This is a post-refereed version of the article published in:

Eur.Food Res.Tech. (2017) 243 (8), 1439-1445

Title: Comparison of the volatile antioxidant contents in the aqueous and methanolic

extracts of a set of commercial spices and condiments

Authors: Rafael Estévez Britoa, José González-Rodríguezb, Mercedes Ruiz Montoyac, José

Miguel Rodríguez Melladoa,*

aDepartamento de Química Física y Termodinámica Aplicada. Facultad de Ciencias. CeiA3,

IUIQFN, Campus Universitario Rabanales, edificio Marie Curie. Universidad de Córdoba. E-

14014-Córdoba (Spain)

bSchool of Chemistry. College of Science. University of Lincoln. Brayford Pool. Lincoln, LN6

7TS (United Kingdom)

cDepartamento de Ingeniería Química, Química Física y Ciencias de los Materiales. Facultad

de Ciencias Experimentales. Campus de “El Carmen”. Universidad de Huelva. E-21071-

Huelva (Spain)

*Corresponding author (Tel: +34 957218647 ; Fax: +34 957218618 ; e-mail: jmrodriguez@uco.es)

Acknowledgements

Financial support from Córdoba University within the framework of the “Ayudas puente para

el desarrollo de proyectos de I+D precompetitivos XXI Programa Propio 2016”, and for the

mobility grant of Dr. Estévez.

1

Abstract

Spices are of great interest because their aromatic properties and to preserve food, with no

or low nutritional value, and also as components of a healthy diet. The composition of the

methanolic and aqueous extracts of commercial samples of Basil, Cinnamon powder,

Cinnamon sticks, Clove, Cumin, Turmeric, Ginger, Nutmeg, Oregano, Rosemary and Thyme

was studied as a first step in the relation of their antioxidant activities with the composition.

Methods used were Gas Chromatography coupled with Mass Spectrometry (GC-MS) and

High Performance Liquid Chromatography (HPLC). Extracts were prepared with an amount of

the sample suspended in ultrapure water preheated at 100 oC or methanol at 60 ºC, stirring

at room temperature and filtering; for GC-MS the extracts were dried and re-dissolved in

methanol. To solve the problem in GC-MS with the flash-points of some compounds, both

techniques have been combined. The contents in antioxidants of the different species are

compared finding spices having much higher antioxidant contents in the methanolic extract

than in water, other with aqueous extracts much rich in antioxidants than the alcoholic

extracts, and spices with low antioxidant content in both extracts. For Clove, Turmeric,

Ginger, Cinnamon, Rosemary and Cumin, it is recommended the use of lipid and/or alcoholic

fractions for the food preparation. For Basil, Oregano, Thyme and Nutmeg, to extract most

of their antioxidant content only water must be used. Knowledge of the composition in

antioxidants can aid the food industry in the design of healthy foods and food preparations.

Keywords

Antioxidant Content; Aqueous and methanolic extracts; GC-MS; HPLC; Spices

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Introduction

Currently, a large part of society is concerned about the health effects of antioxidants. So,

the antioxidant capacity of foods and products having potentially pharmaceutical activity is

under study, especially in relation with their composition. Teas are a main target of such

studies due to the extended consumption of these infusions [1-3], most of the research

being centered in their high polyphenol content [4,5]. On the other hand, spices are

commonly used in the diet because their aromatic properties and to preserve food, with null

or low nutritional value [6], and also as components of a healthy diet. Spices extracts have

been used as an alternative to the use of synthetic antioxidants by preserving oils [7] and

meats, both unprocessed [6] and processed [9], in order to improve their acceptability.

Knowledge of the composition in antioxidants can aid the food industry in the design of

healthy foods and food preparations.

Pharmacological studies on spices revealed a wide range of biological activities, including,

antioxidant, anti-tumor, immunomodulatory and antiradical activities [10-13]. Their

antioxidant constituents are identified as the responsible of the health-promoting properties

of spices, such as anti-atherosclerotic, anti-cancer, and anti-inflammatory activities [14, 15].

Indeed, a diet rich in spices reduces the negative responses of the organism to the

consumption of high-fat meals, a blend of antioxidant spices in the diet increasing the

antioxidant activity in the blood and decreasing insulin response [16].

Herbs and spices are sources of phytochemicals, many of which have antioxidant activity,

though the published investigations were mainly focused on polyphenols [17]. There are

many antioxidants in dietary plants as carotenoids and phenolics, which occur mainly in

fruits and vegetables of human common consumption [18]. The antioxidant content of a

wide number of spices was analysed by the FRAP assay. This assay allowed the quantification

3

of most water- and fat-soluble antioxidants by measuring the antioxidant capacity [19], not

by chemical analysis. But two samples having different antioxidant capacities not necessarily

have different total antioxidant contents. Clove has the highest mean antioxidant value,

followed by peppermint, cinnamon, oregano, thyme, sage and rosemary. The antioxidant

activity of ethanol and water extracts of spices from China has been recently examined by

the FRAP method [20]. In addition, some experimental data have been published for

essential oils of spices [21].

The aim of this paper was to investigate the contents in antioxidants of spices made in

aqueous and alcoholic (methanolic) media as a first step in the relation of their antioxidant

activities, measured by different methodologies, with the composition.

Materials and methods

Samples, chemicals and standards

Spices analyzed were: Basil, Cinnamon powder, Cinnamon sticks, Clove, Cumin, Turmeric,

Ginger, Nutmeg, Oregano, Rosemary and Thyme, all from Hacendado brand.

All the chemicals used were of chromatographic quality. Ultrapure water type I (resistivity

18.2 MΩ·cm at 25 OC) obtained from a MilliporeMilli Q system was used.

HPLC. For the Acid Mobile Phase: HCl (Merck) and Glycine (Sigma) and Ultrapure water

filtered with a 0.45 µm filter. For the Methanolic Phase: Methanol (Merck-Millipore) filtered

with a 0.45 µm filter.

4

GC-MS. Ultrapure water filtered with a 0.45 µm filter, for the aqueous extracts. Methanol

(Merck-Millipore) filtered with a 0.45 µm filter, for the methanolic extracts and the injected

samples.

Extracts preparation

For GC-MS, extracts were prepared with 0.4 g of the sample that was suspended in 10 mL

ultrapure water preheated at 100 oC and stirred for 3 min at 300 r.p.m. at room

temperature. The suspension was filtered through a 0.45 µm filter, 4 mL of the liquid were

passed to vials and the solvent was totally evaporated by gently heating and passing a

nitrogen stream on the surface. Then, 100 µL pure methanol were added and the samples

were sonicated until total dissolution; these samples were introduced in the GC-MS. To

prepare the methanolic extracts the procedure was the same but using 10 mL of pure

methanol at 60 oC for the initial extraction.

For HPLC, extracts were produced by suspending 1 g of the sample in 10 mL ultrapure water

preheated at 100 oC (or pure methanol at 60 oC) and stirring for 3 min at 300 r.p.m. at room

temperature. The suspension was filtered through a 0.45 µm filter and 1.5 mL of the liquid

were passed to the HPLC vials.

Methods

The GC-MS analyses were made on a Gas Chromatography-Mass Spectrum Perkin-Elmer

Clarus 500 with auto-injector, a SLB-5ms (10 m x 0.1 mm x 0.1 µm) column, an injection

volume of 0.5 µL, and with He as carrier. The method consisted of a temperature ramp from

100 oC to a 300 oC (in 4 minutes). This last temperature was constant during 6 minutes to

5

ensure that all the components of the samples passed through both the column and the

mass spectrometer.

HPLC analyses were performed using a High Performance Liquid Chromatography Perkin-

Elmer Flexar with a diode array detector. The method used to improve the separation of the

substances present in the samples was based mainly on their polarity. A fast method

avoiding expensive and not easily accessible solvents was used, consisting of a mobile phase

in two-component gradient formed by aqueous and methanolic parts. The stationary apolar

phase was a C-18 column. The aqueous portion of the mobile phase, Acid Mobile Phase

(AMP), was optimized according to pH, peak resolution and simplicity in composition, and

consisted of a hydrochloric acid aqueous solution of 44·10–3 mol·L–1 and glycine 50·10–3

mol·L–1, being the final pH=2.01. Four methods were designed, depending on whether the

samples were tea (measuring time, tm, 45 min), infusions (tm = 60 min), standards that

showed a signal in less than 12 min (Gallic Acid), or standards that showed a signal before 42

min (Cinnamaldehyde). All methods needed a 5 min equilibration step previous to the run,

with a composition of the mobile phase of a 60% AMP and 40% methanol. After this step the

instrument automatically injected 10 μL of sample and the run steps began, the mobile

phase gradually changing from a majority percentage in AMP to a majority percentage in

methanol.

Results and discussion

The abundant information provided by GC-MS technique is limited by the thermal

decomposition of some components of the samples. This happened in this work with some

of the antioxidants most widespread in nature, and present in some samples, such as

6

Cinnamaldehyde in Cinnamon, Vitamin E in many samples as cumin, turmeric, oregano and

rosemary and other characteristic compounds such as Camphor in Sage and Rosemary. But,

in compensation, many others interesting antioxidant compounds were monitored.

For Basil, the water extract showed in the chromatogram a main peak (more than 22% of the

extract, denoted as “main” in figure 1) corresponding to the mixture of the 5-methylated

isomers of 4-, 5- and 6-hydroxybenzaldehydes, which have contrasted antioxidant activity

[22]. The three isomers of benzenediol, recognized natural antioxidants, were also found but

in much lower proportion. The methanolic extract contained a great variety of aromatic

compounds, among they α-bergamotene, a typical compound of Basil, and eugenol, one of

the most ubiquitous natural antioxidants [22]. Non-aromatic antioxidants, as squalene [23],

and some pro-oxidants as β-Sitosterol [24] were also found. The total phenolic content of

water and ethanol extracts of basil was reported to be equivalent [22].

FIGURE 1

The three isomers of benzenediol and a very low quantity of eugenol were also found in the

aqueous extract of powered cinnamon, in addition to cinnamaldhyde and cinnamic acid,

characteristic of this spice, which provide high antioxidant capacity [22, 25, 26]. The most

abundant compound extracted in water was coumarin (benzopiran-2-one) previously

detected in Cinnamomum cassia but not reported in extracts of Cinnamomum zeylanucum

[27]. Supporting the low solubility of coumarins in apolar media, it was reported that

essential oil of cinnamon does not include coumarins [28]. In the methanolic extract the

proportion of coumarin and cinnamic acid decrease with respect to the aqueous extract,

while the proportion of cinnamaldehyde increases due to the lowering in polarity. For

cinnamon sticks the extracts were of much lower concentration than those obtained for the

7

powered spice because the ligneous character of the sample. Cinnamic acid was not found

probably due to the absence of oxidation of cinnamaldehyde caused by the cellular rupture

originated in the powdering process.

The ligneous character of clove is also the responsible for the low concentration of aqueous

extracts, being eugenol the main compound found. In methanol the abundance of this

antioxidant was higher than 90%. It was found that the content of redox-active compounds

was much greater for powdered clove essential oil than for basil essential oil [22], supporting

this finding.

For cumin, a variety of hydroxylated and polyhydroxylated aromatic compounds were found.

The main compound found in methanol was 4-(1-methylethyl)-benzaldehyde, or

cuminaldehyde, antioxidant typically found in cumin as major antioxidant [29, 30], which

was also detected in water in this work but in very lower concentration due to its lower

solubility in more polar solvents.

Turmeric extracts contained antioxidants as eugenol, thymol, Vitamin E, dihydroxy- and

hydroxymethoxy- derivatives of benzaldehyde etc, but the main compounds extracted were

Turmerone, Ar-Turmerone and curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-

Heptadiene-3,5-dione), three antioxidant compounds that act as superoxide radical

eliminators and unactivators of molecular oxygen [31, 32]. As it can be seen in figure 1, the

aqueous extract contains a greater number of compounds than the methanolic extract.

Major components of the essential oils of turmeric rhizomes were ar-turmerone and α- and

β-turmerone [31].

Ginger extracts contained a great variety of aromatic compounds, mainly phenolics, being

the main antioxidant found gingerol, the active component of fresh ginger, as reported in

8

the literature mainly for essential oils [30, 31, 33, 34]. In the methanolic extract it was also

identified gingerone (also named zingerone) used as additive in spice oils and in perfume

industry [35].

In the water extract of nutmeg the major component found is 2,6-dimethoxy-4-(2-propenyl)-

phenol, the reduced form of 4-methoxy-6-(2-propenyl)- 1,3-Benzodioxol (myristicin), this last

being the main compound in the extract in methanol together 1,2,3-trimethoxy-5-(2-

propenyl)-Benzene (elemicin). Only low contents in myristicin and elemicin were reported in

essential oils of nutmeg [36]. The structures of all these compounds ensure their antioxidant

activity.

Oregano extracts have antioxidant, antifungal, antibacterial and antimicrobial properties as

has been widely reported in the literature [8, 13, 14, 37]. Concerning antioxidant

compounds, in this work stands out the high content in the three isomers of benzenediol,

together carvacrol and Vitamin E in lower quantities. Due to the very low solubility in water

of carvacrol and Vitamin E, these compounds appeared in higher proportions in the

methanolic extracts. It was shown that hydroquinone is the responsible antioxidant

compound in the ethyl acetate extract of Oregano [37].

For rosemary the aqueous extract was poor in compounds, whereas the alcoholic extract

was more rich in antioxidants. The main compounds in this case were eucalyptol, camphor

and β-pinene. Carnosol, carnosic acid rosmarinol and rosmarinic acid, found previously in

the literature in essential oils as main components [26, 38], were not determined by GC-MS

due to their high boiling points. However, totarol was quantified in the extract in methanol.

Finally, the main antioxidants identified for thyme were carvacrol and thymol that were

extracted from this condiment by several methods including supercritical fluids [26, 33, 39].

9

In this work, these antioxidants were quantified in less proportion in water due to their

lower solubility in this medium than in methanol. In the aqueous extract, hydroxy-

benzaldehydes were also obtained.

Some interesting compounds do not reach the boiling point in the temperature range

allowed by the equipment in GC-MS, this being a limitation and, in addition, many of them

decomposed before reaching that point, causing errors by default. For this reason, HPLC was

used to identify and quantify some compounds.

On the basis of GC-EM data and the standards used, the three isomers of benzenediol (1,2-

benzenediol, resorcinol and hydroquinone) were measured from the HPLC peaks appearing

in water (or methanol, but in this case in very low proportions) at 2-5 min retention times.

This is illustrated in figure 2 with clove.

FIGURE 2

Cinnamaldehyde was quantified in the sample of powered cinnamon, obtaining 2.37·103

ppm in water and 12.68·103 ppm in methanol, the difference being due to the higher

solubility in this medium. These values are around 500 ppm in water and 600 ppm in

methanol for the cinnamon sticks. Nevertheless, the compositions of extracts in water and

methanol are similar. This implies that the pulverization process improves the extraction of

antioxidant compounds. Moreover, the quantity of cinnamaldehyde found was much higher

than that found in GC-MS due to the thermal decomposition of this compound taking place

at relatively low temperatures [40].

In agreement with the results mentioned above, eugenol was reported as the main

compound of clove [41]. This compound was quantified using HPLC, the signal obtained for

the extract in methanol showing much more intensity (see figure 2). This compound was also

10

found in nutmeg and cinnamon, but in much lower proportion. The contents for clove were

1.21·103 ppm for the alcoholic extract and 540 ppm for the aqueous extract.

Cuminaldehyde was quantified for cumin [29, 30, 42], and curcumin for turmeric. For this

last spice, before 10 min the signals are virtually inexistent, because the methanolic extract

contains a very low proportion of aromatic alcohols and aldehydes. Gingerol is detected in

both extracts of ginger but in much more quantity in methanol, as occurred in essential oils

[30, 31, 33, 34]. Conversely to that observed for GC-MS, gingerone is observed in the

aqueous extracts in addition to extracts in methanol.

The results for oregano strengthen the above said in the sense that they show a high content

in the three isomers of benzenediol as main antioxidants. In addition, eucalyptol is one of

the main components of the rosemary extracts. In the cases of both carvacrol and thymol,

found in oregano and thyme, were found in much lower quantities in water than in

methanol extracts, due to the higher solubility in polar media.

Because the strong differences found between the contents in antioxidants of the different

spices, it is impossible to summarize the results in tables for the comparison. So, only the

main characteristics of the extracts will be commented. Figure 3 presents the total

antioxidant content of the extracts.

FIGURE 3

As it can be seen, the total antioxidants are very different from one spice to another. The

main comments to these results are the following:

In some cases, the antioxidant content is much higher in the extract made in methanol than

in the extract made in water due to the low solubility of the antioxidant compounds in

11

water. This is the case of clove (the most extreme), which main antioxidant is eugenol,

turmeric, with turmerone, Ar-turmerone and curcumin, and ginger, with gingerol and

gingerone. It is also the case for cinnamon, both powered and in sticks, with

cinnamaldehyde. Cinnamic acid is also found, being much more soluble in water, but present

in much lower proportion than the aldehyde. In the case of cinnamon, the pulverization of

the sample facilitates the extraction of their components. Finally, for rosemary the water

extract is also less rich in antioxidants than the alcoholic extract.

Conversely to the preceding group, there is a second group of spices that have aqueous

extracts much rich in antioxidants than the alcoholic extracts. This is due to the greater

polarity of the antioxidants extracted. In this group are included basil, oregano and thyme,

three green-leaved spices that have a great variety and quantity of hydroxyl-substituted

aromatic compounds soluble in water. The most representative case is basil, being the

content in antioxidants of the water extract c.a. sevenfold of the methanolic extract.

Oregano present the higher antioxidant content in the aqueous extract of all the studied

spices, being 2.5 times greater than the content of the alcoholic extract. In the case of thyme

the difference between the two extracts is around a 20%.

Finally, the third group is integrated by spices that have low antioxidant contents in both

extracts: cumin and nutmeg. In the case of cumin, the richest extract is that made in

methanol whereas for nutmeg the case is the contrary. It must be taken in mind that a low

antioxidant contents do not imply necessarily a low antioxidant activity.

So, it can be said that for spices as clove, turmeric, ginger, cinnamon, rosemary and cumin, if

one wants to take advantage of their antioxidant properties, it is recommended to use

alcohol in their food preparations, as wine, beer or whisky, for example, whereas for the

12

other spices, basil, oregano, thyme, and nutmeg, it is enough to use water to extract the

antioxidants to a greater extent.

Conclusions

Gas Chromatography coupled with Mass Spectrometry (GC-MS) and High Performance

Liquid Chromatography (HPLC) allowed the determination of the composition of methanolic

and aqueous extracts of commercial samples of Basil, Cinnamon powder, Cinnamon sticks,

Clove, Cumin, Turmeric, Ginger, Nutmeg, Oregano, Rosemary and as a first step in the

relation of their antioxidant activities with the composition. To solve the problem in GC-MS

with the flash-points of some compounds, both techniques have been combined. The

contents in antioxidants of the different species are compared finding three group of spices:

i) those having much higher antioxidant contents in the methanolic extract than in water; ii)

those with aqueous extracts much rich in antioxidants than the alcoholic extracts; iii) and

those with low antioxidant content in both extracts. For Clove, Turmeric, Ginger, Cinnamon,

Rosemary and Cumin, it is recommended the use of lipid and/or alcoholic fractions for the

food preparation. For Basil, Oregano, Thyme and Nutmeg, to extract most of their

antioxidant content only water must be used.

13

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study, J Sci Food Agric 91:2100-2107.

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Figures

Figure 1. GC-MS chromatograms of the aqueous and methanolic extracts of selected spices with the

identification of some components.

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Figure 2. HPLC chromatograms of the aqueous (up) and methanolic (down) extracts of clove.

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Figure 3. Total antioxidant content of water and methanolic extracts of spices.

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