Antimicrobial and lipase inhibition of essential oil and solvent extracts of Cota tinctoria var. tinctoria and characterization of the essential oil

The essential oil (EO) of Cota tinctoria var. tinctoria was analyzed using GC-FID / MS. A total of 51 compounds were determined from this taxon, accounting for 99.79% in hydrodistillation. Monoterpenes were the primary chemical class for the volatile organic compounds in the EO (36.1%, 13 compounds). Borneol (18.1%), camphor (14.9%), and β-pinene (11.3%) were the major components in the EO of C. tinctoria var. tinctoria. The antimicrobial activities of EO and n-hexane, acetonitrile, methanol, and water solvent extracts of the taxon were screened in vitro against ten microorganisms. The EO yielded the best activity (15 mm, 372.5 MIC, 59600 μg/μL) against Mycobacterium smegmatis. The acetonitrile extract was the most active against the Staphylococcus aureus and Bacillus cereus with 274 μg/mL MIC value. IC50 values for the lipase enzyme inhibitory activity of EO and solvent extracts (n-hexane, acetonitrile, methanol, and water) were found to be 59.80 ± 4.3285 μg/mL 68.28 ± 3.1215 μg/mL, 52.60 ± 3.7526 μg/mL, 48.73 ± 2.8265 μg/mL, and 99.50 ± 5.5678 μg/mL, respectively.

Obesity is a long-term problem which causes a series of psychological and physiological problems. Many kinds of chronic metabolic diseases are caused by obesity. Porcine pancreatic lipase (PPL) inhibitors have been of interest in obesity treatment research in recent years. PPL inhibitors from natural products have a wide range of sources and low toxicity and have therefore attracted interest. Because of these advantages, they could lead to new health products in the pharmaceutical industry [35].
To the best of our knowledge, total phenolic and flavonoid contents, antioxidant activity, butyrylcholinesterase, acetylcholinesterase, and tyrosinase enzyme inhibition of methanol extract of C. tinctoria var. tinctoria were mentioned [36]. Cytotoxic activity of ethanol extract of C. tinctoria flowers was investigated [37]. In another study, phenolic constituents, cytotoxic activity, and dyeing properties for the ethanol and aqueous extracts of stem, flower, and root of the plant were studied [38]. However, antimicrobial activity and lipase enzyme inhibition for the essential oil and solvent extracts (n-hexane, acetonitrile, methanol, and water) of C. tinctoria var. tinctoria were not studied. Hence, we focused our study on the essential oil composition with GC and GC-MS analysis, antimicrobial and lipase enzyme inhibition activities of the EO and the solvent extracts of C. tinctoria var. tinctoria were screened in vitro against ten microorganisms and porcine pancreatic lipase, respectively.

Plant materials
Aerial part of C. tinctoria var. tinctoria (160 g, wet) was harvested from Şiran, Gümüşhane at a height of 1650 m in May 2018. The plant was collected by Prof. Nurettin Yaylı and authenticated by Prof. Salih Terzioğlu. Voucher specimen (KATO: 19258) has been deposited in the Herbarium of the Faculty of Forestry, Karadeniz Technical University, Turkey.

Chemicals and reagents
All solvents (methanol, acetonitrile, and n-hexane) and chemicals (Tris-HCl and p-nitrophenyl butyrate) used were purchased from Sigma-Aldrich in analytical grade.

Hydrodistillation procedure for the obtaining of EO
The aerial part of C. tinctoria var. tinctoria (106 g, dry) was grounded with plant mill into small pieces and then hydrodistillated (HD) with a modified Clevenger-type apparatus with cooling bath (-15 °C, 3 h), yield (v/w): 0.079%. After the HD, EO was extracted with n-hexane (0.5 mL) and dried over anhydrous Na 2 SO 4 and stored in a dark glass bottle in the refrigerator at 4 °C prior to the GC-MS analysis. 2.4. Solvent extractions (n-hexane, acetonitrile, methanol, and water solvents) of C. tinctoria var. tinctoria The aerial parts of the plant (40 g, dry) were blended into small pieces. Blended material (5 g, each) was extracted (25 mL × 3; 12 h each) using maceration method at room temperature with analytical grade n-hexane, acetonitrile, methanol, and water solvents in flasks (50 mL) separately. After the suction filtration, the same extracts were combined and solvents were evaporated or lyophilized to yield crude n-hexane (0.0722 g), acetonitrile (0.0439), methanol (0.5234 g), and water extract (0.0435 g) [39,40].

Gas chromatography-mass spectrometry (GC-FID/MS)
GC-MS analysis of the EO was carried out by a Shimadzu QP2010 ultra, having Shimadzu 2010 plus FID, PAL AOC-5000 plus auto sampler and Shimadzu Class-5000 Chromatography Workstation software. Restek Rxi-5MS capillary column (30 mm × 0.25 mm × 0.25 μm) (USA) was used for the analysis. Sample (1 μL, in HPLC grade n-hexane) injection was performed in split mode (1:30) at 230 °C. Initial column temperature was 60 °C for 2 min, then increased to 240 °C with a 3 °C/min heating ramp. The final temperature for the oven was held at 250 °C for 4 min. Helium (99.999%) was the carrier gas with 1 mL/min flow rate. MS detection was implemented in electronic impact mode (EI, 70 eV, and scan mode 40-450 m/z). Sample was analyzed and mean was reported.

Identification of volatile constituents
RI values of the volatile components in EO of C. tinctoria var. tinctoria were determined by Kovats method (Table 1) [39][40][41][42][43][44]. Volatile compounds of the EO were identified by comparisons of RI values with those reported in the literature RI  and MS data matching mass spectral libraries (NIST, Wiley7NL, FFNSC1.2, and W9N11).

Antimicrobial activity assessment (agar-well diffusion method)
All test microorganisms: Escherichia coli ATCC35218, Yersinia pseudotuberculosis ATCC911, Pseudomonas aeruginosa ATCC43288, Enterococcus faecalis ATCC29212, Staphylococcus aureus ATCC25923, Bacillus cereus 709 Roma, Mycobacterium smegmatis ATCC607, Candida albicans ATCC60193, Candida tropicalis ATCC 13803; and Saccharomyces cerevisiae RSKK 251 were obtained from the Refik Saydam Institute of Hygiene and Public Health (Ankara, Turkey). The adapted antimicrobial screening test (agar-well diffusion method) was used earlier [76][77]. Each tested microorganism was suspended in Brain Heart Infusion (BHI) and diluted approximately 10 6 colony-forming units (per mL), which were "flood-inoculated" onto the surface of BHI agar and Sabouraud Dextrose Agar (SDA), then dried. SDA was used for C. albicans. Wells (5 mm diameter) were cut from the agar, and the extracts (100 μL, each) were delivered into the wells. The plates were incubated (35 °C, 18 h) and antimicrobial activity was evaluated by measuring the inhibition zone against the test organism. The EO dissolved in n-hexane, and other solvent extracts (acetonitrile, methanol, and water solvents) were dissolved in dimethyl sulphoxide to prepare stock solutions (43.500-523.400 μg/mL). n-Hexane and dimethyl sulphoxide were used as solvent control with dilution of 1:2. Ampicillin, streptomycin, and fluconazole were used as positive controls at 10 µg/mL, 10 µg/mL, and 5 µg/mL concentrations, respectively ( Table 2).

EO composition of C. tinctoria var. tinctoria
Volatile components in the EO of the C. tinctoria var. tinctoria were analyzed by GC-FID/MS using Rxi-5MS capillary column. Identification of the volatile constituents in EO was made by comparison of RI and MS data with literature . The chemical profile of volatiles, the percentage content, and calculated retention indices of the constituents of the taxon are presented in Table 1. Borneol (18.11%), camphor (14.90%), β-pinene (11.26%), camphene (10.69%), eucalyptol (6.67%), valencene (6.37%), and α-pinene (6.35%) were the major compounds in the EO of C. tinctoria var. tinctoria (Table  1). Monoterpenes (36.11%) and oxygenated monoterpenes (23.22%) were the main constituents of the EO obtained from aerial parts of C. tinctoria var. tinctoria., and sesquiterpenes (10.75%) and oxygenated sesquiterpenes (4.62 %) were the second major components in the EO of C. tinctoria var. tinctoria.
A literature review has revealed that phytochemical analysis of C. tinctoria var. tinctoria had shown lipophilic extract, and GC-MS analysis was mentioned that content was rich in saturated fatty acids [83]. Isolation of 3-glucoside, and 3rutinoside of patuletin from acetone extract of the leaves of A. tinctoria var. subtinctoria was reported [84].
In a study, the total flavonoid and phenolic contents, antimicrobial, the antioxidant, antibiofilm activities, and anticholinesterase of A. stiparum subsp. sabulicola aerial parts methanolic extract and essential oil were reported and 72 constituents (99.02%) were exhibited, and major compounds were determined as germacrene D, t-cadinol, camphor, spathulenol, and isoamyl salicylate with percentage of 11.13%, 11.01%, 6.73%, 6.50%, 6.45%, respectively [82]. The volatile constituents of the EOs obtained from the aerial parts of A. cretica subsp. messanensis (Brullo) Giardina & Raimondo, from the aerial parts of the rock-grown form and the cultivated of A. arvensis L. subsp. arvensis, and from flowers and leaves of A. cretica subsp. columnae (Ten.) Frezén were reported.
Torreyol (85.4 %) from A. arvensis subsp. arvensis, (E)-chrysanthenyl acetate (28.8%) from A. cretica subsp. messanensis, and 18-cineole (13.3% and 12.2 %) from both flower and leaf oils of A. cretica subsp. columnae were reported as main constituents [34]. The essential oil analysis of A. fungosa and its antioxidant (IC 50 = 3000 ± 8.3 μg/mL, compared with the standard, quercetin, with an IC 50 of 33.3 ± 1.3 μg/mL) and antimicrobial activities against nine microorganisms (six bacteria and three fungi) were found to have strong antimicrobial activity (MIC = 32 μg/mL) against K. bacteria, S. epidermidis, and all of the tested fungi [85]. The hepatoprotective and anti inflammatory activities of the methanolic extract of A. scrobicularis herbs were reported; A. scrobicularis was toxicologically safe when orally taken and possessed very important hepatoprotective and anti inflammatory activities and it had the potential to be used in inflammatory and hepatic diseases [86]. As for the antimicrobial and antioxidant activities of methanol extracts of A. cretica subsp. argaea and A. fumariifolia, they showed 59.10 % and 55.41% inhibition against linoleic acid oxidation, respectively. The study demonstrated that the methanol extracts of A. fumariifolia and A. cretica subsp. argaea had strong antibacterial activity against many tested bacteria (14 mm and 15 mm inhibition zone against B. cereus) [87].
The literature comparison of this study showed that similar compounds were found at different rates. However, more terpene components (74.70%) were characterized in this work. In addition, borneol was detected as major compound in the EO of the C. tinctoria var. tinctoria that may be used as taxonomical marker for the future classification of the C. tinctoria var. tinctoria. The variations in the volatile organic compounds on aerial parts of C. tinctoria var. tinctoria with other species may be due to environmental and analysis conditions. Thus, it could be pointed out that qualitative and quantitative results of this study were quite different from the previous reports.

Antimicrobial activity of EO and solvent extracts
The antimicrobial properties of the EO, n-hexane, acetonitrile, methanol, and aqueous extracts of C. tinctoria var. tinctoria were tested by an in vitro agar-well diffusion method using P. aeruginosa, Y. pseudotuberculosis, E. coli, E. faecalis, B. cereus, S. aureus, M. smegmatis, C. tropicalis, S. cerevisiae, and C. albicans (Table 2). After the inhibition diameters were observed in mm, the MIC values (µg/mL) were calculated [76][77] (Table 2). EO of C. tinctoria var. tinctoria was the most active against M. smegmatis with 15 mm inhibition (MIC 372.5 µg/mL). The acetonitrile extract of C. tinctoria var. tinctoria showed the best zone diameters as 14 mm, 15 mm, and 12 mm against S. aureus, B. cereus, and M. smegmatis with MIC values of 274 µg/mL, 274 µg/mL, and 548 µg/mL, respectively. EO and solvent extracts were more active for the gram-positive bacteria, while EO extract was only active for the gram-negative Y. pseudotuberculosis. These antimicrobial activities indicate the presence of active components in these extracts. None of the extracts were active against E. coli, P. aeruginosa, and E. faecalis. n-Hexane, methanol, and water extracts of C. tinctoria var. tinctoria were not active against tested fungi. No correlation was observed between solvent polarities and antimicrobial activity.
In the previous antimicrobial evaluation of Anthemis species; essential oils of A. pseudocotula A. pectinata var. pectinata and A. dipsacea were reported against eight microorganisms but did not affect the growth of E. faecalis, Enterobacter cloacae, Salmonella thyphimurium, and Staphylococcus epidermidis. When compared with standard antibiotics such as ceftazidime, sulbactam, ampicillin, and nystatin; the essential oils of A. pseudocotula, A. pectinata var. pectinata, and A. dipsacea at a concentration of 20 μL/disc had inhibitory effect on E. coli, P. aeruginosa, and S. aureus [19]. EO of A. cretica subsp. messanensis showed quite a good antimicrobial activity towards E. coli, Streptococcus faecalis, and S. epidermidis with MIC values of 25 μg/mL, 25 μg/mL, and 12.5 μg/mL, respectively [34]. Antimicrobial activities of aerial parts methanolic extract and essential oil of A. stiparum subsp. sabulicola were reported, and methanol extract displayed better antimicrobial activity than EO of A. stiparum subsp. sabulicola, being active against S. aureus and Bacillus subtilis, with MIC of 1.56 mg /mL [82]. Methanol extract of A. fumariifolia and A. cretica subsp. argaea were reported against 13 bacteria and two yeasts. Test results showed that the methanol extract had great potential of antibacterial activity against many bacteria which were tested. The inhibition zones for bacterial strains were found to be in the range of 6-14 mm and 7-15 mm, respectively. Nevertheless, they had no inhibitory effect on S. cerevisiae and C. albicans [87].

Lipase enzyme activity of EO and solvent extracts
The literature has shown that some of the common natural sources with lipase inhibitors contain active ingredients including polyphenols, flavonoids, terpenoids, and other active ingredients [35]. Terpenoids were the major constituents of the EO; thus, we investigated the lipase activity of EO and solvent extracts of C. tinctoria var. tinctoria. Essential oil and solvent extracts of C. tinctoria var. tinctoria were evaluated for lipase enzyme inhibition activities compared with orlistat as positive control (IC 50 : 13.49 ± 1.2262 µg/mL). The highest activity was found in the methanol extract (IC 50 : 48.73 ± 2.8265 µg/mL). Afterwards, the best activities were determined with IC 50 values of 52.60 ± 3.7526 µg/mL, 59.80 ± 4.3285 µg/mL, and 68.28 ± 3.1215 µg/mL in acetonitrile, essential oil, and n-hexane, respectively (Figure). The lowest activity was found in the water extract (IC 50 : 99.5 ± 5.5678). No correlation was observed between solvent polarities and lipase activity. In another study, enzyme inhibitions of ethyl acetate, methanol, and water extracts of A. chia L. flowers were reported. MeOH extract of it showed the highest activity in tyrosinase inhibitory and α-amylase activity with 290.22 mg kojic acid equivalents (KAEs)/g extract and 413.66 mg acarbose equivalents (ACEs)/g extract, respectively [88]. In a study, key enzyme inhibitory potentials for the ethyl acetate, methanol, and aqueous extracts obtained from aerial parts of A. cretica subsp. tenuiloba and A. tinctoria var. pallida were mentioned. Ethyl acetate and methanol extracts showed potent activity against AChE with the highest activity observed for methanol extract (3.28 ± 0.43 mg GALAE/g) of A. tinctoria var. pallida and ethyl acetate extract of A. cretica subsp. tenuiloba (4.68 ± 0.21 mg GALAE/g). In case of BChE inhibitions of extracts; ethyl acetate extract of A. tinctoria var. pallida (3.48 ± 0.21 mg GALAE/g) and ethyl acetate (2.51 ± 0.34 mg GALAE/g), and methanol (1.15 ± 0.05 mg GALAE/g) extract of A. cretica subsp. tenuiloba were found to be more promising. Furthermore, enzyme inhibitory effects against α-glucosidase and tyrosinase were given, as well [89].

Conclusion
The composition of the EO obtained from aerial part of C. tinctoria var. tinctoria characterized and lipase enzyme and antimicrobial activities for the EO and solvent extracts were investigated for the first time. Monoterpenes were the main chemical class in the EO. Borneol (18.1%), camphor (14.9%), and β-pinene (11.3%) were the major components in the EO of C. tinctoria var. tinctoria. The EO showed the best activity against M. smegmatis (372.5 μg/μL MIC value). The acetonitrile extract was the most active against the S. aureus and B. cereus (274 µg/mL MIC value). The best activity for the lipase enzyme inhibitory of EO and solvent extracts (n-hexane, acetonitrile, methanol, and water) was found to be methanol extract with 48.73 µg/mL IC 50 value. Therefore, the overall results of observed lipase enzyme and antimicrobial activities suggest that EO and solvent extracts of C. tinctoria var. tinctoria could be promising for pharmaceutical and other industrial applications. In a further study, Bio-guided activity isolation and purification could be carried out on C. tinctoria var. tinctoria for the bioavailability.