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Assessment of Endemic Cota fulvida ( Asteraceae ) for Phytochemical Composition and Inhibitory Activities against Oxidation ,-Amylase , Lipoxygenase , Xanthine Oxidase and Tyrosinase Enzymes

In the present work, chemical compositions of essential oil and methanol extract of endemic Cota fulvida (Grierson) Holub were investigated as well as their antioxidant, antidiabetic, antiinflammatory and antimelanogenesis potent. The phytochemical analyses have been performed with GC-MS/FID and LC-MS/MS techniques. The essential oil was characterized with hexadecanoic acid (25.6 %), camphor (6.1 %), caryophyllene oxide (5.3 %), 1,8-cineole (4.9 %) and humulene epoxide (3.9 %). In the extract, phenolic acids, phenylpropanoid dimer and flavonoids were detected. The major constituents of the extracts were found to be 5-feruloylquinic acid, caftaric acid, 3,5-O-dicafeoylquinic acid and quercetin rutinoside. The antioxidant activities of the oil and extract were evaluated through scavenging of free radicals, inhibition of linoleic acid peroxidation and superoxide anion radical (O2-) generated by xanthine xanthine oxidase (XO) system. The extract showed free radical scavenging activity (IC50 0.131 mg/mL), Trolox equivalent antioxidant capacity (1.33 mM) and inhibited (Inh. 36.3 %) peroxidation of lipids. The oil and extract demonstrated significant hypoglycemic activity via inhibition of porcine pancreatic -amylase. The antiinflammatory effects of the oil and extract via inhibition of 5-LOX enzyme were found as 53.7 % and 23.9 %, respectively. The extract demonstrated moderate inhibitory effect (23.3 %) on oxidation of L-DOPA via inhibition of tyrosinase enzyme.


Introduction
The genus Cota J. Gay is represented by 63 taxa in the world and is mainly distributed in Europe (excluding northern Europe), North Africa, Caucasia and Central Asia [1]. Cota was earlier classified as a section in the genus Anthemis L. in Flora of Turkey [2]. According to the last reports, the Anthemis section Cota has been accepted as a generic name, Cota [3,4]. In Turkey, the genus consists of 22 taxa, nine of which are endemic [5]. Recently, a new species Cota hamzaoglui Özbek & Vural has been described [1]. DOPA) and nordihydroguaiaretic acid (NDGA), allopurinol, -amylase from porcine pancreas (Type VI-B, EC 3.2.1.1), tyrosinase from mushroom (EC 1.14.18.1), xanthine oxidase from bovine milk (Grade IV), lipoxidase from Glycine max (Type I-B) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Starch soluble extra pure, iodine and potassium iodide were purchased from Merck (Darmstadt, Germany). Sodium phosphate, disodium phosphate, aluminum chloride, water and methanol were extra pure analytical grade. A C8-C40 n-alkane standard solution was purchased from Fluka (Buchs, Switzerland). All solvents were purchased from Sigma Aldrich (Germany) and were of analytical grade.

Plant Material
The aerial parts of C. fulvida were collected on Dedegöl Mountain, above Melikler pastures in Yenişarbademli (Isparta) province, on 24 August 2012, and dried under the shade. Botanical identification was performed by Dr. M. Arslan and Dr. M. U. Özbek. The voucher specimen is keep in Gazi University Herbarium under the herbarium code GAZI.

Essential Oil Isolation
The aerial parts of C. fulvida were subjected to hydrodistillation (3 h) to yield essential oil in Clevenger-type apparatus according to European Pharmacopeia [30]. The oil was dried over anhydrous sodium sulfate and stored in sealed vials in refrigerator (4°C), until GC-FID/MS analyses and biological activity testing. The oil was dissolved in n-hexane (10 %, v/v) to conduct chromatographic determination of the composition.

Extract Preparation
Aerial parts of C. fulvida were subjected to maceration in methanol with continuous shaking (3000 per min) at room temperature for 24 h. The obtained liquid extract was filtered and evaporated until dryness in vacuo. The dried extract was kept at 4°C before phytochemical analysis and biological activities tests.

Gas Chromatographic Analysis
The oil was analyzed with GC-FID and GC/MS techniques simultaneously. Details of chromatographic analysis conditions are given in S1 (Supporting information).

Identification and Quantification of Volatile Compounds
Identification of the volatile constituents was given in Table 2. Identification methods are given in S2 (Supporting information)

LC-MS/MS Analysis
LC-MS/MS analysis was carried out using an Absciex 3200 Q trap MS/MS detector. The experiment's conditions are given in S3 (Supporting information).

Free Radical Scavenging Activity (DPPH assay)
The free radical scavenging ability of the essential oil and the extract of C. fulvida were evaluated according to bleaching of purple colored methanol solution of DPPH stable radical using a method of Brand-Williams [31] with slight modifications. Details of the experiments are given in S4 (Supporting information). The free radical scavenging activity of the samples was calculated on base of triplicate experiments and expressed as percentage of inhibition calculated according to equation (1) where, Abscontrol is the absorbance of the control (containing all reagents except the test compound), Abssample is the absorbance of the sample with added DPPH. The IC50 values were obtained by plotting the DPPH scavenging percentage of each sample against the sample concentration. Data were analyzed using the SigmaPlot software (Version 12.0).

Trolox Equivalent Antioxidant Capacity (TEAC assay)
ABTS •+ free radical cation scavenging activity of the essential oil and extract were tested according to the procedure described by Re et al. [32] with slight modifications. Details of the experiments are given in S5 (Supporting information). ABTS •+ scavenging activity of the samples was expressed as Trolox equivalent antioxidant capacity and calculated using linear equation obtained for Trolox (y = 33.644x + 2.6523, r² = 0.9942).

2.10.-Carotene / Linoleic Acid Peroxidation Inhibition Assay
Inhibition of lipid peroxidation by C. fulvida essential oil and methanol extract was measured according to method of Marco [33] with slight modifications. -Carotene / linoleic acid peroxidation inhibition assay uses a linoleic acid in an emulsified form (with Tween-20) as the model lipid substrate. Briefly, the method lies in between methods employing only model substrates (e.g. DPPH) and those using real lipids. Details of the experiments are given in S6 (Supporting information). The rate of -carotene bleaching was monitored by measuring the absorbance at 15 min periods at 470 nm sing an ELISA microplate reader (Biotek Powerwave XS) [33,34]. The experiment was performed in triplicate and the results were expressed as average of inhibition percentage values calculated according to equation (2): where, AA is antioxidant activity, Abs0sample and Abs120sample are the absorbance of the sample at 0 min and 120 min, Abs0control and Abs120control are the absorbance values of the control at 0 min and 120 min.

Microtiter Assay for Determination of Xanthine Oxidase Inhibition
The xanthine oxidase inhibition assay was carried out according to procedure reported by Chen [35] with slight modifications. The stock solutions of the essential oil and the extract of C. fulvida (5 mg/mL) were prepared in methanol, then diluted with phosphate buffer (0.1 M, pH=7.5) up to 1 mg/mL. Details of the experiments are given in S7 (Supporting information). Absorbance at 295 nm was recorded with spectrophotometer. The percentage inhibition was calculated according to equation (3) where Abscontrol and Abscontrol blank are the absorbance values of the control and its blank, Abssample and Abssample blank are the absorbance values of the sample and its blank.

Microtiter Assay for Determination of -Amylase Inhibition
The activity of-amylase was measured using iodine/potassium iodide (IKI) method [36] with slight modifications. The substrate solution (0.05 %) was prepared as follow: soluble potato starch (10 mg) was dissolved in 20 mL ultrapure water then boiled for 10 min and cooled to room temperature before use. Details of the experiments are given in S8 (Supporting information).The sample and blank absorbance values were read at 630 nm. The percentage inhibition of the -amylase activity (Inh %) was calculated according to equation (3).

Microtiter Assay for Determination of Lipoxygenase Inhibition
Inhibition of lipoxygenase activity was measured with spectrophotometric method reported by Albano et al. [37] with slight modifications. The increase in absorbance at 234 nm was recorded for 6 min. The percentage inhibition was calculated according to equation (3). Details of the experiments are given in S9 (Supporting information).

Microtiter Assay for Determination of Tyrosinase Inhibition
An inhibitory activity of C. fulvida essential oil and methanol extract against tyrosinase was measured using the modified 96-well microplate method reported by Masuda [38]. The oil and the extract were dissolved in DMSO (10 %) and then diluted with buffer (pH 6.8) to get concentration 1 mg/mL. Details of the experiments are given in S10 (Supporting information).The percentage inhibition of the tyrosinase activity (Inh %) was calculated according to equation (4):

Statistical Analysis of Data
Data obtained from antioxidant and enzyme inhibition experiments were expressed as mean standard error (±SEM). IC50 values were estimated using a nonlinear regression algorithm.

Results and Discussion
In literature, it could be found highlighting promising phytochemical properties and biological activities of diverse Anthemis species. However, there is no information about phytochemistry and biological potential of endemic species C. fulvida. The main goal of the present work was to evaluate chemical composition and biological properties of endemic C. fulvida volatile and non-volatile metabolites.

Essential Oil Composition
In the present work, the essential oil of Cota fulvida has been hydrodistilled and investigated for chemical profile for the first time. The hydrodistillation of the herb of C. fulvida resulted with yellowish essential oil (0.1 % yield) with specific odor.
Gas-chromatographic analysis of the oil resulted with 70 compounds, which belong to diverse phytochemical groups, namely, monoterpene hydrocarbon, oxygenated monoterpene, sesquiterpene hydrocarbon, oxygenated sesquiterpene, fatty acids, aliphatic aldehydes and alkanes. The list of detected compounds with their relative retention indices, relative percentages and method of identification is given in Table 1 in order of their elution on the HP-Innowax FSC column. Gas-chromatographic profile of C. fulvida essential oil is presented on Figure S11 (Supporting information).
In general, the essential oil of C. fulvida was characterized with high abundance of the fatty acids (>30.0 %). Hexadecanoic acid (25.6 %) was found as predominate fatty acid in the oil. It is noteworthy to mark that the monoterpenes as hydrocarbons (12.6 %) and oxygenated (15.9 %) constituents were found as the second important group after fatty acids in the essential oil. Camphor (6.1 %), 1,8-cineole (4.9 %) and -pinene (3.0 %) were the major monoterpenes in the oil. The sesquiterpenes comprised almost all of oxygenated constituents (19.0 %) with caryophyllene oxide (5.3 %), humulene epoxide (3.9 %) and spathulenol (2.4 %) as major constituents. Distribution of the main compound groups detected in the essential oil of Cota fulvida is presented on Figure S12 (see supporting information).  It was interesting to compare the chemical profile of C. fulvida essential oil with those reported earlier for different Anthemis species. Actually, there are several reports in the literature dealing with the essential oils of diverse Anthemis species. The fatty acids and especially hexadecanoic acid have earlier been observed to be the major constituents in previously studied essential oils obtained from aerial parts of A. dipsacea Bornm.  [57] and A. segetalis Ten (6.1 %) [58]. Observation of the main constituents detected in different Anthemis species, it can be concluded that the oil of C. fulvida was found to be similar to many Anthemis species.

Methanol Extract Composition
In scope of the research, the total phenolics content and total flavonoids content of C. fulvida methanol extract were determined with Folin Ciocalteau reagent and AlCl3, respectively, via spectrophotometric measurements. Subsequent phytochemical analysis via LC-MS/MS technique of the extract led us to identification of phenolics acids, flavonoids and phenylpropanoid dimer. The list of the constituents detected in C. fulvida methanol extract with MS detector is summarized in Table 2.

Antioxidant Activity
The essential oil and methanol extract of C. fulvida have been investigated for an antioxidant activity by using non-enzymatic and enzymatic systems employing model substrates: stable free radical DPPH • and cation radical ABTS +• as well as real substrates: linoleic acid peroxides and superoxide anion radicals (O2 -• ). Such approach allowed obtaining rather realistic results about antioxidant potent of the samples. According to antioxidant activity experiments, the methanol extract of C. fulvida demonstrated the highest antioxidant activity in all applied assays. Namely, the extract scavenged DPPH radicals with IC50 0.131 mg/mL. It was more effective than BHT (IC50 0.50 mg/mL) but less effective than ascorbic acid (IC50 0.03 mg/mL) and gallic acid (IC50 0.003 mg/mL). The essential oil did not demonstrated activity in this assay. The extract prevented bleaching of -carotene by inhibition of linoleic acid peroxidation at 36.3 %, while the essential oil was inactive. In the TEAC assay, the methanol extract showed ABTS •+ bleaching activity equivalent to 1.33 mM of Trolox (water soluble tocopherol), while the oil demonstrated weak activity (0.44 mM of Trolox). In the xanthine/ xanthine oxidase system, the extract demonstrated the highest inhibitory effect (69.36 %). Therefore, as can be seen from the antioxidant activity determination results (Table 2), the essential oil and the extract possess different potentials in diverse oxidation systems.
In the extract of C. fulvida polyphenols have been presented by phenolic acids as well as flavonoids and their glycosides. The phenolic acids in the extract comprised of 3,4-O-dicafeoylquinic, 3,5-Odicafeoylquinic, 1,5-dicaffeoyl-3-succinoylquinic, caftaric and chicoric acids. In literature there is information that caffeoylquinic acid derivatives display more potent free radical-scavenging activity than the most commonly used antioxidants (such as vitamin C, vitamin E, and caffeic acid) [65].
On the other hand, the biological activity of phenolic compounds is known to be due to the activity of nonsubstituted hydroxyl groups. In flavonoid's structure, the presence of a C2-C3 double bond on the ring C, a dihydroxyl group (catechol-type) or three adjacent hydroxyl group (pyrogallol-type) on the ring B, and the presence of C-5, and C-7 hydroxyl group on the ring A are mentioned as requirements for antioxidant activity [66,67]. Luteolin, quercetin rutinoside and glucoside detected in the extract of C. fulvida have all mentioned requirements. Therefore, noteworthy antioxidant activity of the extract may be due to those compounds. In conclusion, the methanol extract of C. fulvida may be considered as potential antioxidant agent in preventing of oxidative damage.

Inhibitory Effects of the Plant on Pancreatic -Amylase Activity
The essential oil and methanol extract of C. fulvida were in vitro evaluated for hypoglycemic activity via inhibition of the pancreatic -amylase. As can be seen in Table 2, both of the tested extracts demonstrated inhibitory activity. The oil inhibited the enzyme's activity up to 75.11 % at concentration 0.5 mg/mL. The extract demonstrated significant inhibiton and prevented activity of -amylase with IC50 0.35 mg/mL. It seems to be that the plant of C. fulvida can be source of an effective hypoglycemic phytochemicals.
One of prominent inhibitors of amylase in herbal products are the flavonoids. The molecular structures that influence the inhibition of -amylase by flavonoids are the following: the hydroxylation of flavonoids improved the inhibitory effect on α-amylase; the presence of an unsaturated 2,3-bond in conjugation with a 4-carbonyl group has been associated with stronger inhibition [67]. The methanol extract of C. fulvida contained flavonoid aglycone as well as flavonoid glycosides. Walle et al. reported that the glycosylation of flavonoids decreased the inhibitory effect on α-amylase depending on the conjugation site and the class of sugar moiety [68].

Inhibitory Effects of the Plant on Lipoxygenase Activity
Antiinflammatory effect of C. fulvida was tested in 5-LOX enzyme inhibition system. Actually, 5lipoxygenase catalyzes the oxidation of arachidonic acid and produces 5(S)-hydroxyperoxyeicosatetraenoic acid (5-HETE) which undergoes dehydration, resulting in the formation of leukotriene. Enzymatic hydrolysis of leukotriene, as well as conjugation with other substances, leads to the formation of inflammatory mediators. In the present study, the oil and extract of C. fulvida were found to be able to inhibit 5-lipoxygenase, particularly the oil (Inh 53.67 % at 1 mg/mL), in comparison to the extract (Inh 23.91% at 5 mg/mL) which showed poor activity ( Table 2).

Inhibitory Effects of the Plant on Mushroom Tyrosinase Activity
In this experiment, tyrosinase inhibitory effects of C. fulvida oil and extract on diphenolase activity of mushroom tyrosinase were evaluated. The extract demonstrated the highest inhibitory activity (45.46 %). However, the oil was found inactive when tested at a concentration 1 mg/mL (Table 3). In general, it can be assumed that C. fulvida have moderate antityrosinase effect.  As a conlusion, we herein disclose the first report on chemical profile of the volatile and non-volatile secondary metabolites obtained from endemic species C. fulvida. This species can be considered as source of valuable metabolites: the oil is rich with diverse mono-and sesquiterpenes, and fatty acids, while the extract contained phenolic acids, flavonoids and phenylpropanoids. In scope of the present study, the biological potential of volatile and non-volatile fractions of C. fulvida was evaluated for the first time. It seems, that C. fulvida can be considered as valuable source of bioactive components useful in combating various diseases such as cell damage, inflammation, skin disease, neurodegenerative problems as well as in the provision of cheap, safe and natural phytoparmaceuticals.

Disclosure statement
The authors declare that there are no conflicts of interest.