NVEO 2018, Volume 5, Issue 1 fileEmail: [email protected] Abstract Essential oils (EOs) are known for their antimicrobial activities against a broad range of microorganisms

NVEO 2018, Volume 5, Issue 1

CONTENTS

1. Comparison between the vapor-phase-mediated anti-Candida activity of conventional and

organic essential oils / Pages: 1-6

Adam F. Feyaerts, Lotte Mathe, Walter Luyten, Patrick Van Dijck

2. A monotypic species from Turkey: Characterization of the essential oil of Berula erecta

(Apiaceae) / Pages: 7-10

Ayla Kaya, Betül Demirci, Muhittin Dinç, Süleyman Doğu

3. Antioxidant Activity of Chamomile Essential Oil and Main Components / Pages: 11-16

Zeynep Fırat, Fatih Demirci, Betül Demirci

4. Essential oil composition of Stachys obliqua Waldst. et Kit. / Pages: 17-22

Betül Demirci, Gülsüm Yıldız, Neşe Kırımer, Atila Ocak, K.Hüsnü Can Başer

5. Major volatile compounds in the essential oil of the aromatic culinary herb Pelargonium

crispum (Geraniaceae) / Pages: 23-28

Nicholas J. Sadgrove, Ben-Erik Van Wyk

Nat. Volatiles & Essent. Oils, 2018; 5(1): 1-6 Feyaerts et al.

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RESEARCH ARTICLE

Comparison between the vapor-phase-mediated anti-Candida activity of conventional and organic essential oils

Adam F. Feyaerts1,2, Lotte Mathé1,2, Walter Luyten3, and Patrick Van Dijck1,2,*

1VIB Center for Microbiology, KU Leuven, 3001, Leuven, BELGIUM 2Laboratory of Molecular Cell Biology, KU Leuven, 3001, Leuven, BELGIUM 3Department of Biology, KU Leuven, 3000, Leuven, BELGIUM

*Corresponding author. Email: [email protected]

Abstract

Essential oils (EOs) are known for their antimicrobial activities against a broad range of microorganisms. In this study we investigated if

EOs obtained from plants grown by organic farming are more potent than those obtained from conventional farming, or vice versa.

Therefore, the aim of this study was to compare pairwise the inhibitory vapor-phase-mediated antimicrobial activity of 33 certified organic

EOs and as many equivalent EOs without such certification against two human pathogenic Candida species using the vapor-phase-

mediated susceptibility assay. Overall, C. glabrata is more susceptible than C. albicans to EOs, but we could not show a significant

difference in EO antimicrobial activity between certified organic and without certification.

Keywords: Essential oil, vapor-phase, antimicrobial, Candida, organic, vapor-phase-mediated susceptibility assay

Introduction

An essential oil (EO) is composed of several components that are principally derived from the methylerythritol

phosphate -, mevalonic acid -, or shikimate pathways (Dewick, 2008). Natural variation in EO composition

between harvests is expected, but is usually limited. However, some botanical species yield EOs with substantial

intra-species differences in composition, which are referred to as chemotypes of that species. Because

chemotypes can have a different biological activity, it is necessary to specify the chemotype of an EO when

applicable (Baser & Buchbauer, 2016; Franchomme & Pénoël, 1996).

A common distinction between EOs is, whether they are obtained from plants grown by organic or conventional

farming. Different countries may have different requirements or procedures to certify a product as organic (EU,

2007, 2008). Organic farming is considered superior over conventional farming by most consumers. One of the

main reasons for consumers to favor organic products (besides lifestyle) is to avoid pesticides, although even in

organic farming a limited number of pesticides are allowed. Furthermore, organic products are often believed to

be more nutritious and/or healthier than conventional products. However, most research on this matter is

inconclusive (Baranski et al., 2014; Hole et al., 2005; Michael & David, 2017; Seufert & Ramankutty, 2017; Smith-

Spangler et al., 2012; Tuomisto, Hodge, Riordan, & Macdonald, 2012; Wilcox, 2011).

EOs are well-known for their activity against fungal pathogens such as Candida albicans and Candida glabrata

(Palmeira-de-Oliveira et al., 2009; Sharifi-Rad et al., 2017). Recently, we showed that EOs can have potent vapor-

phase-mediated anti-Candida activities, and can therefore be considered a novel class of antifungals with distinct

characteristics (A. F. Feyaerts et al., 2018). C. albicans and C. glabrata are the Candida species most commonly


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isolated from humans. While belonging to the same genus, they are genetically and phenotypically very different,

and mostly employ species-specific virulence factors (Brunke & Hube, 2013; Adam F. Feyaerts et al., 2017; Mathé

& Van Dijck, 2013).

In this study, a pairwise comparison was performed between the inhibitory vapor-phase-mediated antimicrobial

activity (iVMAA) of 33 organic EOs (oEOs) and 33 equivalent conventional EOs (cEOs) against the two Candida

species.

Materials and Methods

Material

The EOs used in this work (Table 1) and their chemical analysis were acquired from a commercial source

(Pranarôm International, Belgium). Half of the EOs were certified organic and the other half were obtained from

the same plant species, chemotype, and plant part, but were conventional. The composition of the EOs used in

this study has been published (Feyaerts et al., 2018). The detection of organochlorine and organophosphorus

pesticides was performed using GC-MS-XSD and GC-MS-FPD, respectively, using the internal multi-residue

method validated according to NF V03-110. The maximum residue limit according to EU-legislation was never

exceeded.

Table 1. List of essential oils

Number Essential oil Part of the plant Lot numbers of pair

oEO – cEO 1 Cananga odorata extra/totum Flowers OF10390 – OF9867

2 Cedrus atlantica Wood OF10992 – OF10799

3 Chamaemelum nobile Flowers OF10863 – OF11255

4 Cinnamomum cassia Twigs OF10584 – OF10588

5 Citrus aurantium ssp amara Leaves OF10467 – OF11484

6 Citrus limon Peel OF11188 – OF11178

7 Citrus paradisii Peel OF9436 – OF9722

8 Citrus reticulata Peel OF3457 – OF10644

9 Citrus sinensis Peel OF9238 – OF11321

10 Cupressus sempervirens variety stricta Twigs OF10218 – OF10846

11 Cymbopogon martinii variety motia Aerial Parts OF10011 – OF9950

12 Eucalyptus globulus Leaves OF10646 – OF11274

13 Eucalyptus radiata ssp radiata Leaves OF10865 – OF10720

14 Eugenia caryophyllus Flower buds OF9948 – OF10583

15 Helichrysum italicum ssp serotinum Flowering tops OF9441 – OF10622

16 Lavandula angustifolia ssp angustifolia Flowering tops OF10864 – OF10951

17 Lavandula latifolia Flowering tops OF10007 – OF9693

18 Lavandula x burnatii clone grosso Flowering tops OF1743 – OF6689

19 Lavandula x burnatii clone super Flowering tops OF10869 – OF10745

20 Litsea citrata Fruits OF10225 – OF11261

21 Melaleuca alternifolia Leaves OF11248 – OF10388


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Table 1. List of essential oils (cont.)

Number Essential oil Part of the plant Lot numbers of pair

oEO – cEO

22 Melaleuca cajuputi Leaves OF10662 – OF11405

23 Melaleuca quinquenervia chemotype cineole Leaves OF10731 – OF10956

24 Mentha arvensis Aerial parts OF10883 – OF9728

25 Mentha piperita Aerial parts OF10867 – OF11594

26 Myrtus communis chemotype myrtenyl acetate Leaves OF9391 – OF10882

27 Origanum compactum Flowering tops OF11283 – OF10299

28 Origanum majorana Flowering tops OF10217 – OF9776

29 Pinus sylvestris Needles OF11339 – OF2115

30 Pogostemon cablin Flowering tops OF10211 – OF9954

31 Rosmarinus officinalis chemotype cineole Flowering tops OF10655 – OF10408

32 Salvia officinalis Flowering tops OF10880 – OF9241

33 Thymus satureioides Flowering tops OF10106 – OF10589

oEO = organic EO; cEO = conventional EO; ssp = subspecies

Microorganisms

C. albicans SC5314 (Gillum, Tsay, & Kirsch, 1984) and C. glabrata ATCC 2001 were maintained on YPD agar plates

composed of 10 g/L yeast extract (Merck), 15 g/L DifcoTM agar (Becton, Dickinson & Co.) and 20 g/L bactopeptone

(Oxoid). Prior to experiments, the strains were grown overnight at 35°C on plates containing 47 g/L Sabouraud

agar (Sigma-Aldrich).

Preparation of the cell inocula

The cell density of overnight propagated cells resuspended in 1x phosphate-buffered saline, containing 0.20 g/L

potassium chloride (VWR International), 0.24 g/L potassium dihydrogen phosphate (Merck), 1.44 g/L disodium

hydrogen phosphate (Merck) and 8 g/L sodium chloride (Sigma-Aldrich), was estimated by measuring the optical

density at 600 nm (OD600). The cell suspension was prepared in Roswell Park Memorial Institute 1640 (RPMI)

medium (Sigma-Aldrich) in accordance with CLSI guidelines (CLSI, 2012). Briefly, the RPMI medium was buffered

with 3-(N-morpholino) propanesulfonic acid (MOPS; Sigma-Aldrich), and filter-sterilized over a 0.20 µm non-

pyrogenic NalgeneTM filter (Fisher Scientific).

Vapor-phase-mediated susceptibility (VMS) assay

The VMS was set up and performed as described before (Feyaerts et al., 2018). Briefly, a 200 µL inoculum

containing 5 x 103 cells was added to each well of a polystyrene 96-well microtiter plate (Greiner Bio-One), except

for wells H1 and H12 which served as blanks and contained 200 µL medium. Next, 20 µL of the EOs to be tested

was added on top of the cell suspension in wells D/E3-4 and D/E9-10. The microtiter plate was covered with a lid

and statically incubated for 24 hours at 35°C while limiting air draughts. After resuspending the cells, the OD600

was measured with a multi-well plate reader (Synergy H1, BioTek Germany). Wells in which growth was visually

absent (OD600 ≤ 0.07) were counted, excluding wells to which the EO was added and blanks, and categorized to

determine the iVMAA, with a higher category corresponding with a stronger iVMAA. If no iVMAA was detected,


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category zero was assigned. All EOs with an iVMAA larger than zero against at least one of the two Candida

species were tested in 3 independent experiments.

Statistical analysis

GraphPad Prism version 7.04 was used for statistical analysis. Figures show categorized averages of the biological

repeats. The population-wide susceptibility of both Candida species to EOs was compared using the Wilcoxon

matched-pairs signed rank test.

Results and Discussion

The 33 EO pairs shown in table 1 represent all possible organic-conventional EO combinations of the previously

described EO collection (A. F. Feyaerts et al., 2018). The iVMAA of these EOs was determined against C. albicans

and C. glabrata using the VMS assay.

Only for a few EOs we found intra-pair differences in iVMAA against one or both Candida species under study

(Figure 1). For instance, Cinnamomum cassia oEO (Table 1; pair 4) has a higher iVMAA compared to

Cinnamomum cassia cEO against both C. albicans (iVMAAcEO = 2.5; iVMAAoEO = 3.5) and C. glabrata (iVMAAcEO =

2.5; iVMAAoEO = 4.5). In contrast, Thymus satureioides oEO (Table 1; pair 33) has a lower iVMAA compared to

Thymus satureioides cEO against both C. albicans (iVMAAcEO = 1; iVMAAoEO = 0.5) and C. glabrata (iVMAAcEO = 1;

iVMAAoEO = 0.5).

Figure 1. Categorized average inhibitory vapor-phase-mediated antimicrobial activity of EO pairs against two Candida

species

oEO = organic EO; cEO = conventional EO; iVMAA = inhibitory vapor-phase-mediated antimicrobial activity

Despite these apparent individual differences, a pairwise comparison including all EOs pairs did not show a

significant difference between the iVMAA of cEO and oEO against C. albicans [p = 0.51; npairs = 33; sum of signed

ranks (W) = 64], against C. glabrata (p = 0.91; npairs = 33; W = -14), or against both Candida species combined (p =


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0.74; npairs = 66; W = 94). Hence, our results, which are based on a large set of EO pairs, show that the iVMAA of

an EO likely does not depend in general on whether it is certified as “organic” or not.

Additionally, we showed that the overall iVMAA of cEOs (p < 0.0001; npairs = 33; W = 371), oEOs (p < 0.0001; npairs

= 33; W = 441), and cEOs and oEOs combined (p < 0.0001; npairs = 66; W = 1616) is significantly higher against C.

glabrata than against C. albicans. This is in line with a previous report showing that C. glabrata is significantly

more susceptible to the VMAA of EOs and their components (n = 212) compared to C. albicans (Feyaerts et al.,

2018).

Together, we conclude that the iVMAA of an EO generally does not depend on whether the EO is certified organic

or not. However, it is possible that our study failed to detect a difference in iVMAA between the two categories

of EOs because of several reasons. (i) Some EOs lacking a certified organic label might have been derived from

plants grown under organic conditions. While this seems unlikely considering that organic farming requires extra

measures and costs, which producers try to recover by obtaining an organic label that assures a higher selling

price, we cannot exclude it. (ii) It is also possible that our sample did not show a difference due to selection bias,

despite the inclusion of all possible oEO-cEO pairs available in our large EO collection. (iii) We may have missed

a difference because it was too small to detect because e.g. our sample lacks power. However, this raises the

question of whether such a minimal difference would be relevant. (iv) Lastly, it is possible that a we did not

observe a difference because we only tested two yeast species using a single antimicrobial test. Therefore, we

encourage similar studies using multiple micro-organisms and different assays.

ACKNOWLEDGMENT

We thank Jean‐François Baudoux for logistical support. This work was supported by grants from the Fund for Scientific

Research Flanders (FWO projects WO.009.16 N and G.0D48.13 N). WL largely supported himself. AUTHOR CONTRIBUTION:

A.F.F. conceptualized the study. A.F.F. and L.M. designed the experiments. A.F.F. and L.M. performed the experiments.

A.F.F., L.M., W.L. and P.V.D. contributed to manuscript preparation. P.V.D. supervised the project.

REFERENCES

Baranski, M., Srednicka-Tober, D., Volakakis, N., Seal, C., Sanderson, R., Stewart, G. B., Leifert, C. (2014). Higher antioxidant

and lower cadmium concentrations and lower incidence of pesticide residues in organically grown crops: a systematic

literature review and meta-analyses. British Journal of Nutrition, 112(5), 794-811.

Baser, K. H. C., & Buchbauer, G. (2016). Handbook of Essential Oils: Science, Technology, and Applications; Second Edition:

Boca Ranton, FL. CRC Press.

Brunke, S., & Hube, B. (2013). Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiology,

15(5), 701-708.

Dewick, P. M. (2008). Medicinal Natural Products :A biosynthetic approach (Third Edition ed.). Chichester: Wiley.

EU Council Regulation (EC) No 834/2007 of 28 June 2007 on organic production and labelling of organic products and

repealing Regulation (EEC) No 2092/91, (2007).

EU Commission Regulation (EC) No 1235/2008 of 8 December 2008 laying down detailed rules for implementation of Council

Regulation (EC) No 834/2007 as regards the arrangements for imports of organic products from third countries, (2008).


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Feyaerts, A. F., Mathe, L., Luyten, W., De Graeve, S., Van Dyck, K., Broekx, L., & Van Dijck, P. (2018). Essential oils and their

components are a class of antifungals with potent vapour-phase-mediated anti-Candida activity. Scientific Reports, 8(1),

3958. doi:10.1038/s41598-018-22395-6

Feyaerts, A. F., Mathé, L., Luyten, W., Tournu, H., Van Dyck, K., Broekx, L., & Van Dijck, P. (2017). Assay and recommendations

for the detection of vapour-phase-mediated antimicrobial activities. Flavour and Fragrance Journal, 32(5), 347-353.

doi:10.1002/ffj.3400

Franchomme, P., & Pénoël, D. (1996). L'Aromathérapie Exactement. Encyclopédie de l'utilisation thérapeutique des huiles

essentielles. Limoges: Roger Jollois.

Gillum, A. M., Tsay, E. Y., & Kirsch, D. R. (1984). Isolation of the Candida albicans gene for orotidine-5'-phosphate

decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Molecular and General Genetics MGG,

198(2), 179-182.

Hole, D. G., Perkins, A. J., Wilson, J. D., Alexander, I. H., Grice, P. V., & Evans, A. D. (2005). Does organic farming benefit

biodiversity? Biological Conservation, 122(1), 113-130.

Mathé, L., & Van Dijck, P. (2013). Recent insights into Candida albicans biofilm resistance mechanisms. Current Genetics,

59(4), 251-264.

Michael, C., & David, T. (2017). Comparative analysis of environmental impacts of agricultural production systems,

agricultural input efficiency, and food choice. Environmental Research Letters, 12(6), 064016.

Palmeira-de-Oliveira, A., Salgueiro, L., Palmeira-de-Oliveira, R., Martinez-de-Oliveira, J., Pina-Vaz, C., Queiroz, J. A., &

Rodrigues, A. G. (2009). Anti-Candida activity of essential oils. Mini reviews in medicinal chemistry, 9(11), 1292-1305.

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Sharifi-Rad, J., Sureda, A., Tenore, G. C., Daglia, M., Sharifi-Rad, M., Valussi, M., Iriti, M. (2017). Biological Activities of

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organic foods safer or healthier than conventional alternatives? A systematic review. Ann Intern Med, 157(5), 348-366.

Tuomisto, H. L., Hodge, I. D., Riordan, P., & Macdonald, D. W. (2012). Does organic farming reduce environmental impacts?

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Wilcox, C. (2011). Mythbusting 101: Organic Farming > Conventional Agriculture. Retrieved from

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Nat. Volatiles & Essent. Oils, 2018: 5(1): 7-10 Kaya et. al.

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RESEARCH ARTICLE

A monotypic species from Turkey: Characterization of the

essential oil of Berula erecta (Apiaceae)

Ayla Kaya1,*, Betül Demirci2, Muhittin Dinç3 and Süleyman Doğu3

1 Department of Pharmaceutical Botany, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, TURKEY 2 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, TURKEY 3 Department of Biology, Ahmet Keleşoğlu Faculty of Education, Necmettin Erbakan University, Konya, TURKEY


Abstract

The genus Berula W. Koch belongs to the Apiaceae family and it is only represented by B. erecta (Huds.) Coville in Turkey. B. erecta

was collected in June, 2014 from Konya province. The chemical composition of essential oil obtained by hydrodistillation from the

dried aerial parts of B. erecta was analysed by GC-FID and GC-MS. Thirty-five compounds, constituting about 79.4% of the total oil,

were identified. The major components were found as hexahydrofarnesyl acetone (14.1%) and α-bisabolol oxide A (12.8%).

Keywords: Berula erecta, essential oil, GC-FID, GC-MS, Turkey

Introduction

The Apiaceae is one of the best known families of flowering plants because of its characteristic inflorescences

and fruits, and the diverse chemistry reflected odour, flavour and even toxicity of many of its members. It

contains about 300 genera and 2500-3000 species throughout the world (Heywood, 1979). The genus Berula

W. Koch belongs to the Apiaceae family (subfam. Apioideae, tribus Oenantheae). It is only represented by B.

erecta (Huds.) Coville (syn. Sium erectum Huds, Sium angustifolium L. Berula angustifolia (L.) Mert. & W.D.J.

Koch) in Turkey (Peşmen, 1972).

B. erecta, water-parsnip, is an aquatic plant, probably a monotypic species distributed in damp places in

Europe, Asia, East and South Africa, North America, and many parts of Iran and Turkey (Javidnia et al., 2011).

The local name of plant is “gendeme” (Güner et al., 2012). The plant is toxic, and capable of causing death to

grazing animals. The leaves and flowers have been used for food. The plant is used externally in the treatment

of rheumatism. An infusion of the whole plant can be used as a wash for swellings, rashes and athletes foot

infections (http, 2018). B. erecta is a perennial glabrous, 40-100 cm in height, leaves 3-4-pinnate, rays 8-20

erecto-patent, 1-4 cm, bracts lanceolate, mericarps c. 2x1.5 mm. It grows on marshy places by streams, and

at sea level-1750 m altitudes in Anatolia (Peşmen, 1972).

There are two previous papers on the chemical composition of B. erecta from Serbia (Lazarevic et al., 2010)

and Iran (Javidnia et al., 2011). Here we report on the composition of the aerial parts essential oil of B. erecta

which has not been reported previously from Turkey.


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Plant material

B. erecta was collected during the flowering period (June, 2014) from Konya province. B4 Konya: Sille, 1170

m, hills, 26.06.2014. The voucher specimens (M Dinç 3588 & S Doğu) are deposited at the Herbarium of the

Department of Biology, Necmettin Erbakan University, Konya, Turkey (NEÜ Herb.).

Isolation of essential oil

The essential oil from air-dried plant materials was isolated by hydrodistillation for 3 h, using a Clevenger-

type apparatus to produce a trace amount of essential oil which was trapped in n-hexane. The yield of oil

was 0.01%. The obtained oil was dried over anhydrous sodium sulphate and stored at +4°C in the dark until

analyzed and tested.

GC-MS analysis

The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 0.25

mm, 0.25 m film thickness) was used with helium as carrier gas (0.8 ml/min). GC oven temperature was

kept at 60C for 10 min and programmed to 220C at a rate of 4C/min, and kept constant at 220C for 10

min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector

temperature was set at 250C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450.

GC analysis

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300C. To

obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same

column applying the same operational conditions. Relative percentage amounts of the separated compounds

were calculated from FID chromatograms. The analysis results are given in Table I.

Identification of the components

Identification of the essential oil components were carried out by comparison of their relative retention times

with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes.

Computer matching against commercial (Wiley GC/MS Library, MassFinder Software 4.0) (McLafferty &

Stauffer, 1989; Hochmuth, 2008) and in-house “Başer Library of Essential Oil Constituents” built up by

genuine compounds and components of known oils.


The identified volatile constituents of B. erecta are reported in Table I. Analysis of the essential oil of B. erecta

led to the identification of 35 compounds, which represented 79.4% of the total oil. Components of the oils

can be grouped into six main chemical classes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes,

fatty acids, diterpenes, alkanes and others. The oil was characterized by a high content of others (27.7%),

oxygenated sesquiterpenes (23.9%) and alkanes (18.5%). The main components of the oil were found as

hexahydrofarnesyl acetone (14.1%) and α-bisabolol oxide A (12.8%).


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Table 1. The Composition of the Essential Oil of Berula erecta

RRIa Compound %b IMc

1244 2-Pentyl furan 2.3 MS

1296 Octanal tr tR, MS

1400 Nonanal tr tR, MS

1500 Pentadecane 0.9 tR, MS

1548 (E)-2-Nonenal 0.8 MS

1600 Hexadecane 0.8 tR, MS

1695 (E)--Farnesene 3.6 MS

1700 Heptadecane 0.3 tR, MS

1715 2-Dodecanone 1.9 MS

1766 Decanol 3.7 tR, MS

1773 -Cadinene 0.8 MS

1815 Hexyl octanoate 1.2 MS

1827 (E,E)-2,4-Decadienal 0.4 MS

1868 (E)-Geranyl acetone 1.5 MS

1900 Nonadecane 0.1 tR, MS

1958 (E)--Ionone 0.2 MS

1992 Neophytadiene 0.6 MS

1973 Dodecanol 0.2 tR, MS

2000 Eicosane 1.3 tR, MS

2100 Heneicosane 3.7 tR, MS

2131 Hexahydrofarnesyl acetone 14.1 MS

2156 -Bisabolol oxide B 2.0 MS

2200 Docosane 2.0 tR, MS

2200 α-Bisabolon oxide A 2.5 MS

2232 -Bisabolol 5.4 tR, MS

2300 Tricosane 4.4 tR, MS

2384 Farnesyl acetone 1.2 MS

2430 Chamazulene 1.4 MS

2438 α-Bisabolol oxide A 12.8 MS

2500 Pentacosane 2.8 tR, MS

2622 Phytol 1.4 MS

2670 Tetradecanoic acid 1.1 tR, MS

2700 Heptacosane 0.7 tR, MS

2900 Nonacosane 1.5 tR, MS

2931 Hexadecanoic acid 1.8 tR, MS

Sesquiterpene Hydrocarbons 4.4

Oxygenated Sesquiterpenes 23.9

Fatty acids 2.9

Diterpenes 2.0

Alkanes 18.5

Total 79.4 aRRI: Relative retention indices calculated against n-alkanes; b%: calculated from FID data; cIM: Identification Method; tR, identification based on the retention times (tR) of genuine standard compounds on the HP Innowax column; MS, tentatively identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data. tr Trace (< 0.1 %)


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According to literature survey, there are two study on the essential oil from aerial parts of B. erecta from

Serbia (Lazarevic et al., 2010) and Iran (Javidnia et al., 2011). Lazarevic et al. (2010) studied essential oils of

B. erecta subsp. erecta and they reported one hundred and twenty-five compounds and identified accounted

for 96.2% of the total oil. The yield of oil was 0.004%. The oil was characterized by the presence of (Z)-

falcarinol (21.5%), -sesquiphellandrene (17.2%),-caryophyllene (14.9%) and -terpinene (14.8%).

Terpenoids (66.2%) constituted the main fraction of the oil, with monoterpene (19.3%) and sesquiterpene

hydrocarbons (39.2%) as the most abundant compound class.

Javidnia et al. (2011) reported 44 compounds which represent 94.0% of the total oil from B. angustifolia (syn.

B. erecta). The yield of oil was 0.04%. They found piperitenone oxide (14.6%), limonene (13.9%), -

zingiberene (12.8%), and (E)--farnesene (9.6%) as main compounds.

Various factors, both endogenous and exogenous, can affect the composition of the essential oil of B. erecta.

We believe that the time of flowering, geographical, and climatic factors may be very important. Several

papers have reported on the variation in the essential oil composition induced by environmental,

physiological, and edaphic factors, which can induce changes in biosynthesis accumulation or metabolism of

given compounds of the essential oil (Senatore et al., 1997).

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Javidnia, K., Miri, R., & Assadollahi, M. (2011). Constituents of the essentıal oil of Berula angustifolia from Iran,

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Lazarević J., Radulović N., Palić R., & Zlatković B. (2010). Chemical analysis of volatile constituents of Berula erecta

(Hudson) Coville subsp. erecta (Apiaceae) from Serbia. Journal of Essential Oil Research, 22(2), 153-156.

McLafferty, F.W., & Stauffer, D.B. (1989). The Wiley/NBS Registry of Mass Spectral Data. New York: J. Wiley and Sons.

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Edinburgh University Press.

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the essential oils from Salvia glutinosa L. growing wild in southern Italy. Journal of Essential Oil Research, 9, 151–157.

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Nat. Volatiles & Essent. Oils, 2018; 5(1): 11-16 Fırat et al.

11

RESEARCH ARTICLE

Antioxidant Activity of Chamomile Essential Oil and Main Components

Zeynep Fırat1,2*, Fatih Demirci3 and Betül Demirci3

1 Graduate School of Health Sciences, Anadolu University, 26470 Eskisehir, TURKEY 2 Vocational School of Health Services, Department of Medical Services and Techniques, Anadolu University, 26470

Eskisehir, TURKEY 3 Faculty of Pharmacy, Department of Pharmacognosy, Anadolu University, 26470 Eskisehir, TURKEY


Abstract

The objective of this study was to characterize the antioxidant capacity of the chamomile (Matricaria recutita L.) essential oil (EO) as

well as its major constituents. For this purpose, Pharmacopoeia grade Matricariae aetheroleum was used after the analytical

evaluation, which was confirmed by GC/MS and GC/FID, respectively. The main components were identified as α-bisabolol oxide A

and B, (E)--farnesene, α-bisabolone oxide A, chamazulene, and α-bisabolol according the European Pharmacopoeia, respectively.

The major constituents α-bisabolol oxide A and chamazulene were purified using prep-TLC from the EO, and the identification and

quantification of the constituents were confirmed by GC/MS and GC/FID analyses. Chamomile EO, α-bisabolol oxide A,

(E)- - farnesene, chamazulene, and α-bisabolol were evaluated comparatively for their in vitro antioxidant activities using the

spectrophotometric 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging microdilution assay. The highest free radical

inhibitory activity was observed for chamazulene followed by α-bisabolol oxide A, the chamomile EO and (E)--farnesene,

respectively.

Keywords: Chamomile essential oil, Matricaria recutita L., DPPH antioxidant activity, chamazulene, sesquiterpene

Introduction

In general, antioxidant substances present in foods or in the biological system at low concentrations

compared with oxidative substrates may delay or prevent the oxidation process. Assessments of antioxidant

properties of natural compounds including essential oils (EOs) are important due to their uses in food,

medicine, and cosmetics (Halliwell et al., 1995; Mishra et al., 2012).

It is well known that EOs have various properties, and the use of EOs as natural antioxidants is common

practice in several cases. Essential oils are mainly composed of terpenes such as monoterpenes and

sesquiterpenes (Amorati et al., 2013; Baser and Demirci, 2007). The sesquiterpenes are subject for a vast

number of biological evaluations and activities including antioxidant potential (Bartikova et al., 2014).

Chamomile, Matricaria recutita L. of the Asteraceae family, is an annual herbaceous plant, growing in

Germany, Hungary, France, Russia, Yugoslavia, India including Turkey, which is cultivated in several countries

such as Egypt, Algeria, Hungary, Germany, among others (Das, 2015). Chamomile EO is highly demanded,

which is obtained by steam distillation, and is also described in several pharmacopoeias for its specifications.

Pharmaceutically it is commonly used for the treatment of various diseases associated to inflammation,

infection, and spasms among others. The EO is characterised by the presence of sesquiterpenes such as

chamazulene, farnesene and α-bisabolol and oxygenated sesquiterpenes were the most characteristic

chamomile EO such as bisabolone oxide A and bisabolol oxide A and B. The sesquiterpenes are the major

biologically active substances in the EO (Rhind, 2012; Schilcher, 2005; Singh et al., 2011; Srivastava et al.,


12

2010). Among other uses chamomile EO has been reported as a natural antioxidant (Abdoul-Latif et al., 2011;

Ayoughi et al., 2011; Owlia et al., 2007; Stanojevic et al., 2016) and also is dark blue coloured oil that contains

chamazulene, which is an important factor for the antioxidant power of chamomile EO (Buckle, 2015;

Capuzzo et al., 2014; Ornano et al. 2013; Rekka et al., 1996).

The aim of this present study was to evaluate the biological activity of Pharmacopoeia grade chamomile EO.

For this purpose, the EO was subjected to in vitro antioxidant activity by microdilution spectroscopy. In

addition to the EO the major components α-bisabolol oxide A, (E)--farnesene, chamazulene, α-bisabolol;

where the purified bisabolol oxide A and chamazulene were used and evaluated using the DPPH radical

scavenging activity.


EO and chemicals

Pharmacopeia (PhEur) grade chamomile EO (Matricaria recutita L.) was acquired from (Phatrade, Cairo in

Eygpt). α-Bisabolol and (E)--farnesene are purchased from Sigma-Aldrich. All chemicals used were of high

purity and analytical grade which were supplied by Sigma Aldrich, Fluka and Merck and used without further

purification unless otherwise stated. The chamomile EO composition was previously reported in detail (see

Goger et al., 2018).

Gas chromatography/flame ionization detection (GC/FID) and gas chromatography/mass

spectrometry (GC-MS) analyses

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 °C. In

order to obtain the same elution order with GC-MS, simultaneous auto-injection was done by using same

column and an appropriate operational conditions. The GC/MS analysis was carried out with an Agilent 5975

GC/MSD system as previously described in detail (Demirci et al, 2015; Goger et al., 2018).

Analytical and preparative thin layer chromatography (TLC, prep-TLC)

Chamomile EO was analysed by TLC. TLC analysis was performed on silica gel 60 GF 254 (Merck) using

chloroform-toluene (3:1, v/v) as a mobile phase by spraying with anisaldehyde/sulphuric acid and heating for

5 minutes at 110 °C. α-Bisabolol oxide A and chamazulene were isolated using successive prep-TLC

techniques from the chamomile EO and the identification and quantification of the constituents were by

GC/MS and GC/FID analysis.

The DPPH free radical scavenging activity

The DPPH free radical scavenging activity is based on the ability of 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), a

stable free radical, to be decolorized in the presence of antioxidants (Kumarasamy et al., 2007). Briefly, stock

solutions and DMSO (control) were prepared and added 80 μg/ml DPPH solution in MeOH. The mixtures were

shaken vigorously and left to stand in the dark for 30 min at room temperature, then absorbance was read

at 517 nm. Radical scavenging capacity was expressed as percentage inhibition (I %) and calculated using the

following equation:

(I %) =[(Abs control – Abs sample) / Abs control] × 100 (formulae)

Ascorbic acid was used as a positive control. The DPPH IC50 values (IC50 value is the concentration of the

sample required to inhibit 50 % of radical) of the samples were calculated and are listed in Table 2.

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Chamomile EO composition

The chemical composition of the study material chamomile EO was recently reported where α- bisabolol

oxide A (1) (47.7 %), (E)--farnesene (2) (21.5 %), α-bisabolol oxide B (3) (6.2 %), α-bisabolone oxide A (4)

(5.7 %), chamazulene (5) (4.1 %) and α-bisabolol (6) (2.1 %) (Goger et al., 2018), were identified as the main

components. The MS fragmentation of the main components are shown in Figure 1. which was detailed and

listed in Table 1.

Stanojevic et al. (2016), identified 52 components consisting of -farnesene (29.8 %), α-farnesene (9.3 %), α-

bisabolol and its oxide (15.7 %), chamazulene (6.4 %), germacrene D (6.2 %) and spiroether (5.6 %) in

chamomile EO. In another study, major compounds were identified as chamazulene (31.2 %), 1,8-cineole

(15.2 %), -pinene (10.1 %), α-pinene (8.14 %), α-bisabolol (7.5 %) and terpinen-4-ol (4.1 %) (Farhoudi, 2013).

Also the chamomile EO consisted of α-bisabolol oxide A (48.2 %), α-bisabolol oxide B (23.31 %), α-bisabolol

(12.1 %) and -farnesene (5.2 %), chamazulene (2.4 %) sesquiterpenes as major constituents (Roby et al.,

2013). Ayoughi et al. (2010), reported that (E)- -farnesene (24.2 %), guaiazulene (10.6 %),

α- bisabolol oxide A (10.2 %), α-farnesene (8.7 %) and α-bisabolol (7.3 %) were present in M. recutita EO.

Table 1. GC/MS analysis results of chamomile EO

Compounds Rt (min) m/z (Relative intensity, %) %

(E)--Farnesene 33,66 M+ 204 (4), 189 (3), 175 (1), 161 (18), 148 (4), 133 (33), 120 (23), 107 (11),

93 (67), 79 (26), 69 (100), 55 (16), 41 (52) 21.5

α-Bisabolol oxide B 46,38 M+ 238 (2), 220 (5), 205 (3), 179 (27), 161 (55), 143 (100), 134 (39), 125 (34), 107 (24), 105 (74), 95 (27), 85 (58), 71 (35), 59 (47), 43 (55)

6.2

α-Bisabolone oxide A 47,45 M+ 236 (2), 218 (3), 200 (5), 178 (11), 169 (6), 150 (28), 141 (96), 132 (16),

121 (54), 107 (38), 93 (100), 79 (34), 67 (46), 55 (20), 43 (47) 5.7

α-Bisabolol 48,06 M+ 222, 204 (33), 189 (6), 161 (15), 147 (6), 134 (11),119 (97), 109 (100),

93 (46), 79 (20), 69 (85), 55 (23), 43 (63) 2.1

Chamazulene 52,73 M+ 184 (97), 169 (100), 153 (34), 141 (13), 128 (19), 115 (13), 89 (6), 77 (6),

63 (3), 45 (6) 4.1

α-Bisabolol oxide A 52,96 M+ 238 (1), 220 (2), 180 (3), 159 (3), 143 (100), 134 (18),125 (36), 107 (26),

93 (37), 81 (12), 71 (24), 59 (17), 43 (40) 47.7

Rt: Retention time; m/z: Mass-to-charge ratio; % calculated from FID data.

Figure 1. Chamomile EO major constituents

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Isolation of the EO components

α-Bisabolol oxide A and chamazulene were purified by prep-TLC eluting with chloroform-toluene (3:1, v/v)

from the chamomile EO (Figure 2), and the identification-quantification of the constituents were performed

by GC/MS and GC/FID analyses.

Figure 2. (a) TLC of analytical standard, (1) chamomile EO, (2) α-bisabolol, (3) α-bisabolol oxide A, (4) chamazulene, (5)

(E)--farnesene; (b) preparative TLC of chamomile EO; (c) isolation of high-purity chamazulene.

As previously reported (Ashnagar et al., 2009), three major components were separated, purified and

identified namely as chamazulene (Rf = 0.93), bisabolonoxide (Rf = 0.75) and bisabololoxide A (Rf = 0.3) from

Matricaria recutita EO. The chemical structures of the components were determined by their IR, 1H NMR and

MS spectra. These data also comply with our results.

Results of the DPPH free radical scavenging assay

In the DPPH assay the radical scavenging ability of the chamomile EO, and its four main components as listed

in (Table 2), and also the positive control (ascorbic acid) was compared spectrophotometrically.

Table 2. Antioxidant activity of chamomile EO and its four main components on DPPH• assay

Samples DPPH IC50 (mg/mL)

Chamomile EO 2.20

α-Bisabolol 43.88

α-Bisabolol oxide A 1.50

(E)--Farnesene 7.45

Chamazulene 0.27

Ascorbic acid (reference) 0.015

The results showed that the scavenging activity of the main substances were in the following order;

chamazulene > α-bisabolol oxide A > chamomile EO > (E)--farnesene > α-bisabolol, respectively, accordingly

to their IC50 values compared to the positive control ascorbic acid, which exhibited high antioxidant activity

with IC50 value of 0.015 mg/mL in the same experimental system.


15

Previous antioxidant activity of chamomile EO showed the best antioxidant properties after 90 minutes of

incubation with the EC50value of 2.07 mg/mL by DPPH free radical method (Stanojevic et al., 2017). In another

antioxidant activity study by DPPH free radical scavenging and β-carotene/linoleic acid methods, the EO EC50

value was determined as 5.63 ± 0.20 mg/mL as reported by Ayoughi et al. (2010). In the -carotene bleaching

test, the EO gave the best inhibition result of 82.5 % after 120 minutes, supporting the antioxidant activity

(Owlia et al., 2007).

Interestingly, Capuzzo et al. (2014), reported that chamazulene was unable to react with DPPH.. According

to the literature, it is necessary to avoid DPPH assay to test chamazulene radical scavenging activity owing to

its interference with the nitrogen-centred DPPH, colour factor and thus suggests the use of ABTS assay

instead. Actually when chamazulene was evaluated using ABTS., a strong and significantly higher free radical

scavenging activity was observed (IC50 3.7 ± 0.7 µg/mL).

Conclusion

The results showed that the in vitro scavenging activity of chamazulene was the highest after α-bisabolol

oxide A, and the chamomile EO followed by the other chamomile major volatile constituents. To the best of

our knowledge this is the first report on the antioxidant activity of α-bisabolol oxide A and (E)--farnesene

constituents, which may responsible of the total activity of the EO.

ACKNOWLEDGEMENTS

This work was supported by the Anadolu University Scientific Research Projects (BAP-1401S008) fund, which is part of

the PhD thesis of ZF. Part of this work was also presented in ISEO 2014, Istanbul.

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Goger, G., Demirci, B., Ilgın, S. & Demirci, F. (2018). Antimicrobial and toxicity profiles evaluation of the chamomile

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17

RESEARCH ARTICLE

Essential oil composition of Stachys obliqua Waldst. et Kit.

Betül Demirci1, Gülsüm Yıldız1,*, Neşe Kırımer1, Atila Ocak2 and K. Hüsnü Can Başer3

1 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, 26470, TURKEY 2 Department of Biology, Faculty of Science and Literature, Osmangazi University, Eskişehir, 26480, TURKEY 3 Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia (Lefkoşa), N. CYPRUS, Mersin 10,

TURKEY


Abstract

The genus Stachys L. (Lamiaceae) is represented in Turkey by 83 species and altogether 109 taxa. The rate of endemism in Turkey is

43.4 with 33 species. Stachys. obliqua Waldst et Kit was collected in July, 2015 in Yarımca to Eskişehir. The essential oil from air-dried

aerial parts was isolated by hydrodistillation using a Clevenger apparatus. Chemical composition of the oil was investigated using GC-

FID and GC/MS techniques. In total, 97 compounds were identified making up 70.1% of the total volatile constituents. Hexadecanoic

acid (10.1%), germacrene D (8.2%), hexahydrofarnesyl acetone (4.4%), β-bourbonene (2.1%) were found as main constituents in the

oil.

Keywords: Stachys obliqua, essential oil, GC-MS, GC-FID.

Introduction

The genus Stachys L., one of the largest genera of the Labiatae (Lamiaceae) family, is widely distributed in

the Mediterranean countries, South Western Asia, North and South America and South Africa (Bhattacharjee,

1980). It is represented in Turkey by 83 species and altogether 109 taxa (Akcicek, 2010). The rate of endemism

in Turkey is 43.4 with 33 species (Davis et al., 1988, Guner et al., 2000).

Stachys are used as wild tea in Anatolia and Iran (Ozturk et al., 2009) and in Anatolia they are known as

'Adaçayı', `Dağ çayı' and `Balbaşı' (Sezik & Basaran, 1985). Ethno-botanical notes are accessible showing the

utilization of species like thyme in Mediterranean cultures (Akcicek et al., 2012, Goren, 2014).

Decoctions or infusions of Stachys are utilized as tonics to treat skin disorders or taken internally for

gastrointestinal problems (Ozturk et al., 2009), cold, fever and cough (Cakir et al., 1997).

In the literature, some members of the genus have been reported for their anti-inflammatory (Khanavi et al.,

2005, Skaltsa et al., 2000), anti anxiety (Rabbani et al., 2003), antibacterial (Grujic‐Jovanovic et al., 2004),

anti-nephritic (Hayashi et al., 1994), anti-Helicobacter pylori (Stamatis et al., 2003) and antioxidant effects

(Khanavi et al., 2009).

Our study deals with the analysis of essential oil isolated from the aerial parts of S. obliqua Waldst. & Kit

growing in Eskişehir, Turkey.


Plant material

S. obliqua was collected in July, 2015 in Yarımca to Eskişehir 2nd km by the road side (voucher specimen

code: K.H.C. Baser 1867). A voucher specimen is also deposited at the Herbarium of Faculty of Pharmacy of

the Anadolu University, Eskişehir, Turkey (ESSE No: 15022).


18

Isolation of essential oil

The essential oil from air-dried aerial parts was isolated by hydrodistillation using a Clevenger apparatus.

Obtained oil was stored in a dark coloured vial at low temperature before analysis. Oil yield was calculated

as 0.01% on moisture-free basis.

GC-MS analysis

The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 0.25

mm, 0.25 m film thickness) was used with helium as carrier gas (0.8 ml/min). GC oven temperature was

kept at 60C for 10 min and programmed to 220C at a rate of 4C/min, and kept constant at 220C for 10

min and then programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector

temperature was set at 250C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450.

GC analysis

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300C. To

obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same

column applying the same operational conditions. Relative percentage amounts of the separated compounds

were calculated from FID chromatograms. The analysis results are given in Table 1.

Identification of components

Identification of the essential oil components were carried out by comparison of their relative retention times

with those of authentic samples or by comparison of their relative retention indices (RRI) to the series of n-

alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) (McLafferty

&Koenig, 1989, Koenig et al., 2004) and in-house “Başer Library of Essential Oil Constituents” built up by

genuine compounds and components of known oils, as well as MS literature data (Joulain & Koenig, 1998,

ESO 2000, 1999) was used for the identification.


Oil yield was calculated as 0.01% (w/w) from the hydrodistillation of the dried aerial parts of S. obliqua using

a Clevenger-type apparatus.

In total, 97 compounds were identified by GC and GC/MS analysis, amounting to 70.1% of the whole oil. The

composition of the essential oil of S. obliqua is reported in Table 2. The chemical composition of S. obliqua

essential oil showed hexadecanoic acid (10.1%), germacrene D (8.2%), hexahydro farnesyl acetone (4.4%), β-

bourbonene (2.1%) as main constituents.

In a previous study, the major components of S. obliqua collected from Fethiye were reported as germacrene

D (25.4%), thymol (16.4%), borneol (4.9%), α-pinene (4.7%) and isomenthol (3.4%) (Harmandar et al., 1997).

According to Goren et al. (2011) germacrene D (45%) and β-caryophyllene (17%) were the main components

of the oil from plants gathered in Balıkesir.

Table 1. Chemical composition of S. obliqua essential oil.

RRIa Compound %b

1032 α-Pinene tr

1203 Limonene 0.5

1244 2-Pentylfuran 0.1

1280 p-cymene 0.1


19

1393 3-Octanol 0.1

1400 Nonanal 0.8

1443 Dimethyl tetradecane* 0.5

1452 1-Octen-3-ol 1.2

1466 α-Cubebene 0.1

1497 α-Copaene 0.3

1499 α-Campholene aldehyde 0.4

1506 Decanal 0.2

1528 α-Bourbonene 0.3

1535 β-Bourbonene 2.1

1553 Linalool 1.4

1562 Octanol 0.2

1589 β-Ylangene 0.8

1597 β-Copaene 0.6

1600 β-Elemene 0.9

1604 2-Undecanone 0.2

1611 Terpinene-4-ol 0.2

1612 β-Caryophyllene 1.0

1638 β-Cyclocitral 0.3

1648 Myrtanal 0.4

1655 (E)-2-Decenal 0.8

1659 γ-Gurjunene 0.6

1670 trans-Pinocarveol 0.4

1683 trans Verbenol 1.0

1687 α-Humulene 0.2

1688 Selina-4,11-diene 0.5

1694 p-Vinyl anisole 0.3

1704 γ-Muurolene 0.3

1706 α-Terpineol 0.7

1715 (E,E)-2,4-Nonadienal 0.2

1726 Germacrene D 8.2

1740 α-Muurolene 0.3

1744 α-Selinene 0.3

1751 Carvone 0.4

1764 (E)-2-Undecenal 0.9

1773 δ-Cadinene 0.6

1776 γ-Cadinene 0.2

1779 (E,Z)-2,4-Decadienal 0.3

1786 ar-Curcumene 0.3

1804 Myrtenol 0.3

1808 Nerol 0.1

1815 2-Tridecanone 0.1

1827 (E,E)-2,4-Decadienal 0.7

1838 (E)-β-Damascenone 0.5

1845 trans-Carveol 0.5

1849 Calamenene 0.2

1857 Geraniol 0.2

1868 (E)-Geranyl acetone 1.4

1878 1-Methyl naphthalene 0.4


20

1925 2-Methyl naphthalene 0.3

1945 1,5-Epoxy salvial-4(14)-ene 0.1

1958 (E)-β-Ionone 1.2

1965 2-Ethyl hexanoic acid 0.2

1972 1-Ethyl napthalene 0.3

2000 Eicosane 0.1

2008 Caryophyllene oxide 1.5

2009 trans-β-Ionone-5,6-epoxide 0.3

2037 Salvial-4(14)-en-1-one 0.3

2041 Pentadecanal 0.7

2046 Norbourbonone 0.3

2050 (E)-Nerolidol 0.2

2084 Octanoic acid 0.2

2130 Salviadienol 0.7

2131 Hexahydrofarnesyl acetone 4.4

2145 Valeranone 0.4

2174 Fokienol 0.5

2179 3,4-Dimethyl-5-pentylidene-2(5H)-furanone 1.5

2192 Nonanoic acid 0.2

2200 Docosane 0.5

2209 T-Muurolol 0.4

2226 Methyl hexadecanoate 0.3

2255 α-Cadinol 0.5

2273 Selin-11-en-4α-ol 1.5

2278 Torilenol 0.6

2298 Decanoic acid 0.3

2300 Dibenzofuran 0.4

2300 Tricosane 0.4

2324 Caryophylla-2(12),6(13)-dien-5α-ol (=Caryophylladienol II) 0.4

2345 Galaxolide-I 0.3

2373 4-oxo-α-Ylangene 0.4

2384 Farnesyl acetone 0.3

2384 1-Hexadecanol 0.3

2500 Pentacosane 0.2

2503 Dodecanoic acid 0.7

2607 1-Octadecanol 1.8

2607 14-Hydroxy-δ-cadinene 0.2

2622 Phytol 0.8

2655 Benzyl benzoate 0.1

2670 Tetradecanoic acid 0.9

2700 Heptacosane 1.4

2740 Anthracene 0.2

2822 Pentadecanoic acid 0.1

2931 Hexadecanoic acid 10.1

Total 70.1 aRRI Relative retention indices calculated against n-alkanes; % calculated from FID data; tr Trace (< 0.1 %); * Correct isomer not identified


21

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Royal Botanic Garden Edinburgh, 38(1), 65-96.

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of Botany, 34(2), 131-136.

Davis, P.H., Mill, R., Tan, K. (Eds.), (1988). Flora of Turkey and East Aegean Islands, Vol. 10. Edinburgh University Press,

Edinburgh, pp. 204–206.

Guner, A., Ozhatay, N., Ekim, T., & Baser, K. H. C. (2000). Flora of Turkey and the East Aegean Islands, Vol. 11. Second

Supplement, Edinburgh.

Ozturk, M., Duru, M. E., Aydogmus-Ozturk, F., Harmandar, M., Mahlicli, M., Kolak, U., & Ulubelen, A. (2009). GC-MS

analysis and antimicrobial activity of essential oil of Stachys cretica subsp. smyrnaea. Natural product communications,

4(1), 109-114.

Sezik, E., & Basaran, A. (1985). Phytochemical investigation on the plants used as folk medicine and herbal tea in Turkey;

essential oil of Stachys lavandulifolia Vahl. var. lavandulifolia. Journal of Faculty of Pharmacy Istanbul, 21, 98.

Akcicek, E., Dirmenci, T., & Dundar, E. (2012). Taxonomical notes on Stachys sect. Eriostomum (Lamiaceae) in Turkey.

Turkish Journal of Botany, 36(3), 217-234.

Goren, A. C. (2014). Use of Stachys species (Mountain Tea) as herbal tea and food. Records of Natural Products, 8(2),

71.

Cakir, A., Duru, M. E., Harmandar, M., Izumi, S., & Hirata, T. (1997). The volatile constituents of Stachys recta L. and

Stachys balansae L. from Turkey. Flavour and fragrance Journal, 12(3), 215-218.

Khanavi, M., Sharifzadeh, M., Hadjiakhoondi, A., & Shafiee, A. (2005). Phytochemical investigation and anti-

inflammatory activity of aerial parts of Stachys byzanthina C. Koch. Journal of Ethnopharmacology, 97(3), 463-468.

Skaltsa, H., Bermejo, P., Lazari, D., Silvan, A. M., Skaltsounis, A. L., Aurora, S. A. N. Z., & Abad, M. J. (2000). Inhibition of

prostaglandin E2 and leukotriene C4 in mouse peritoneal macrophages and thromboxane B2 production in human

platelets by flavonoids from Stachys chrysantha and Stachys candida. Biological and Pharmaceutical Bulletin, 23(1), 47-

53.

Rabbani, M., Sajjadi, S. E., & Zarei, H. R. (2003). Anxiolytic effects of Stachys lavandulifolia Vahl on the elevated plus-

maze model of anxiety in mice. Journal of Ethnopharmacology, 89(2), 271-276.

Grujic‐Jovanovic, S., Skaltsa, H. D., Marin, P., & Sokovic, M. (2004). Composition and antibacterial activity of the essential

oil of six Stachys species from Serbia. Flavour and Fragrance Journal, 19(2), 139-144.

Hayashi, K., Nagamatsu, T., Ito, M., Hattori, T., & Suzuki, Y. (1994). Acteoside, a component of Stachys sieboldii MIQ,

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pylori activity of Greek herbal medicines. Journal of ethnopharmacology, 88(2), 175-179.

Khanavi, M., Hajimahmoodi, M., Cheraghi-Niroomand, M., Kargar, Z., Ajani, Y., Hadjiakhoondi, A., & Oveisi, M. R. (2009).

Comparison of the antioxidant activity and total phenolic contents in some Stachys species. African Journal of

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Joulain, D., Koenig, W.A. (1998). The Atlas of Spectra Data of Sesquiterpene Hydrocarbons, EB-Verlag, Hamburg.

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from Turkey. Flavour and fragrance Journal, 12(3), 211-213.

Goren, A. C., Piozzi, F., Akcicek, E., Kılıç, T., Çarıkçı, S., Mozioğlu, E., & Setzer, W. N. (2011). Essential oil composition of

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Nat. Volatiles & Essent. Oils, 2018; 5(1): 23-28 Sadgrove & Van Wyk

23

RESEARCH ARTICLE

Major volatile compounds in the essential oil of the aromatic culinary herb Pelargonium crispum (Geraniaceae)

Nicholas J. Sadgrove and Ben-Erik Van Wyk*

Department of Botany and Plant Biotechnology, University of Johannesburg, Johannesburg 2006, SOUTH AFRICA


Abstract

The aromatic culinary herb Pelargonium crispum is used as a condiment across the world to confer a lemony aroma to food. The

phytochemistry of one cultivar of unknown origin has been tentatively described in earlier studies, with only the dominant volatile

components assigned as neral and geranial and with chrysin as the major flavone. In the current study a more detailed chemistry of

the essential oils is given, and the major flavanone in the leaves is assigned as (2S)-(-)-pinocembrin, a dihydro derivative of chrysin.

The predominance of neral and geranial is confirmed and a number of related oxide and phenylpropanoid ester derivatives are

assigned. A major outcome is the realization that the wild specimens sampled by us displays different chemistry to the chemotype

previously described.

Keywords: Neral, geranial, (2S)-(-)-pinocembrin, phytochemistry, wild form

Introduction

Pelargonium crispum (L.) L’Her. is a native of South Africa, occurring from Worcester to Bredasdorp in the

south of the Western Cape Province. It is a small aromatic shrub that is valued for the lemony aroma that it

confers to food or tea (Lim, 2014). It is widely cultivated in herb gardens and several cultivars (some

possibly of hybrid origin) are known. Studies have demonstrated that the volatiles are not merely the

flavor principle and that tartaric acid that is accumulated in the leaves also contributes a sour taste

(Stafford, 1961). Various biosynthetic studies of tartaric acid demonstrated routes from simple sugars via

ascorbic acid (Lim, 2014).

As a food condiment, P. crispum is recognized for more than its aesthetic contribution to dishes. The

flavone chrysin is implicated in therapeutic or health boosting outcomes, particularly related to anti-

inflammatory, antiviral and anticancer activities (Lim, 2014). In addition to chrysin, Williams et al. (1997)

identified a C-methyl flavanone that was suggested to be either strobopinin or cryptostrobin, which are

structurally very similar to pinocembrin but differ markedly by their masses, due to an aromatic methyl

moiety.

An earlier investigation of the chemistry of the essential oils under the vernacular names ‘Crispum

variegatum’ and ‘Lemon fancy’ merely identified neral, geranial and ‘sesquiterpenes’ (Lis-Balchin et al.,

1998). Antimicrobial activities of these essential oils were measured by agar well diffusion, so it is difficult

to generalize about the outcome, but in relative terms, neral and geranial-rich essential oils were among

the most active tested in that study (Lis-Balchin et al., 1998).

Evidently, the chemistry of the essential oils needs to be more comprehensively assessed, as there are

many essential oils dominated by neral and geranial. It is also necessary to confirm if chrysin predominance

in the leaf exudate is consistent across the species distribution, to provide backing to the health boosting

claims that are now made for this species when used as a food condiment. Thus, the current study aims to


24

provide a more comprehensive chemistry to the essential oils from three specimens collected in their

endemic distribution. Isolation of the major flavonoid in this population aims to confirm or nullify the

occurrence of chrysin in randomly selected specimens.


Plant material

Plant samples from three different individuals were provided by the property owners of the farm

Klipbokkop in the vicinity of Worcester, near Brandvlei Nature Reserve (33.80487093844676˚ S;

19.37562882900238˚ E). The material was collected from strongly aromatic shrubs of ca. 1 m high, growing

in rocky sandy soil in mountainous terrain. A herbarium voucher specimen (Sadgrove 545) was deposited in

the University of Johannesburg Herbarium (JRAU). Careful examination of the material by one of us (B EVW,

a taxonomist) confirmed the identity of the species as P. crispum, as is evidenced by the small leaves and

single-flowered inflorescences.

Isolation of the essential oil

Hydrodistillations were performed using a Clevenger-type apparatus and essential oils were dried over

anhydrous sodium sulfate and stored at 4 °C away from light until analysis.

Analysis

Essential oils were characterised by GC-MS and NMR, and quantified with GC-FID. Only components higher

than 1% by MS were identified.

GC-MS operating conditions were as follows: Shimadzu 2010 with detector interface at 250 °C; ion source

200; injector temperature 200 °C; carrier gas helium; 1 µl injections with a split ratio (1:20); fitted with an

OV-1 (WCOT) (non-polar) column; column flow at 1 ml/min; column ramp: 60 °C (no hold), 5 °C per min

then held at 280 °C for 5 min. Identification of compounds was made by comparing the mass spectra and

retention indices (calculated relative to n-alkanes) with the NIST library and Adams (2007) and some major

components confirmed by NMR. GC-FID operating conditions were identical to GC-MS and n-alkanes were

used to guide peak assignment. NMR assignments were made using a 500 Mhz Bruker Avance (Bruker,

Germany) in CDCl3. Spectra for neral and geranial were matched to shifts included in Kelm et al. (1997) and

linalool to shifts in Blanc et al. (2005).

(2S)-(–)-Pinocembrin

100 g of plant material was extracted in methanol then the volume reduced under pressure. The residue

was re-extracted into dichloromethane, filtered and the residue (760 mg) was subjected to column

chromatography over silica gel in mobile phase 1:9 ethyl acetate:petroleum ether. A total of 203 mg was

isolated and first dissolved into CDCl3 for NMR analysis then re-dissolved into d-DMSO for comparison to

literature values. The spectra were identical to shifts included in Tanaka et al. (1985). Nowhere in the

literature are the spectra for (2S)-(–)-pinocembrin given in CDCl3 so 13C shifts are provided here; 13C-NMR

(CDCl3) δ: 196.1 (C-4), 164.6’, 164.59’, 163.4’ (C-5, C-7 or C-8a)’, 138.5 (C-1’), 129.1 (C-4’), 129 (C-3’and C-

5’), 126.3 (C-2’and C-6’), 103.5 (C-4a), 96.9’’, 95.6’’ (C-6 or C-8)’’, 79.4 (C-2), 49.5 (C-3). Optical rotations

were calculated on a Polartronic H532 and the negative enantiomer assigned. Absolute stereochemistry

was inferred from Napal et al. (2009).


25


Confirmation of the dominant components in the essential oil, neral and geranial, is unsurprising (Table 1).

However, previous reports of ‘sesquiterpenes’ is not applicable to the chemical character in the current

study. Sesquiterpenes were observed but comprise only trivial relative abundance compared to other

monoterpenes, such as linalool and hydroxycitronellol, and monoterpene alkane and phenylpropanoid

esters, such as geranyl hexanoate or geranyl benzoate respectively. Z- and E-linalool oxides and epoxy

oxides are also present, as well as the two methyl cinnamate isomers. The dominant two sesquiterpenes

were β-bourbonene and α-curcumene, but these are in only minor quantities.

The lemony aroma of the essential oil from P. crispum is without a doubt derived from the terpenoid

mixture of the two dominant components geranial (citral A; α-citral) and neral (citral B; β-citral), which are

merely cis/trans isomers of 3,7-dimethyl-2,6-octadienal. It is noteworthy that geranial and neral were

detected in only one of 13 shrubby species of Pelargonium studied by Lalli et al. (2006), namely P.

citronellum (the reported yields respectively 27.2 and 17.4%).

Table 1. Major essential oil compounds in three individual plants (A, B and C) of Pelargonium crispum sampled from a

natural population of the species.

AI Pub AI A B C

Yield (% g/g wet wt)

0.56 0.49 0.55

p-Cymene 1024 1020 1.8 0.5 0.9

Z-Linalool oxide 1070 1067 3.0 2.3 4.1

E-Linalool oxide 1087 1084 2.9 2.1 3.8

Linalool 1105 1100 12.1 4.5 5.0

Nonenol 1144 1164 - - 1.6

Terpinen-4-ol 1188 1174 1.7 1.9 0.9

Neral 1250 1235 14.9 18.6 13.3

Geranial 1283 1264 24.3 30.9 20.7

Z-Epoxy-linalool oxide 1290 * 2.0 7.5 10.9

E-Epoxy-linalool oxide 1295 * 2.2 5.9 9.9

Z-Methyl cinnamate 1312 1299 2.1 1.6 -

Hydroxycitronellol 1342 1359 3.4 1.5 -

E-Methyl cinnamate 1388 1376 5.5 7.0 7.0

β-Bourbonene 1398 1387 2.3 1.9 1.1

Funebrene, 2-epi-β- 1426 1411 0.8 0.7 1.0

β-Cedrene 1434 1419 0.5 0.8 0.5

α-Curcumene 1490 1479 1.7 2.2 3.1

Spathulenol 1593 1577 1.0 0.3 0.6

Globulol 1600 1590 0.6 1.5 0.4

Geranyl tigliate 1703 1696 1.6 - -

Neryl hexanoate 1730 1732 - 1.3 2.1

Geranyl hexanoate 1757 1755 3.4 2.3 3.2

Neryl benzoate 1955 1946 3.3 0.5 1.8

Geranyl benzoate 1973 1951 0.8 0.6 1.6

Total

92.1 96.5 93.4


26

The mixture of geranial and neral is often called ‘citral’ or ‘lemonal’, which earned its name in the 1800s

after it was isolated from a lemon Citrus species, but today the major supply of citral comes from

Cymbopogon (Akhila, 2009). Due to poor separation of the isomers, citral was initially treated as a pure

isolate, but was subsequently demonstrated to be a mixture, with their structures established as early as

1947, but confirmed later by NMR (Ohtsuru et al., 1967). The antimicrobial activity of ’citral’ is already

known and reported to be noteworthy, with the general value of 0.05% v/v (<0.5 mg/ml) across a range of

Gram-positive and Gram-negative bacteria (Onawunmi, 1989). Citral is also regarded as a prominent insect

pheromone (Kuwahara & Suzuki, 1983) and it is claimed to have antiadipogenic activity in rats (Modak &

Mukhopadhaya, 2011).

Trace amounts of similar flavonoids to pinocembrin were observed but could not be isolated in sufficient

quantity for proper chemical assignment. However, the assignment of pinocembrin as the overwhelmingly

dominant flavonoid is in contrast to previous reports of chrysin as predominating (Williams et al., 1997).

This suggests that chemotypes exist within the species or that cultivars grown in Europe may have a hybrid

origin.

The realization of pinocembrin as the major flavonoid in the leaves came as a surprise since it was not

assigned earlier (Williams et al., 1997). The possibility of a misidentification in the study by Williams et al.

(1997) of the C-methyl flavone was initially contemplated by the authors of the current study. Since we

identified pinocembrin, which is not a C-methyl flavanone, there is a possibility that it was observed in the

study by Williams et al. (1997), but incorrectly characterized. However, this is not the case because the

masses of the compounds do not overlap. It is evident that pinocembrin was not present in the material

studied by Williams et al. (1997), which indicates that at least two chemically distinct variants occur within

P. crispum. It is not yet clear if this will also correlate to essential oil chemotypes, but such an outcome will

not be surprising.

Much like chrysin, pinocembrin has also demonstrated biological activities that implicate positive human

health benefits. It is present in honey and propolis; but well known plant sources include ginger roots

(Zingiber officinale Radix), Eucalyptus sieberi, wild marjoram (Origanum vulgare) and Eriodictyon

californicum; the latter is from where pinocembrin was first isolated (Lan et al., 2016). In 2008 the

compound was approved in China for use in the treatment of stroke, which is thought to at least partly

derive from its cognition improving activity via neurovascular unit protection (Liu et al., 2014). Other

noteworthy benefits include anti-inflammatory activity, modulation of mitochondrial function, regulation of

apoptosis, protection of the blood-brain barrier (Lan et al., 2016; Rasul et al., 2013) and cardioprotection

(Lungkaphin, 2015). Furthermore, citral is not the only pheromone identified in the current study. The

active antifeedant component in an extract from Flourensia oolepsis, which was used against pest larvae of

Epilachna paenulata, was in fact pinocembrin (Napal et al., 2009).

There is some overlap in biological activities of chrysin and pinocembrin. Chrysin also has neuroprotective

and anti-inflammatory effects (Oh, 2016). However, mediation of insulin resistance has been somewhat

overlooked for the two flavonoids. It is evident from a study of Chinese propolis that both chrysin and

pinocembrin should be examined more comprehensively for management of insulin resistance (Zhao et al.,

2014). Due to much overlap in biological activities of both chrysin and pinocembrin, it is feasible that health

claims associated with use of P. crispum as a food condiment are warranted in both flavonoid chemotypes.

At this stage it is not known if essential oil chemotypes occur in nature but the current study constitutes

the first comprehensive investigation of three plants from a wild population of the species, which assigns


27

mainly geranial/neral (citral), linalool and their related derivatives at a yield of approximately 0.5% g/g wet

leaves.

ACKNOWLEDGMENT

We thank the University of Johannesburg and the National Research Foundation of South Africa for financial support

(post-doctoral fellowship to NJS and grant number UID 8442 to BEVW).

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intense diet model of obesity. Indian Journal of Pharmacology, 43(3), 300-305.

Napal, G. N. D., Carpinella, M. C., & Palacios, S. M. (2009). Antifeedant activity of ethanolic extract from Flourensia

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Oh, Y. S. (2016). Bioactive compounds and their neuroprotective effects in diabetic complications. Nutrients, 8, 472-

492.

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Rasul, A., Millimouno, F. M., Eltayb, W. A., Ali, M., Li, J., & Li, X., (2013). Pinocembrin: A novel natural compound with

versatile pharmacological biological activities. BioMedical Research International, 379850, 1-9.

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Documents

NVEO 2018, Volume 5, Issue 1 fileEmail: [email protected] Abstract Essential oils (EOs) are known for their antimicrobial activities against a broad range of microorganisms