Phenolic compounds, essential oil composition, and antioxidant activity of Angelica purpurascens (Avé-Lall.) Gill

In this study, methanol extracts (MEs) and essential oil (EO) of Angelica purpurascens (Avé-Lall.) Gill obtained from different parts (root, stem, leaf, and seed) were evaluated in terms of antioxidant activity, total phenolics, compositions of phenolic compound, and essential oil with the methods of 2,2-azino-bis(3ethylbenzo-thiazoline-6-sulfonic acid (ABTS•+), 2,2-diphenyl-1-picrylhydrazil (DPPH•) radical scavenging activities, and ferric reducing/antioxidant power (FRAP), the Folin–Ciocalteu, liquid chromatography−tandem mass spectrometry (LC−MS/MS), and gas chromatography-mass spectrometry (GC−MS), respectively. The root extract of A. purpurascens exhibited the highest ABTS•+, DPPH•, and FRAP activities (IC50: 0.05 ± 0.0001 mg/mL, IC50: 0.06 ± 0.002 mg/mL, 821.04 ± 15.96 µM TEAC (Trolox equivalent antioxidant capacity), respectively). Moreover, EO of A. purpurascens root displayed DPPH• scavenging activity (IC50: 2.95 ± 0.084 mg/mL). The root extract had the highest total phenolic content (438.75 ± 16.39 GAE (gallic acid equivalent), µg/mL)). Twenty compounds were identified by LC−MS/MS. The most abundant phenolics were ferulic acid (244.39 ± 15.64 μg/g extract), benzoic acid (138.18 ± 8.84 μg/g extract), oleuropein (78.04 ± 4.99 μg/g extract), and rutin (31.21 ± 2.00 μg/g extract) in seed, stem, root, and leaf extracts, respectively. According to the GC−MS analysis, the major components were determined as α-bisabolol (22.93%), cubebol (14.39%), α-pinene (11.63%), and α-limonene (9.41%) among 29 compounds. Consequently, the MEs and EO of A. purpurascens can be used as a natural antioxidant source.

The literature review reveals that although there are many studies on the composition of the essential oils and biological activities of Angelica species, the data about A. purpurascens essential oil composition and antioxidant activities are limited. However, to the best of our knowledge, there are no reports in the literature regarding its phenolic constituents.
The current study aims (i) to study the antioxidant capacity of A. purpurascens methanol extracts prepared from root, stem, leaf, and seed and the essential oil obtained from the root of A. purpurascens using three common methods DPPH•, ABTS• + , and FRAP, (ii) to explore the total phenolic contents (TPC) of methanol extracts, (iii) to evaluate the phenolic composition in different parts (root, stem, leaf, and seed) of A. purpurascens by LC-MS/MS, and (iv) to determine the chemical composition of essential oil of A. purpurascens root by gas chromatography-mass spectroscopy (GC-MS). , all HPLC standards (pyrogallol, gallic acid, protocatechuic aldehyde, chlorogenic acid, syringic acid, caffeic acid, 4-hydroxybenzaldehyde, vanillin, syringaldehyde, ferulic acid, hesperidin, luteolin-7-glucoside, rutin, oleuropein, benzoic acid, resveratrol, myricetin, apigenin, naringenin, ellagic acid) were supplied from Sigma Aldrich. The root, leaf, seed, and stem parts of the plant were dried at 40 °C for 2 days in an oven and powdered with a laboratory blender. Each part of the plant was weighed 5 or 10 g. All weighed samples were extracted with 50 or 100 mL methanol under reflux at 200 rpm and 40 °C for about 2 h [33], and the extracts were filtered with filter paper and then were centrifuged at 6000 rpm for 15 min. Supernatant fractions were filtered by using 0.45-µm syringe filters (International Ltd., Kent, England) to produce clear extract solutions. A rotary evaporator was used to evaporate the solvents, and final concentrations were adjusted to 10 mg/mL. Extraction yields of methanol extracts of different parts of the plant were 0.4888 g (9.776%) for root, 0.4781 g (9.562%) for leaf, 0.3145 g (6.29%) for seed, and 0.1951 g (3.902%) for stem. The prepared extracts were stored at -18 °C until used for analysis. The EO was hydrodistilled from the ground roots (80 g) of the plant for 5 h by using a Clevenger type apparatus and dehydrated with anhydrous sodium sulfate. The EO obtained with a yield of 0.8125% was stored at +4 °C for further studies.

In vitro antioxidant activity
The antioxidant activities of A. purpurascens MEs for each part (root, leaf, seed, and stem) and EO were tested by using three common methods ABTS• + and DPPH• radical scavenging assays and ferric reducing/antioxidant power (FRAP) assay.

Ferric reducing/antioxidant power (FRAP) assay
The FRAP reagent solution was prepared by mixing 10 mM TPTZ, 300 mM, acetate buffer (pH 3.6), and 20 mM FeCl 3 (1:10:1) [34]. The bottle was then wrapped in aluminum foil and stored at room temperature until analysis was performed. The FRAP reagent was prepared fresh. All extracts were diluted to a concentration of 5 mg/mL. After that, 1.5 mL FRAP reagent was pipetted into a 50-μL sample and mixed. The absorbances were read at 595 nm (UV−Vis spectrophotometer, ATI/Unicam UV2) after incubation of samples for 20 min at room temperature. The calibration graph was obtained by using Trolox (62.5−1000 μM), and antioxidant activity was given as Trolox Equivalent Antioxidant Capacity (μM TEAC).

DPPH• radical scavenging assay
A 100 µM solution of DPPH• was prepared in methanol, and the solution bottle was wrapped in aluminum foil. Next, the solution was mixed in the magnetic stirrer for at least 1 h. The DPPH• radical scavenging method was used as described by Cuendet et al. (1997) with a few modifications [35]. Five different concentrations of MEs and standard solutions were prepared, and 750 µL of them were mixed with an equal volume (750 µL) of DPPH• solution by vortexing. The reaction mixtures were incubated at room temperature for 50 min. Absorbance values were determined at 517 nm in the UV−Vis spectrophotometer (ATI/Unicam UV2). Triple measurements were conducted throughout the experimental study. The calibration graph was plotted using absorbance versus concentration to determine the unknown sample concentration (IC 50 ), and IC 50 value was identified as the reduced amount of DPPH• by 50%. A low IC 50 value means high radical scavenging potential and thus high activity.

ABTS• + radical scavenging assay
Stock ABTS• + solution was prepared by mixing 7 mM ABTS and 2.45 mM potassium persulfate and left in a dark environment for about 16-20 h at room temperature until the day of the analysis. The ABTS• + radical solution was diluted with 60% ethanol to show an absorbance reading of 0.70 ± 0.02 at 734 nm. In this method, 1950 µL ABTS• + radical solution was mixed with 50 µL of the sample, vortexed, and incubated at room temperature for 20 min. Absorbance measurement was carried out at 734 nm (UV−Vis spectrophotometer, ATI/Unicam UV2). The results were given as IC 50 [33,36].

Determination of total phenolic contents (TPC)
The total phenolics of all MEs were tested with some minor modifications according to the method of Slinkard and Singleton [37,38]. Catechin and gallic acid were used as standard. A 50 μL of the sample was diluted by using 2.5 mL of distilled water. Then, Folin-Ciocalteu reagent (0.2 N) diluted with 250 µL of pure water at intervals of 20 s was added, vortexed, and incubated at room temperature for 3 min. Then, 750 µL (7.5%) Na 2 CO 3 was added again in 20 s. It was vortexed again by pipetting and left for incubation for 2 h at room temperature. Besides, one blank for each concentration of sample and standard (sample/standard + Folin−Ciocalteu reagent solvent [pure water]) was studied. All the experiments were performed three times. The absorbances were measured at 765 nm (UV−Vis spectrophotometer, ATI/Unicam UV2). A calibration graph was drawn (62.5−1000 μg/mL), and TPC was given as microgram catechin (CE) and gallic acid equivalent (GAE) per mL sample.

Gas chromatography−mass spectrometry (GC-MS) analysis
About 10 mg of root sample was taken and dissolved in hexane. A Thermo Scientific GC−MS was used to identify partial components of EO. The oils were analyzed using the TG−5MS column (film thickness 0.25 µm, 30.0 m × 0.25 mm i.d.). The injection port temperature was set at 250 °C, whereas the oven temperature was arranged as the first temperature was 50 °C with the GC oven temperature was held at 220 °C for 0.67 min and programmed with a rate of 5 °C/min to 250 °C and then held constant at 250 °C for 5 min. The ionization mode was at 70 eV. The carrier gas was helium with a flow rate of 1.0 mL/min. The components were determined by comparison of their relative retention times and mass spectra with those of standards, reported in the literature [39] and available on Wiley and NIST mass spectral libraries.

Liquid chromatography-tandem mass spectrometry (LC−MS/MS) analysis
Phenolic compounds found in various parts of the A. purpurascens plant were determined by using LC−MS/MS (Thermo Scientific/TSQ Quantum Access Max) technique. Twenty phenolic compounds were used as standard. Initially, the optimization of the MS program was made, for which different collision energies were used to generate a qualifier ion and a quantifier ion for each standard. Serial dilutions of the standards (0.25−0.5−1−2−4−6 mg/L) were used to obtain a linear standard curve (r 2 > 0.99). A. purpurascens phenolics were identified by matching the retention time and MS spectra with those of the standards.
A reversed-phase Hypersil™ ODS C 18 column (4.6 × 250 mm 5µm) was used, and 0.1% formic acid in water (A) and 100% methanol (B) were used as the mobile phase solutions with a flow rate of 0.7 mL/min. A 20-μL injection volume and 30 °C column temperature were used. The gradient program included an initial 0-1 min of 100% A and the following compositional changes: 1−22 min, 100% A; 22−25 min, 5% A; 25−30 min 100% B. Mass spectrometry signals were acquired by maintaining the temperature for capillary at 300 °C and for vaporizer at 350 °C; spray voltage of positive and negative polarity was set to 4000 and 2500 V; the pressure of sheath gas and aux gas were kept at 30 arb and 13 arb, respectively; discharge current 4 μA.

Statistical analysis
Antioxidant test results and total phenolic contents were statistically analyzed by using a one-way analysis of variance ANOVA with Tukey post hoc test using SPSS 22.0 software. Test results were expressed as mean ± standard error (SD) of three experiments, and the differences were considered significant at p < 0.01.

Antioxidant activity and total phenolic content of A. purpurascens
There are many antioxidant assays in the literature based on methodological differences to screen antioxidant capacities of samples from natural sources, including extracts and essential oils from plants. In this study, DPPH•, ABTS• + , and FRAP methods were used to determine the antioxidant potentials of A. purpurascens MEs and EO ( Table 1).
The ABTS• + and DPPH• assays result expressed as IC 50 means the effective concentration of test samples required for 50% antioxidant activity under the experimental conditions. Lower IC 50 values indicate higher radical scavenging activity. The root extract showed higher values than other parts. The root methanolic extract of A. purpurascens demonstrated the highest ABTS• + and DPPH• radical scavenging activities (IC 50 : 0.05 ± 0.0001 a mg/mL and IC 50 : 0.06 ± 0.002 a mg/mL, respectively) while the stem methanolic extract showed the lowest antioxidant activities (IC 50 : 0.19 ± 0.003 d mg/mL and IC 50 : 1.23 ± 0.001 d mg/mL, respectively). Moderate antioxidant activity was observed in seed and leaf extract (IC 50 : 0.35 ± 0.003 c and 0.09 ± 0.0007 b mg/mL for DPPH• and IC 50 : 0.18 ± 0.004 c and 0.09 ± 0.003 b mg/mL for ABTS• + , respectively). Moreover, the root EO of A. purpurascens displayed DPPH• scavenging activity (IC 50 : 2.95 ± 0.084 mg/mL). Essential oil and all MEs showed significant radical scavenging activities, though lower than those of standard antioxidants ( Table 1).
The FRAP test results were expressed in comparison to the activity of Trolox, and the Trolox equivalent antioxidant capacity (TEAC, μM) values obtained from the calibration graph were used to express antioxidant potentials. According to all antioxidant assays, all extracts displayed an antioxidant activity with the order of activity root > leaf > seed > stem.
Significant differences were observed in the total phenolic content and antioxidant activities of the root, stem, seed, and leaf parts of A. purpurascens ( Table 1). The root extract was observed to have the highest total phenolic content (438.75 ± 16.39 d GAE, µg/mL) compared with the stem, seed, and leaf parts. The stem extract had a significantly lower phenolic content (68.33 ± 1.90 a GAE, µg/mL) than the extracts from other plant parts. The order of the total phenolic content of A. purpurascens MEs was root > leaf > seed > stem. Good positive correlations were observed between the results of the phenolic content and antioxidant assays (ABTS• + , DPPH•, and FRAP; r 2 values were 0.8927, 0.9212, and 0.8587, respectively).
Although the antioxidant potentials of some Angelica species essential oil or extracts have been reported in the literature, it was found only one study reporting on the antioxidant activity of A. purpurascens. Karakaya et al. (2020) evaluated MEs of different parts of (root, fruit, and aerial) A. purpurascens in terms of antioxidant activity [16]. There was no study in the literature attempting to determine the antioxidant capacity of A. purpurascens EO using the DPPH•, ABTS• + , and FRAP methods. In this study, the antioxidant activity of A. purpurascens EO was also determined for the first time. In a previous research, DPPH• scavenging activity for the ME of A. gigas aerial part was a dose-dependent antioxidant activity, and it was lower than that of synthetic antioxidants vitamin C and BHT [40]. Similarly, A. glauca oil was noted to increase the DPPH• scavenging capacity in a concentration-dependent manner with an IC 50 value of 32.32 µg/mL, but showed lower activity compared to BHT [41]. Moreover, Pervin et al. (2014) reported that A. dahurica root extracts showed a dosedependent increasing DPPH• and ABTS• + scavenging activities [42]. Roh and Shin (2014) demonstrated that A. koreana root EO and its two main components showed less scavenging activity than butylated hydroxyl anisole (BHA) at 1 mg/mL [43]. In some previous studies, A. archangelica L. seed EO [44] and A. sinensis extracts [45] exhibited moderate DPPH• scavenging activity. Moreover, ME of A. officinalis L. fruits did not have radical scavenging activity against DPPH• while it had moderate FRAP activity [46]. Leaf extract of A. keiskei showed significant DPPH• scavenging activity close to the rutin standard [47]. The MEs of two species of Angelica (A. pancicii and A. sylvestris) exhibited a positive correlation between antioxidant activity and polyphenol content [48]. A. sylvestris var. sylvestris EOs from dried roots, leaves, flowers, and fruits mentioned by Ağalar et al. (2020) had similarly low antioxidant activity [23]. Finally, Zhang et al. (2020) reported that among the antioxidant activities of different solvent extracts of A. amurensis root, methanol, and ethanol extracts exhibited high antioxidant activity [49]. In this study, A. purpurascens MEs and EO had low antioxidant activity, similar to the activity results of other Angelica species mentioned in the literature. Although the antioxidant activity of A. purpurascens MEs and EO was weaker than that of the standard compounds, its use would prevent the toxicity problems of the synthetic standards. However, further studies are recommended before the usage of A. purpurascens MEs and EO as antioxidant additives.

Identification and quantification of phenolic compounds in A. purpurascens
The identification of the phenolics was accomplished by comparing retention times and MS fragments with those of reference standards. Molecular ions of phenolic standard compounds were determined with both negative and positive ion modes in LC−MS/MS (Table 2). In LC−MS/MS analysis, 20 phenolic compounds were identified and quantified ( Table  3). The seed extract of A. purpurascens was found to have the highest value in terms of phenolic compound concentration compared with the extracts from other plant parts. The major phenolic compound of seed extract was found as ferulic acid (244.39 μg/g extract). Benzoic acid (138.18 μg/g extract), oleuropein (78.04 μg/g extract), and rutin (31.21 μg/g extract) were found as the most abundant phenolic compounds in the stem, root, and leaf extracts, respectively. Ferulic acid, benzoic acid, and rutin were found in one part of the plant. In addition, pyrogallol, gallic acid, chlorogenic acid, myricetin, and ellagic acid were found in different amounts in one part of the plant. 4-Hydroxybenzaldehyde, vanillin, syringic acid, and hesperidin phenolic compounds were detected in all plant parts. Oleuropein, which is generally found in some Oleaceae species, has been documented by in vitro and in vivo studies to have antitumor, antifungal, antimicrobial, anticancer, and cardioprotective properties, besides its strong antioxidant activity as a free radical scavenger [50]. In this study, oleuropein, which is abundant in A. purpurascens root, may be responsible for the high antioxidant activity. In a previous paper involving the phenolic composition of A. purpurascens, four coumarin derivatives (ostruthol, phellopterin, xanthotoxin, and biakangelicin) were isolated from roots, and the last three of them were isolated for the first time [25]. The previous study of characterization of phenolic compounds has led to the identification of furocoumarins, including imperatorin, phelloptorin, and isoimperatorin, in the roots of A. dahurica by HPLC/DAD/ESI-MS [51]. Coumarins including psoralen and xanthontoxin, and chalcones have been reported as phenolic compounds found in A. keiskei [52].
Analysis of phenolic compounds in some Angelica species has been previously reported, but quantitative analysis of methanol extracts from different parts of A. purpurascens has not been reported previously. This novel study is the first report of the identification and quantification of phenolic components found in all four parts of A. purpurascens, root, stem, leaf, and seed, by LC−MS/MS.

GC−MS analyses of essential oil of A. purpurascens root
Gas chromatography-mass spectrometry (GC−MS) is one of the most widely used methods in determining the chemical composition of EOs [53,54]. According to the literature, the EOs of different Angelica species grown in different geographical regions have been obtained by various extraction techniques such as hydrodistillation, steam distillation, supercritical liquid extraction, and solvent-free solid injection. Besides, it was reported that the EOs of Angelica species show various biological activities, including antibacterial, antifungal, insecticidal activities, and pronounced antioxidant activity due to their volatile compositions [29].

Conclusion
The root essential oil and the methanol extracts of A. purpurascens exhibited a remarkable antioxidant potential. The strong antioxidant activity of A. purpurascens with high total phenolic content indicated a great potential for its use in the production of functional foods. The biochemical compositional data thus obtained for the extracts from different parts of A. purpurascens (root, stem, seed, and leaf) and essential oil can form a background for further investigations to develop new formulations or products by the use of Angelica species. Retention indices (RIs) relative to n-alkanes (C 7 -C 30 ) on the same capillary column.