Composition and Antimicrobial Activity of Celery _Apium graveolens ..

Composition and Antimicrobial Activity of Celery (Apium graveolens)

Leaf and Root Extracts Obtained with Liquid Carbon Dioxide

A. Sipailiene and P.R. Venskutonis A. Sarkinas and V. Cypiene

Department of Food Technology Lithuanian Food Institute

Kaunas University of Technology Kaunas

Lithuania Lithuania



Keywords: essential oil, extract yield, carvone, limonene, phthalides



Abstract

Dried roots and leaves of celery were extracted with liquid carbon dioxide in

the pilot plant scale equipment. In total, 9 extractions of roots and 4 extractions of

leaves were performed at ambient temperature and 60 bar pressure by using

different operation cycle programs. Extract yields were up to 1.6% both from roots

and leaves. Volatile compounds were hydrodistilled from the extracts in a Clevenger

type apparatus and their composition was analysed by capillary gas

chromatography and mass spectrometry (GC/MS). The main constituents in the oil

of roots were limonene, carvone and 3n-butylphthalide. The essential oil of leaves

contained higher amount of limonene comparing to the roots, and very small

amount of carvone. Other differences in the volatile oil composition between roots

and leaves also were observed. The extracts were tested on the following

microorganisms: Escherichia coli, Listeria monocytogenes, Citrobacter freundii,

Hafnia alvei, Salmonella typhimurium, Bacillus cereus, Enterococcus faecalis,

Enterobacter aerogenes, Staphylococcus aureus and Proteus vulgaris. The

antimicrobial effect was assessed using agar diffusion method by applying ethanolic

solutions of extracts. It was found that all the investigated leaf extracts were effective

inhibitors of H. alvei, S. aureus, E. coli, Bac. cereus, E. faecalis and E. aerogenes,

however the extracts isolated from the roots were less effective; all of them possessed

high activity only against B. cereus and E. faecalis. C. freundii and P. vulgaris were

resistant against celery extracts isolated both from roots and leaves.



INTRODUCTION

The antimicrobial properties of spices and aromatic herbs have been tested in

numerous studies, including their role as a ‘natural’ method of food preservation.

Biological properties of different anatomical parts of celery and its extracts were reported

in various literature sources (Bedin et al., 1999). Celery had potent antimicrobial activity

against Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae (Krishna and

Banerjee, 1999); celery seed oil was active against Campylobacter jejuni (Friedman et al.,

2002), celery essential oil inhibited various pathogenic and saprophytic microorganisms

(Elgayyar et al., 2001). However, in some cases it was reported that essential oil of celery

was inactive or had little activity (Kivanc and Akgul, 1986).

Various biologically active compounds were identified in celery essential oils and

extracts. For instance, sedanolide and two senkyunolides were active against nematode,

mosquito larvae and fungi (Momin and Nair, 2001), furanocoumarins inhibited Listeria

monocytogenes, Escherichia coli O157:H7, and Micrococcus luteus (Ulate-Rodrigues et

al., 1997).

Plant extracts can be obtained by different extraction methods with various

solvents. Extraction with liquid and supercrititical gases provides several important

advantages, which were thoroughly discussed in numerous literature sources (Moyler,

1993). This method has also been used with Umbelliferae family plants, including celery

(Catchpole et al., 1996; Dauksas et al., 2002). It is interesting to note that the yields of

steam distilled oil and isolated by liquid CO2 extracts from celery seeds were almost

equal and constituted 2.5-3.5% and 3% respectively, whereas in case of parsley seeds 2.0-

3.5% of oil were distilled by steam during 5 h, and 9.8% of the extract were obtained by







Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry

Eds. K.H.C. Başer, G. Franz, S. Cañigueral, F. Demirci, L.E. Craker and Z.E. Gardner 71

Acta Hort. 677, ISHS 2005

CO2 during 2 hours of extraction at 58 bar and 20°C (Moyler, 1994).

The goal of this study was to prepare celery leaf and root extracts with liquid

carbon dioxide and to determine extract yields, composition and antimicrobial activity.



MATERIALS AND METHODS

The company “Meta” provided dried celery (Apium graveolens L.) leaves and

roots. They were comminuted in a hammer mill and extracted in a pilot plant at company

“Stumbras”, Lithuania. Carbon dioxide extraction unit (Fig. 1) operated at ambient

temperature and a pressure of 60 bar. The main extraction cycle consisted of the three

steps: (i) filling of the extractor containing ground raw material with liquid CO2; (ii)

maceration of raw material with solvent; (iii) solvent removal and extract collection at

40°C.

The antimicrobial properties were evaluated by diffusion method into the agar.

Ten undesirable in food products species, namely Escherichia coli, Listeria

monocytogenes, Citrobacter freundii, Hafnia alvei, Salmonella typhimurium, Bacillus

cereus, Enterococcus faecalis, Enterobacter aerogenes, Staphylococcus aureus and

Proteus vulgaris, were used as test cultures. Bacteria were grown on the plate count agar

(LAB 98, LAB M) 24 hours at 37°C. After cultivation test culture cells were washed with

saline and cell suspensions were prepared according to McFarland standard. The

suspension of bacteria cells was introduced into the dissolved and cooled to 47°C media,

10 ml of which were pipetted into a 90 mm diameter Petri plate. Nine-millimetre diameter

wells were pushed in the agar and filled with 1, 5 or 10% solutions of extracts. The plates

were incubated overnight at 37°C temperature, and the clear zone of inhibition (mm) was

measured.

The content of the essential oil in ground roots, leaves and CO2-extracts was

determined in European Pharmacopoeia apparatus. Composition of essential oils was

analysed by capillary gas chromatography (GC) and coupled gas chromatography-mass

spectrometry (GC/MS). Essential oils were diluted in diethyl ether (20 µl in 1 ml) and

analysed with Fisons 8261 gas chromatograph equipped with flame ionisation detector (FID)

on a fused silica capillary column DB-5, 25 m length, 0.32 mm i.d., and 0.5 µm film

thickness. Helium was used as a carrier gas with a flow rate of 1.6 ml/min; detector

temperature was 260°C, oven temperature was programmed from 40°C to 250°C at the rate

of 4°C min-1. Split injector was heated at 250°C, split ratio was 15:1. Data was processed on

a DP 800 integrator.

For the identification essential oils were analysed on a HP 5890 (II) instrument

equipped with a 5971 series mass selective detector in the electron impact ionisation

mode at 70eV, and the following GC parameters: split inlet 1:10; helium as a carrier gas

at a flow rate of 2 ml min-1; fused silica HP5 MS column (Hewlett Packard, crosslinked

5% phenyl methyl silicone) 30 m length, 0.25 mm id, 0.25 µm film thickness,

temperature program from 40 to 250°C increasing at 4°C min-1. Identification was based

mainly on the comparison of retention indices (Adams, 2001) and mass spectra

(NIST/EPA/NIH Mass Spectral Database NBS75K).



RESULTS AND DISCUSSION

Extraction results are summarised in Table 1. Extract yields varied from 0.52 to

1.59% from the roots and from 0.56 to 1.26% from the leaves. So far as it was not

possible to change the pressure and the temperature in the pilot extractor, three main

factors could have an influence on the extraction yield: quality of raw material, fineness

of particles and extraction procedure. It was observed that additional grinding of raw

material in the hammer mill increased the yield. It is known that hydrophobic essential oil

substances are soluble in liquid carbon dioxide, therefore it can be expected that major

part of the oil will be isolated from the plant material. The content of the essential oil in

celery roots was very low, actually it was difficult to distil measurable amount of oil from

100 g of ground roots by the standard method. Celery root and leaf extracts contained

higher amounts of oil and it confirms that extraction with liquid carbon dioxide is suitable









72

method to concentrate essential oil components.



Composition of Volatile Compounds in the Extracts

The composition of volatile constituents in root and leaf extracts is provided in

Table 2. The main constituents in celery essential oils were terpenes, sesquiterpenes and

phthalides. Limonene was the major constituent both in root and leaf volatile oil, however

it was more abundant in the leaves (>30%). Very high amount of other aliphatic terpene,

myrcene was characteristic to leaf oils, while in the root oil it constituted less than 0.2%.

The content of 3n-butylphthalide, which was the major phthalide group component was

almost equal in root and leaf extracts. Considerable amounts of other phthalides, mainly

sedanenolide and sedanolide were determined in the roots. These two phthalides were not

detected in the leaf extract. The content of carvone was the main quantitatively

differentiating factor in the composition of two oils. This compound was abundant in the

root extracts (>17%), while in the leaves it was a trace constituent (<0.2%). Higher

content of sesquiterpenes in the leaf essential oil has to be mentioned; for instance, the

content of three identified selinenes exceeded 9% in the leaf oil. In the root oil, β-

selinene, which was a single selinene isomer constituted up to 1.5%. Also, the contents of

α-humulene and β-caryophyllene were considerably higher in the leaf oil.



Antibacterial Effect of Celery Extracts

Results presented in Table 3 show that celery leaf and root extracts in most cases

inhibited bacterial growth in the test cultures. However, P. vulgaris and C. freundii were

resistant to all celery extracts at the applied concentrations. B. cereus was the most

sensitive bacteria to celery extracts; inhibition was observed at the all applied

concentrations of the all extracts used in the study. In general, higher concentrations of

the extracts had bigger effect on bacteria. In some cases concentration was not an

important factor, even comparing 1 and 10% concentrations (e.g., B. cereus, leaf extract,

batch no. 3; S. aureus, leaf extract, batch no. 1). The differences between extraction

batches have also been observed. For instance, leaf extracts from batches no. 1 and no. 2

did not have any effect on L. monocytogenes, whereas, batch no. 3 inhibited the growth of

these pathogens at the all concentrations applied. Root extract from the batch no. 1 had

the effect only on L. monocytogenes and E. faecalis (10%), while root extract from the

batch no. 3 inhibited all tested bacteria except for C. freundii and P. vulgaris. Crude

extracts are complex mixtures containing various compounds, both active and having no

effect on microorganisms.



CONCLUSIONS

Liquid carbon dioxide is suitable solvent for the extraction of celery volatile

constituents, however, the yields of the extracts were not high, most likely due to a low

amount of essential oil and other soluble constituents at the CO2 parameters applied.

Limonene and 3n-butylphthalide were important components of volatile fractions distilled

from celery roots and leaves. High amounts of myrcene and some sesquiterpenes were

characteristic to leaf extracts, root extracts contained high percentage of carvone. Leaf

extracts were more active antimicrobial agents compared to root extracts. P. vulgaris and

C. freundii were most resistant to celery extracts, while the growth of B. cereus was

inhibited by all extracts at the all applied concentrations.



Literature Cited

Adams, R.P. 2001. Identification of Essential Oil Components by Gas Chromatography/

Quadrupole Mass Spectroscopy. Allured Publishing, USA.

Bauermann, U., Ehrich, J. and Thomann, R. [Herb essential oils.] Aetherische Krautoele.

Lebensmitteltechnik 26:36-38.

Bedin, C., Gutkoski, S.B. and Wiest, J.M. 1999. Antimicrobial activity of spices. Higiene

Alimentar. 13:26-29 (Portuguese).

Catchpole, O.J., Grey, J.B. and Smallfield, B.M. 1996. Near-critical extraction of sage,









73

celery, and coriander seed. J. Supercrit. Fluids 9:273-279.

Dauksas, E., Venskutonis, P.R., Sivik, B. and Nillson, T. 2002. Effect of fast CO2

pressure changes on the yield of lovage (Levisticum officinale Koch.) and celery

(Apium graveolens L.) extracts. J. Supercrit. Fluids 22:201-210.

Elgayyar, M., Draughon, F.A., Golden, D.A. and Mount, J.R. 2001. Antimicrobial

activity of essential oils from plants against selected pathogenic and saprophytic

microorganisms. J. Food Protect. 64:1019-1024.

Friedman, M., Henika, P.R. and Mandrell, R.E. 2002. Bactericidal activities of plant

essential oils and some of their isolated constituents against Campylobacter jejuni,

Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J. Food Protect.

65:1545-1560.

Momin, R.A. and Nair, M.G. 2001. Mosquitocidal, nematicidal, and antifungal

compounds from Apium graveolens L. seeds. J. Agric. Food Chem. 49:142-145.

Moyler, D. 1993. Extraction of flavours and fragrances with compressed CO2. p.140-183.

In: M.B. King and T.R. Bott (eds.), Extraction of Natural Products Using Near-

Critical Solvents. Blackie A&P, London.

Moyler, D. 1994. Spices - recent advances. p.1-70. In: G. Charalambous (ed.),

Developments in Food Science 34, Spices, Herbs and Edible Fungi, Elsevier Science

Publishers, Amsterdam.

Kivanc, M. and Akgul, A. 1986. Antibacterial activities of essential oils from Turkish

spices and citrus. Flav. Fragr. J. 1:175-179.

Krishna, de A.M. and Banerjee, A.-B. 1999. Antimicrobial screening of some Indian

spices. Phytother. Res. 13:616-618.

Ulate-Rodrigues, J., Schafer, H.W., Zottola, E.A. and Davidson, P.M. 1997. Inhibition of

Listeria monocytogenes, Escherichia coli O157:H7, and Micrococcus luteus by linear

furanocoumarins in culture media. J. Food Protect. 60:1046-1049.







Tables





Table 1. Celery extraction parameters and extract yields.



Number of Extraction Extract yield, Essential oil

Extraction extraction cycle* time, % content in the

cycles min extract, %

Roots 1 batch 6 30+15 0.52 3.5

Roots 2 batch 6 30+15 0.84 3.0

Roots 3 batch 12 30+15 0.61 0.9

Roots 4 batch 1 30+300 1.30 2.3

Roots 5 batch 1 30+300 0.67 12.4

Roots 6 batch 1 30+300 0.75 1.2

Roots 7 batch 1 30+300 1.25 0.9

Roots 8 batch 4 30+240 0.87 0.6

Roots 9 batch 6 30+60 1.59 2.0

Leaves 1 batch 1 30+120 0.56 10.5

Leaves 2 batch 6 30+15 1.26 7.5

Leaves 3 batch 1 30+240 0.96 9.6

Leaves 4 batch 7 30+30 0.92 4.0

* Extraction cycle = extractor filling with carbon dioxide + raw material maceration









74

Table 2. Composition (%) of volatile fraction of celery root and leaf extracts.



Constituent Root Leaf Constituent Root Leaf

α-Pinene 0.10 0.15 Myrtenyl acetate (t) 0.14 nd

Camphene 0.16 nd E-Carvyl acetate nd 0.15

β-Pinene 0.46 0.16 γ-Nonalactone (t) 0.17 nd

Myrcene 0.18 9.14 α-Cubebene nd 0.11

Ethyl hexanoate 0.10 nd α-Copaene nd 0.24

Octanal 0.25 nd 1-Tetradecene 0.39 nd

Methyl heptanoate nd 0.63 Methyl undecanoate nd nd

Limonene 21.87 32.16 Tetradecane 0.35 nd

cis-β-Ocimene 0.46 nd β-Caryophyllene 0.53 1.84

trans-β-Ocimene nd 0.28 α-Humulene 0.26 4.34

γ-Terpinene 0.47 0.12 allo-Aromadendrene 0.24 nd

Methyl octanoate nd 0.12 9-epi-trans- 0.13 nd

Caryophyllene (t)

Fenchone 0.17 nd Germacrene D nd 0.68

Linalool 1.63 nd β-Selinene 1.47 1.84

neo-allo-Ocimene nd 0.15 δ-Selinene nd 5.98

Limonene oxide nd 0.13 α-Selinene nd 1.34

Camphor 0.13 nd Neryl propanoate (t) nd 0.42

Pentyl benzene 0.80 0.09 γ-Cadinene nd 0.48

Menthol 0.47 nd δ-Cadinene nd 0.62

Ethyl octanoate 0.25 nd Caryophyllene oxide 1.02 0.67

trans-Carveol nd 0.32 3n-Butylphthalide 11.55 12.64

Carvone 17.71 0.20 cis-3n- 1.80 0.40

Butylidenephthalide

2-trans-Decenal 0.23 nd Unknown 3.39 nd

Methyl nonanoate nd 0.08 Sedanenolide 4.77 nd

Isobornyl acetate 1.31 nd Sedanolide 7.01 nd

2-trans,4-cis-Decadienal 0.30 nd Octadecane 0.31 nd

2-Undecanone nd 0.13 Ethyl hexadecanoate 2.38 nd

2-trans,4-trans- 0.38 nd cis-Falcarinol (t) 1.69 nd

Decadienal

Methyl decanoate nd 0.21 Methyl linoleate 1.61 nd

nd = not detected; t = tentatively identified (based on a good match of mas spectrum)









75

Table 3. Inhibition of various microorganisms by celery leaf and root extracts, mm inhibition zone.



Extract Concen- Hafnia Salmonella Staphylococcus Escherichia Listeria Bacilus Enterococcus Citrobacter Enterobacter Proteus

traction alvei typhimurium aureus coli monocytogenes cereus faecalis freundii aerogenes vulgaris

Leaves 10% 17.5±0.7 27.5±0.7 23.5±0.7 40.0±0.0 - 40.0±0.0 30.0±0.0 13.0±0.0 19.5±0.7 -

batch 5% - 15.5±0.7 20.0±0.0 30.0±0.0 - 30.0±0.0 30.0±0.0 - 15.0±1.4 -

no. 1 1% - 11.5±0.7 25.0±0.0 20.0±0.0 - 32.0±0.0 20.0±0.0 - 9.5±0.7 -

Leaves 10% 17.5±0.7 19.0±1.4 25.0±0.0 19.5±0.7 - 27.0±1.4 22.5±0.7 - 17.0±0.0 -

batch 5% 13.0±0.0 12.0±0.0 21.0±0.0 15.5±0.7 - 24.0±0.0 16.0±1.4 - 13.5±0.7 -

no. 2 1% 9.0±0.0 - 17.0±0.0 12.5±0.7 - 23.0±0.0 12.5±0.7 - - -

Leaves 10% 25.5±0.7 - 25.5±0.7 24.0±0.0 30.0±0.0 25.0±1.4 20.0±0.0 - 18.5±2.1 -

batch 5% 16.5±2.1 - 22.0±0.0 20.0±0.0 25.0±0.0 31.0±1.4 20.0±0.0 - 14.5±0.7 -

no. 3 1% - - 12.0±1.4 11.0±0.0 20.0±0.0 24.5±0.7 11.5±0.7 - - -

Roots 10% - - - - - 40.0±0.0 25.0±0.0 - - -

batch 5% - - - - - 30.0±0.0 - - - -

no. 1 1% - - - - - 30.0±0.0 - - - -

Roots 10% 11.0±0.0 - 33.0±0.0 - 32.0±0.0 29.0±0.0 12.5±0.7 - 15.0±0.0 -

batch 5% - - 31.0±1.4 - 23.5±0.7 28.0±0.0 - - - -

no. 2 1% - - 25.0±1.4 - 24.5±0.7 23.0±1.4 - - - -

Roots 10% 12.0±0.0 12.5±0.7 20.0±0.0 20.0±0.0 30.0±0.0 27.0±0.0 18.0±0.0 - 18.0±0.0 -

batch 5% 11.0±0.0 11.5±0.7 20.0±0.0 18.0±0.0 30.0±0.0 25.0±0.0 18.0±0.0 - - -

no. 3 1% - - 20.0±0.0 12.5±0.71 24.0±0.0 21.0±0.0 15.0±0.0 - - -









76

Figures









Condenser





CO2 tank









Extractor





Extract separator









Fig. 1. Carbon dioxide extraction equipment.









77