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Bioactivity of Rosmarinus officinalis essential oils against Apis mellifera,


Varroa destructor and Paenibacillus larvae related to the drying treatment
of the plant material
M. Maggiab; L. Gendeabc; K. Russod; R. Fritzc; M. Eguarasab
a
Laboratorio de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar
del Plata, Funes 3350, 7600 Mar del Plata, Argentina b Consejo Nacional de Investigaciones Científicas
y Técnicas (CONICET), Rivadavia 1917, C1033AJ Buenos Aires, Argentina c Departamento de Química,
Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350, 7600
Mar del Plata, Argentina d Instituto Zooprofilatico Sperimentale Regione Lazio e Toscana, Via Appia
Nuova 1411, 00178 Roma, Italia

First published on: 08 July 2010

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

Bioactivity of Rosmarinus officinalis essential oils against


Apis mellifera, Varroa destructor and Paenibacillus larvae related
to the drying treatment of the plant material
M. Maggiab*, L. Gendeabc, K. Russod, R. Fritzc and M. Eguarasab
a
Laboratorio de Artrópodos, Facultad de Ciencias Exactas y Naturales, Universidad
Nacional de Mar del Plata, Funes 3350, 7600 Mar del Plata, Argentina; bConsejo Nacional
de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Rivadavia 1917, C1033AJ Buenos
Aires, Argentina; cDepartamento de Quı´mica, Facultad de Ciencias Exactas y Naturales,
Universidad Nacional de Mar del Plata, Funes 3350, 7600 Mar del Plata, Argentina;
d
Instituto Zooprofilatico Sperimentale Regione Lazio e Toscana, Via Appia Nuova 1411,
Downloaded By: [Maggi, Matías] At: 17:27 14 February 2011

00178 Roma, Italia


(Received 14 September 2009; final version received 20 March 2010)

In this study, chemical composition, physicochemical properties and


bioactivity of two essential oils of Rosmarinus officinalis extracted from
plant material with different drying treatments against Apis mellifera,
Varroa destructor and Paenibacillus larvae were assessed. The lethal
concentration 50 (LC50) for mites and bees was estimated using a
complete exposure method test. The broth microdilution method was
followed in order to determine the minimum inhibitory concentrations
(MICs) of the essential oils against P. larvae. Physicochemical properties
were similar in both the essential oils, but the percentage of components
showed certain differences according to their drying treatment. -Myrcene
and 1,8-cineole were the main constituents in the oils. The LC50 for
complete exposure method at 24, 48 and 72 h was minor for mites
exposed to R. officinalis essential oil dried in oven conditions. MIC values
were 700–800 mg mL1 and 1200 mg mL1 for R. officinalis dried in air and
oven conditions, respectively. The results reported in this research show
that oil toxicity against V. destructor and P. larvae differed depending
on the drying treatment of the plant material before the distillation of
essential oil.
Keywords: essential oils; LC50; Apis mellifera; Varroa destructor;
Paenibacillus larvae; drying treatment

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

*Corresponding author. Email: biomaggi@gmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online


ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2010.481261
http://www.informaworld.com
398 M. Maggi et al.

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

2. Results and discussion


The compounds -myrcene and 1,8-cineol were the main components of the oils.
These results were similar to those reported by Larran et al. (2001). The main
difference between the compositions of both essential oils was that R. officinalis dried
in oven conditions showed the presence of camphor, while this component was
absent in the air-dried oil; another difference is regarding the percentage of 1,8-
cineole (Table 1).
Physicochemical properties of the oils are related to the chemical composition,
which can be used as an approach of purity, identification and verification. In this
study, R. officinalis essential oil dried in air conditions presented a higher acid index
(AI) (0.63 mg KOH g1 oil  01) and yield (3.15%  0.65) than R. officinalis dried in
oven conditions (0.38 mg KOH g1 oil  0.1 and 2.35%  0.5). The differences
between the distillation yields of the oils could be because of the solvation
phenomena of the water. It is important to know about the drying process to which
the plant material is subjected, since the yield extraction of the oil could be varied.
The drying process is recommended because it improves performance (Sanda, Koba,
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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

Table 1. Main compounds, expressed as percentage of chromato-


graphic area, of R. officinalis essential oils, obtained from material
treated with different techniques: air dried and oven dried.

Components RA RO

-Pinene 6.3 (0.2) 7.3 (0.5)


-Pinene 0.8 (0.3) 0.3 (0.1)
-Myrcene 22.1 (1.7) 20.5 (2.2)
Camphene 3.3 (0.4) 3.7 (0.7)
1,8-Cineole 16.6 (1.1) 24.8 (1.9)
Camphor – 18.8 (2.1)

Notes: RA is R. officinalis dried in air conditions and RO is


R. officinalis dried in oven conditions. Values (area percentage)
represent averages of three determinations. Standard deviations
(SDs) are given within parentheses.
400 M. Maggi et al.

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.

LC50 mite (mL) LC50 honeybee (mL)

Treatment drying techniques 24 h 48 h 72 h 24 h 48 h 72 h

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).

of the constituents of an oil can determine different bactericidal and acaricidal


properties. These results could help to improve the control of P. larvae and
V. destructor in future field trials, where the essential oil of R. officinalis with
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different drying treatments will be used.

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.

3.2. Essential oil analysis


The oils were analysed by gas chromatography (GC)–flame ionisation detection
(FID)–mass spectrometry (MS), using a Perkin Elmer Clarus 500 model
chromatograph equipped with one split injector (split ratio 1 : 100) connected with
flow dividers in two fused silica capillary columns: (1) polyethylene glycol (DB-Wax,
J&W Scientific) and (2) 5% phenyl–95% dimethylpolysiloxane (DB-5, J&W
Scientific) both measuring 60 m  0.248 mm and film thickness 0.25 mm. The polar
column was connected to an FID detector, and the non-polar column to another
FID detector and to an MS quadrupole detector (70 eV), using a vent system
TM
(MSVent ). The oven temperature was programmed at 90.00 C, then 3.00 C min1
until 225 C (15 min). The injector and detector temperatures were set at 255.00 C
and 275.00 C, respectively, with helium as carrier gas at a flow rate of
1.87 mL min1. Diluted essential oil of 0.2 mL was injected to 10% ethanol at a
temperature line transfer of 180.00 C. The identification of components was based
402 M. Maggi et al.

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.

3.3. Physicochemical properties determinations


Yield was calculated as the ratio between the mass of the extracted oil and the
mass of the plant. To estimate the d20, 1 mL of essential oil was weighed in
triplicate (Montes, 1981). The n20  
D was determined at 20.00  0.05 C with an Abbe
refractrometer, in compliance with the AOAC official method 921.08 (AOAC,
1999). The AI was obtained by titrating with an aqueous solution of 0.1 M NaOH.
For this titration, 1 g of oil was dissolved in alcohol at 96 C, previously neutralised
with NaOH using thymol blue as indicator.
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3.4. P. larvae bioassay


Bacterial strains of P. larvae, collected from five cities in Buenos Aires province, La
Plata, Mar de Cobo, Sierra de los Padres, Mar del Plata and Vidal, were isolated
from brood combs of beehives with clinical symptoms of AFB. Isolation was made
on MYPGP agar (Dingman & Stahley, 1983) and in order to inhibit the growth of
Paenibacillus alvei, it was supplemented with 9 mg mL1 of nalidixic acid. Plates were
incubated under microaerobic conditions (5–10% of CO2), and the strains were
identified using biochemical tests (Alippi, 1991, 1992; Gordon, Haynes, & Pang,
1973). The pure strains were maintained on MYPGP agar with 15% (v/v) glycerol
until used.
Vegetative cells of P. larvae previously cultivated on MYPGP agar during 48 h at
35.00  0.5 C were suspended in double-distilled sterile water and the suspension was
standardised according to the Food and Drug Administration (FDA) method (FDA,
1998). The concentration was adjusted to 0.5 of the McFarland scale for measuring
the antimicrobial activity with the serial dilution method.

3.5. Determination of minimal inhibitory concentrations


The minimal inhibitory concentration (MIC) is defined as the lowest concentration
of an antimicrobial agent capable of inhibiting the visible growth of a microorganism
after incubation (Lennette, Balows, Hansler, & Shadony, 1987). MIC individual
determination was directly evaluated by turbidity observation. The oils were mixed
in water and emulsified with 8% (v/v) propylene glycol (1,2-propanediol). For broth
microdilution, 100 mL of MYT broth (Gende, Eguaras, & Fritz, 2008a) was placed in
each of the 96-well microtiter plates and then diluted to obtain serial dilutions.
Microbial biomass suspension was added to each serial dilution. The final serial
dilution concentrations ranged between 2000 and 12.5 mg mL1. Positive and
negative controls (with microorganisms and water, respectively) were used.
Microtiter plates were incubated at 35.00  0.5 C for 48 h so as to determine the
MIC values. Antimicrobial activity was tested by triplicate analyses for oil and
Natural Product Research 403

strains. The MICs of oxytetracycline were also determined in parallel experiments as


a way of controlling the sensitivity of the microorganisms tested.

3.6. Mite and bee lethality test


The bioactivity of the essential oils of R. officinalis against V. destructor was
established using a complete exposure method (Ruffinengo et al., 2005). Treatments
were assayed in unmodified Petri dishes (60  20 m) using new dishes for each
experiment. Each essential oil was diluted in ethanol to a desirable concentration,
and 1 mL solution was then applied to the bottom of the Petri dish. Concentrations
of 2.5, 5, 10 and 20 mL per cage of essential oils were used. Ethanol was evaporated
from the dishes by exposing it to airflow for 3–5 min. Five newly emerged adult bees
(between 0 and 3 days old) and five Varroa mite females obtained from brood cells
were placed into each Petri dish. Bees and mites were exposed to essential oils for
72 h. The bees present in Petri dishes were fed with 3 g of candy and incubated at
30.00 C and 70% relative humidity during the test. Control treatments consisted of
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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.

3.7. Statistical analysis


To determine if the bioactivity of R. officinalis essential oils against V. destructor
varied with different drying treatments, statistical analyses were conducted in
accordance with the specific software for the calculation of LC50 values, using a 95%
confidence interval (United States Environmental Protection Agency, 1986) and
EPA software (Version 1.5) as proposed by Lindberg et al. (2000). Mortality values
were adjusted as a function of natural mortality in agreement with Abbott (1925).
Comparisons between LC50 value pairs of the different R. officinalis oil in
V. destructor were carried out at each point of time (24, 48 and 72 h) by means of
LC50 greater/LC50 lower quotient. Statically significant differences were detected
when the statistical value was higher than the corresponding critical value set forth
by APHA (1992).

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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