CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF VOLATILE

OILS OF ARABIS ALPINA L. SSP. ALPINA

O. Ucuncu*

Department of Food Engineering, Faculty of Engineering and Natural Sciences, Gümüşhane University, TR-29100 Gumuşhane, Turkey

Corresponding Author’s E-mail: osmanucuncu@yahoo.com

ABSTRACT

This work reports volatile constituents, antimicrobial and antioxidant activity of volatile oils from the air-dried flower and aerial parts (APs) of Arabis alpina L. ssp. alpina. The volatile components of flower and aerial parts (stem + leaf) were investigated by means of hydrodistillation in Clevenger type apparatus and GC/MS/FID analysis. Fifty-one and fifty-three compounds in the volatile oils of flower and APs of A. alpina were identified. Terpene derivatives were the major volatiles in both oils. Hexahydrofarnesyl acetone was the main component of flower oil and APs oil in ratios of 16.27% and 26.94%, respectively. Additionally, both flower and APs essential oils were investigated for their antimicrobial activity against twelve bacteria and five fungi, using agar dilution method and antioxidant activities by using DPPH˙, ABTS˙⁺ and Folin-Ciocalteu assays. Flower oil was effective against B. subtilis and B. cereus even at 100µg/mL. The amount of total phenolic, %DPPH˙ scavenging activity and ABTS˙⁺ scavenging activity were found as 485.60±7.28 mg/mL GAE, 49.85±1.22% and 166.43±12.05µM Trolox equivalent, respectively. Flower oil exhibited antimicrobial effect against gram positive bacteria especially, and moderate antioxidant activity.

Keywords: Antimicrobial activity, Antioxidant activity, Arabis alpina L. ssp. alpina, Essential oil, GC-MS/FID, Hexahydrofarnesyl acetone

https://doi.org/10.36899/JAPS.2021.5.0352
Published online January 24, 2021

INTRODUCTION

Being a significant group of vegetables and considered to be a staple food all over the world, Brassicaceae (also known as Cruciferae) species are highly consumed due to their nutritional value (Singh et al., 2017). Brassicaceae vegetables, which can be used as a salad, fresh or dried as a spice, cooked, fried, baked or fermented, contain many minerals, fibers, vitamins and phytochemical compounds. They contain antioxidant compounds such as carotenoids, ascorbic acid, isothiocyanates and phenolics. There are many species in the Brassicaceae family that are used in traditional medicine and foods these are also recognized as the functional food. Brassicaceae family has received a great deal of attention in recent years because of its antioxidative and antimicrobial properties (Balpinar, 2018). The main metabolite of Brassicaceae is glucosinolates that contain sulfur. The phytochemicals obtained from the flower, seed, leaf and stem of these plants provide a great source for medicinal purposes (Shankar et al., 2019). Consumption of Brassicaceae vegetables has preventive role against a variety of chronic diseases, for example, several cancers etc. (Samec et al., 2017).

Arabis L. is a genus within the family Brassicaceae (Koch et al., 2006). Arabis L. (Brassicaceae) consists of nearly 128 species (The Plant List, 2018), and most of them are widespread in Northern Hemisphere, also including some part of Africa (Stevens, 2011 2001) and characterized by rosette leaves at the base of plants. Arabis alpina is found mostly subalpine and alpine regions and prefers mesophytic areas (Koch et al., 2006). In Turkey, genus Arabis is represented by 22 taxa. Ten of these 22 taxa is endemic to Turkey (Cullen, 1965; Mutlu, 2012). A. alpina L. is a very variable taxon, which is previously known as Arabis caucasica Willd. in Turkey. With renewed Flora of Turkey, A. caucasica is now accepted as synonym of A. alpina (Mutlu, 2012). There are economical and culinary uses of A. alpina. While A. alpina is used as ornamental plant in Erzincan (Turkey) province (Korkmaz et al., 2016), shoots and leaves of this plant is eaten freshly in Himalaya (India) region (Bhoyar et al., 2011). In addition to these, some Arabis species are used for medicinal purposes traditionally. A. tibetica is used for wound healing (Kala, 2006), whereas A. glandulosa is benefited for treatment of abdominal pain (Ballabh and Chaurasia, 2009).

Although there are lots of studies on plant morphology and Arabis mosaic virus of Arabis taxa in the literature (Park et al., 2017; Abelleira et al., 2010), the phytochemical studies are limited. Some phytochemical studies on the Arabis taxa have been reported, which describe the isolation and identification of a number of glucosides (Kjaer, 1960; Kjaer and A. Schuster, 1972). In these studies, on glucosinolates in Arabis, sulfur containing hirsutin, 8-methylsulphinyl-3-oxooctyl isothiocyanate, 8-methylthio-3-oxooctyl glucosinolate, and 9-(methylsulfinyl) nonylisothiocyanate were separated from the seeds of A. alpina L. and A. hirsute (Kjaer, 1960; Kjaer and A. Schuster, 1972). In a study of GC analyses with 14 wild Arabis species seeds, the Arabis species were characterized by longer chain homologues (hexyl to decyl) and methoxyphenyl oxazolidine-2-thiones (Daxenbichler et al., 1991). 3-methylthiopropyl, 6-methylthiohexyl, 7-methylthioheptyl glucosinolates, all of which have sulfur, were isolated from A. purpurea and A. kennedyae seeds (Hasapis et al., 1981). In another study biological activities of the extracts of ethanol and methanol for A. alpina L. subsp. brevifolia against different food pathogens were investigated. The extracts were shown to have polyphenols, such as quercetin and rutin, and phenolic acids, such as 2,5-dihydroxybenzoic, vanillic and caffeic acid (Balpinar, 2018).

A previous phytochemical study on A. caucasica has shown the presence of different heterosides derived from quercetin and kaempferol. Quercetin derivatives were 3-glucoside, 3-β-glucosido-7-α-rhamnoside and 3-diglucosido-7-glucoside of quercetin, and kaempferol derivatives were 7-β-glucoside, 7-rhamnoside, 3-β-glucosido-7-α-rhamnoside, 7-arabinosido-glucoside, and 7-xylosidoarabinosido-glucoside of kaempferol (Matlawska et al., 1992; Matlawska et al., 1991). Five acylated anthocyanins were isolated and identified from flowers of A. blepharophylla with HPLC (Ito et al., 2013). In a related study analysis of the HPLC results of A. alpina seeds revealed some derivatives of cinnamoyl choline (Bouchereau et al., 1991). Additionally, C₈-C₁₀ methylsulfinyl alkyl and methylsulfonyl alkyl glucosinolates were identified in the seed of A. turrita by HPLC-ESI/MS analysis of intact glucosinolates (Blaževićet al., 2015). Only two volatile oil studies on Arabis genus have been reported. First report includes six Arabis species (A.holboellii, A. demisa, A. crandalii, A. ligniferia, A. drummondii, and A. gunisoniana) (Raguso and Roy, 1998). According to this report, Arabis species contain caryophyllene (A. holboellii) as terpene, indole (A. demisa) and benzyl alcohol (A. holboellii) as aromatics, and especially n-hexane derivatives that (Z)-3-hexenylacetate, (Z)-3-hexenyl-3-methylbutylate and(Z)-3-hexenal (A.holboellii, A. demisa, A. crandalii, A. ligniferia, A. drummondii) as fatty acid derivatives. Also A. holboellii, A. demissa and A. crandallii contain isopropyl isothiocyanate. Second report includes volatile oil composition of two Arabis species (A. purpurea and A. cypria) from Cyprus (Polatoğlu et al., 2017). The major ingredients of the essential oils were found as nonacosane, heptacosane, and hexahydrofarnesyl acetone. As mentioned above Arabis species contains interesting natural phytochemicals.

Till today, there is only one report on the volatile oil composition of two Arabis species (A. purpurea and A. cypria) in scientific publications. Literature survey did not reveal any publication related to the chemical composition, biological activities of the volatile oil of the aerial parts of A. alpina plant.

Nowadays, 80% of the materials used in the treatment of diseases in developed countries are of plant origin. In order to take advantage of the properties of plants such as antimicrobial and antioxidant many researchers conduct research to discover new active substances. In this paper, we have reported volatile constituents and biological properties of the volatile oil of the flower and aerial parts (stem + leaf) of Arabis alpina L. ssp. alpina wild-growing in Turkey. This study is the first attempt in the literature on antioxidant and antimicrobial properties of essential oil of any Arabis species.

MATERIALS AND METHODS

Plant Material: Arabis alpina L. ssp. alpina plant at the blooming stage were gathered from nearby Karaçukur village of Torul, Gümüşhane: (40°36ꞌ27ꞌꞌN, 39°17ꞌ08ꞌꞌE at 960 m above sea level) in Turkey(A7) during March 2017. The taxonomic identification of plant materials was done by Assoc. Prof. Mutlu Gültepe, in Programme of Forestry, Dereli Vocational School, Giresun University, Giresun, Turkey. Flowers and aerial parts (stem + leaf) of A. alpina were separated from plant and shade-dried at room temperature. The voucher specimen has been deposited with the number KTUB744 in the Department of Biology, KTU, Trabzon-Turkey.

Volatile Oil Isolation: The volatile oils from minced and shade-dried plant parts (flower-157g, leaf+stem-181g) of A. alpina were obtained by hydrodistillation in Clevenger apparatus (4h, yields: 0.04 and 0.03 %(v/w), respectively). The tests and analyses were performed with GC-FID/MS (İskender et al., 2009; Pino et al., 2005).

GC/FID/MS Analysis of the Volatile Oil: The GC analysis of the volatile oils was performed using an Agilent-5975 Network Gas Chromatography System equipped with a FID and MS Detectors. HP-5MS capillary column (30 m × 0.25 mm ID, film thickness 0.2 µm) was used for GC-FID and GC-MS analyses. Helium was used as carrier gas at a flow rate of 1.2 mL/min. 1 μL volatile oil solution in hexane was injected and analyzed with HP-5MS column. Each sample was injected at split ratio 1:1. Operation conditions were as follows: oven temperature at 50°C (5 min), 50 to 260°C at 4°C/min and 260°C (15 min), manual injection 250°C, and MS detector temperature 230°C (Pino et al., 2005).

Identification of Volatiles: Retention indices (RI or Kovats Index, KI) of all of the volatiles were determined through the retention times (RT) of n-alkanes C₆-C₃₂ with linear interpolation. Identification of essential oils compositions was completed through comparing of retention indices(RI) values obtained with the published values and with data of mass spectral libraries (Wiley 275,NIST 05 and Adams Essential Oil Mass Spectral Library) (Pino et al., 2005; Adams 2007; Andriamaharavo 2014; Zhao et al., 2009; Fanaro et al., 2012; Forero et al., 2008; Hammami et al., 2011; Saroglou et al., 2006; Pérez et al., 2007; Demyttenaere et al., 2002; Zhao et al., 2006; Kallio et al., 2006; Sarikurkcu et al., 2008; Kukic et al., 2006). The essential oil samples were analyzed twice and the percentages of components calculated from the GC results. Chemical compositions of essential oils are presented in Table 1 and Table 2.

Antimicrobial activity: Strains of twelve bacteria, which are four gram-positive bacteria and eight gram-negative bacteria and five fungi, were provided by Food Engineering Laboratories of Gümüşhane University. The antimicrobial activities of the volatile oils were determined against bacteria and fungi with agar-well diffusion method (Maksimovic and Mraovic, 2005; Sağdıç and Özcan, 2003). Antimicrobial results of essential oils were presented Table 3.

Antioxidant activity test:

DPPH˙ assay: The DPPH (2,2-diphenyl-2-picryl-hydrazyl) radical scavenging test is the most common method among antioxidant determination assays. The antioxidant activities of volatile oils were determined by DPPH method with some modifications (Ekici and Özaltın, 2018). Pure methanol was used for blank instead of essential oils of A. alpina. Both reaction mixture and reactive blank measurements were repeated in three parallels and averaged. Antioxidant activity was shown through scavenging the DPPH radical percentages. Trolox and ascorbic acid were used as standard antioxidants at 200 μg/mL concentration for comparison.

The result of % scavenging was calculated using the following formula: 

%DPPH Radical Scavenging = ((A_blank – A_sample) / A_blank) x 100

A_blank: Absorbance value of the blank DPPH solution

A_sample : Absorbance value of the sample tube

Determination of Total Phenolic: The amount of total phenolic contents of essential oils was determined using Folin-Ciocalteu reagent according to the method from the literature, with some modification (Agbor et al., 2014). The total phenolic content was calculated from the calibration curve (obtained with gallic acid standard), and the results were expressed as gallic acid equivalent (GAEmg/mL).

ABTS˙⁺ radical cation scavenging assay: The radical scavenging capacity of essential oils was evaluated by using ABTS radical cation scavenging assay according to the method from the literature, with some modification (Miller and Rice-Evans, 1997). Results were expressed as µM Trolox equivalent.

For this analysis, 200 μL samples were added to the tubes and mixed thoroughly with 2850 μL ABTS˙⁺ solutions and vortexed. This mixture was let to stand for 120 minutes, and absorbance values were measured at 734 nm. The same procedures were performed with standard solutions of ascorbic acid and Trolox. Instead of samples, 150 μL pure methanol was used as a blank. Antioxidant activity was expressed as scavenging percentage of the ABTS˙⁺radical (Miller and Rice-Evans, 1997).

%Scavenging =((A_blank – A_sample)/A_blank)×100

A_blank: Absorbance value of the blank ABTS˙⁺solution

A_sample : Absorbance value of the sample tube

Statistical analysis: The data were analyzed using the SPSS 17.0 statistical version software program (SPSS Inc., Chicago, IL, USA) for Windows. All the experimental results were presented as mean values ± SD of triplicate measurements. The data were evaluated by using the one-way analysis of variance (ANOVA). Significant differences in groups were indicated at p < 0.05.

RESULTS AND DISCUSSION

The chemical composition, antimicrobial and antioxidant activities of essential oils from the flower and aerial parts (leaf + stem) of A. alpina ssp. alpina were investigated for the first time. According to our literature survey, the present study is the second report on the essential oil composition of any Arabis species.

In this study, we evaluated the chemical composition, antimicrobial and antioxidant activities of essential oils from flower and aerial parts (stem and leaf) of Arabis alpina L. ssp. alpina, which is used as a culinary and ornamental plant. Fifty one and fifty three components were identified constituting 86.64 and 92.98% of the essential oil composition of the flower and APs of A. alpina, respectively. Chemical compositions of essential oils are presented in Table 1. Identified volatile compounds were classified as terpene derivatives, hydrocarbons, aldehyde and ketones, fatty acids, alcohols and others (Table 2). The major components of the oils of A. alpina were hexahydrofarnesyl acetone (flower 16.27 and APs 26.94%), 2,6,10-trimethyltridecane (flower 9.98%), nonanal (flower 3.19 and APs 4.06%) and geranylacetone (APs 3.70%). Terpene derivatives were the major volatiles in both flower (38.58%) and APs (46.76%). These compounds trigger pharmacological activities through antioxidant action (González-Burgos and Gómez-Serranillos, 2012).

Table 1. Identified components and chemical class distribution of the essential oils A. alpina

APs: Aerial parts(leaf + stem) of A. alpia,, Exp. RI: Experiment Retention Index, Lit. RI: Literature Retention Index

^aPercentages obtained by FID peak-area normalization.

^bRetention index calculated from retention times relative to n-alkanes (C₆-C₃₂) on the non-polar HP-5MS column.

^cIncluded as authentic compound

Table 2. The chemical class distribution in the essential oils of A. Alpina

NC^a: Number of compounds

α-Pinene, carvacrol, eugenol, geranylacetone, β-ionone, and hexahydrofarnesyl acetone were some of the common terpenes in all parts of the A. alpina. When compared to literature results of essential oils of Arabis species (A. purpurea and A. cypria) (Polatoğlu et al., 2017). β-(E)-caryophyllene, geranyl acetone, β-ionone, caryophyllene oxide, hexahydrofarnesyl acetone, phytol, 1-octanol, 1-nonanol, 1-octadecanol, nonanal, (E,E)-2,4-decadienal, pentadecanal, hexadecane, heptadecane, heneicosane, docosane, tetracosane, pentacosane, heptacosane and nonacosane were similarly detected in A. alpina oils. The major components of essential oils of A. purpurea and A. cypria have been reported as hexahydrofarnesyl acetone, nonacosane, heptacosane and phytol. These compounds were also detected in considerable amounts in oils of A. alpina in the present research. However, some differences within the chemical composition of essential oils from Arabis species were identified, and it is probably related not only to the species difference but also to environmental factors, time of collection of the plant and climatic conditions.

In this work, essential oils of flower and APs (leaf + stem) of A. alpina were studied separately. This situation enlightened us for the information about sulfur containing components of the plant, particularly. In the composition of the investigated APs oils, we encountered some isothiocyanates. Thus, sulfur-containing compounds such as 4-isothiocyanato-1-butene, 2,4,5-trimethyl thiazole, 2,5-thiophenedicarboxaldehyde and phenethyl isothiocyanate were only detected in aerial parts (leaf + stem) at various amounts. Isothiocyanates are the hydrolysis products of glucosinolates (Vaughn and Berhow, 2005). It has been reported in the literature that some sulfur containing glycosinates are obtained from Arabis species (Kjaer, 1960; Kjaer and A. Schuster, 1972; Daxenbichler et al., 1991; Hasapis et al., 1981). GC-MS analysis of seed A. turrita L. showed the presence of long-chain olefinic isothiocyanates along with other long-chain thio functionalized glucosinolate breakdown products (Blaževićet al., 2015). Isothiocyanates have been shown to possess high bactericidal activity against various food pathogens and food spoilage microorganisms (Luciano and Holley, 2009). In the literature survey has reported that isothiocyanates can lower the incidences of different cancers (Dinkova-Kostova and Kostov, 2012).

According to literature A. purpurea and A. cypria had very low essential oil yields (<0.01%v/w) (Polatoğlu et al., 2017). A Clevenger apparatus was used in the current work as in theirs. Likewise, yields of the essential oils of flower and APs were found low (0.04 and 0.03%v/w, respectively).

No biological activity data on antimicrobial or antioxidant properties of essential oils of Arabis species in the literature was found. Essential oils are active against most of the microorganisms, including gram positive bacteria such as Bacillus subtilis depending on amount and type of terpene derivatives and phenolic compounds (Bakhtiary et al., 2018). Essential oils of A. alpina exhibited different inhibition levels against selected eight-gram negative bacteria, four-gram positive bacteria and five fungi (Table 3). According to antimicrobial activity results, the inhibition zone increased with increased concentration of A. alpina oils.  Four different concentrations (50,100, 500 and 1000 µg/mL) were studied in this work. No antimicrobial activity was observed at 50 µg/mL concentrations. On the other hand, at 1000, 500 and 100 µg/mL concentrations, samples, especially flower oil, exhibited moderate inhibition activity against the bacteria. Bacterial inhibition by essential oils of A. alpina was stronger than the inhibition of fungi. The essential oil of flowers showed antibacterial activity against all of the gram-positive bacteria (B. cereus, B. subtilis, L. monocytogenes and S. Aureus) and the gram-negative bacteria E. coli O157:H7at 1000 µg/mL concentration. It also showed activity against B. cereus and B. subtilis and E. coli O157:H7 at 500 µg/mL concentration. Flower oil was effective against B. cereus and B. subtilis even at100 µg/mL. However, Aps oil was effective only for one-gram negative bacteria (E. coli O157:H7) at 1000 and 500 µg/mL. Results indicate that there is better antimicrobial activity for the essential oil of A. alpina flower than that of APs. Balpinar reported that methanol and ethanol extracts of A. alpina L. subsp. brevifolia exhibited antimicrobial activity against S. typhimurium (Balpinar, 2018). In that study extracts had no activity against Bacillus subtilis, Staphylococcus aureus, Salmonella Typhimurium, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, and the fungus Candidaalbicans. The essential oils in our study showed no activity against S. typhimurium and all tested fungi at all concentrations. There may be differences in antimicrobial effects between related species of the same genus. This is most likely due to the different chemical contents of essential oils and extracts.

Brassica vegetables exhibit biological activities like antibacterial, anticancer activity, antiviral and for the innate immune response system (Shankar et al., 2019). Our study showed that the essential oil of A. alpina flower has a moderate antibacterial activity against all tested gram positive bacteria and also essential oils have significant antimicrobial activity against E. coli. As known, gram positive bacteria are capable of causing serious and sometimes fatal infections in newborn infants (Mhairi, 2015). E. coli O157:H7 is among well-known foodborne pathogens that can cause severe diseases (Campion et al., 2017). Probably, the essential oils of A. alpina may find uses as food preservative or food additive. In the proceeding studies, the antimicrobial activity of the flower oil of A.alpina should be investigated towards other microorganisms. The fact that these essential oils have an inhibitory effect on both gram-negative and gram-positive bacteria can be used as a source in the search for alternative drugs to replace existing antibiotics.

Table 3. Antimicrobial activity results (in mm) of the essential oils of A. alpina

(-): no activity.* Aerial parts(APs) of Arabis alpina, NT: Not tested

The antioxidant activities of essential oils (flower and APs) were investigated by using DPPH˙, ABTS˙⁺and Folin-Ciocalteu assays (Table 4). For the determination of total phenolic content (TPC) gallic acid curve with an equation (y = 0.0059x + 0.0093 R²=0.9997) was used (Figure 1). According to this equation, the amount of total phenolic contents of flower and APs essential oils were found 485.60±7.28 and 140.00±3.24 mg GAE/g, respectively. The TPC capacity of flower oil was higher than APs oil.

The total antioxidant activities of essential oils were determined by DPPH method. Trolox and Vitamin C were used as standard antioxidants in %DPPH scavenging tests. %DPPH scavenging value of Trolox and Vitamin C was found 97.23±0.92 and 97.41±0.98%, respectively, at 200 μg/mL concentration. DPPH˙ scavenging activities of flower and APs essential oils were measured as 49.85±1.22 and 23.20±0.76%, respectively. Balpinar determined the highest DPPH scavenging activity (76.3%) in the flower-fruit-seed ethanol extract of A. alpina L. subsp. brevifolia (Balpinar, 2018). Due to both the use of a different species, difference of composition of flower-fruit-seed and difference in concentrations of samples and DPPH solution, %DPPH scavenging results of A. alpina L. subsp. brevifolia could be measured higher than the present results. In all commonly used antioxidant assay methods, the antioxidant activity of the flower essential oil was found to be higher than APs oil.

DPPH˙ scavenging activities of flower and APs essential oils were measured as 102.46±9.54 and 47.68±1.23 µM Trolox equivalent, respectively. ABTS˙⁺scavenging activity of flower and APs essential oils were found 166.43±12.05 and 78.67±5.12µM Trolox equivalent, respectively. Thus, ABTS˙⁺scavenging activity values were higher than DPPH˙ scavenging activity values. According to literature sources, there is a strong correlation between isothiocyanate and phenolic compound contents in essential oils and DPPH and ABTS (Fusari, et al., 2020). Isothiocyanates and phenolic compounds in essential oils may be the cause of the antioxidant effect.

Table 4. Values of antioxidant activity of Arabis alpina

Means ±standard deviations. Different letters (a-c) on the same lines are significantly different at the 5% level (P < 0.05).

The evaluation of the antioxidant capacity of essential oils is complex due to the diversity of oxidants and possible different mechanisms. There is not single test that right reflects the antioxidant capacity of the samples. Therefore, different tests have been used for a complete assessment of antioxidant capacity.

According to results, the essential oil of flower had moderate antioxidant activity. Due to its moderate phenolic content and antioxidant capacity, the essential oil of flower can be used in daily diet or in functional foods. We could not find any reports on the antioxidant properties of the Arabis species. Therefore, the results have not been compared with any data. This report is the first study in the literature on antioxidant properties of essential oil of any Arabis species.

This study may contribute to future researches on antimicrobial and antioxidant properties of the similar plants (especially Arabis species). Hexahydrofarnesyl acetone was the main component of both essential oils. This component demonstrated antimicrobial effect, allopathic and pest control potential (Balogun et al. 2017). The allopathic and pest control potential of volatile oils should be investigated individually. Probably, the essential oils and various extracts of A. alpina and component compounds may find uses as pesticide and allopathic medicine. The essential oils obtained from A. alpina have various isothiocyanates. As known isothiocyanates can lower the incidences of different cancers (Dinkova-Kostova and Kostov, 2012). In further studies, anti-cancer tests can be performed for essential oils and extracts.

Acknowledgments: Thanks to Assoc. Prof. Mutlu Gültepe for the taxonomic identification of plant, and Research Assistant Ş. Merve Karataş for biological activity tests.

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