Apple Scab Control With Grapefruit Seed Extract No Alternative To Chemical Fungicides

Apple scab control with grapefruit seed extract:
no alternative to chemical fungicides
Renate Spitaler1
, Klaus Marschall2
, Christian Zidorn1
, Kelderer Markus2
, Roland
Zelger2
, Hermann Stuppner1
Key words: apple scab, Venturia inaequalis, grapefruit seed extract, preserving
agents, benzethonium chloride, benzalkonium chloride, methyl parabene,
propyl parabene.
Summary
The growth inhibiting effect of four commercially available grapefruit seed extracts
on the causal organism of apple scab Venturia inaequalis was tested. Germination of
the conidia of Venturia inaequalis was pronouncedly inhibited by all tested extracts.
The commercial products were analyzed by high pressure liquid chromatography and
thin layer chromatography. All samples contained at least one preserving agent.
These substances were identified as either benzethonium chloride, benzalkonium
chloride, methyl parabene or propyl parabene. Freshly prepared extracts from seeds
of grapefruits (Citrus paradisi) did not inhibit the germination of Venturia inaequalis. It
was therefore concluded that the antifungal effect of grapefruit seed extracts is
caused by the added preservatives.
Introduction
Apple scab is the most important disease affecting apple growing in temperate
climates. Especially in organic cultures only a few permitted substances are able to
control scab effectively. These compounds include lime sulphur, sulphur and copper
witch. A large number of alternative products (250) were compared with regards to
their fungicidal activity against apple scab. These assays were performed by means
of a standardized apple seedling greenhouse test. Several GSE-products revealed a
pronounced antifungal activity.
For many years the use of grapefruit seed extracts (GSE) as highly effective
antimicrobial agents has been praised by lay media. The supposed activity
encompasses inhibition of bacteria, fungi and viruses as well as several unicellular
parasites (Uhlenbrock, 1996). However, only a limited number of scientific reports
deal with these effects (Woedtke et al., 1999, Takeoka et al., 2001).
In 1997 the Central Laboratory of German Pharmacists (Zentrallaboratorium
Deutscher Apotheker) detected benzethonium chloride in several commercial GSE.
Based on these findings, the Drug Commission of the German Pharmacists
Association (Deutsche Arzneimittelkommission, AMK) issued a statement, that GSE
1
Institut für Pharmazie, Abt. Pharmakognosie, Universität Innsbruck, 6020, Austria.
2
Land- und forstwirtschaftliches Versuchszentrum Laimburg, Pfatten, 39040 Auer, Italy.
Archived at http://orgprints.org/14543/

potentially containing benzethonium chloride must not be sold by pharmacies any
more (AMK, 1998).
Woedtke et al. (1999) examined six commercial grapefruit seed extracts and
detected high activity against Bacillus subtilis, Micrococcus flavus, Staphylococcus
aureus, Serratia marcescens, Escherichia coli, Proteus mirabilis and Candida
maltosa in five of the products investigated. All active samples contained one to three
preserving agents, namely benzethonium chloride, triclosan, and methyl parabene. In
contrast, freshly prepared extracts from seeds and juiceless pulp of Citrus paradisi
didn’t show any antimicrobial activity. Thus, the authors concluded that the
antimicrobial activity of commercially available grapefruit seed extracts was due to
the added preservatives.
The commercial products are labeled by the manufacturers as glyceric extracts
containing ascorbic, lactic and citric acid as antioxidants. No other preserving
additives are indicated and no data about the extraction method are given. According
to Nutri Team Inc. (Vermont, USA) GSE is obtained from an aqueous solution of
dried and ground grapefruit seeds and pulp, which is distilled, then spray dried and
re-dissolved in glycerin. The obtained liquid is mixed with ammonium chloride,
ascorbic acid and catalysts, which allegedly convert the polyphenols of the seeds
and pulp into quaternary ammonium compounds (www.nutriteam.com/gsewhat.html).
The supporters of commercial GSE products claim that these GSE-derived
quaternary ammonium compounds differ from the chemically identical synthetic
compounds benzethonium chloride and benzalkonium chloride by their lack of toxicity
to animal life. However, none of these pseudo-scientific statements has ever been
validated by scientific data.
The presence of quaternary ammonium compounds in a commercial grapefruit
seed extract was proven by Takeoka et al. (2001) employing high pressure liquid
chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS),
nuclear magnetic resonance (NMR) spectroscopy and proton-induced X-ray emission
(PIXE) analysis. The main compound of the chloroform layer of the commercial
product was identified as benzethonium chloride.
Results
In the present study, four commercial grapefruit seed extracts were tested against
conidia of Venturia inaequalis by means of a standardized germination test (Olaya
and Köller, 1999). All four GSE products were partitioned successively with petrol
ether, dichloromethane and methanol, obtaining a total of 12 phases (three for each
product). After evaporating the solvents in vacuo, each phase was tested in
concentrations of 2.5 mg/ml, 0.125 mg/ml, 0.013 mg/ml and 0.001 mg/ml. After an
incubation period of 48 h at room temperature conidial germination was examined by
microscopy. Additionally, an untreated control was analyzed. All samples were
analyzed in triplicate. Inhibition rates of each tested concentration were determined
employing Abbott’s formula. All tested dichloromethane phases showed 100% growth
inhibition in concentrations down to 0.125 mg/ml. In the same concentration range,
petrol ether and methanol phases were less active.

To characterize and compare the extracts, analytical HPLC and TLC methods
were established. The obtained chromatograms indicated the presence of preserving
agents in all four commercial products. The identity of these substances was
confirmed by comparison with reference compounds.. Two products contained both
methyl parabene and propyl parabene as main compounds (Fig. 1), one
benzethonium chloride, and one benzalkonium chloride.
Fig. 1: HPL-chromatogram of a commercial grapefruit seed extract containing
methyl parabene and propyl parabene
HPLC-method: FM A: H2O, FM B: Acetonitril; linear gradient: 0 min. 20% B; 5 min 20% B; 20
min. 75% B; 25 min. 95% B. 35 min. STOP; flow rate 1.000 ml/min.; column Zorbax SB-C18
4.6 x 150 mm, 3.5 µm; injection volume 10 µl; detection wave length 205 nm.
To clarify, whether the antifungal activity of the extracts was exclusively an effect
of the added preserving agents, two different accessions of Citrus paradisi seeds
were extracted and the extracts analyzed for antifungal activity. The first accession
was successively extracted with dichloromethane and methanol, the second one
successively with petrol ether, dichloromethane and methanol. Venturia inaequalis
bio-assay results showed no inhibiting activity against the fungus for any of the
extracts investigated in concentrations up to 2.5 mg/ml.
Discussion
The presented results clearly indicate that the antifungal activity of commercial
grapefruit seed extracts is due to the contents of well known chemical preserving
agents. No hint was found that natural compounds from the seeds of Citrus paradisi

have any antifungal activity. These results fit perfectly well with those by Woedtke et
al. (1999), who analyzed the antibacterial and antifungal activity of five commercial
GSE and who were also able to demonstrate, that the activity was due to a number of
added synthetic preservatives and not to natural compounds contained in grapefruit
seeds. The presence of preserving substances was in no case indicated on the GSE
product labels.
Conclusively, the usage of GSE in alternative farming is to be discouraged. All
products investigated so far are of dubious origin and composition and the
accompanying product data of the liquids are misleading.
Literature
AMK (1996): Information der Arzneimittelkommission Deutscher Apotheker. Pharm.
Ztg. 143, 2612.
Olaya, G., Köller, W. (1999): Diversity of kresoxim-methyl sensitivities in baseline
populations of Venturia inaequalis. Pesticide Science 55,1083-1088
Takeoka, G., Dao, L., Wong, R.Y., Lundin, R., Mahoney, N. (2001): Identification of
benzethonium chloride in commercial grapefruit seed extracts. J. Agric. Food
Chem. 49, 3316-3320.
Uhlenbrock, S. (1996): Grapefruitkernextrakt: natürlich gut für alles? Pharm. Ztg. 141,
3882-3886.
Woedtke, Th. von, Schlüter, B., Pflegel, P., Lindequist, U., Jülich, W.-D. (1999):
Aspects of the antimicrobial efficacy of grapefruit seed extracts and its relation to
preservative substances contained. Pharmazie 54 (6), 452-456.

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Apple Scab Control With Grapefruit Seed Extract No Alternative To Chemical Fungicides