Volatile oils typically contain a complex mixture of low boiling components which are composed predominantly of terpenoid compounds derived by the mevalonate pathway or composed predominantly of aromatic substances derived by the shikimate pathway. These oils are widely used in the whole world in aromatherapy, in perfumery, to produce cosmetic (hygiene and health care formulations as toothpastes, mouthwashes) as natural flavouring, as spice blends and as medicines due to their therapeutic properties (generally they have a broad spectrum of bioactivity) due to the presence of several active compounds or they are added on it for its flavouring purposes. Those oils are still used in various human activities including religious ceremonies, adornments, sensory perception and others personal uses (Sangwan et al. 2001;Schnaubelt 2005).

Material and Methods
This is a no human subject research, all experimental protocols used in this work were approved and performed in accordance with the recommendations of Commission of Ethics for Use of Animals (CEUA/FIOCRUZ) under numbers 002/08 and 033/09.

Post-harvest processing
Medicinal plants in post-harvest processing should be dried between 30 and 60 o C, and medicinal plants containing essential oils should be dried at temperatures below 40 o C (Azar et al. 2012). Aerial parts of S. chilensis were dried in electric oven inflated at 37 o C with air velocity of 0.45 m/s and relative humidity not exceeding 30%, for 48 hours. The mass of S. chilensis for essential oil extraction was approximately 310 g of fresh plant or the equivalent when dry. The moisture content in flowers usually varies between 15 and 80%. Were found 40% of moisture content in inflorescences from S. chilensis.

Headspace solid phase micro-extraction
An SPME (Solid Phase Micro-Extration) holder (Supelco, Bellefonte, PA) was used in performing the experiments. A fused silica fiber coated with a 50/30 µm layer of polydimethylsiloxane (PDMS) was chosen to extract the volatile components from the S. chilensis. The fiber was conditioned following the manufacturer's instructions previous to its use. Approximately 1 g of sample was placed in a 4 mL vial closed with a plastic film. Once the desired temperature had been reached in a water bath (60 o C), the vial was placed inside the bath and was allowed to condition for the equilibrium time (no fiber exposition). After the equilibrium time, the fiber was introduced into the vial and exposed to the headspace of the sample during 20 minutes.

Extraction of essential oil
The aerial parts from Solidago chilensis Meyen were separated to steam distillation of the essential oil and they are cut into small pieces. The essential oil extraction was carried out by steam distillation, using the modified apparatus Clevenger adapted to a round bottom flask with 2 liters of capacity. Triplicate samples of portions of 130 g of plant material, were extracted separately by the same technique. The extraction process was performed during 3 hours, maintaining the boiling water. After that, the essential oil has been separated from hydrolate obtained following centrifugation for 10 min, 4,000 rpm, with the aid of Pasteur pipette. The organic phase was filtered through anhydrous sodium sulfate. The samples were stored at -20 o C until analysis.

GC-MS
The chromatographic separation was performed on a column of 30 m × 0.32 mm and 0.2 mm thick, with 5 % diphenyl 95 % dimethylpolysiloxane as stationary phase. The chromatographic conditions were: linear heating from 30 to 240 o C with a rate of 10 o C/min; gun "split-splitless" rate "split" of 1/100, and flow rate of 1 mL/min. SCAN mode was used for m/z 15 to 300, helium as mobile phase, manual injection and flow rate of 1.00 mL/min. The heating was linear from 40 o C to 300 o C with a rate of 10 o C/min.

Analysis and identification of essential oil by GC/MS
Shimadzu GC/MS QP-2010 (Kyoto, Japan) spectrometer was used in the electron impact (EI) mode for the quantitative analysis. The ionization voltage and temperature of injector and ion source were 70eV, 250 and 280 o C, respectively. The mass spectrometer scanned from 30 to 400 m/z. A DB5 capillary column (30 m × 0.25 mm; 0.25 µm film thickness) was used for the separation. The oven temperature was programmed at 60 o C (isothermal for 2 min) which was ramped to 250 o C at 4 o C/min. Helium was used as the carrier gas with a flow rate of 1.0 mL/min with an injector volume of 1 µL using a 1:20 split ratio.
Compounds of interest were identified by comparing their retention times with that of authentic standards injected separately under the same GC-MS conditions and according with literature values (Fig.  1). Peaks compounds were identified by comparing its mass fragmentation pattern with that of standard compounds as well with data available spectral library (Wiley NIST Libraries) of the instrument. All analysis were performed in triplicate and quantification of each constituent obtained by standardization of the areas (%).

Pharmacological assays Animals
Male Swiss mice and Wistar rats weighing 23-30 and 130-200 g respectively were used. The animals were obtained from the animal-breeding colony at the Oswaldo Cruz Institut Foundation (FIOCRUZ) and maintained in the animal room of the Department of Physiology and Pharmacodynamics, in Oswaldo Cruz Institut (IOC/FIOCRUZ), with free access to pelleted diet and water and a controlled period of light/darkness of 12/12 h. The animals were allowed to adapt to the laboratory for at least 2 hours before testing and were used only once. The experiments in this study received prior approval from the Oswaldo Cruz Institut Foundation's Animal Welfare Committee. The license for use of the animals was granted by Center for the Study of Animal Use (CEUA/FIOCRUZ) under numbers 002/08 and 033/09. After the experimental procedures animals were euthanized in CO 2 chamber.

Antinociceptive effect
The EOCS, EODI and EOFI samples were evaluated for their analgesic activity through Collier's test acetic acid induced writhes in mice to evaluate their antinociceptive effect (Sousa 2011). The amount of contortions was evaluated for 10 minutes, beginning the registers after 5 minutes of the injection of the stimulus. The doses of EOCS, EODI and EOFI (1 mg/Kg), and the control analgesic, diclofenac (50 mg/Kg), were administrated orally (p.o.), always an hour after the induction of pain. The control group was treated with saline solution + DMSO (diluent).

Statistical analysis
Statistical significance was determined by a one-way analysis of variance, followed by Student´s-

Results and Discussion
In order to identify the largest possible number of volatile compounds of the S. chilensis by HS-SPME (Headspace Solid Phase Microextraction) the PDMS (Polydimethylsiloxane) fiber was chosen. The PDMS fiber is more suitable for the analysis of low molecular weight polar and non polar compounds. Table 1 lists the volatiles, obtained by SPME, of the analyzed samples CSV (Central Stem Volatiles), DIV (Dry Inflorescences Volatiles) and FIV (Fresh Inflorescences Volatiles) of S. chilensis.
Germacrene D is the major constituent of the CSV and DIV volatiles samples, and it represents 12% of the CSV and 23%, of the DIV. In spite of FIV sample Germacrene D has been found in large amounts (20.8%), limonene is the compound with the highest concentration of 20.95%. This hydrocarbon is a widely distributed metabolic compound in S. chilensis plant species and it is considered to be a key intermediate in the biosynthesis of many sesquiterpenes from farnesyl diphosphate (Bülow et al. 2000;Steliopoulos et al. 2002;Shah et al. 2012). According to Vila and Gressler (Vila et al. 2002;Gressler et al. 2003), the pumiloxide, a labdane diterpene, was found as the major compound in the essential oil from leaves and inflorescences of Solidago chilensis Meyen collected in Pérez (Rosario, Argentina). In the S. chilensis cultivated at Phytomedicine Agroecological Platform (PAF), FIOCRUZ/RJ, there was no pumiloxide found. The diterpene compounds found were phytol and solidagenone (also a labdane diterpene) (Tab. 2) . The difference in concentration of germacrene D can be explained   (Collier et al. 1968;Him et al. 2008). The comparison of the major compounds in the analyzed samples from S. chilensis is showed in Table 3. The antinociceptive effect was observed in the three oils samples, the EOCS have inhibited about 35% of number of writhings (29.42 + 4.6), the EOFI have inhibited about 53% of number of writhings (21.25 ± 4.2), EODI have inhibited about 49% of number of writhings (23.08 ± 1.4) and the standard substance diclofenac have inhibited about 67% of number of writhings (14.83 ± 3.1). A difference in the antinociceptive activity of about 6% between dry and fresh inflorescences was observed (Tab. 4). Chemically, there was the loss of the most volatile substances in the dry sample due to their evaporation of the some monoterpenes as camphene, β-mircene, α-phellandrene that are present only in the EOFI and limonene, (Z)-β-ocimene, β-cubebene, cedren-13-ol and α-cadinol which are present in only one half of the EODI sample concentration.

Conclusions
Germacrene D is also previously reported as the main compound of many species of the Solidago genus. The limonene, that is widely used as pharmaceutical ingredient in industry to produce bath products as soap and room sprays due to their pleasant smell, comprises about 10% of the essential oil of fresh inflorescences. The antinociceptive activity of many terpenes present in essential oils, combined with the chemical complexity of these  1 EOCS = central stem S. chilensis essential oil; EODI = inflorescences of S. chilensis essential oil; EOFI = fresh inflorescences of S. chilensis essential oil; n = 12. 2 Values represent mean ± S.E.M.; **P < 0.01; ***P < 0.001. oils, shows the synergistic potential that the Solidago chilensis essential oil could exerts, acting through different mechanisms of action, in addition to their potential in developing new topical analgesics, even as adjuvants in pharmaceutical formulations. This is the fisrt report about the SPME analysis and about the pharmacological activity of the Solidago chilensis Meyen essential oil.