Lianying Fang, Xiangxing Wang, Limin Guo and Qiang Liu

Antioxidant, Anti-microbial Properties and Chemical Composition of Cumin Essential Oils Extracted by Three Methods

De Gruyter | 2018


The purpose of this study is to evaluate the chemical composition, antioxidant and anti-bacterial activity of cumin essential oils (CEOs) extracted by different techniques, including supercritical carbon dioxide extraction (SCE), subcritical butane extraction (SBE) and traditional solvent extraction (SE). Our results indicated that CEOs are a valuable source of bioactive compounds, including cumin aldehyde, γ-terpinene and β-pinene. The most abundant components found in CEOs obtained by SCE and SBE were similar, while the abundant components in SE, β-Cumic aldehyde (19.31%) and α-phellandrene (9.49%), were distinctive. CEOs obtained by SCE exhibited higher antioxidant activity, followed by those extracted by SE and SBE. Moreover, the anti-microbial properties of CEOs obtained by SCE and SBE were higher than that of CEOs collected by SE. In conclusion, CEOs exhibit strong antioxidant and anti-microbial properties, which suggests a potential role of CEOs in preventing diseases associated with aging and oxidative stress, and our results highlight the potential usage of CEOs in the food industry.

1 Introduction

Cumin, an important commercial seed spice belonging to the umbelliferae family, is native to south and southwest Asia [1], and it is particularly abundant in the Xinjiang region of China. Cumin is widely consumed throughout the world due to its pleasant aroma [2,3]. The seeds of the cumin plant are used extensively as a spice for flavoring and preserving food, especially in bakery products and cheese [4]. Due to its bioactive constituents, cumin possesses multiple biological functions that are important in the use of traditional Chinese herbs for preventing chronic diseases, such as blood hyperviscosity, hypertension, high cholesterol and arteriosclerosis [5,6,7]. Moreover, cumin essential oils (CEOs) have been shown to have anti-diabetic, antimicrobial, antiseptic and antioxidant properties, and the oils can inhibit blood platelet aggregation [8,9,10].

CEOs are an aromatic oily liquad obtained from cumin seeds via various extraction techniques [11]. The most commonly used techniques to extract and purify essential oils include solvent extraction (SE), supercritical CO2 extraction (SCE), and subcritical butane extraction (SBE). Previous research has investigated the chemical compositions and bioactive functions of CEOs [10]. However, few studies have focused on the identification of bioactive compounds and the potential anti-microbial and antioxidant effects of extracts from Xinjiang cumin. A complete understanding of CEOs function requires further analyses of antioxidant and anti-microbial activities and the bioactive compound content of CEOs obtained by different extraction methods. Our results provide insight into the potential impact of extraction techniques on the chemical composition and biological function of CEOs, and they suggest that the choice of extraction method is critical in order to obtain the desired composition in a CEOs product.

2 Experimental

2.1 Chemicals and strains

Chemicals of analytical grade, such as 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH), butylated hydroxytoluene (BHT), vitamin C (Vc), crystal violet, ferrous sulfate and 2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), were obtained from Solarbio Chemical (Beijing, China). The bacterial strains, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa, were preserved at the College of Biotechnology and Food Science, Tianjin University of Commerce. Saccharomyces cerevisiae (BY4741) was purchased on Euroscarf. The bacterial strains were stored as glycerol stocks at -80°C.

2.2 Preparation of CEOs

Samples of cumin were obtained from Xinjiang China. Extracts were prepared from the seeds of cumin. In brief, ground cumin seeds were clarified through a 40 mesh filter, followed by supercritical CO2 extraction, subcritical butane extraction or solvent extraction. For SCE, a laboratory scale extractor was employed under the following conditions: temperature of 50°C, pressure of 35 MPa, CO2 flow rate of 25 L/h and extraction time of 4 hours. When 30g of cumin sample was loaded into the extraction vessel, the average yield from SCE was 13.51%. For SBE, 30 g of sample was added into the extractor with 300mL butane on a laboratory scale. The extraction was performed under a pressure of 0.4-0.6MPa at 50°C for 1.5 hours, and it resulted in a yield of 17.01%. For SE, the extraction process was performed with n-hexane as the extracting solvent and using a pressure of 0.05-0.08 MPa and a temperature of 65°C. In brief, 30 g of the cumin sample was soaked in 150 mL solvent, and the extraction was performed for 3 hours with regular stirring. The yield of SE was 10.12%. The yield of cumin oil was expressed as g of oil per 100 g of cumin seed. The distillate oil was collected and stored in dark sealed vials at 4°C prior to characterization of their chemical composition.

2.3 Chemical content analysis of CEOs

The total chemical content of the extracts was quantified using Gas chromatography/mass spectrometry (GC/MS) as described by Anastasia et al [13]. GC/MS was carried out in a GC/MS-QP2010 (Shimadzu Corporation, Kyoto, Japan) with a DB-5MS capillary column (30.0 m×0.25 mm×0.25 μm). The carrier gas helium was delivered at a constant flow of 1.04 mL/min, and the interface temperature was 265°C, the split ratio was 1:10, and the injection volume was 8 μL. The column oven temperature program was set to start at 60°C, then gradually increase from 60°C to 240°C with a final hold of 13 min at 240°C. The scanning range of mass spectrometer ionization was 35-500 m/z. The identification of the components was carried out using Xcalibur® software, NIST Mass Spectral Library, and MS literature data. Retention indices (RIs) of the compounds were determined relative to the retention times of a standard solution of n-alkanes for GC (C8–C20). The relative percent of individual components was calculated based on the GC peak areas without the use of correction factors.

2.4 Determination of antioxidant activity

2.4.1 DPPH scavenging assay

The ability of CEOs to scavenge DPPH was assessed using the method described by Yen et al. with some modifications [14]. The CEOs were diluted with ethyl alcohol to the tested concentrations of 1, 2, 4 and 8 μg/ mL. Vitamin C and butylated hydroxytoluene (BHT) were used as reference compounds. CEOs (2.0mL) were treated with 2.0 mL of DPPH solution for 30 min in the dark. The blank sample consisted of 2.0 mL ethyl alcohol and 2.0 mL DPPH solution at a concentration of 1.0×10–4 mol/L. The absorbance of the reaction mixture was evaluated at 517 nm. Radical scavenging activity was expressed as an inhibition percentage and was calculated using the following formula: Scavenging activity (%) ={1-(A-A0)/ A1×100%, where A0 was the absorbance of blank, A was the absorbance in the presence of CEOs of different concentrations, and A1 was the absorbance of CEOs in ethyl alcohol.

2.4.2 ABTS•+ scavenging assay

The ability of CEOs to scavenge ABTS was measured using the method described by Re et al. with some modifications [15]. In brief, ABTS•+ was produced by reacting 2 mmol/L ABTS in aqueous solution with 70 mmol/L potassium persulfatein (K2S2O8) in the dark for 12-16 h at room temperature. Prior to the assay, the solution was diluted in PBS to achieve an absorbance of 0.70±0.02 at 734 nm. Then, the CEOs were diluted in ethyl alcohol to concentrations of 2, 4, 6 and 8 μg/mL. Subsequently, 0.1 mL of CEOs were treated with 1.9 mL ABTS•+ solution for 30 min at room temperature in the dark. The absorbance was measured at 734 nm. Radical scavenging activity was expressed as inhibition percentage and was calculated using the following formula: Scavenging activity (%) = (A0-A)/A0×100%. A0 was the absorbance of blank, A was the absorbance in the presence of CEOs of different concentrations.

2.4.3 Hydroxyl scavenging assay

The scavenging activity for hydroxyl radicals was measured according to the method described by Smirnoff with slight modifications [16]. In brief, CEOs were diluted with ethyl alcohol to final concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 μg/mL. Vitamin C and BHT were used as reference compounds. The reaction mixture contained 1.2 mL of 1.0 mmol/L FeSO4, 0.3 mL of 0.4 mmol/L crystal violet, 2.5 mL of phosphate buffer (pH 7.8), 0.6 mL of 2.0 mmol/L H2O2 and 0.4 mL of a CEO solution. The reaction was initiated with the addition of H2O2. After incubation at room temperature for 5 min, the absorbance of the mixture was measured at 580 nm. Hydroxyl radical scavenging activity was expressed as a percentage, using the formula: (A1–A0)/A1)×100, where A0 is the absorbance of the control and A1 is the absorbance in the presence of the tested compound.

2.4.4 Ferric reducing antioxidant power (FRAP) assay

The reduction capacities of different CEOs were estimated according to the procedure described by Amarowicz with minor modifications [17]. In brief, CEOs was diluted with ethyl alcohol concentrations of 5, 10, 20 and 40 μg/mL. Vitamin C and BHT were used as reference compounds. The FRAP solution consisted of 1% K3Fe(CN)6, 0.2 mol/L PBS, and the tested sample solution at a ratio of 10:1:1 (v/v), respectively. The FRAP solution was incubated for 20 min at 50°C. Subsequently, 2.5 ml of 10% trichloroacetic acid was added to each FRAP solution. The supernatant was obtained after centrifugation at 3000 r/min for 10 min. The final tested solution consisted of the supernatant solution, distilled water and 0.1% FeCl3 at the ratio of 5:5:1 (v/v). The absorbance was measured at 700 nm after incubation at room temperature for 10 min.

2.5 Determination of antimicrobial activity

Antimicrobial activity of CEOs was assayed using the microorganisms Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae (BY4741). Bacteria were cultured and maintained on beef extract peptone medium, and yeast were cultured on solid 2% YPD (1% yeast extract, 2% glucose, 2% peptone, and 2% agar).

Antimicrobial activity was measured using the filter paper dispersion method as described by Cosentino with slight modifications [18]. Briefly, CEOs were diluted with ethyl alcohol to concentrations of 20 and 40 μg/ mL. Gentamicin (40 μg/mL in saline solution) was used as a positive control. Ten millimeter filter paper discs were soaked with 10 μL of cumin extract stock solution of different doses and then air-dried at room temperature. The test microorganisms (105 bacteria or yeast cells/mL) were seeded onto the respective medium containing the filter paper discs. Measurement of the zone of inhibition was taken after 24 h at 37°C (bacterial) or 48 h at 28°C (yeast).

2.6 Statistical analysis

Each experiment was performed at least three times, and results are shown as means± SEM. Ethical approval: The conducted research is not related to either human or animals use.

3 Results and discussion

3.1 Comparative analysis of CEO components

The essential oil samples prepared by SCE, SBE and SE were analyzed using GC/MS. Chemical compositions were determined based on their retention index values and mass spectra. The chemical compositions of the essential oils are shown in Table1. GC/MS results indicated that CEOs represented more than 90% of the total oil. The main compounds identified were monoterpene hydrocarbon, sesquiterpene hydrocarbon, oxygenated monoterpene, oxygenated sesquiterpenoid and aromatic compounds. Some variations in the compositions of CEOs from different extraction methods were observed.

Table 1

Chemical composition of essential oils extracted from cumin seeds by 3 methods as determined by GC/MS.

NO Compounds Relative percent content%
1 α-Pinene 0.49 0.35 0.27
2 2,2-Dimethyl-3-methylenebicyclo heptane - - 0.56
3 β-Pinene 10.76 8.21 0.04
4 α-Phellandrene - - 9.49
5 o-Isopropyltoluene 3.52 2.46 0.01
6 trans-Sabinene hydrate - 0.32 3.67
7 D-Limonene 0.3 0.18 0.06
8 β-Phellandrene 1.2 0.52 0.4
9 (+)-4-Carene - - 8.92
10 γ-Terpinene 11.51 9.09 3.04
11 2-methyl-5-(1-methylethyl)-cyclohexanon 0.34 - -
12 Limonene epoxide 0.34 - 3.41
13 Phellandral 1.7 1.62 1.02
14 1,3,4-Trimethyl-3-cyclohexenyl-1-carboxaldehyde - - 1.71
15 Pterin-6-carboxylic acid 2.18 0.04 -
16 β-Cumic aldehyde 25.49 22.17 19.13
17 1-Phenyl-1-hexanol 23.38 21.96 1.32
18 2-(Methylsulfanyl)-5-pyrimidinol - - 0.49
19 β-Mentha-1,4-dien-7-ol 0.74 0.84 -
20 γ-Cadinene 0.3 0.26 0.24
21 Caryophyllene 0.26 - 0.21
22 β-(Z)-Farnesene 0.49 0.47 0.44
23 Cedr-8-ene 0.7 0.59 -
24 2,2,4,4,7,7-Hexamethyl-2,3,3a,4,7,7a-hexahydro-1H-indene 0.71 0.66
25 Hexadecanoic acid - 0.5 -
26 Carotol 0.19 0.2 -
27 Acetic acid 0.32 0.12 -
26 2-Butenoic acid - 0.21 0.91
29 (E)-β-Ionone 0.93 0.24 -
30 Dodecylcyclobutanone - 0.44 0.25
31 Octadeamethyl-cyclononasiloxane 1.31 0.36 -
32 1-Cyclohexene-1-carbaldehyde 0.63 0.69 0.23
33 (Z)-11-Hexadecenal - 2.51 9.71
34 Sarverogenin - 0.36 -
35 Lineoleoyl chloride 0.3 0.16 0.3
36 cis-13-Octadecenal 2.23 2.51 1.13
37 (2E)-3-2-propenoic acid 0.59 0.17 0.15
38 Tricyclo[,8)] undecan-1-amine 0.17 0.23 0.11
39 13-Octadecenal 0.17 1.34 -
40 N-(1-Cyclohexylethyl) acrylamide 0.1 - -
41 2-Methyl-Z, Z-3,13-octadecadienol 0.2 0.57 -
42 (Z)-9-Tetradecenal 0.75 0.17 -
43 cis-9-Hexadecenal - 3.9 -
44 2-(Acetyloxy)-1-[(acetylox methyl]ethyly) 0.11 - -
45 14-Methyl-8-hexadecyn-1-ol - 2.12 2.82
46 2,3-Bis[(9E)-9-octadecenoyloxy] propyl(9E)-9-octadecenoate - 5.96 -
47 9-Octadecenoic acid - - 7.1
48 9,12,15-Octadecatrienoic acid - 0.69 -
49 Tricyclo triacontane - - 1.08
50 Tetracosamethyl-cyclododecasiloxane 1.31 1.63 -
51 Heptadecyl cyclohexanecarboxylate - - 1.73
52 9-Methyl-Z-10-tetradecen 1-ol acetate 0.9 - -

In this study, CEOs from SCE and SBE contained similar major components, while the most abundant components in SE were β-Cumic aldehyde (19.13%) and α-Phellandrene (9.49%). The main contents of CEOs extracted by SCE were β-Cumic aldehyde (25.49%), γ-Terpinene (11.51%), and β-Pinene (10.76%). Similarly, the main components of CEOs obtained by SBE were β-Cumic aldehyde (22.17%), γ-Terpinene (9.09%) and β-Pinene (8.21%). Overall, the levels of β-Cumic aldehyde were highest, with an average of 22.26%, across CEOs from all three extraction methods. The levels of β-Pinene extracted were different among the methods, with SCE extracts containing the highest level of β-Pinene (10.76%), followed by extracts from SBE (8.21%) and SE (0.04%). In addition, SE extract contained the highest concentration of 4-Carene (8.92%). These findings indicated that SCE and SBE are the preferred methods for extracting β-Pinene and γ-Terpinene, while SE is more appropriate for the extraction of α-Phellandrene, 4-Carene and (Z)-11-Hexadecenal. Taken together, we conclud that different extraction methods lead to variations in the compositions of extracted essential oils.

3.2 Antioxidant capacity of CEOs

Next, we evaluated whether the antioxidant capacity of CEOs could be affected by the extraction technique utilized. The antioxidant activities of CEOs were determined by DPPH, OH, and ABTS free radical scavenging activity assays and the Ferric reducing antioxidant power assay.

DPPH, OH, and ABTS free radical scavenging activity assays and the Ferric reducing antioxidant power assay. The DPPH scavenging activities of the tested CEOs were observed at concentrations of 1-8μg/mL. All of the CEOs, regardless of extraction method, showed DPPH scavenging capacity that increased in a concentration dependent manner. As shown in Figure 1-B, all of the CEOs, including the reference antioxidants butylated hydroxytoluene (BHT) and Vitamin C (Vc), exhibited intense DPPH radical scavenging activity at the concentration of 2 μg/mL. Vc was the most effective scavenger, followed by BHT. The SCE and SBE extracts had roughly equivalent activities, while the SE sample was the least potent in terms of scavenging activity.

Figure 1 DPPH radical scavenging activities of (A) CEOs and (B) compared with Vc and BHT at the concentration of 2μg/mL. Each value is expressed as mean ± SEM.

Figure 1

DPPH radical scavenging activities of (A) CEOs and (B) compared with Vc and BHT at the concentration of 2μg/mL. Each value is expressed as mean ± SEM.

As shown in Figure 2, the CEOs exhibited significant ABTS•+ scavenging capacity, with CEOs from SBE demonstrating higher scavenging activity than those extracted by SCE and SE. At 2 μg/mL, the reference antioxidants BHT and Vc demonstrated the highest ABTS•+ scavenging activities. The SCE sample demonstrated the highest activity among CEOs, followed by the SBE sample and then the SE sample.

Figure 2 ABTS•+ radical scavenging activities of (A) CEOs and (B) compared with Vc and BHT at the concentration of 2μg/mL. Each value is expressed as mean ± SEM.

Figure 2

ABTS•+ radical scavenging activities of (A) CEOs and (B) compared with Vc and BHT at the concentration of 2μg/mL. Each value is expressed as mean ± SEM.

As shown in Figure 3, the OH scavenging activity of the CEOs increased in a concentration dependent manner, especially in CEOs extracted by SCE, which showed the strongest antioxidant capability compared to the other extracts. At 0.5 μg/ml, Vc was the most potent OH scavenger, but the SCE sample was more potent than the reference antioxidant BHT. The SBE sample was more potent than the SE sample under these conditions. The scavenging abilities of the SCE, SBE, and SE CEOs were 98.6%, 44.5%, and 33%, respectively.

Figure 3 OH radical scavenging activities of CEOs, Vc and BHT at the concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 μg/mL. Each value is expressed as mean ± SEM.

Figure 3

OH radical scavenging activities of CEOs, Vc and BHT at the concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 μg/mL. Each value is expressed as mean ± SEM.

As shown in Figure 4, all the CEOs exhibited reducing power compared to the reference antioxidants Vc and BHT. Based on absorbance at 700 nm, the CEOs obtained by SCE showed higher reducing power than those of the SE and SBE extracts. At the treatment concentration of 40 μg/mL, the descending order of reducing power was as follows: Vc>SCE>BHT>SBE>SE.

Figure 4 Reducing power of CEOs, Vc and BHT at the concentrations of 5, 10, 20, 40 μg/mL. Each value is expressed as mean ± SEM.

Figure 4

Reducing power of CEOs, Vc and BHT at the concentrations of 5, 10, 20, 40 μg/mL. Each value is expressed as mean ± SEM.

Among the tested examples, the SCE extract exhibited the strongest antioxidant activity, followed by the SBE and SE extracts. CEOs extracted by SCE, SBE and SE all exhibited antioxidant capacity, but the quantitative evaluation of this activity showed that variations exist in the antioxidant potencies among the extracted CEOs, which could be attributed to the differences in the main chemical compositions of the CEOs.

3.3 Antimicrobial activities of CEOs

The antimicrobial activities of CEOs were measured based on the diameter of the inhibition zone in the disc-diffusion test. As shown in table 2, all extracts exhibited different levels of antibacterial activity against gram negative bacteria, gram positive bacteria and fungi. In general, the CEOs extracted by SCE and SBE showed higher antimicrobial activity than that of SE, except in the case of Bacillus subtilis, for which the diameter of inhibition zone was largest when treated with SE extract (26.9±2.3 mm at the concentration of 40 μg/mL). Among the tested bacterial strains, Bacillus subtilis was the most sensitive to CEOs. Treatment of this organism with 40 μg/mL CEOs for 24 h resulted in inhibition zone diameters of 23.6±2.7 mm (SCE), 22.7±1.6 mm (SBE) and 26.9±2.3 mm (SE), respectively. On the other hand, Staphylococcus aureus was the most resistant strain, with inhibition zones of 12–15 mm in diameter after treatment with CEOs for 24 h. Gentamicin (40 μg/mL in saline solution) was used as a positive control. Treatment with 40 μg/mL CEOs for 24h resulted in inhibition zone diameters that were lower than that of the standard antibiotic gentamycin. Moreover, the CEOs extracted by SE exhibited the best inhibitory activity against Bacillus subtilis; the SE zone was close in size to that of gentamycin. Our results demonstrated the antibacterial and antifungal activities of the CEOs, which could be attributed to the volatile oil substances present in the extracts. The inhibitory effects of volatile compounds include adsorption to cell membranes, interaction with enzymes and substrates, and deprivation of metal ions [12].

Table 2

Antimicrobial activities of essential oils extracted by 3 methods from cumin.

Strains 20 μg/mL SCE SBE SE 40 μg/mL SCE SBE SE 40 μg/mL Gentamicin
Staphylococcus aureus 14.9±0.9 15.4±2.5 12.7±0.3 17.3±0.3 17.3±1.3 14.6±2.9 22.49±5.9
Escherichia coli 17.6±0.7 17.4±0.6 14.4±1.5 19.0±1.19 16.3±0.9 15.4±3.5 23.67±2.3
Bacillus subtilis 16.2±1.5 17.6±1.7 20.4±0.8 23.6±2.7 22.7±1.6 26.9±2.3 25.32±4.7
Pseudo monas 15.6±1.3 15.5±2.7 14.1±1.0 21.2±0.9 18.3±3.1 17.6±2.5 21.28±3.3
Saccharomyces cerevisiae 17.6±0.6 19.6±3.74 18.4±0.7 22.4±0.6 23.3±4.8 20.8±2.8 24.21±6.6

4 Conclusions

Collectively, the results presented in our study confirmed that chemical composition, antioxidant activity and antimicrobial properties of CEOs are affected by extraction methods. Our findings also indicated that SCE and SBE were the ideal methods for extraction of β-Pinene and γ-Terpinene, and n-hexane was the appropriate solvent for the extraction of α-Phellandrene, 4-Carene and (Z)-11-Hexadecenal. Knowledge of these conditions are important for future studies of the extraction and isolation of different chemical compounds from CEOs. In addition, our results suggested that the extraction technology significantly affects biological activities of CEOs. Among the tested CEOs, SCE was the most effective extraction for generating extracts with greater antioxidant and antibacterial activity. The efficacy of these extracts against bacteria could in part be attributed to their volatile oil content.

CEOs demonstrate great economic, medicinal, and nutritional values due to their wide-spectrum biological activities. For examples, CEOs could be utilized in the food and pharmaceutical industries, or as a therapeutic and preventive agent against a variety of diseases. However, the complexity of the chemical compositions of CEOs might have contributed to the variations in biological activities we observed. Therefore, further investigations are necessary to explore the biological activities of CEOs in vivo and to identify the molecular mechanisms behind these biological activities.


This study was supported by the National Natural Science Foundation of China (31670859), Fundamental Research Funds for CAMS&PUMC (2016ZX310198), PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (3332016100, 10023201601602), Research Funds for the Innovation Team of IRM-CAMS (1650), Pre-research Special Grant in key Projects of Xinjiang Academy of Agricultural Sciences (No.xjzdy-2017-005).


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