Characterization of Antioxidants in Nutmeg (Myristica fragrans

Mar 18, 2010 - 2 Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA ... Lanka often produce nutmeg oils in 10-15% of ...
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Characterization of Antioxidants in Nutmeg (Myristica fragrans Houttuyn) Oil H. J. Kim,1,4 F. Chen,*,1 X. Wang,2 Y. Wang,1 J. McGregor,1 and Y. M. Jiang3 1Department

of Food Science and Human Nutrition, of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA 3South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P. R. China 4Food Convergence Technology Division, Korea Food Research Institute, Bundang, Sungnam, Gyeonggi 463746, Korea *[email protected] 2Department

Antioxidant capacity of nutmeg (Myristica fragrans Houttuyn) oil was investigated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical scavenging assay and the β-carotenelinoleic acid assay. The antioxidant EC50 values of the crude nutmeg oil dissolved in methanol were 2.4 μL/mL and 0.4 μL/mL, respectively. The former value was approximately equivalent to the free radical scavenging capacities of 462 μM BHT and 656 μM α-tocopherol, and the latter one was comparable to the inhibitive capacities of 43 μM BHT and 9 μM α-tocopherol against the oxidation of β-carotene and linoleic acid. Further investigations discovered three major antioxidant constituents of the nutmeg oil (i.e., eugenol, isoeugenol, and methoxyeugenol), which were sequentially separated and identified by silica open column chromatography, HPLC and GC-MS. Their antioxidant activities in the DPPH• assay decreased in the following order: eugenol > methoxyeugenol > BHT > isoeugenol > α–tocopherol, while in the β-carotene-linoleic acid assay, the antioxidant activities of the chemicals were in the following order: α–tocopherol > BHT > isoeugenol > methoxyeugenol > eugenol. © 2010 American Chemical Society In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Introduction Essential oils extracted from various herbs and spices have been widely used throughout history as cosmetics and aromatherapeutic agents as well as flavoring agents in food (1). Since it has been discovered that essential oils have many other biological properties such as antimicrobial, antioxidant, and anticancer activities, research on bioactive principles of essential oils has become increasingly popular (1–4). Nowadays many research groups are focusing their investigations on the pharmacological actions of essential oils from aromatic and medicinal plants (3, 5, 6). Among them, nutmeg (Myristica fragrans Houttuyn) oil is one of the most intensively studied objects because of its wide utilization in food and pharmaceutical industries (7–9). Nutmeg oil, which is commercially obtained by steam distillation from kernels of nutmeg, usually contains more than 50 components. Nutmegs cultivated in tropical countries such as Indonesia and Sri Lanka often produce nutmeg oils in 1015% of the seed weight (10, 11). Regardless of some concerns about the toxicity of myristicin, elemicin, and safrole in nutmeg (12, 13), nutmeg powder and its essential oil are still commonly and widely used as the spicy and flavoring agents in foods, and as medicinal agents (7–9, 14, 15). Although nutmeg oil has been reported with various biological properties such as antimicrobial, antibacterial, antiplatelet, hepatoprotective, and hypolipidaemic effects (8, 9, 16, 17), its antioxidant capacity has not been investigated thoroughly (18–21), let only the characterization of its inherent antioxidant constituents. Therefore, this study aimed to reveal the antioxidant capacity of nutmeg oil and its antioxidant components that were hypothesized to be linked with nutmeg’s biofunctional activities. In this research, components with strong antioxidant activity were separated from the crude nutmeg oil through chromatographic techniques, and identified by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). In addition, the antioxidant capacities of nutmeg oil and its identified major constituents were assessed by two in vitro assays: DPPH• free radical scavenging assay and β-carotene-linoleic acid assay.

Experimental Materials 2,2-Diphenyl-1-picrylhydrazyl (DPPH•) and α-tocopherol were purchased from Sigma Chemical Co. (St. Louis, MO). Eugenol (4-allyl-2-methoxy-phenol), isoeugenol (2-methoxy-4-propenyl-phenol), methoxyeugenol, myristicin, and silica gel (70-230 mesh, 60Å) were purhased from Aldrich Chemical Co. (Milwakee, WI). Nutmeg oil, linoleic acid, β-carotene, and butylated hydroxytoluene (BHT) were obtained from the Good Scents Co. (Oak Creek, WI), TCI (Portland, OR), Fluka Chemical Co (Milwakee, WI), and Acros (NJ),

240 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

respectively. Tween 20 and all HPLC analytical grade solvents were from Fisher Scientific (Suwanee, GA).

Separation and Identification of Antioxidants from Nutmeg Oil

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Separation by Silica Gel Open Column Chromatography Six milliliters of the crude nutmeg oil dissolved in hexane was loaded on a column (60 cm × 2.5 cm) packed with silica gel (70-230 mesh, 60Å) and equilibrated with hexane. Then the sample was eluted by solvents sequentially in the following order: hexane (200 mL), hexane/DCM (1:1, 200 ml), DCM (200 mL), and DCM/methanol (1:1, 200 mL). The flow rate was controlled at 4 mL/min. Each collected fraction was in size of 7 mL.

Separation by Normal-Phase HPLC The Spherisorb silica column (250 mm × 4.6 mm, 5 μm; Waters, MA, USA) was installed on a LC-10AT HPLC system (Shimadzu, Kyoto, Japan) and equilibrated with hexane. Fifty microliters of each fraction separated by the silica gel open column was injected into the HPLC column, and eluted with a linear gradient of a mixture solvent of DCM/methanol (1:1) at a flow rate of 1 mL/min. The absorbance of the eluant was scanned from 200 to 500 nm by a SPD-M10V photodiode array detector (Shimadzu, Kyoto, Japan).

Chemical Identification by Gas Chromatography−Mass Spectrometer (GC/MS) To further separate and identify antioxidants from silica-HPLC, GC-MS (GC17A-QP 5050 MS) system (Shimadzu, Kyoto, Japan) installed with a DB-5 capillary column (60 m × 0.25 mm, thickness 0.25 μm; J&W Scientific, Folsom, CA, USA) was used. The oven temperature was programmed from 60°C to 280°C at 10°C/min and held at 280°C for 7 min. The injector and ion source temperatures were 220°C and 290°C, respectively. The detector voltage was 70 eV and the MS spectra were recorded in the mass range of m/z 43-350. Helium was used as a carrier gas and its flow rate was 1.1 mL/min. The injection volume was 1 μL and a split rate was 1:5. Identification of compounds was based on comparison with mass spectra and retention index (RI) of the authentic standards. Mass spectrum of each target compound was also compared with that of Wiley and NIST mass spectral databases.

241 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Determination of Antioxidant Activity The antioxidant capacities of nutmeg oil and its antioxidant components were evaluated by two methods: the DPPH• free radical scavenging assay and the βcarotene-linoleic acid assay. Standard antioxidants, BHT and α-tocopherol, were used as the controls.

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DPPH• Free Radical Scavenging Assay Scavenging activities on DPPH• free radicals by the crude nutmeg oil and its components were determined according to the method of Yamaguchi et al. (22) with minor modification. The reaction mixture containing 0.1 mL of sample, 0.3 mL of 0.1 M Tris-HCl (pH 7.4), 0.1 mL of methanol, and 0.5 mL of 0.3 mM DPPH• was vigorously shaken and incubated in the darkness at room temperature for 10 min. After incubation, the absorbance of the reaction mixture was spectrophotometrically measured at 517 nm and the scavenging activity of DPPH• free radical was calculated by the following formula:

β-Carotene−Linoleic Acid Assay Antioxidant capacities of the crudenutmeg oil and its components were also determined by measuring the β-carotene bleaching (23). Three milliliters of β-carotene solution that was prepared by dissolving 5 mg of β-carotene in 50 mL of chloroform was mixed with 50 mg of linoleic acid and 500 mg of Tween 20. The chloroform in the mixture solution was then purged off by high purity nitrogen gas before 100 mL of distilled water was added to the mixture. Sample (0.1 mL) was mixed with 1 mL of the emulsion and incubated at 50°C for 30 min. The reaction mixture without sample was used as a blank. The absorbance of the reaction mixture was spectrophotometrically measured at 470 nm and the antioxidant activity was calculated by the following formula:

Where S0 and B0 are the absorbance of the sample and blank, respectively, before incubation, while S30 and B30 are the absorbance of the sample and blank, respectively, after incubation at 50°C for 30 min.

242 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Figure 1. Antioxidant capacities of crude nutmeg oil and antioxidant standard controls were evaluated by: (A) DPPH• free radical scavenging assay; and (B) the β-carotene-linoleic acid assay, which were spectrophotometrically measured at 517 nm and 470 nm, respectively. BHT and α-tocopherol were used as standards. EC50 values that were used to evaluate the antioxidant capacities of the crude oil, identified antioxidant compounds, and standards were the effective concentrations at which DPPH• radicals were scavenged or the β-carotene oxidation was inhibited by 50%.

Statistical Analysis The data on the antioxidant activities of the crude nutmeg oil and antioxidants identified from the nutmeg oil were subjected to the analysis of variance and analyzed with nonlinear regressions. Statistical analysis was conducted on the SAS V8 software for Windows (SAS Institute Inc., Cary, NC). Differences among all sample means were determined by analysis of variance (AVONA) at p methoxyeugenol > BHT > isoeugenol > 247 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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α–tocopherol. Unlike the DPPH• scavenging capacities shown by the compounds, in the β-carotene-linoleic acid assay, the inhibitory activities of three compounds against the oxidation of β-carotene were significantly weaker than the two standard antioxidants (Table 2). Their antioxidant activities were in the following order: α–tocopherol > BHT > isoeugenol > methoxyeugenol > eugenol. The EC50 values of isoeugenol (144 μM) and methoxyeugenol (163 μM) were about 3 times lower than that of eugenol (526 μM), but were 3 times and 16-18 times higher than that of BHT (43 μM) and α–tocopherol (9 μM), respectively.

Table 1. Chemical compositions of the crude nutmeg oil and antioxidant peaks (A, B, C, and D) separated by silica-HPLC Composition (%) Peak no.

Components

RT (min)

RIa

Crude oil

Peaks separated by silica-HPLC A

1

α-thujene

7.98

932

3.5

2

α-pinene

8.16

942

12.5

3

sabinene

8.75

981

13.5

4

β-pinene

8.90

990

12.0

5

α-terpinene

9.44

1025

4.1

6

cymene

9.56

1033

1.4

7

limonene

9.64

1038

6.1

8

β-phellandrene

9.70

1042

3.5

9

γ-terpinene

10.07

1065

5.4

10

cis-sabinene hydrate

10.28

1080

0.9

11

α-terpinolene

10.53

1094

2.9

12

β-terpineol

11.25

1141

>0.1

13

terpinene-4-ol

12.06

1194

6.6

14

α-terpineol

12.25

1207

1.3

15

safrole

13.73

1309

3.5

16

eugenol

14.49

1364

0.7

3.9

17

isoeugenol

15.78

1462

0.9

79.8

18

myristicin

16.69

1535

10.6

7.5

5.3

B

C

D

17.2

26.3

5.8

5.9

56.1 16.8

17.3

30.3

Continued on next page.

248 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Table 1. (Continued). Chemical compositions of the crude nutmeg oil and antioxidant peaks (A, B, C, and D) separated by silica-HPLC Composition (%) Peak no.

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a

Components

RT (min)

RIa

Crude oil

19

elemicin

16.85

1548

0.8

20

methoxyeugenol

17.50

1604

0.4

Peaks separated by silica-HPLC A

B

D

C 69.7

4.2

50.4

RI was calculated based on the index of a series of n-alkanes (C8-C30).

Table 2. Antioxidant capacities of antioxidants identified from the crude nutmeg oil Antioxidant capacity, EC50 (µM) Scavenging activity

*

Antioxidant activity

Eugenol

329e

526a

Isoeugenol

615b

144c

Methoxyeugenol

355d

163b

BHT

462c

43d

α-Tocopherol

656a

9e

*

Different superscript symbols in the same column represent significant difference at P