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To cite this Article Maggi, M. , Gende, L. , Russo, K. , Fritz, R. and Eguaras, M.(2011) 'Bioactivity of Rosmarinus officinalis
essential oils against Apis mellifera, Varroa destructor and Paenibacillus larvae related to the drying treatment of the
plant material', Natural Product Research, 25: 4, 397 — 406, First published on: 08 July 2010 (iFirst)
To link to this Article: DOI: 10.1080/14786419.2010.481261
URL: http://dx.doi.org/10.1080/14786419.2010.481261
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Natural Product Research
Vol. 25, No. 4, February 2011, 397–406
1. Introduction
The bees belonging to Apis mellifera species are key insects for the pollination of wild
plants as well as crops (Goulson, 2003). In the recent years, bee populations are on
the decline and their numbers are at a critical level (Goulson, Lye, & Darvill, 2008;
Kearns, Inouye, & Waser, 1998). Several studies have focussed on the conservation
of bees with the aim to elucidate the causes of their decline (Stokstad, 2007a, 2007b).
American foulbrood (AFB), caused by the bacterium Paenibacillus larvae
(Genersch et al., 2006), and varroosis, caused by the ectoparasitic mite Varroa
destructor (Anderson & Trueman, 2000), are the main pests affecting A. mellifera.
Much effort has been made to control these diseases, including the use of preventive
and curative treatments with antibiotics and synthetic acaricides. Unfortunately, the
extensive use of these leads to an accumulation of residues in the beehive
products (Bogdanov, 2006; Wallner, 1999), decreasing their quality and making
their marketing more difficult. In addition, it was reported that the inappropriate use
of miticides could generate resistant mite populations (Maggi, Ruffinengo,
Damiani, Sardella, & Eguaras, 2009; Maggi, Ruffinengo, Gende, Eguaras, &
Sardella, 2008).
Natural acaricides and bactericides are preferred alternatives to synthetic ones,
because generally, they have low toxicity in mammals, little environmental effect
and wide public acceptance (Isman, 2001). Several natural products, especially
organic acids and essential oils, have shown bactericidal and acaricidal effect
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(Albo, Cerimene, Re, De Giusti, & Alippi, 2001; Eguaras, Del Hoyo, Palacio,
Ruffinengo, & Bedascarrasbure, 2001; Floris, Carta, & Moretti, 1996; Gende,
Floris, Fritz, & Eguaras, 2008b; Maggi et al., 2010; Ruffinengo et al., 2005). Many
of the tested oils have been effective against V. destructor and P. larvae in
laboratory experimental conditions and in field trials (Calderone & Shimanuki,
1994; Gende et al., 2007, 2008b; Lindberg, Melathopoulus, & Winston, 2000;
Ruffinengo et al., 2001, 2002). Although these products are effective in controlling
these pests, they have shown a marked variability in their final efficacy in the hives
(Mutinelli, Cremasco, & Irsara, 1994; Rickli, Imdorf, & Kilchenmann, 1991). The
essential oil composition of each plant species tends to be unique. However, some
species have different chemotypes with varying essential oil compositions (Imdorf,
Bogdanov, Ibañez Ochoa, & Calderone, 1999). The chemical composition of an
essential oil often depends upon the cultivation and climatic conditions. Vapour
distillation, cold pressing and solvent extractions produce oils of varying
composition. The lack of consistency in the chemical composition of essential
oils undoubtedly contributes to the variation in the results obtained by different
studies (Imdorf et al., 1999) and consequently, to an unsuccessful control strategy
of the main pests of A. mellifera.
Rosemary (Rosmarinus officinalis) is an aromatic plant belonging to the Labiates
family. It is characterised by its medium height, and simple and opposite leaves with
3.5 cm of longitude. Its flowers are small and clumped, and they appear from the end
of the spring to the beginning of the summer (Alonso, 1998). There are many studies
on the chemical composition of the essential oil obtained from the leaves of
R. officinalis (Angioni et al., 2004; Diab, Auezova, Chebib, Chalchat, & Figueredo,
2002; Jaganmohan, Meenakshi, Raghavan, & Abraham, 1997; Larran et al., 2001;
Masatoshi & Hiroaki, 1997; Porte et al., 2000), showing that the main components of
the essential oil differ according to the geographical locations from which they were
collected.
The main objective of this work was to compare the bioactivity of R. officinalis
essential oils extracted from plant material with different drying treatments against
A. mellifera, V. destructor and P. larvae.
Natural Product Research 399
Akpagana, & Tchepan, 2001), but it should not be carried out in oven conditions
(50.00 C, 2 h) because the percentage of essential oil obtained by distillation
decreases as the drying temperature increases. Here, the values reported for the oil
density at 20.00 C (d20) and the refractive index (n20 D ) were similar for both oils
(d20 ¼ 0.88 g mL1 and n20 D ¼ 1.47). The values of density and refractive indices were
in agreement with those reported for oils with a majority composition of terpenic
compounds (Montes, 1981). AI valuation is a quantitative determination of the total
acidity of oil and is related with its conservation, increasing their value with the
ageing of the oil (Retamar, 1982).
In this study, it was confirmed that R. officinalis dried in oven conditions
contained camphor, while the oil that was dried in air conditions did not. Other
studies reported that the chemical composition of an essential oil depends upon
cultivation, climatic conditions and method of extraction (Imdorf et al., 1999).
In addition, Colin, Ducos De Lahitte, Larribau and Boue (1989) found that the
Components RA RO
Table 2. Antimicrobial activity of R. officinalis essential oils against P. larvae strains isolated
from different geographic origins.
Strains
1 2 3 4 6
RA 700 (100) 800 (50) 700 (100) 750 (50) 750 (50)
RO 1200 (200) 1200 (150) 1200 (100) 1200 (100) 1200 (200)
Notes: Data are MIC (mg mL1) range values for the five strains. The name of each strain
corresponds to the geographic zone from which they were isolated: 1, La Plata; 2, Cobo;
3, Sierra de los Padres; 4, Mar del Plata; 5, Vidal. The antimicrobial activity was determined
by quintupled analyses for oil and strains. SDs are given within parentheses.
thymol content of thyme oils of different origin and chemotypes varies between 5%
and 40%.
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MIC values for the five strains of P. larvae in the presence of both essential oils
are given in Table 2. For R. officinalis classified as RA, the MIC values were in the
range of 700–800 mg mL1 while that of the oil obtained from kiln-dried material was
1200 mg mL1. These results were similar to those reported for the same vegetal
species against P. larvae (Alippi, Ringuelet, Cerimele, Re, & Henning, 1996). RA oil
presented a higher concentration of -mircene, showing better inhibitory activity
against P. larvae with MIC values between 600 and 700 ppm, while the rosemary oil
classified as RO presented a high percentage of 1,8-cineol. The latter also showed
lower antimicrobial activity, with MIC values between 1000 and 1200 ppm. This
could be attributed to the delocalised system of electrons resulting from the presence
of three double bonds in -myrcene that allow proton exchange, thereby rendering
the substances more active against microorganisms (Ben Arfa, Combes, Preziosi-
Belloy, Gontard, & Chalier, 2006) in relation to the other two major components of
these essential oils.
The essential oil of R. officinalis dried in oven conditions (RO) was more toxic
against V. destructor for each period of exposition ( p50.001). The estimated LC50
values for V. destructor obtained at each time interval for each treatment and its
comparison are given in Table 3. For A. mellifera, the estimated LC50 values
obtained at each time interval for each treatment were superior to 20 mL per cage
because of the low bee mortality at all assayed concentrations. For mites and bees,
LC50 of fluvalinate decreased with time, and significant differences were detected
regarding treatments in each time ( p50.001). Taking into account that the main
difference between the two oils was camphor, the rest of the constituents being
similar for both essential oils (Table 1), for V. destructor, it is possible that this
constituent was responsible for the highest toxicity of RO when both oils were
compared. The acaricidal properties of camphor have been reported by Higes,
Suárez and Llorente (1997) and Imdorf, Bogdanov, Kilchenmann and Maquelin
(1995), who showed that camphor caused high mite mortality at concentrations
which were not toxic for bees. In addition, Liebig (1991) has shown how the different
compositions of an essential oil are responsible of the final result for a lethality test.
In this study, it was demonstrated that the drying treatment of a plant material
can vary the composition of its essential oil, and that the difference in the percentage
Natural Product Research 401
Table 3. LC50 (mL per Petri dish) estimated for V. destructor mites and A. mellifera bees for
both oils.
Rosemary
RO 16.94a 19.51a 7.07a 420b 420b 420b
RA 420b 420b 420b 420b 420b 420b
Fluvalinate 2.82c 1.97c 1.49c 1512c 1389c 1027c
Note: Different letters indicate significant differences inside each time ( p50.001).
3. Experimental
3.1. Plant material and oil isolation
The leaves of Rosmarinus officinalis were collected during March 2005 in Sierra de la
Ventana (38 90 S–61 480 W), Buenos Aires province (Argentina). The same vegetable
material was treated by different drying techniques: (1) in air conditions (RA)
(20.00–27.00 C and 50% relative humidity for 2–8 days); (2) in oven conditions
(RO) (50.00 C and 50% relative humidity for 2 h). The oils were obtained by
hydrodistillation using a Clevenger-type European Pharmacopoeia apparatus
(Larran et al., 2001) for 2 h. On average, 100 g of leaves was used in each
experiment, and several distillations were performed until the volume required to run
all trials was reached. The oils were dried over anhydrous sodium sulphate
and stored in screw-capped dark glass vials at 5.00–8.00 C until further tests.
on the comparison of their mass spectra with those reported in the literature (Adams,
2007) and by a computerised search of their 70 eV mass spectra with those stored in
the library of the GC/MS data system, as well as by retention indices. Quantitative
data was obtained by electronic integration of FID area percentages without the use
of collection factors.
Petri dishes that were treated only with ethanol and dishes treated with technical
grade of tau-fluvalinate. Five replicates were used for each experimental unit. The
number of dead Varroa and bees was determined after visual inspection of the dish
bottoms after 24, 48 and 72 h.
Acknowledgements
The authors would like to thank Lic. Alfredo Romeo for providing the vegetable material.
This work was supported by UNMdP, CONICET and the grant PICT Redes 890/06
ANPCyT. We thank the two anonymous referees for their criticisms and suggestions.
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