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Purity, Antimicrobial Activities and Olfactoric Evaluations of Geraniol/Nerol and Various of Their Derivatives

June 7, 2007

By Jirovetz, Leopold; Buchbauer, Gerhard; Schmidt, Erich; Stoyanova, Albena S; Et al

Abstract

Commercially available geraniol and nerol, as well as some derivatives, were analyzed for their purity using GC and GC/MS. The olfactoric quality of the samples was evaluated by professional perfumers. Antimicrobial testings using an agar dilution and an agar diffusion method were done to obtain information about their activities against some Gram-(+) and Gram-(-) bacteria, as well as the yeast Candida albicans. The effects were compared with those of the phenolic compound eugenol and some synthetic antibiotics. Most of the investigated compounds were found to have a characteristic, pleasant odor and a high activity against all strains of microorganisms used.

Key Word Index

Geraniol, nerol, geranial, geranyl acetone, geranic acid, geranyl esters, geranyl geraniol, geranyl linalool, geranyl

Introduction

For their especially pleasant sweet-floral impressions, geraniol, nerol and many of their derivatives are used in perfumery and cosmetic products, e.g. perfumes, lotions, soaps, deodorants (1-3) and as flavors in food products, such as non-alcoholic beverages, ice creams, candies, baked goods, puddings, chewing gums and syrups (1,4,5).

Although, the antimicrobial activity of many mono- and sesquiterpenes using various different testing methods has been reported in a large number of scientific papers over the last few years, this study is a continuation of our systematic investigation of such effects of aroma samples by means of an optimized agar dilution and agar diffusion method. The aim of this study is to obtain antimicrobial data of geranol/nerol and their derivatives, to increase the input to a corresponding databank (6-10).

Experimental

Investigated compounds: Ceraniol, nerol and their derivatives, are products from Kurt Kitzing Co., Wallerstein (Germany), Sigma- Aldrich Co., Vienna (Austria) and/or Symrise Co. (former Dragoco Co.), Holzminden (Germany), only partly commercially available (Table 1).

Reference compounds: Eugenol (W24.670-0) and tetracycline hydrochloride (achromycine hydrochloride – 25g, T3383-25G) are products from Sigma-Aldrich Austria Co., Vienna (Austria); Ciproxin” 500mg-tablettes (1 tablet = 582 mg ciprofloxacine hydrochloride/ water) from Bayer Austria Co., Vienna and LidaprimR-infusion-bottle (250 mg contain 0.8 g sulfametrol and 0.16 g trimethoprim) from Nycomed Austria Co., Vienna.

GCYFiD: GC/FID analyses were carried out using a GC14A with split/ splitless injector, FID and C-R6A-Chromatopac integrator (Shimadzu, Japan), a GC-3700 with FID (Varian, Germany) and C-RlB-Chromatopac integrator (Shimadzu). The carrier gas was H; injector temperature, 250C; detector temperature,320C.Thetemperatureprogramwas:40C/5rnm to 280C/5 min, with a heating rate of 6C/min. The columns were 30 m 0.32 mm bonded FSOT-RSL-200 fused silica, with a film thickness of 0.25 m (Biorad, Germany) and 30 m 0.32 mm bonded Stabilwax, with a film thickness of 0.50 m (Restek, USA). Quantification was achieved using peak area calculations, and compound identification was carried out partly using correlations between retention times (11- 15).

GC/SfS;ForGC/MSmeasurementsaGC-17AwithQP5000 (Shimadzu), split/ splitless injector and Compaq-ProLinea data system (classSk- software), a GC-HP5890 with HP5970-MSD (Hewlett-Packard, USA) and ChemStation software on a Pentium PC (Bhm, Austria), a GCQ (Finnigan- Spectronex, Germany-USA) andGateway-2000-PS75 data system (SiemensNixdorf, Germany, GCQ-software) were used. The carrier gas was H; injector temperature, 25O0C; interface-heating at 300C, ion- source-heating at 200C, El-mode was 70 eV, and the scan-range was 41- 450 amu. For other parameters, see description of GC/FID, above. Mass spectra correlations were done using Wiley, NBS, NIST and our own library as well as published data (11,13,14).

Olfactoric evaluations: All investigated samples were olfactorically evaluated by professional perfumers and aroma chemists and the aroma described as mentioned in Table I as well as correlated with odor impressions published elsewhere (1-5,16,17) and private libraries

Antimicrobial testings: As test microorganisms, gram-(+)bacteria Stavhulococcus aureus ATCC 6538P and Enterococcus faecalis (clinically isolated); gram-(-)-bacteria Escherichia coli ATCC 8739, Proteus vulgaris (clinically isolated), Pseudomonas aeruginosa C 28 and Klebsiella pneumoniae and Salmonella sp. (clinically isolated) as well as the yeast Candida albicans ATCC 10231 – all products from the National Bank of Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria – were used.

The antimicrobial activity was studied by two methods: Agar diffusion disc method using 6 mm paper discs and quantities of 6 L of the sample. After cultivation of the bacteria and the yeast at 37C for 24h the diameter of the inhibition zone (IZ) was measured. Agar serial tube dilution method with results as minimum inhibitory concentration (MIC) as follows: The pure and reference compounds were added to brine, containing 1.0% (v/v) Tween 80 at the appropriate volumes to produce final concentrations of the samples in the range of 100-1,000 ppm; the Petri dishes were inoculated by pipetting O.1cm3 of the desired culture and 0.6 L, of the samples as well as the reference compounds (the tablets of Ciproxin were added as solution in saline at a quantity of 300 g) on paper discs (6 mm) and then incubated at 37C for 24h.

Table I. Investigated aroma compounds – Purity and olfactorlc evaluations

Results and Discussion:

For all of the investigated compounds a high purity in a range of 87.5% (geranyl geraniol) up to 99.4% (neryl isovalerate) and characteristic odor impressions with a pleasant floral top-note were found (Table I).

The antimcrobial testing using an agar dilution and agar diffusion method showed strong effects against the Gram(+)-bacteria by geraniol and nerol as well as nearly all of their derivatives, with only weak activity of neryl butyrate and neryl isobutyrate against Staphylococcus aureus.

Table II. Antimicrobial activities of geraniol/nerol and their derivatives as well as reference compounds (-no inhibition observed).

In the case of the testings against different strains of Gram(-)- bacteria medium up to high, but also low to no antimicrobial activities were found for the aroma chemicals. While geranial, geraniol, geranic acid, geranyl formate, geranyl benzoate, geranyl phenylacetate, geranyl tiglate, geranyl valerate, geranyl geraniol, geranyl Iinalool, geranyl amine, geranyl chloride and nerol were effective against all Gram-(-)-bacteria, no inhibition was observed by the following compounds: geranyl propionate against Escherichia coli; geranyl acetone, geranyl acetate, geranyl butyrate and geranyl bromide against Proteus vulgaris; geranyl acetate, neryl butyrate, neryl isobutyrate and neryl isovalerate against Pseudomonas aeruginosa; neryl isobutyrate against Salmonella sp.; geranyl acetone, geranyl acetate, geranyl isobutyrate, geranyl bromide and neryl isobutyrate against Klebsiella pneumoniae (Table II).

The compounds geranyl benzoate, geranyl chloride and neryl isobutyrate showed no antimicrobial activity against the yeast Candida albicans.

Eugenol, a phenolic aroma component with well-known antimicrobial effects against many microorganisms showed high activity against all strains using both methods.

The results obtained from testings of synthetic antibiotics against Gram-(+)-bacteria were as expected, while Lidaprim surprisingly showed no activity against the Gram-(-)-bacteria P, aeruginosa and K pneumoniae (Table II).

In summary, it is to report that geraniol and nerol as well as their derivatives are very effective compounds against nearly all microorgansims tested. In general, geranial, geraniol, nerol and the aromatic esters as well as geranyl amine and geranyl chloride were found to have a high activity in all testings. Geranyl acetone, geranyl bromide and lower esters (acetates, butyrates and isobutyrates) showed no activity against some Gram-(-)-bacteria.

This group of aroma chemicals are therefore potential antimicrobial substances to be added into our databank, which is in preparation.

Acknowledgment

The authors acknowledge the reference compounds from Kurt Kitzing Co. and Symrise Co., the olfactoric evaluations by Wolfgang Hi)ppner(retiredperfumerofSymrise Co.)andby the team of perfumers at Kurt Kitzing Co., as well as the valuable discussion about the used antimicrobial testing methods with Heinz Schikher, emeritus of the “Freie Universitt Berlin”.

References

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Leopold Jirovetz* and Gerhard Buchbauer

University of Vienna, Department of Clinical Pharmacy and Diagnostics, Althanstrasse 14, A-1090 Vienna, Austria

Erich Schmidt

Kurt Kitzing Co., Hinterm Alien Schloss, D-86757 Wallerstein, Germany

Albena S. Stoyanova

University of Food Technologies, Department of Essential oils, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria

Zapriana Denkova and Radosveta Nikolova

University of Food Technologies, Department of Microbiology, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria

Margit Geissler

SHIMADZU-Germany, Department of GC and GC/MS, Albert-Hahn- Strasse 6-10, D-47269 Duisburg, Germany

*Address for correspondence

Received: February 2006

Revised: July 2006

Accepted: July 2006

1041-2905/07/00030 -0288$14.00/00 -2007 Allured Publishing Corp.

Copyright Allured Publishing Corporation May/Jun 2007

(c) 2007 Journal of Essential Oil Research : JEOR. Provided by ProQuest Information and Learning. All rights Reserved.




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