Journal of Apicultural Research

Amaicha del Valle (Tucumán, Argentina) propolis: volatile compounds and chemical
relationships with Zuccagnia punctata Cav. (Fabaceae)
--Manuscript Draft--

Full Title: Amaicha del Valle (Tucumán, Argentina) propolis: volatile compounds and chemical
relationships with Zuccagnia punctata Cav. (Fabaceae)

Manuscript Number: TJAR-2018-0077

Article Type: Original research article

Keywords: Propolis; palynology; volatile fraction; Zuccagnia punctatae; Apis mellifera.

Manuscript Classifications: Biochemistry and chemical ecology; Hive product science; Sociobiology and behaviour

Abstract: Propolis samples from Amaicha del Valle (Tucumán, Argentina) were evaluated by
palynology, FT-IR/UV spectra, and RP-HPTLC. In addition, the volatile fraction was
studied by HS-SPME GC-MS. The botanical species most visited by Apis mellifera L.
near the apiaries were collected and their extracts' RP-HPTLC profiles were compared
with propolis samples. In addition, GC-MS was performed for volatile compounds from
Zuccagnia punctata (Zp) (Fabaceae). The frequency of pollen type occurrence varied
significantly between samples, mainly due to changes in pollen flow from plants near
the apiaries. Only 11 pollen types occurred with a frequency higher than 3%, and only
9 pollen types had frequencies between 3% and 1.6%. Prosopis pollen was the best
regional representative, as it was the only taxon that occurred with 20% to 25%
frequency. Other native species from Amaicha del Valle such as Zp, Larrea divaricata
and L. cuneifolia, Schinus areira and S. fasciculatus, Acacia praecox, and Ligaria
cuneifolia were found to be less significant. FT-IR/UV spectra and RP-HPTLC
fingerprints of propolis samples showed similar profiles. In RP-HPTLC analyses, only
Zp presented a similar fingerprint to Amaicha propolis. The major volatile compounds
present in both were trans-linalool oxide (furanoid), 6-camphenone, linalool, trans-
pinocarveol, p-cymen-8-ol, and 2,3,6-trimethylbenzaldehyde. The presence of other
compounds in the volatile fraction of propolis suggests that the contribution of other
resin-producing plant species also contribute to propolis.

Order of Authors: Mariela González, Ph. D.

María Elena García, Ph. D.

Alberto Slanis

Ana C. Bonini

Stephanie Fiedlerd

Eduardo Dellacassa, Ph. D.

Concetta Condurso, Ph. D.

Daniel Lorenzo

Marcos Russo

María Laura Tereschuk, Ph. D.

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1 Amaicha del Valle (Tucumán, Argentina) propolis: volatile compounds

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and chemical relationships with Zuccagnia punctata Cav. (Fabaceae)
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7 Mariela Gonzáleza*, María Elena Garcíab, Alberto Slanisc, Ana C. Boninid,
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9 Stephanie Fiedlerd, Eduardo Dellacassad, Concetta Condursoe, Daniel Lorenzof,
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11 Marcos Russog and María Laura Tereschuka.
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a
14 Departamento de Ingeniería de Procesos y Gestión Industrial, Facultad de Ciencias
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16 Exactas y Tecnología. Universidad Nacional de Tucumán, San Miguel de Tucumán, 4000,
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18 República Argentina, Tel. 543814364093 int. 7724; bLaboratorio de Palinología, Fundación
19 Miguel Lillo, Miguel Lillo 251, San Miguel de Tucumán, 4000, República Argentina;
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21 c
Facultad de Ciencias Naturales e IML, Universidad Nacional de Tucumán, Miguel Lillo
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23 250, San Miguel de Tucumán, 4000, República Argentina; dLaboratorio de Biotecnología de
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25 Aromas. Facultad de Química, UdelaR, Gral. Flores 2124, 11800-Montevideo, Uruguay;
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e
Universitá di Messina. Viale Stagno d’Alcontres. 98166. Messina. Italy. fTaller de
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instrumentos DETEMA, Facultad de Química UdelaR, Montevideo, Uruguay; gEstación
30 Experimental Agroindustrial Obispo Colombres, San Miguel de Tucumán, Av. William
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32 Cross 3150, T4101XAC, República Argentina.
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35 *Corresponding author. E-mail: magonzalez@herrera.unt.edu.ar
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 1 Amaicha del Valle (Tucumán, Argentina) propolis: volatile compounds
2
3
4
and chemical relationships with Zuccagnia punctata Cav. (Fabaceae)
5
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7 Propolis samples from Amaicha del Valle (Tucumán, Argentina) were evaluated by
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9 palynology, FT-IR/UV spectra, and RP-HPTLC. In addition, the volatile fraction was
10 studied by HS-SPME GC-MS. The botanical species most visited by Apis mellifera L.
11
12 near the apiaries were collected and their extracts’ RP-HPTLC profiles were compared
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14 with propolis samples. In addition, GC-MS was performed for volatile compounds
15 from Zuccagnia punctata (Zp) (Fabaceae). The frequency of pollen type occurrence
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17 varied significantly between samples, mainly due to changes in pollen flow from
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19 plants near the apiaries. Only 11 pollen types occurred with a frequency higher than
20 3%, and only 9 pollen types had frequencies between 3% and 1.6%. Prosopis pollen
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22 was the best regional representative, as it was the only taxon that occurred with 20%
23
24 to 25% frequency. Other native species from Amaicha del Valle such as Zp, Larrea
25 divaricata and L. cuneifolia, Schinus areira and S. fasciculatus, Acacia praecox, and
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27 Ligaria cuneifolia were found to be less significant. FT-IR/UV spectra and RP-
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29 HPTLC fingerprints of propolis samples showed similar profiles. In RP-HPTLC
30 analyses, only Zp presented a similar fingerprint to Amaicha propolis. The major
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32 volatile compounds present in both were trans-linalool oxide (furanoid), 6-
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34 camphenone, linalool, trans-pinocarveol, p-cymen-8-ol, and 2,3,6-
35 trimethylbenzaldehyde. The presence of other compounds in the volatile fraction of
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37 propolis suggests that the contribution of other resin-producing plant species also
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39 contribute to propolis.
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45 Propóleos de Amaicha del Valle (Tucumán, Argentina): compuestos
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47 volátiles y correlación química con Zuccagnia punctata Cav. (Fabaceae)
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50 Se evaluaron muestras de propóleos provenientes de Amaicha del Valle (Tucumán,
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52 Argentina) por métodos palinológicos, espectros FT-IR/UV y RP-HPTLC. Por otro
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54 lado, se estudió la fracción volátil mediante HS-SPME GC-MS. Las especies
55 botánicas cercanas a los apiarios, más visitadas por la abeja Apis mellifera L. fueron
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57 colectadas y se compararon sus perfiles cromatográficos en RP-HPTLC con los
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59 correspondientes a las muestras de propóleos. Además se aplicó GC-MS a los
60 compuestos volátiles de Zuccagnia punctata (Zp) (Fabaceae). La frecuencia con la
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 1 que aparecen los distintos tipos polínicos varió significativamente entre las muestras,
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3 principalmente debido a los cambios en el flujo de polen desde las plantas cercanas a
4 las colmenas. Solo 11 tipos polínicos presentaron una frecuencia mayor a 3%, y solo 9
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6 tipos polínicos tuvieron frecuencias entre 3% y 1.6%. El polen de Prosopis fue el
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8 mejor representante regional, ya que fue el único taxón que se presentó con una
9 frecuencia del 20% al 25%. Se encontró que otras especies nativas de Amaicha del
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11 Valle como Zp, Larrea divaricata y L. cuneifolia, Schinus areira y S. fasciculatus,
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13 Acacia praecox y Ligaria cuneifolia son menos significativas. Los espectros FT-
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IR/UV y el perfil en RP-HPTLC de las muestras de propóleos fueron similares. En
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16 RP-HPTLC solo Zp presentó perfil idéntico al propóleos de Amaicha. Los principales
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18 compuestos volátiles en ambos fueron trans-linalool oxide (furanoid), 6-camphenone,
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linalool, trans-pinocarveol, p-cymen-8-ol, and 2,3,6-trimethylbenzaldehyde. La
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21 presencia de otros compuestos en la fracción volátil del propóleos, sugiere la
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23 contribución de otras especies de plantas productoras de resina en su composición.
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26 Keywords: Propolis; palynology; volatile fraction; Zuccagnia punctata; Apis
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mellifera.
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31 Introduction
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33 Propolis is a natural, wax-like resinous substance found in bee hives where it is used by
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35 honeybees as cement, to seal cracks or open spaces and to embalm predators died inside the
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38 hives. It is mostly composed of plant resins, bee wax and secretions of the head glands of
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40 worker bees (Asís, 1991; Guerra & Méndez, 1997; Havsteen, 2002).
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43 The composition of propolis varies considerably according to the region and the plant origin.
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45 The major constituents of propolis from most of the sources are flavonoids, but despite
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numerous studies conducted all over the world so far, the constitution of propolis remains
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50 largely unknown (Bankova, Popov, & Marekov, 1982; Bankova, Popova, & Trusheva, 2014;
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52 Tsimogiannis, Samiotaki, Panayotou, & Oreopoulou, 2007). Being the volatile fraction of
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55 propolis is especially challenging to study.
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57 Propolis is reputed to have antiseptic, antibacterial, antimycotic, astringent,
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60 spasmolytic, anti-inflammatory, anaesthetic, antioxidant, antitumoural, antifungal, antiulcer,
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 1 anticancer, and immunomodulatory effects, leading to its use as a potential active ingredient
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4 in pharmaceutical preparations (Asís, 1991; Falcão, Tomás, Freire, & Vilas-Boas, 2016; Farré,
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6 Frasquet, & Sanchez, 2004; Guerra & Méndez, 1997; Havsteen, 2002; Lupatini, Danopoulos,
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8 Swikidisa, & Alves, 2016; Mujica et al., 2017).
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11 Pollens grains are accidently introduced into propolis, providing a geographical
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13 marker of its origin (Barth, 2004).
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16 Propolis from Amaicha del Valle (Tucumán, Argentina) region has attracted little
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18 scientific attention, however in a recent report Solorzano et. al. (2017) identified the same
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21 chemical fingerprint for Zuccagnia punctata Cav. as for two propolis samples collected where
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23 this plant grows. However, two other propolis samples collected from a nearby location with
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a lower prevalence of that plant were significantly different. In addition, in a preliminary study
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28 we demonstrated that Amaicha del Valle propolis has a composition consistent with bees
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30 foraging on introduced and native plants, including Zuccagnia punctata (Agüero et al., 2010).
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33 In this work we characterized Amaicha del Valle propolis through pollinic and
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35 physicochemical analysis of volatile compounds and chemical profiles associated with
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38 Zuccagnia punctata, in order to provide quality criteria for cultivation and commercialization
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40 by people native to the region.
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45 Materials and methods
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47 Propolis samples
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50 All the tests were performed in triplicate.
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52 Propolis extraction
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55 Eleven propolis samples (AM1 to AM11) were collected from several areas in Amaicha del
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57 Valle (Tucumán, Argentina): Los Zazos, La Aguadita, El Mirador, El Molino, and La Banda.
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60 All samples were frozen and ground prior to extraction according to Sawaya et al. (2006).
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 1 For chemical analysis, 0.1 g of a propolis sample was treated with 20 cm 3 of 80%
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4 ethanol in a 40 ºC thermostatic water bath agitated for 30 minutes. The preparation was then
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6 filtered yielding propolis ethanol extracts (PEE) (Da Silva Cunha et al., 2006; González,
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8 Guzmán, Rudyk, Romano, & Molina, 2003).
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11 Propolis RP-HPTLC
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13 Reversed-phase high-performance liquid chromatography (RP-HPLC) profiles of propolis
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16 were compared to determine their fingerprints (Park et al., 2001). The PEE of each sample
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18 was deposited on RP-18 F254-S silica plates for HPTLC with ethanol-water (55:45 v/v) as a
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21 mobile phase. The detection of flavonoids was carried out by short- and long-wave UV light
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23 (Harborne, 1998; Harborne & Williams, 2000; Mabry, Markham, & Thomas, 1970; Markham,
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1982; Pietta, Gardana, & Pietta, 2002).
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Physicochemical analyses: FT-IR and UV spectra
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32 FT-IR spectra were collected on the propolis extract between 4000 and 400 cm-1 in a Perkin
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35 Elmer FT-IR 1600 spectrophotometer. Determination of the UV-visible spectra of PEE was
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37 performed according to Moţ et al. (2011). UV–vis spectra (200 to 400 nm) were collected on
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propolis extract samples which were diluted 1:100 in absolute ethanol.
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Palynological methods of propolis samples
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46 For palynological study, propolis samples were analysed for morphological elements in their
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49 sediments. Due to the similarity previously observed in the RP-HPTLC fingerprints (Figure
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51 1) and in FT-IR (Figure 2) and UV-Vis spectra of the eleven propolis samples (Figure 3), three
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54 random propolis samples (AM1, AM3, and AM6) were chosen for palynological analysis.
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56 These samples were frozen and subsequently ground. Palynological processing of samples
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58 following the Barth method was performed (Barth, 1998; Barth, Dutra, & Justo, 1999; Barth
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 1 & Da Luz, 2003; Barth & Dutra, 2000), beginning with 0.5 g of propolis extracted with only
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4 ethanol. Propolis tinctures were occasionally shaken, and after a month the sediment was
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6 treated with KOH before adding the acetolysis mixture. Two slides were prepared
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8 immediately prior to acetolysis with unstained and basic fuchsine-stained glycerine-jelly to
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11 provide information about the presence of trichomes or other organic residues that may be
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13 destroyed during further processing. In the final step of the processing, three more slides were
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16 prepared using the same method. Sample sediments were observed using light microscopy
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18 (Erdtman, 1969). Attempts were made to classify the pollen grains to the closest possible
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21 taxon (species, genus, family) using the comparative preparations and available atlases. The
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23 pollen grains and other structured elements were photographed under the microscope with a
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digital Olympus 8 megapixels camera (Figure 4).
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28 Evaluation of propolis samples was carried out in accordance with palynological
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30 criteria (Barth & Da Luz, 2009; Da Luz & Barth, 1996; Louveaux, Maurizio, & Vorwohl,
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33 1978).
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35 Multivariate analysis was performed using principal component analysis (PCA) to
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38 verify pollen occurrence in the samples (Barth & Da Luz, 2009). Principal component analysis
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40 and data centralizing and standardization were done using MATLAB R2014a software.
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HS-SPME GC-MS of the volatile propolis fraction
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45 2 g of each propolis sample were mixed with 10 cm3 ethanol (95%) in a 25 cm3 Erlenmeyer
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47 flask for extraction at room temperature at 150 rpm for 18 h using a shaker. The extract was
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50 filtered twice (Whatman No.1) and the solvent was evaporated under nitrogen (< 40 °C). A
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52 Solid phase microextraction (SPME) device (Supelco, Bellafonte, PA, USA) equipped with a
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55 fibre pre-coated with a 65 μm thick layer of polydimethylsiloxane/divinylbenzene
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57 (PDMS/DVB) was used for extraction of the volatiles. The vial containing the sample was
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60 sealed with a cap and septa, and the fibre was pushed through the film layer, exposing the
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 1 headspace of the extract for 15 min at 50 °C. The fibre was then inserted immediately into the
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4 injection port of the GC-MS.
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6 The experimental conditions were the same as those previously described by
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8 Guarinoni et al. (2014).
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11 Plant samples
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13 Based on propolis palynological analysis, the botanical species most visited by Apis mellifera
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16 were collected at locations across Amaicha del Valle near apiaries. All collected plants were
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18 actively visited by bees, based on beekeepers’ observations. A voucher specimen of each plant
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21 was deposited in the herbarium of Fundación Miguel Lillo (LIL), San Miguel de Tucumán,
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23 Tucumán, Argentina.
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Dried plant samples were macerated in ethanol for 30 minutes with agitation at 40 ºC
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28 in a water bath. Then, the extract was filtered and the solvent was evaporated to obtain each
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30 dry plant extract.
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33 RP-HPTLC of plant samples
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35 Extracts of each botanical species were analysed by RP-HPTLC with the same procedure used
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38 for propolis samples. Detection of flavonoids was carried out by short- and long-wave UV
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40 light for comparison with propolis RP-HPTLC fingerprints.
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45 Extraction procedure for Zuccagnia punctata leaves volatiles
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47 Because only Zuccagnia punctata (Zp) plant extracts exhibited a similar chromatographic
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50 profile as Amaicha del Valle propolis by RP-HPTLC, Zp volatile compounds were analysed
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52 by GC-MS.
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55 Essential oil was obtained from air dried aerial parts by steam distillation for 2 h at
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57 normal atmospheric pressure in a Clevenger type apparatus. The oil was dried with anhydrous
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60 sodium sulphate and kept in a sealed flask at -12 °C until analysis.
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 1 GC-MS analysis of Zp volatiles
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4 GC-MS analyses were conducted using a Shimadzu QP 2020 mass spectrometry equipped
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6 with reference libraries (Adams, 2007; Mc Lafferty & Stauffer, 1991; Mondello, 2015) using
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8 a BP 20 (SGE, Ringwood, Australia) bonded fused silica capillary column (25 m × 0.25 mm
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11 i.d.), coated with poly-(ethylene glycol) (0.25 μm phase thickness), and the following heating
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13 program: column temperature, 40 °C (8 min), rising to 180 °C at 3 °C/min, then to 230 °C at
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16 20 °C/min. The injector temperature was 250 °C, operated in split injection mode with a split
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18 ratio of 1:40 and a 0.2 μl injection volume. The carrier gas was He at 92.6 kPa (55.9 cm/s).
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21 The interface temperature was 250 °C and the acquisition mass range was 40-400 m/z.
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23 Results and discussion
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Propolis samples
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28 RP-HPTLC of propolis samples
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30 HPTLC RP-18 F254-S analysis (Figure 1) clearly suggested that although propolis samples
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33 were collected from different areas in Amaicha del Valle, the samples had similar
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35 compositions, in agreement with others authors that have reported that propolis from the arid
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38 region of Tucumán province had a similar biological activity and fingerprint as measured by
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40 HPLC-DAD (Agüero et al., 2010; Salas et al., 2016).
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[Figure 1 near here]
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45 FT-IR UV spectra of propolis samples
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47 The Amaicha del Valle propolis samples showed the same pattern in the FT-IR spectra (Figure
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50 2). Prominent FT-IR absorption bands within this region and their corresponding functional
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52 groups are listed in Table 1, which are consistent with IR data collected on other propolis
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55 extracts (Moţ, Silaghi-Dumitrescu, & Sârbu, 2011; Oliveira et al., 2016). The 1510 cm-1 band
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57 is specific to the propolis extracts, mostly due to stretching and bending modes associated
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60 with the aromatic rings of polyphenols (Manthey, 2006; Pasto & Johnson, 2008).
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 1 [Figure 2 near here]
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6 The 1510 cm-1, 1637 cm-1, and 1736 cm-1 bands correspond to compounds specific to
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8 propolis extracts which are correlated with the antioxidant capacity of the samples (Gonzalez
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11 et al., 2015; Moţ et al., 2011).
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13 [Table 1 near here]
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16 The UV–vis spectra of Amaicha PEE (Figure 3) are similar to typical high
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18 concentration polyphenol spectra. The primary feature of these spectra is a broad band centred
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21 around 250-400 nm. Moţ et al. (2011) reported that samples with both a 1630 cm-1 band in the
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23 IR region and a 353 nm absorbance band in the UV–vis region have an antioxidant capacity.
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They proposed that these two spectroscopic ‘‘hand-holds’’ can be effectively used for rapid
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28 screening of the antioxidant capacity of ethanol extracts of propolis and other biological
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30 samples (Moţ et al., 2011). In a previous work by our group, DPPH radical scavenging activity
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33 and antioxidant activity toward linoleic acid clearly demonstrated the ability of Amaicha PEE
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35 to defend against oxidizing agents (Gonzalez et al., 2015).
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38 [Figure 3 near here]
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40 Palynological studies
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A variety of organic elements (besides pollen) were detected in the propolis sample sediments,
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45 such as trichomes, vegetable and animal tissue residues, and spores and fungi hyphae.
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47 Trichomes are generally unicellular or bi-cellular, 108 µm long x 13 µm wide, with a 1.5 to 2
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50 µm thick wall. Other glandular spherical elements with a diameter of 100-150 µm were
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52 observed to have several folds and a clear area in the centre, which might be consistent with
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55 an insertion mark. Microscopic images of pollen grains and other structured elements are
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57 presented in Figure 4.
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60 [Figure 4 near here]
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 1 AM1 sample showed fewer pollen grains (705) as well as fewer trichomes and
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4 glandular structures (30). This was the only sample that contained 18% Urtica pollen types.
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6 In addition, AM3 had abundant pollen grains (1755), trichomes, glandular structures, and
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8 tissue residues with stoma or trichomes (183). AM6 contained fewer pollen grains (1143) as
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11 well as trichomes and glandular structures (102). However, it contained Melilotus albus pollen
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13 grains (5.34%). AM6 was notable for the relatively high abundance of whole Zuccagnia
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16 punctata grains (approximately 12 grains per sample). This difference is likely due to this
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18 sample not being ground, as opposed to samples AM1 and AM3 which were ground. These
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21 large-size grains (80 µm) appeared as broken pieces in the other propolis samples. Although
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23 Zuccagnia punctata is a botanical source of propolis in arid regions of Argentina, the limited
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presence of its pollen is probably due to its large size, which makes it difficult for bees to
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28 carry (Agüero et al., 2010; Salas et al., 2016; Solorzano et al., 2017).
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30 AM1, AM3, and AM6 showed significant variability in pollen types, with more than
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33 fifty types identified for each sample. The pollinic types identified were as follows: Prosopis
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35 (P. alba, P. nigra, P. torquata), Myrtaceae (Eucalyptus sp.), Citrus, Brassicaceae, Asteraceae
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38 (Taraxacum officinale, Vernonia sp., Senecio sp., Tessaria sp., Baccharis sp.), Larrea (L.
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40 divaricata, L. cuneifolia), Proteaceae, Amaranthaceae-Chenopodiaceae, Gomphrena,
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Schinus, Clematis, Zuccagnia punctata, Plantago, Salix, Apiaceae, Buddleja tucumanensis,
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45 Ligaria, Melilotus albus, Juglans, Polygalaceae, Populus, Salvia, Morus, Urticaceae, Celtis,
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47 Heliotropium, Anadenanthera colubrina var. cebil, Acacia praecox, Lycium, Justicia,
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50 Cercidium praecox, Liquidambar styraciflua.
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52 Although pollen grains count is not a quantitative evaluation of propolis plant origin,
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55 they were counted for comparison. The total number of pollen grains found and counted for
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57 each sample varied significantly.
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 1 Although Citrus pollens types appeared in a few propolis samples (AM3 and AM6),
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4 Citrus is not a regional species. The presence of its pollen grains can be accounted for by the
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6 fact that the propolis samples were harvested from nomad beehives coming from areas with
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8 Citrus crops. The presence of other pollen types from these places, such as Brassicaceae,
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11 Melilotus, some Asteraceae, and crop plants such as Eucalyptus, Salix, Populus, and
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13 Proteaceae, can be explained similarly.
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16 Pollen grains are an indicative component of propolis phytogeographical origin. The
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18 pollen spectra of propolis is wide-ranging. Nevertheless, only 11 pollen types occurred with
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21 a frequency higher than 3% and only 9 pollen types had a frequency between 3% and 16%.
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23 The pollen type Prosopis is the best regional representative, as it constitutes a unique taxon
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that occurred with a frequency of 20% to 25%. Other native species from Amaicha del Valle
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28 such as Zuccagnia punctata (jarilla macho), Larrea divaricata, and L. cuneifolia (jarilla),
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30 Schinus (molle), Acacia praecox (arca), and Ligaria (liga) were found to be less significant.
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33 Normally, the presence of pollen grains of Schinus is characteristic of red propolis
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35 sediments, but in green - brownish propolis, its proportion was less than 7% (Barth & Da Luz,
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38 2009).
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40 Plots of the first and second scores of the principal component analysis (97.7% of the
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43
data variability) are shown in Figure 5. Three main groups were identified, with the first
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45 corresponding to Prosopis; the second to Brassicaceae, Myrtaceae, and Asteraceae; and the
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47 third to Buddleja tucumanensis, Schinus, Proteaceae, and Larrea.
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49
50 [Figure 5 near here]
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53 HS-SPME of the volatile propolis fraction
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56 Propolis volatiles give it its unique, pleasant fragrance. Similar to reports on the phenolic
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58 composition of hydroalcoholic extracts, variations in the chemical profile of the propolis
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 1 volatile fraction between temperate and tropical regions have been described. (Miguel &
2
3
4 Antunes, 2011). The volatile chemical composition of propolis is also known to be strongly
5
6 dependent on the local flora at the collection site (Fernandes – Silva et al., 2015; Pino, Marbot,
7
8 Delgado, Zumárraga, & Sauri, 2006), making these compounds useful for the chemical
9
10
11 characterization of propolis. In previous studies, volatile propolis compounds extracted by HS
12
13 were studied by GC (Fernandes-Silva et al., 2015; Pellati, Prencipe, & Benvenuti, 2013). In
14
15
16 the present study, powdered raw propolis was analysed by HS-SPME-GC-MS. Twenty-nine
17
18 constituents were identified in the propolis volatile fraction (Table 2).
19
20
21 [Table 2 near here]
22
23 The most abundant compounds were palmitinic acid (36.62 %) and tetracosane (32.39
24
25
26
%), followed by other compounds including oleic acid, eiconasol, octadecanol, tetracosanol,
27
28 hexadecanol, linalool, p-cymen-8-ol, terpinen-4-ol, limonene, α-terpineol, decanoic acid, and
29
30 dodecanoic acid. These components have also been found in the volatile fraction of propolis
31
32
33 samples from Italy, Yemen, Brazil, Bulgaria, and Anatolia (Al-Ghamdi et al., 2017;
34
35 Fernandes-Silva et al., 2015; Pellati, et al., 2013; Prytzyk et al., 2003; Uzel, Önçağ, Çoğulu,
36
37
38 Gençay, 2005). Palmitinic acid and tetracosane have been reported to be present in low
39
40 concentrations in the volatile fraction of propolis samples (Al – Ghamdi et al., 2017; Prytzyk
41
42
43
et al., 2003; Rushdi et al., 2014; Uzel et al., 2005). n-Alkanes are usually present as C25-C31
44
45 compounds in plant waxes, depending on the plant species, season, and locality (Eglinton &
46
47 Hamilton, 1967), which explains the high concentration of tetracosane measured. Moreover,
48
49
50 the high concentration of palmitic acid was probably due to the presence of beeswax as a
51
52 contaminant, since palmitic acid is the most abundant compound in hydrolysed wax
53
54
55 (Bonaduce & Colombini, 2004; Downing, Krantz, Lamberton, & Murray, 1961)
56
57
58 Plant material
59
60
61
62
63
64
65
 1 Fifty-six botanical sample species were registered in Amaicha del Valle (Tucumán,
2
3
4 Argentina), which were identified as shown in Tables 3a, 3 b. Zuccagnia punctata extracts
5
6 had a similar fingerprint by RP-HPTLC as Amaicha del Valle propolis, in agreement with the
7
8 fact that this species is one of the botanical sources of propolis in arid regions of Argentina
9
10
11 (Agüero et al., 2010; Salas et al., 2016).
12
13 [Tables 3a, 3b near here]
14
15
16
17
18 GC-MS analysis of Zp volatiles
19
20
21 Table 4 shows the composition of the Zp volatile fraction, which included the main
22
23 components linalool (5.73%), 2,3,6-trimethylbenzaldehyde (4.32%), p-cymen-8-ol (3.61%),
24
25
26
chrysanthenone (3.41%), cis-linalool oxide (furanoid) (3.36%), trans-linalool oxide
27
28 (furanoid) (3.15%), p-mentha-1,5-dien-8-ol (2.68%), (E)- -terpineol
29
30 (2.35%), and cis-linalyl oxide (pyranoid) (2.15%). This agrees with data obtained by Alvarez
31
32
33 et al. (2012), where the essential oils from aerial parts of nine populations of Zuccagnia
34
35 punctata Cav were investigated.
36
37
38 [Table 4 near here]
39
40
41
42
43
The composition of both Amaicha del Valle propolis and Zp essential oil included
44
45 trans-linalool oxide (furanoid), 6-camphenone, linalool, trans -pinocarveol, p-cymen-8-ol,
46
47 and 2,3,6-trimethylbenzaldehyde, with greater quantities of all of them in propolis.
48
49
50 Zuccagnia punctata has been considered to be the main source of Amaicha del Valle
51
52 propolis, but the presence of other compounds in the volatile fraction of propolis suggests that
53
54
55 the contribution of other resin-producing plant species also contribute to propolis.
56
57
58
59
60
61
62
63
64
65
 1 Conclusion
2
3
4 The FT-IR/UV spectra and RP-HPTLC fingerprints of propolis samples obtained from
5
6 Amaicha del Valle had similar profiles. Pollen from those propolis was varied, with only 11
7
8 pollinic types occurring with a frequency higher than 3%, and only 9 with a frequency between
9
10
11 3% and 1.6%. The Prosopis pollen type was the best regional representative, as it is the only
12
13 taxon with a frequency of 25%. Although Zuccagnia punctata is considered to be a botanical
14
15
16 sources of propolis in arid regions of Argentina, its limited presence may be due to its large
17
18 grain size, which makes it difficult for bees to carry. Only Zp extract showed a similar
19
20
21 fingerprint to Amaicha del Valle propolis by RP-HPTLC analysis. The presence of other
22
23 compounds in the volatile fraction of propolis suggests that the contribution of other resin-
24
25
26
producing plant species also contribute to propolis.
27
28 Acknowledgement
29
30 The authors acknowledge financial support from SCAIT-UNT, PIUNT E/548, National
31
32
33 University of Tucumán, Tucumán, Argentina.
34
35 References
36
37
38 Adams, R. P. (2007). Identification of Essential Oils by Gas Chromatography/Mass
39
40 Spectroscopy. 4th Edition Allured Publishing Corporation, Carol Stream, IL.
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42
43
Agüero, M. B., González, M., Lima, L., Svetaz, L., Sanchez, M., Zacchino, S., … Tapia, A.
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45 (2010). Argentinean propolis from Zuccagnia punctata Cav. (Caesalpinieae) exudates:
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47 Phytochemical characterization and antifungal activity. J. Agric. Food Chem. 58, 194–
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50 201.
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52 Al-Ghamdi, A. A., Bayaqoob, N. I., Rushdi, A. I., Alattal, Y., Simoneit, B. R., El-Mubarak,
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55 A. H., & Al-Mutlaq, K. F. (2017). Chemical compositions and characteristics of organic
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57 compounds in propolis from Yemen. Saudi J. of Biol. Sci., 24, 1094‒1103.
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11 Bankova, V. S., Popov, S. S., & Marekov, N. L. (1982). High performance liquid
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23 Barth, O. M. (2004). Melissopalynology in Brazil: A review of pollen analysis of honeys,
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23 Eglinton, G., & Hamilton, R. J. (1967). Leaf epicuticular waxes. Science, 156, 1322‒1335.
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35 antibacterial activity of green propolis extract and meadowsweet extract against
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47 Manthey, J. A. (2006). Fourier transform infrared spectroscopic analysis of the
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 1 McLafferty, F. W., & Stauffer, D. B. (1991). The important peak index of the registry of mass
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4 spectral data. New York: Wiley.
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6 Miguel, M. G., & Antunes, M. D. (2011). Is propolis safe as an alternative medicine? J.
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11 Mondello, L. (2015). Mass Spectra of Flavors and Fragrances of Natural and Synthetic
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13 Compounds 3rd ed. New York: Wiley.
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16 Moţ, A. C., Silaghi-Dumitrescu, R., & Sârbu, C. (2011). Rapid and effective evaluation of the
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18 antioxidant capacity of propolis extracts using DPPH bleaching kinetic profiles, FT-IR
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23 Mujica, V., Orrego, R., Pérez, J., Romero, P., Ovalle, P., Zúñiga-Hernández, J., …, Leiva, E.
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33 Oliveira, R. N., Mancini, M. C., Oliveira, F. C. S. D., Passos, T. M., Quilty, B., Thiré, R. M.
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35 D. S. M., & McGuinness, G. B. (2016). FTIR analysis and quantification of phenols and
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38 flavonoids of five commercially available plants extracts used in wound healing.
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40 Matéria (Rio de Janeiro), 21(3), 767‒779.
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45 A. (2001). Comparaçao das características Físico-Químicas das Própolis Produzidas na
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47 Regiao Subtropical da América do Sul: Evidência Fitoquímica de sua Origem Botânica.
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52 Pasto, D. J., & Johnson, C.R. (2008). Determinación de estructuras orgánicas. Spain: Reverte
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 1 Pellati, F., Prencipe, F. P., & Benvenuti, S. (2013). Headspace solid-phase microextraction-
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4 gas chromatography–mass spectrometry characterization of propolis volatile compounds.
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6 J. Pharm. Biomed. Anal., 84, 103‒111.
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8 Pietta, P. G., Gardana, C., & Pietta, A. M. (2002). Analytical methods for quality control of
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23 Neto, F. R. A. (2003). Flavonoids and trypanocidal activity of Bulgarian propolis. J.
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28 Rushdi, A. I., Adgaba, N., Bayaqoob, N. I., Al-Khazim, A., Simoneit, B. R., El-Mubarak, A.
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30 H., & Al-Mutlaq, K. F. (2014). Characteristics and chemical compositions of propolis
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33 from Ethiopia. SpringerPlus, 3, 253.
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35 Salas, A., Alberto, M. R., Zampini, C., Cuello, S., Maldonado, L., Ríos, J. L., … Isla, M.I.
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38 (2016). Biological activities of polyphenols-enriched propolis from Argentina arid
39
40 regions. Phytomedicine, 23, 27‒31.
41
42
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Sawaya, A. C. H. F., Cunha, I. B. S., Marcucci, M. C., Oliveira Rodrigues, R. F., & Eberlin,
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45 M. N. (2006). Brazilian propolis of Tetragonisca angustula and Apis mellifera.
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47 Apidologie, 37, 398–407.
48
49
50 Solorzano, E. R., Bortolini, C., Bogialli, S., Di Gangi, I. M., Favaro, G., Maldonado, L., &
51
52 Pastore, P. (2017). Use of a LC-DAD-QTOF system for the characterization of the
53
54
55 phenolic profile of the argentinean plant Zuccagnia punctata and of the related propolis:
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57 New biomarkers. J. Funct. Foods, 33, 425–435.
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 1 Tsimogiannis, D., Samiotaki, M., Panayotou, G., & Oreopoulou, V. (2007). Characterization
2
3
4 of flavonoid subgroups and hydroxxy substitution by HPLC-MS/MS. Molecules, 12,
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6 593‒606.
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8 Uzel, A., Önçağ, Ö., Çoğulu, D., & Gençay, Ö. (2005). Chemical compositions and
9
10
11 antimicrobial activities of four different Anatolian propolis samples. Microbiol. Res., 160,
12
13 189‒195.
14
15
16 Table 1.
17
18
19
20
21 Band (cm-1) Functional group vibration mode Possible compounds
22
23 1044 ν (C-O), δ(C-OH) Primary - secondary alcohols
24
25
26 1163s ν (C-O), δ(C-OH) Lipids, alcohol groups
27
28 1252 ν (C-O) C–O group of polyols
29
30
1450s ν (aromatic), δ(C-H) CH3, CH2, flavonoids, aromatic ring
31
32
33 1510 ν (C=C) (aromatic) Flavonoids, aromatic ring
34
35 1637s ν (C=O), ν (C=C), δas (N-H) Flavonoids, amino acids
36
37
38 1690s ν (C=O) Lipids, flavonoids
39
40 ν – stretching; δ – bending; as – asymmetrical
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
 1 Table 2.
2
3
4 LRIc
Compounda %b
5 BP 20
6
7
8 β-thujene 0.06 1133
9 Limonene 0.21 1198
10
11 Dodecane 0.02 1200
12 p-cymene 0.11 1265
13
14
Cumene 0.05 1270
15 mesitylene 0.01 1271
16 isobutyl benzoate 0.05 1302
17
18 ethyl lactate 0.02 1358
19 thymol methylether 0.19 1456
20
21
trans-linalool oxide (furanoid) 0.01 1454
22 acetic acid 0.27 1450
23 6-camphenone 0.46
24
25 Linalool 0.01 1537
26 2-methylpropanoic acid 0.04 1584
27
28
terpinen-4-ol 0.06 1591
29 trans-pinocarveol 0.04 1632
30 α-terpineol 0.05 1688
31
32 p-cymen-8-ol 0.11 1838
33 2,3,6-trimethyl benzaldehyde 0.03 1929
34
35
tetracosanol 0.07
36 decanoic acid (capric acid) 0.25 2274
37 hexadecanol 2.49 2352
38
39 octadecanol 0.06 2534
40 Tetracosane 32.39 2400
41
42
lauric acid 0.53 2517
43 nonadecanol 2.24 2646
44 Eicosanol 3.74 2755
45
46 palmitinic acid 36.62 2910
47 oleic acid 1.12 3184
48 a
49 Components are reported according to their elution order on BP 20. bRelative proportions of
50
51 the essential oil constituents were expressed as percentages obtained by peak-area
52
53
normalization, with all relative response factors taken as one. cPeak identifications are based
54
55
56 on comparison of LRI values on the BP 20 column with those from pure standards or reported
57
58 in the literature, as well as comparison of MS spectra with library spectra.
59
60
61
62
63
64
65
15
16
17
18
19
20
21
22
23 Table 3a.
24
25 Area of Area of
26 Plant Family Genus or species collected Plant Family Genus or species collected
27 collection collection
28 Justicia tweediana (Nees) Griseb. Cercidium praecox (Ruiz & Pav. ex
CC, LA LZ
29 Hook.) Harms
30 Acanthaceae
Justicia xylosteoides Griseb. Geoffraea decorticans (Gillies ex Hook.
31 CC, LA EM
& Arn.) Burkart
32
Anacardiaceae Schinus fasciculatus (Griseb.) I.M. Melilotus albus Desr.
33 CC, LA LA
34 Johnst.
35 Altingiaceae Liquidambar styraciflua L. Prosopis chilensis (Molina) Stuntz
EM LA
36 emend. Burkart
37 Amaranthaceae Gomphrena sp. LZ Fabaceae Prosopis alba Griseb. LZ, LA
38 Acanthostyles buniifolius Hook. & Prosopis nigra (Griseb.) Hieron.
39 CC LA
Arn.) R.M. King & H. Rob.
40
Austrobrickellia arnottii (Baker) Prosopis torquata (Cav. ex Lag.) DC.
41 CC LZ
42 R.M. King & H. Rob.
43 Baccharis sp. Senna rigida (Hieron.) H.S. Irwin &
CC LZ
44 Barneby
45 Baccharis salicifolia (Ruiz & Pav.) Zuccagnia punctata Cav.
46 LA LZ
Pers.
47 Cyclolepis genistoides Gillies ex D. Lamiaceae Salvia cuspidata Ruiz & Pav. ssp. gilliesii
48 LA LA
Don (Benth.) J.R.I. Wood
49 Asteraceae
50 Flourensia fiebrigii S.F. Blake CC Loranthaceae Ligaria cuneifolia (Ruiz & Pav.) Thieg. LA, CC
51 Gochnatia glutinosa (D. Don) Moraceae Morus alba L.
CC EMo
52 Hook. & Arn.
53 Grindelia pulchella Dunal LA Myrtaceae Eucalyptus sp. LA, CC
54 Senecio sp. CC Plantaginaceae Plantago sp. LZ
55 Senecio rudbeckiiefolius Meyen & Ranunculeaceae Clematis montevidensis Spreng.
56 CC CC
Walp.
57
58 Taraxacum officinale F.H. Wigg. LA Populus sp LZ
59 Tessaria absinthioides (Hook. & Salicaceae Salix sp
LA LZ
60 Arn.) DC.
61
62
63
64
65
15
16
17
18
19
20
21
22
23 Locations: CC: Cuesta de Los Cardones; LA: La Aguadita; EM: El Mirador; LZ: Los Zazos; EMo: El Molino; A: Arroyo
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
15
16
17
18
19
20
21
22
23 Table 3b.
24
25 Area of Area of
26 Plant Family Genus or species collected Plant Family Genus or species collected
27 collection collection
28 Boraginaceae Heliotropium amplexicaule Vahl LZ Scrophulariaceae Buddleja tucumanensis Griseb. LA
29 Capsella bursa-pastoris (L.) Fabiana punensis S.C.
30 LA A, LA
Medik.
31 Brassicaceae Solanaceae
32 Descurainia sp. LA Lycium chilense Miers ex Bertero LA
33 Lepidium didymum L. LA Lycium tenuispinosum Miers LA
34 Celtidaceae Celtis ehrenbergiana (Klotzsch) Urticaceae Urtica sp.
35 EMo CC
36
Liebm.
37 Capparaceae Capparis atamisquea Kuntze Bulnesia schickendantzii Hieron. ex
LA CC
38 Griseb.
39 Chenopodiaceae Suaeda divaricata Moq. LA Larrea cuneifolia Cav. LZ
40
41
Acacia praecox Griseb. LA Larrea divaricata Cav. LA
42 Acacia visco Lorentz ex Griseb. Zygophyllaceae Plectrocarpa rougesii Descole,
EM LA
43 O’Donell & Lourteig
44 Fabaceae Anadenanthera colubrina (Vell.) Plectrocarpa tetracantha Gillies ex
45
Brenan var. cebil (Griseb.) EM Hook. & Arn. LA
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47 Altschul
48 Heliotropium amplexicaule Vahl LZ
49 Locations: CC: Cuesta de Los Cardones; LA: La Aguadita; EM: El Mirador; LZ: Los Zazos; EMo: El Molino; A: Arroyo
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 Table 4.
1
2 LRIc LRIc
3 Compounda %b Compounda %b
4 SE-52 SE-52
5
6 2-ethyl-
7 α-pinene 0.16 936 benzaldehyde 0.59 1187
8
9 fenchone 0.09 949 p-cymen-8-ol 3.61 1188
10 thuja-2,4(10)-diene 0.43 956 α-terpineol 2.35 1190
11 Benzaldehyde 0.68 963 myrtenal 0.39 1192
12 phenol 0.63 983 chrysanthenone 3.41 1125
13
14 caproic acid 0.26 996 verbenone 0.48 1206
15 α-terpinene 0.04 997 cis-cinnamaldehyde 0.2 1215
16 p-cymene 1.28 1017 trans-carveol 0.34 1217
17 β-phellandrene 0.62 1024 cis-carveol 0.32 1227
18
19 1,8-cineole 0.27 1030 cuminaldehyde 0.15 1238
20 dihydro-5-methyl-5-vinyl-
21 2(3H)-furanone 0.42 1032 benzylacetone 0.33 1240
22 (Z)-β-ocimene 0.3 1039 carvotanacetone 0.24 1245
23
24 (E)-β-ocimene 0.23 1038 piperitone 0.04 1254
25 γ-terpinene 0.17 1048 nerol 1.77 1255
26 Acetophenone 0.26 1060 phellandral 0.37 1275
27
linalool oxide
28
29 cis-linalool oxide (furanoid) 3.36 1067 acetate (pyranoid) 0.49 1286
30 p-cresol 0.29 1075 carvacrol 0.24 1300
31 trans-linalool oxide
32 (furanoid), 3.15 1077 thymol 0.86 1301
33
34 6-camphenone 2.38 1083 (E)-anethole 2.61 1320
35 dehydro sabina ketone 0.28 1093 myrtenyl acetate 0.2 1329
36 linalool 5.73 1098 trans-carvyl acetate 0.28 1337
37
38
2,3,6-trimethyl
39 Hotrienol 0.89 1099 benzaldehyde 4.32 1338
40 4-phenyl-3-buten-2-
41 endo-fenchol 0.25 1101 one 0.05 1346
42 cis-p-menth-2-en-1-ol 0.22 1115 geranyl acetate 0.72 1380
43
44 α-campholenal 0.04 1123 endo-arbozol 0.41 1418
45 trans-pinocarveol 0.71 1124 trans-caryophyllene 0.09 1420
46 trans-verbenol 0.44 1140 aromadendrene 0.28 1441
47
48
p-mentha-1,5-dien-8-ol 2.68 1144 γ-muurolene 0.31 1476
49 cis-caran-trans-2-ol 0.29 1171 α-muurolene 0.22 1498
50 myrtenal 0.12 1177 δ-cadinene 0.95 1523
51 safranal 0.45 1179 viridiflorol 0.3 1591
52
53
cis-linalyl oxide (pyranoid) 2.15 1186 torreyol 0.43 1645
54 Borneol 0.32 1180 guaiazulene 0.72 1772
55 terpinen-4-ol 0.24 1181 phytol 0.04 2116
56 a b
Components are reported according to their elution order on BP 20. Relative proportions
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59 of the essential oil constituents were expressed as percentages obtained by peak-area
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 normalization, with all relative response factors taken as one. cPeak identifications are
1
2 based on comparison of LRI values on the BP 20 column with those from pure standards
3
4
5 or reported in the literature, as well as comparison of MS spectra with library spectra.
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 Captions Tables and Figures
1
2 Table 1. FT-IR bands found in PEE.
3
4
5 Table 2. Percentage composition of the volatile propolis fraction and linear retention
6
7 indices (LRI) of the components.
8
9
10 Table 3a. Plants species registered in Amaicha del Valle (Tucumán, Argentina) district.
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12 Table 3b. Plants species registered in Amaicha del Valle (Tucumán, Argentina) district.
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Table 4. Percentage composition of the essential oil of Zuccagnia punctata and linear
16
17 retention indices (LRI) of the components.
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19
20
21
22 Figure 1. RP-HPTLC of 11 propolis samples from Amaicha del Valle.
23
24 Figure 2. FT-IR spectrum of Amaicha del Valle propolis.
25
26
27 Figure 3. UV-Vis spectrum of Amaicha del Valle propolis.
28
29 Figure 4. Microscopy images of pollinic types and other plant structures founds in
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31
32
samples.
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34 Figure 5. Score plot of the first two principal components of AM samples. Percentage of
35
36 variance explained by each principal component are given in parentheses.
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Figure 1. RP-HPTLC of 11 propolis samples from Amaicha del
Valle.

AM1 AM2 AM3 AM4 AM5 AM6 AM7 AM8 AM9 AM10 AM11

Figure 1. RP-HPTLC of 11 propolis samples from Amaicha del Valle.

Figure 2. FT-IR spectrum of Amaicha del Valle propolis.

Figure 2. FT-IR spectrum of Amaicha del Valle propolis.

Figure 3. UV-Vis spectrum of Amaicha del Valle propolis.

Figure 3. UV-Vis spectrum of Amaicha del Valle propolis.

Figure 4. Microscopy images of pollinic types and other plant
structures founds in samples.

Figure 4. Microscopy images of pollinic types and other plant structures founds in
samples.

1-Bulnesia: polar view 2-Larrea: polar view. 3-Salix: equatorial view .4-Populus 5-Melilotus
albus: equatorial view 6-Schinus: equatorial view.7-Asteraceae: equatorial view 8-Asteraceae:
optical cross section 9-Lamiaceae: optical view 10-Plantago: optical cross section 11-Apiaceae:
equatorial view. 12-Brassicaceae: polar view optical cross section 13-Myrtaceae: polar view
optical cross section 14-Alnus acuminata: polar view 15-Acacia: polyad 16-Glandular structure
17-Trichome 18-Zuccagnia punctata: polar view 19-Prosopis: polar view.
Figure 5. Score plot of the first two principal components of AM
samples. Percentage of variance explained by each principal

Pollen: 1-Prosopis, 2-Myrtaceae, 3-Asteraceae, 4-Brassicaceae, 5-Proteaceae, 6-Larrea, 7-

Schinus, 8-Salix, 9-Melilotus, 10-Populus, 11-Amaranthaceae, 12-Clematis, 13-Juglans, 14-
Plantago, 15-Loranthaceae, 16-Urticaceae, 18-Heliotropium, 19-Morus, 20-Acacia, 21-
Buddleja tucumanensis, 22-Zuccagnia punctata.

Figure 5. Score plot of the first two principal components of AM samples. Percentage of
variance explained by each principal component are given in parentheses.

5 7 8

10

16