Volatile Compounds and Bioactivity of Eremurus spectabilis (Ciris),a Turkish Wild Edible Vegetable

Abstract

Eremurus spectabilis grows in the spring as a wild vegetable and for many years has been used both as a food or food additive and for therapeutic purposes. This study investigated the total phenolic content and the antimicrobial, antioxidant, and antiradical activities of methanol, ethanol, and aqueous extracts of E. spectabilis (obtained from the Antalya region of Turkey). In addition, volatile compounds of E. spectabilis were characterized by using gas chromatography/mass spectrometry. Major components of E. spectabilis volatile compounds were carvone (44.64%), carvacrol (14.45%), pentane, 2-methyl- (7.34%), (E)-caryophyllene (5.57%), valencene (5.11%), cis-calamenene (2.01%), cadalene (1.10%), and acetic acid (1.12%). The highest total phenolic content was seen with methanol extract (mean±standard deviation, 31.92±0.48 mg gallic acid equivalents/g dry extract). The ethanol extract showed the highest antiradical activity, with a 50% inhibitory concentration of 35.14 μg/L in the 1,1-diphenyl-2- picrylhydrazyl assay. The strongest antioxidant activity was detected in methanol extract (81.72±0.62 mg ascorbic acid equivalents/g). Twelve bacteria species were used to analyze the antimicrobial activity of extracts. The 1% concentrations of all extracts showed no inhibitive effect on any bacterium. The most resistant bacterium was Yersinia enterocolitica, and the most sensitive bacterium was Pseudomonas aeruginosa. A positive correlation was seen between concentrations and inhibition zones, and some differences occurred between antimicrobial activity of other concentrations.

Introduction

W ith its 10,000 different species, Turkey has a rich flora compared with other countries in Europe and the Middle East because of its climate and geographic position. Wild edible plants (spices or vegetables) have been used various purposes as medicine or food.¹ Wild edible plants are influential in treating various diseases caused by oxidative stress, including microbial infections.² The medical properties of these plants are based on chemical substances, such as bioactive compounds.³ Chemical structures and functions of these compounds are classified according to their type.⁴ Phenolic compounds, including simple phenolics, phenolic acids, anthocyanins, hydroxycinnamic acid derivatives, and flavonoids, are commonly found in plants. Many studies have evaluated the physiologic functions of all the phenolic compounds, including free radical–scavenging, antioxidant,⁵ and antimicrobial activities.⁶ During processing and storage of food products, lipid oxidation is a significant measure of deterioration, and free radicals are the most important cause of lipid oxidation. Free radicals may also cause some health problems.⁷ Antioxidants play an important role in preventing lipid oxidation, and they are used frequently in food industry. However, synthetic antioxidants can adversely affect human health.⁸

Natural products with antioxidant activity have recently received increasing interest and have been investigated as alternatives to synthetic products.⁹ Negative consumer perceptions of synthetic preservatives have prompted increased attention to natural antimicrobial agents, including essential oils and plant extracts. Eremurus spectabilis (Bieb.) Fedtsch is a perennial plant belonging to the Liliaceae family. This plant grows in the spring as a wild vegetable and has been used both as a food or food additive and for therapeutic purposes for many years. The young body of the plant, along with the rhizome and root nodules, can be consumed as a meal after cooking, and it can be dried and preserved for consumption in the winter. Karaman and Kocabas¹⁰ reported that E. spectabilis has been used to treat scabies in folk medicine. Some authors indicated that E. spectabilis is used in the production of otlu (herby) cheese in Turkey.^11,12

Published reports on E. spectabilis are limited, and to our knowledge no study on the bioactive properties and volatile compounds of this plant has appeared in the literature. Therefore, we sought to determine the volatile compounds, total phenolic content, and antimicrobial and antiradical activities of methanol, ethanol, and aqueous extracts of E. spectabilis.

Material and Methods

Plant material

E. spectabilis (Bieb.) Fedtsch (Liliaceae) specimens, including the young stem, leaves, and root (rhizome and root nodules), were collected from the Cimi village in Akseki, located 160 km north of Antalya, Turkey, in March 2009. The plant samples were left on a bench to dry and then preserved in plastic bags at room temperature until analyses were performed.

Preparation of the herb extracts

To obtain E. spectabilis extract, we used the modified extraction method of Gu and Weng.¹³ Ten grams of dried and ground plant were extracted in a Soxhelet extractor with 100 mL methanol, ethanol, and water (60°C for 6 hours) and evaporated at reduced pressure (rotavator; temperature < 40°C) to obtain crude extract.

Determination of volatile compounds by gas chromatography/mass spectrometry

The volatile composition of E. spectabilis was analyzed by using gas chromatography (Agilent 7890A gas chromatography system, Agilent, Avondale, CA, USA); gas chromatography/mass spectrometry coupled to a mass selective detector (Agilent Technologies) and HP-5MS column (60 m×0.250 mm internal diameter; film thickness, 0.25 μm) was used. The oven temperature was held at 40°C for 10 minutes and was then increased as follows: to 95°C at 3°C/min, from 95 to 210°C at 10°C/min, and finally to 210°C/min. Temperature was then held for 10 minutes. Helium was used as the carrier gas, with a flow rate 0.5 mL/min. The detector electron ionization voltage was 70 eV. The compounds adsorbed by the fibers were desorbed from the injection port for 15 minutes at 50°C in the splitless mode. The compounds were identified by comparison with spectra from the libraries Flavor 2, NIST 05a, and Wiley7n and by using internal standards.

Determination of total phenolic content

The amount of phenolic compounds in the extract was determined by the Folin–Ciocalteu colorimetric method.¹⁴ Estimations were done in triplicate and calculated from a calibration curve obtained with gallic acid. The total phenolic content was determined as mg gallic acid equivalents (GAE)/g extract.

Determination of antiradical activity

A 1.0-mL methanol solution of the extract at 100 ppm (methanol for the control) was placed in a test tube, and 2.0 mL of a 1,1-diphenyl-2-picrylhydrazyl (DPPH) methanol solution (10 ppm) was added. The absorbance was measured at 517 nm after 5 minutes of reaction. The percentage of DPPH decoloration of the samples was calculated according to the method of Lee et al.¹⁵ as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \hbox{Antiradical activity} =& 100 \times (1-\hbox{absorbance of sample/} \\ & \hbox{absorbance of blank}).\end{align*} \end{document}

Evaluation of antioxidant activity

The antioxidant activity of the extract was evaluated by the formation of phosphomolybdenum complex method described by Prieto et al.¹⁶ An aliquot of 0.4 mL of the sample solution (100 ppm in methanol) was combined in a vial with 4 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The blank was prepared with 0.4 mL of methanol in place of the sample. The vials were capped and incubated in a water bath at 95°C for 90 minutes. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank. The antioxidant activity was expressed relative to that of ascorbic acid.

Determination of antibacterial effect

Twelve bacteria were used as test organisms: Aeromonas hydrophila ATCC (American Type Culture Collection) 7965, Bacillus brevis FMC 13, Bacillus cereus FMC 19, Bacillus subtilis ATCC 6633, Escherichia coli O157, Escherichia coli O157:H7 ATCC 33150, Klebsiella pneumoniae FMC 5, Listeria monocytogenes 1/2b, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium NRRLE 4463, Staphylococcus aureus ATCC 29213, and Yersinia enterocolitica ATCC 1501. These bacteria were supplied by the Department of Biology, Sutcu Imam University, Kahramanmaras, Turkey.¹⁷ The agar diffusion method was used to detect the antibacterial activities of the extracts. Four equidistant holes were made in the agar by using sterile cork borers (4 mm diameter). Fifty microliters of 1%, 2.5%, 5%, or 10% extract concentrations in methanol, ethanol, and aqueous solutions was added to the holes by using a micropipette. At the end of the incubation period, inhibition zones were measured in millimeters.

Results and Discussion

Thirty-four volatile compounds were identified in E. spectabilis (Table 1). Carvone (44.64%), carvacrol (14.45%), pentane, 2-methyl- (7.34%), (E)-caryophyllene (5.57%), valencene (5.11 %), cis-calamenene (2.01%), cadalene (1.10%), and acetic acid (1.12%) were the major components. Carvone is a chemical substance classified as a terpenoid and is most abundant in the oil seeds of caraway and dill. It is the source of herbal aromas, especially in spearmint and caraway.¹⁸ Carvacrol, the second main component of E. spectabilis volatiles, is a monoterpenoid phenolic compound and is present in the essential oil of Origanum vulgare, thyme, pepperwort, and wild bergamot oil.¹⁹ It is a natural aromatic compound and has a potential bactericidal ability; as a result it can be used as a natural food additive in food preservation.²⁰ Caryophyllene, the third main component, is a natural bicyclic sesquiterpene that is a constituent of many essential oils. It contributes to the spiciness of black pepper and has local anesthetic activity.²¹ No study has assessed the volatile compounds of E. spectabilis.

Table 1.

Volatile Compounds of Eremurus spectabilis

Number	Volatile component	RPM	Composition (%)
1	Pentane, 2-methyl-	6.49	7.34
2	Trans-2-hexenal	29.78	0.62
3	Cyclohexasiloxane	33.4553	0.17
4	Hexenol	35.2272	0.24
5	ρ-Cymenene	36.5344	0.29
6	Acetic acid	36.6425	1.12
7	α-Copaene	37.2205	0.53
8	α-Ylangene	37.6202	0.28
9	Germacrene D	38.9167	0.19
10	(E)-Caryophyllene	39.365	5.57
11	Cis-dihydro carvone	39.5055	0.84
12	γ-Amorphene	40.3266	0.67
13	Heptadecane	40.6507	0.18
14	Valencene	40.80	5.11
15	Germacrene D	40.8992	0.54
16	Γ-Muurolene	40.9749	0.52
17	Trans-β-guaiene	41.1369	0.53
18	Zonarene	41.2773	0.59
19	α-Muurolene	41.4556	0.87
20	Carvone	41.5745	44.64
21	δ-Cadinene	41.9202	1.07
22	γ-Cadinene	42.066	0.53
23	α-Cadinene	42.5198	0.59
24	Transcarveol	42.7683	0.17
25	Cis-calamenene	43.1518	2.01
26	Cis-carveol	43.2491	0.63
27	α-Dehydro-ar-himachalene	44.189	0.20
28	α-Calacorene	44.5671	0.60
29	(E)-jasmone	44.9831	0.69
30	Benzothiazole	45.5125	0.37
31	Cubenol	47.1709	0.69
32	Spathulenol	48.543	0.50
33	Carvacrol	50.1744	14.45
34	Cadalene	51.4655	1.10

t_R/min=RPM (revolution per minute).

t_R, retention time; t_R/min, retention time/minute.

Total phenolic content and antioxidant, antiradical, and antimicrobial activities of methanol, ethanol, and aqueous extracts of E. spectabilis were determined. Table 2 shows the antiradical and antioxidant activity and total phenolic content of the samples. The total phenolic content of the extracts ranged from a mean (±standard deviation) of 7.15±0.42 to 31.92±0.48 mg GAE/g. The highest total phenolic content was found in methanol extract (31.92±0.48 mg GAE/g), and the lowest was found in aqueous extract (7.15±0.42 mg GAE/g). Ozturk et al.²² investigated methanol, ethanol, and aqueous extract of pistachio hulls for antioxidative, antiradical, and antimicrobial activities. They determined that methanol extracts (167.49 mg GAE/g) of the hulls showed the highest total phenolic content compared with ethanol (89.87 mg GAE/g) and aqueous (31.73 mg GAE/g) extracts. Another study investigated the antioxidant properties of different cultivars of Portulaca oleracea and used different solvents for the extraction. In this study, the methanol extracts yielded the highest total phenolic content (295 mg GAE/100 g) compared with 50% ethanol (172 mg GAE/100 g) and aqueous (141 mg GAE/100 g) extracts.²³ Yao et al.²⁴ reported that methanol is the most effective solvent for the extraction of plant polyphenolics because it can inhibit the activity of polyphenol oxidase–induced oxidation of polyphenols. In addition, P. oleracea, like E. spectabilis, is a commonly consumed plant, and consumption of both plants in Turkey is similar. Our study showed that E. spectabilis had higher total phenolic content and greater biological activity than P. oleracea.

Table 2.

Total Phenolic Content and Antioxidant and Antiradical Activities of Eremurus spectabilis Extracts Obtained by Using Different Solvents

Solvent type	Total phenolic content (mg GAE/g)	Antiradical activity (IC₅₀) (μg/mL)	Antioxidant activity (mg AAE/g)
Methanol	31.92±0.48	49.90±0.44	81.72±0.62
Ethanol	13.75±0.14	35.14±0.21	12.53±0.47
Aqueous	7.15±0.42	80.18±0.18	21.01±0.54

Values in the first and third columns are expressed as the mean±standard deviation of 3 experiments.

AAE, ascorbic acid equivalents; GAE, gallic acid equivalents; IC₅₀, 50% inhibitory concentration.

The free radical–scavenging activity of E. spectabilis extracts was determined by using the DPPH method, and results are given in Table 2. The IC₅₀ (μg/mL) value of the ethanol extract was lower than that of the other extracts. As the IC₅₀ value of the extract decreases, the free radical–scavenging activity increases. Thus, the best antiradical activity was observed in ethanol extract of E. spectabilis (IC₅₀, 35.14 μg/mL). Mavi et al.²⁵ reported that free radical–scavenging activities of methanol extract of Urtica dioica L. and Malva neglecta (which are commonly consumed as tea or as a meal in Turkish folk medicine) were 1012 and 2242 mg/L, respectively. It is clear that E. spectabilis has strong antiradical activity compared with U. dioica L. and Malva neglecta. In another study of P. oleracea, antiradical activity was determined to be 1.19–1.72 mg/mL depending on the collection dates.²³ El-Agbar et al.²⁶ found that IC₅₀ values of methanol extracts of Thymus vulgaris, Nigella sativa, and Matricaria chamomilla (all edible plants) were 54.2, 168.8, and 65.8 μg/mL, respectively. According to these results, E. spectabilis has important free radical–scavenging activity compared with several wild plants.

Phosphomolybdenum assay is a simple test that can be done independently of other antioxidant assays. Prieto et al.¹⁶ reported that this assay is an alternative method for evaluating total antioxidant capacity of plant species. Thus, we used both DPPH and phosphomolybdenum assays in the present study. As illustrated in Table 2, the highest antioxidant activity was detected in the methanol extract of E. spectabilis (81.72±0.62 mg ascorbic acid equivalents [AAE]/g), and the lowest was found in ethanol extract (12.53±0.47 mg AAE/g). Aqueous extracts were approximately twice as effective as ethanol extracts. Zengin et al.²⁷ reported that total antioxidant capacities of methanol extracts of Centaurea patula, C. pulchella, and C. tchihatcheffi (plants used in traditional folk medicine) were 43.80, 67.89, and 41.30 mg AAE/g, respectively. Thus, it can be said that methanol extract of E. spectabilis had strong antioxidant capacity compared with several other plants. No relationship was seen between antioxidant and antiradical activity.

As noted in Table 2, the ethanol extract showed the highest antiradical activity but the lowest antioxidant activity. This finding might be attributable to the high content of water-soluble compounds. To support this theory, Simirnova et al.²⁸ reported that the genus Eremurus has high levels of water-soluble glucomannan polysaccharides and that these glucomannans are bioactive additives in the food and medicinal forms of these species. Antioxidant and antiradical activities do not depend on total phenolic content of the sample. In our study, total phenolic content of the ethanol extract was higher than that of aqueous extract but the antioxidant activity was lower (Table 2). Albayrak et al.⁷ observed a similar tendency. They found no correlation between total phenolic content and antioxidant activity. For example, total phenolic content and antioxidant activity of Helichrysum artvinense and H. chionophilum were 83.98 and 106.97 mg GAE/g extract and 171.02 and 140.43 mg AAE/g extract, respectively. The lack of correlation between them is clear.

Prior et al.²⁹ reported that the Folin–Ciocelteu procedure provides only an estimate of the total phenolic compounds in the extract; it cannot establish a definitive measure because it is not specific to polyphenols, and many interfering compounds may react with the reagent. As a result, the phenolic concentrations may be inaccurately elevated with this method. In addition, the responses of various phenolic compounds differ from each other with this method depending on the number of phenolic groups. Thus, phenolic compounds that do not incorporate all the antioxidants may be present in an extract.²⁷

We used the agar diffusion method to determine antimicrobial activities of extracts. Table 3 summarizes these activities at 1%, 2%, 5%, and 10% concentrations against the test microorganisms. As was seen with antioxidant activity, the extract most effective against some bacteria was the aqueous extract. This finding might also be attributable to the high content of water-soluble bioactive compounds.²⁸ Hajji et al.³⁰ investigated antioxidant and antimicrobial activities of various solvent extracts from Mirabilis jalapa tubers and found that the aqueous extract showed important antibacterial activity against Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, K. pneumoniae, B. cereus, and Enterococcus faecalis compared with methanol extracts. The inhibition zone clearly increased with increasing of extract concentration, as expected.

Table 3.

Antibacterial Activities of Eremurus spectabilis Extract Obtained by Using Different Solvent and Percentage Concentrations

	Methanol				Ethanol				Aqueous
Microorganisms	1%	2%	5%	10%	1%	2%	5%	10%	1%	2%	5%	10%
Aeromonas hydrophila	–	–	–	8.5±0.5	–	–	–	10±0.0	–	–	–	–
Bacillus brevis	–	–	–	–	–	–	–	12±0.0	–	–	–	–
B. cereus	–	–	–	8.5±0.5	–	–	7.5±0.5	12±0.0	–	–	–	–
B. subtilis	–	–	7.5±0.5	9.5±0.5	–	–	–	9.5±0.5	–	–	–	–
Escherichia coli O157	–	–	–	–	–	–	–	–	–	–	–	18±1.0
E. coli O157:H7	–	–	–	8±0.0	–	–	–	8.5±0.5	–	–	–	18.5±0.5
Klebsiella pneumoniae	–	–	–	13.5±0.5	–	–	–	–	–	10.5±0.5	13±1.0	16±1.0
Listeria monocytogenes	–	–	–	8±0.0	–	–	–	11.5±0.5	–	–	–	14±1.0
Pseudomonas aeruginosa	–	–	–	24.5±0.5	–	–	–	–	–	12.5±0.5	16.5±0.5	20±1.0
Salmonella typhimurium	–	–	–	–	–	–	–	11.5±0.5	–	–	–	–
Staphylococcus aureus	–	–	–	–	–	–	–	–	–	7.5±0.5	13±0.0	16.5±0.5
Yersinia enterocolitica	–	–	–	–	–	–	–	–	–	–	–	–

Values are expressed as mean±standard deviation inhibition holes in millimeters. Includes diameter of hole (4 mm). Dashes indicate that extract was ineffective.

The 1% concentrations of all extracts were not effective any bacteria species. The 2% concentrations of methanol and ethanol extracts showed no inhibitive effect on any bacteria, and the 2% concentrations of aqueous extract were effective on Staphylococcus aureus, Pseudomonas aeruginosa, and K. pneumoniae. The highest inhibitory effect of 2% aqueous extract was found on Pseudomonas aeruginosa, with the highest inhibition zone (12.5 mm), and the lowest was in Staphylococcus aureus, with the lowest inhibition zone (7.5 mm). Whereas the 5% concentrations of extracts generally seemed inadequate, the 10% concentrations showed the best antimicrobial activity for almost each microorganism. The 5% methanol extract showed an inhibitory effect on B. subtilis, and the ethanol extract of the same concentration was effective on B. cereus. However, only Staphylococcus aureus and E. coli O157:H7 were not inhibited by any concentration of methanol or ethanol extract; Y. enterocolitica was not inhibited by any concentration of E. spectabilis extracts. The most sensitive bacterium against this plant was Pseudomonas aeruginosa in all extracts; the highest inhibition zone was determined in 10% methanol extract for this organism (24.5 mm) (Table 3).

Albayrak et al.⁷ investigated the antimicrobial activity of methanol extracts of 16 different Helichrysum species and reported that the most resistance bacterium was Y. enterocolitica. Only 1 species showed low antibacterial effect, with 6 and 7 mm for 5% and 10% concentration of extract, respectively. Ozkan et al.³¹ reported that the bacterium most resistant to grape pomace extracts was Y. enterocolitica. Several researchers reported similar observations.^22,32,33 Several research reports indicated that Gram-negative bacteria are more resistant to different antimicrobial agents compared with Gram-positive organisms because of the lipopolysaccharide membranes in their cell wall structures.^18,34
–36 In contrast to these reports, we found no definite correlation. For example, although Pseudomonas aeruginosa is a Gram-negative bacterium, the greatest inhibition zone diameter for this bacterium was seen in 10% methanol extract of E. spectabilis. In addition, the Gram-positive organisms L. monocytogenes and B. cereus showed important resistance to 10% methanol extract of E. spectabilis compared with Pseudomonas aeruginosa.

Conclusion

E. spectabilis is an uncultivated, commonly consumed wild vegetable. Our results indicate that E. spectabilis can be used to protect against oxidation and bacterial infection in food industry and pharmacology. In general, aqueous extracts showed important antibacterial activity. As a result of their strong antioxidant and antiradical activity, the extracts can be considered a natural source of antioxidants for improving human health. A positive relationship was seen between antibacterial activity and volatile compounds, as determined by using gas chromatography/mass spectometry.

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