Chemical composition, antimicrobial, and lipase enzyme activity of essential oil and solvent extracts from Serapias orientalis subsp. orientalis

The volatile components of essential oil (EO), SPME, and SPME of solvent extracts ( n -hexane, methanol, and water) obtained from fresh Serapias orientalis subsp. orientalis ( Soo ) were analyzed by GC-FID/MS. EO of Soo gave 11 compounds in the percentage of 99.97%; capronaldehyde (37.01%), 2-( E )-hexenal (23.19%), and n -nonanal (19.05%) were found to be major constituents. SPME GC-FID/MS analyses of fresh plant and solvent extracts of Soo revealed 7, 12, 7, and 4 compounds within the range of 99.7% to 99.9%. Limonene (76.5%, 41.7%, and 61.3%) was the major compound in SPMEs of the n -hexane and methanol extracts. α -Methoxy- p -cresol (52.9%) was the main component in its water extract. The antimicrobial activity of EO and the solvent extracts of Soo were screened against 9microorganisms. EO showed the best activity against Mycobacterium smegmatis , with 79.5 µg/mL MIC value. The n -hexane, methanol, and water extracts were the most active against the Staphylococcus aureus within the range of 81.25–125.0 µg/mL (MIC). IC 50 values for the lipase enzyme inhibitory activity of EO and solvent extracts ( n -hexane, methanol, and water) were determined to be 59.87 µg/mL, 64.03 µg/mL, 101.91 µg/mL, and 121.24 µg/mL, respectively.

g/day [17]. It is known that supplementary food products containing glucomannan can help control weight and fight the urge to overeat. Thus, Serapias orchids could be used as a healthy food supplement.
In the literature, there has been limited work related to the volatile constituents (EO, SPME, and n-hexane extract) of some Serapias species (Serapias vomeracea (Burm. F.) B.; Serapias vomeracea subsp. longipetala (Ten.) H. Baumann & Künkele; Serapias cordigeraL. subsp. cordigera; Serapias cordigera subsp. lucana R. Lorenz & V.A. Romano; Serapias parviflora Parl.; Serapias lingua L.; Serapias lingua L.) [18][19][20]. The essential oil of S. vomeracea has been shown to have dose-dependent antioxidant effects through DPPH assay [18]. Some orchid species are known to exhibit antimicrobial and antioxidant biological activity [18,21]. However, to the best of our knowledge from the literature survey, no data about chemical compositions and biological activities of S.orientalis subsp. orientalis (Greuter) H.Baumann & Künkele have been reported to date. The subject of this study is to investigate the effect of different extraction methods. Therefore, in this paper, we report various volatile constituents of EO, SPME, and SPMEs of n-hexane, methanol, and water extracts analyzed with GC-FID/MS.Antimicrobial activities of the extracts (n-hexane, methanol, and water) and EO against 9 microorganisms are evaluated. In addition, porcine pancreatic lipase (PPL) enzyme activity of EO and solvent extracts was investigated. The results of antimicrobial and porcine pancreatic lipase activities of EO and the solvent extracts (n-hexane, methanol, and water) may be useful in foods, pharmaceuticals, and other industries.

Hydrodistillation (HD) procedure for the isolation of EO
The fresh aerial part of Soo (115 g) was ground into small pieces and subjected to hydrodistillation (HD) using a modified Clevenger-type apparatus with a cooling bath (-15 °C) system (3h) [yield (w/w): 0.09%]. The obtained oil was extracted with n-hexane (0.5 mL), dried over anhydrous Na 2 SO 4 , and kept in sterilized dark glass bottles in the refrigerator at 4 °C prior to analysis.

Solvent extractions
The fresh plant (120 g) was ground into small pieces using a plant mill. Blended material was weighed (40 g each sample) into 3 different flasks (100 mL) and extracted 3 times with analytical-grade n-hexane, methanol, and water solvents (25 mL × 3; 12 h each). After the suction filtration, the same extracts were combined and evaporated at 40 °C to produce crude n-hexane (0.0445 g) and methanol (0.7356 g) extracts. The water extract was lyophilized to produce crude water extract (0.2470 g).

Gas chromatography-mass spectrometry (GC-FID/MS)
EO analysis was carried out using a Shimadzu QP2010 ultra GC-FID/MS, Shimadzu 2010 plus FID fitted with a PAL AOC-5000 plus auto sampler, and Shimadzu Class-5000 Chromatography Workstation software (Shimadzu Corp., Kyoto, Japan). The separation was analyzed by means of a Restek Rxi-5MS capillary column (30 mm × 0.25 mm × 0.25 μm) (Restek Corp., Bellefonte, PA, USA). The essential oil injections to GC-FID/MS wereperformed in split mode (1:30) at 230 °C. The essential oil solution (1 μL) in n-hexane (HPLC grade) was injected and analyzed with the column held initially at 60 °C for 2 min and then increased to 240 °C with a 3 °C/min heating ramp. The oven program was as follows: the initial temperature was 60 °C for 2 min, which was increased to 240 °C for3 min; the final temperature of 250 °C was held for 4 min. Helium (99.999%) was used as the carrier gas, with a constant flowrate of 1 mL/min. Detection was implemented in electronic impact mode (EI); ionization voltage was fixed at 70 eV and scan mode (40-450 m/z) was used for mass acquisition. Each sample was analyzed and the mean of each reported.

Solid phase microextraction (SPME)
The blended fresh plant (2 g), n-hexane (0.0241 g), methanol (0.3802), and water extracts (0.1450) of Soo were placed into a sealed SPME vial (10 mL) with a silicone-rubber septum cap, then submitted to a solid-phase microextraction device (Supelco Inc., Bellefonte, PA, USA). A DVB/Carboxen/PDMS coating fiber was used to obtain volatile components. The SPME fibers were conditioned for 5 min at 250 °C in the GC injector. Extraction was achieved with magnetic stirring at 80 °C using an incubation time of 5 min and an extraction time of 10 min. Fibers with extracts of volatile compounds were subsequently injected into the GC injector. GC-FID/MS analysis was performed using a Shimadzu QP2010 Ultra mass selective detector attached to the 2010 Plus chromatograph. The carrier gas used was helium, at a flow rate of 1 mL/ min. The injection was performed in split mode (1:30) at 230 °C. Samples were analyzed and the results recorded. The temperature, incubation, and extraction times were set according to the reported experiment [22].

Antimicrobial activity assessment (agar-well diffusion method)
All test microorganisms were obtained from the Hıfzıssıhha Institute of Refik Saydam (Ankara, Turkey) and were as follows: Escherichia coli ATCC35218, Yersinia pseudotuberculosis ATCC911, Pseudomonas aeruginosa ATCC43288, Enterococcus faecalis ATCC29212, Staphylococcus aureusATCC25923, Bacillus cereus 709 Roma, Mycobacterium smegmatis ATCC607, Candida albicans ATCC60193, and Saccharomyces cerevisiae RSKK 251. The antimicrobial screening test using the agarwell diffusion method as adapted was used earlier [37,38]. Each microorganism was suspended in brain heart infusion (BHI) broth (Difco Laboratories Inc., Detroit, MI, USA) and diluted to approximately 106 colony forming units (cfu) per mL. They were flood-inoculated onto the surface of BHI agar and Sabouraud dextrose agar (SDA) (Difco Laboratories Inc.), and then dried. For C. albicans, SDA was used. Five-millimeter-diameter wells were cut from the agar using a sterile cork-borer, and 50μL of the extract substances was delivered into the wells. The plates were incubated for 18 h at 35 °C. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organism. Extract stock solutions were prepared at different concentrations (10,000-155,400 μg/mL) according to the amount of material obtained. A 1/10 dilution of each solvent was used as a control.

Minimal inhibition concentration (MIC) assay
The antimicrobial properties of EO, n-hexane, methanol, and water extracts of Soo were investigated quantitatively in respective broth media by using double microdilution; the minimal inhibition concentration (MIC) values (μg/mL) were examined [37,38] and used in our previous work [25,26]. The antibacterial and antifungal assays were carried out in Mueller-Hinton broth (MH) (Difco Laboratories Inc.) at pH. 7.3 and buffered yeast nitrogen base (Difco Laboratories Inc.) at pH 7.0, respectively. The microdilution test plates were incubated for 18 h at 35 °C. Brain heart infusion broth (BHI) (Difco Laboratories Inc.) was used for M. smegmatis, and incubated for 48-72 h at 35 °C. The MIC was defined as the lowest concentration that showed no growth. Ampicillin (10 mg/mL), streptomycin (10 mg/mL), and fluconazole (2 mg/mL) were used as standard antibacterial and antifungal drugs, respectively. The 1/10 dilution of each solvent was used as a control. 2.10. Lipase inhibitory effect assay EO, n-hexane, methanol, and water extracts of the Soo species studied were evaluated for lipase (PPL) inhibitory assay by the modified method, using p-nitrophenyl butyrate (p-NPB) as the substrate [39][40][41]. First, all fractions were dissolved with buffer solution (0.1 M Tris-HCl buffer, pH = 8.0) in 25, 50, 100, and 200 µg/mL concentrations. Orlistat, used as a positive control, was prepared in 6.25, 12.5, 25, 50, and 100 µg/mL concentrations. The test procedure was designed as I, II, III, and IV wells, described as follows:

Analysis of bioactive compounds
The VOCs in EO, SPME, and SPME of extracts (n-hexane, methanol, and water) of fresh Soo were investigated by GC-FID/ MS analysis. The chemical names of the identified volatile constituents of Soo, together with their retention indices and percentages, are given in Table 1, where the compounds have been listed in the order of elution on the Restek Rxi-5MS column used [42]. Volatile compounds were identified by comparison of the registered mass spectrum libraries (NIST, Wiley7NL, FFNSC1.2, and W9N11), and by using the Kovats index [23][24][25][26][27][28][29][30][31][32][33][34][35][36]. GC-FID/MS analysis for the EO of Soo gave 11 natural compounds totaling 99.97%. EO of Soo was characterized by a high percentage of aldehydes (88.6%), with capronaldehyde (37.01%), 2(E)-hexenal (23.19%) pelargonaldehyde (19.05%), and decane (9.03%) as the major constituents. The terpene-type compound was not detected in the essential oil ofthe Soo. The composition of EO of Soo differed from that of EO of the other Serapias species. The main represented volatile compounds were reported to be saturated hydrocarbons (53.29%) in EO of S. vomeracea and pentacosane (17.59%); n-tricosane (14.21%), n-heneicosane (5.68%), and n-heptacosane (4.99%) were the most abundant compounds from the Italian orchid [18]. SPME GC-FID/MS analysis for the fresh plant and solvent extract (n-hexane, methanol, and water) of Soo revealed 7, 12, 7, and 4 natural compounds totaling 99.7%-99.9%. Limonene (76.6%, 41.8%, and 61.6%) was found to be the major compound in the SPME of the fresh plant and the SPMEs of the n-hexane and methanol extracts of Soo, respectively. SPME of the water extract revealed α-methoxy-p-cresol (52.9%) as the main component. Limonene is widely used as a flavor and fragrance additive in perfumes, beverages, and cleaning products [43]. Considering its medicinal potential and cosmetic uses, it is important that Soo might be a good source for limonene. Monoterpenes (76.6% and 61.3%) werethe major constituent in the SPME of the fresh plant and the SPME of methanol extract. Aldehydes and aromatic compounds accounting for 57.4% and 52.9% of the SPMEs of n-hexane and water extracts respectively were the most abundant constituents (Table 1). Major constituents were found to be limonene (76.5%, 41.7%, 61.3%), pelargonaldehyde (7.0%, 21.5%, and 12.3%), undecanal (3.2%, 15.1%, and 4.2%), and heptanal (10.4% and 7.9%) in the SPMEs of n-hexane and methanol extracts, respectively. α-methoxy-p-cresol (52.9%) and limonene (45.3%) were the most abundant constituents of the SPME of the water extract.

Antimicrobial and lipase enzyme activities
Antimicrobial activity of EO and solvent extracts (n-hexane, methanol, and water) obtained from Soo were screened by using the agar well diffusion method with the microorganisms seen in Table 2. In general, EO and n-hexane extract showed moderate antimicrobial activities with inhibition zones of 8/12 mm, 9/12 mm, and 20/8 mm against S. aureus, B. cereus, and M. smegmatis, respectively. Water extract gave better activity against E. faecalis, S. aureus, and B. cereus with 15-, 20-, and 10-mm inhibition zones, respectively. Methanol extract showed moderate antimicrobial activity against all tested microorganisms except for C. albicans and S. cerevisiae. The results indicate that antimicrobial activity of EO, n-hexane, and water extracts are more susceptible to gram-positive bacteria. The MIC of the methanol extract of S. orientalis subsp. orientalis was within the range of 121.4-3885.0 µg/mL against E. coli, Y. pseudotuberculosis, P.aeruginosa, E. faecalis, S. aureus, B. cereus, and M. smegmatis. EO and solvent extracts which contain limonene, α-methoxy-p-cresol, and aldehydes as major components of the samples have been reported to have antibacterial properties [43][44][45][46]. Therefore, the bactericidal activity of the EO and solvent extracts obtained from Soo may be mainly related to these compounds.
Other compounds which were also present in the samples, including capronaldehyde, 2(E)-heptenal, octanal, heptanoic acid, pelargonaldehyde, caprylic acid, decanal, methyl nonanoate, undecanal, dodecanal, tridecanal, hexadecanal, palmitic acid, octadecanal, and ethyl oleate, have been reported to have antibacterial properties, and may also collectively have a remarkable contribution to the bactericidal activities of the EO and solvent extracts. Recently, a series of aliphatic aldehydes from olive has been shown to exhibit noticeable activity against several microfungal and bacterial strains [45,46].The antibacterial activity investigation showed variations which may be due to factors such as composition and concentration of the EO and solvent extracts. However, the investigated methanol extract of this plant with its major compound of α-methoxy-p-cresol (52.9%) and limonene (45.9%) showed the highest antibacterial activity against S. aureus, with 121.4 µg/mL MIC value [38].The greatest lipase enzyme inhibitions [39][40][41] were obtained for EO and n-hexane extract of Soo with IC 50 values of 59.87 µg/mL and 64.03 µg/mL, respectively (Figure). Literature RI; b RI calculated from retention times relative to that of n-alkane (C 6 -C 32 ) series; c Percentage peak area obtained by FID peak-area normalization; EO: Essential oil; Hydrodistillation by Clevenger method of S. orientalis subsp. orientalis ; SPME: SPME of S. orientalis subsp. orientalis; A1: SPME of hexane extract; A2: SPME of methanol extract; A3: SPME of water extract.

Conclusion
VOC composition of Soo has been analyzed, and their antimicrobial and lipase enzyme activities investigated for the first time. Limonene was the major compound of allextracts (SPME, SPMEs of EO, n-hexane, and methanol extracts) obtained from fresh Soo within the range of 41.7% to 76.5%, respectively. However, α-methoxy-p-cresol (52.9%) was found to be a major component only in the SPME of the methanol extract of the plant. This clearly showed that various extraction methods used in this work resulted in the identification of different components as in the literature. n-Hexanal was the major compound obtained in EO of fresh Soo. Aldehydes were found to be the major class of components (88.6%) in the EO. The amount of limonene and α-methoxy-p-cresol is so high that Soo could be a source for the production of these compounds. In general, the greatest activity of EO was against M. smegmatis with 79.5 µg/mL MIC value. According to the agar diffusion method, the most antimicrobial activity was observed against Gram-positive and Gram-negative bacteria in methanol extract, which had better antimicrobial activity againstP. Aeruginosa and S. aureus with 19-mm and  21-mm inhibition zones, respectively. The MIC values were found to be 489.6 µg/mL and 121.4 µg/mL, respectively. The EO of the Soo was found to be most effective against the lipase enzyme (59.87 µg/mL, IC 50 ). Therefore, the overall results of antimicrobial and lipase enzyme activities suggest that EO and solvent extracts of Soo may be promising prospects for pharmaceutical, food, and other industrial applications. In further study, activity guided isolation and purification could be carried out on Soo.