Comparative Chemistry of Propolis from Eight Brazilian Localities

Propolis is a complex honeybee product with resinous aspect, containing plant exudates and beeswax. Their color, texture, and chemical composition vary, depending on the location of the hives and local flora. The most studied Brazilian propolis is the green (alecrim-do-campo) type, which contains mainly prenylated phenylpropanoids and caffeoylquinic acids. Other types of propolis are produced in Brazil, some with red color, others brown, grey, or black. The aim of the present work was to determine the chemical profiles of alcohol and chloroform extracts of eight samples of propolis, corresponding to six Brazilian regions. Methanol and chloroform extracts were obtained and analyzed by HPLC/DAD/ESI/MS and GC/MS. Two chemical profiles were recognized among the samples analyzed: (1) black Brazilian propolis, characterized chiefly by flavanones and glycosyl flavones, stemming from Picos (Piauí state) and Pirenópolis (Goiás state); (2) green Brazilian propolis, characterized by prenylated phenylpropanoids and caffeoylquinic acids, stemming from Cabo Verde (Bahia state), Lavras and Mira Bela (Minas Gerais state), Pariquera-Açu and Bauru (São Paulo state), and Ponta Grossa (Paraná state). The present work represents the first report of prenylated flavonoids in Brazilian propolis and schaftoside (apigenin-8-C-glucosyl-6-C-arabinose) in green propolis.


Introduction
Propolis is a resinous hive product containing bee secretions and plant resins. The wide diversity of plant species used by bees as resin sources for propolis production determines its chemical diversity. More than 300 constituents have already been identified [1], among which phenolic compounds such as flavonoids, phenolic acids, and phenolic acid esters have been reported as major constituents of propolis from the temperate zone [2].
The use of propolis with medicinal purpose dates back to 300 BC [3] and has been extensively used in folk medicine in the east and the west for centuries. It has been established that propolis possesses a wide spectrum of biological activities, such as antibacterial [4,5], anti-inflammatory [6,7], antioxidant [8,9], hepatoprotective [10], and antitumoral [11]. The medicinal properties of propolis are due to a large variety of substances of plant origin, acting alone and/or synergistically. The high medicinal value of many propolis constituents stimulates chemical studies also of the corresponding plant sources [12].

Propolis Sampling and Processing.
Samples of propolis produced by Apis mellifera were obtained from eight Brazilian localities: Picos (state of Piauí, North Central Brazil) (black propolis), Cabo Verde (Bahia state, northeast) (green propolis), Pirenópolis (Goiás state, Central Brazil) (black propolis), Lavras and Mira Bela (Minas Gerais state, Southeast) (green propolis), Pariquera-Açu and Bauru (São Paulo state, southeast) (green propolis), and Ponta Grossa (Paraná state, south) (green propolis) ( Figure 1). The samples were grounded with a mortar and pestle to a fine powder. Extracts were successively prepared in Soxhlet for 3 h with 5 g of each propolis sample, first with chloroform and then with methanol. Each extract was concentrated under reduced I   II   III  IV   V  VI  VII  VIII   I   II   III I I I I I I I I I  IV   V  VI  VII I I I I I I  VIII I I I I I I I I I I I  pressure and the residue evaporated on a steam bath to constant weight. The chloroform extracts were dissolved in diethyl ether and treated with diazomethane [36]. The dried methanol extracts were dissolved in methanol at 0.1 mg mL −1 prior to HPLC analysis.

Total Polyphenol Contents.
Total polyphenol contents were determined according to the Folin-Ciocalteu colorimetric method [37], using p-coumaric acid as reference. The analyses were carried out in triplicates of each sample.

Total Flavonoid Contents.
Total flavonoid contents were determined by the aluminium chloride [37] and dinitrophenylhydrazin [38] methods. Calibration curves were made using quercetin (aluminium chloride method) and pinocembrin (dinitrophenylhydrazin method) as reference. Total flavonoid contents were assumed to be the sum of the values obtained by each method. Analyses were performed in triplicates.

GC/EIMS Analysis.
The diethyl ether solutions of the diazomethane-treated chloroform extracts were diluted to the 1000 ppm concentration. Ether solutions (1 L) of each extract was injected into a Hewlett Packard 5890 series II plus gas chromatography coupled to Chem Station System Mass Spectrometer 5989B operating with the EI mode at 70 eV. The GC conditions were as follows: DB-5HT fused silica capillary column (30 m × 0.32 mm internal diameter, 0.25 m film thickness) held at 100 ∘ C for 1 min and then heated to 300 ∘ C at 6 ∘ C min −1 , the final temperature being held constant for 2 min; He was used as a carrier gas with flux of 1.5 mL min −1 , Evidence-Based Complementary and Alternative Medicine 3
On the other hand, samples from Bauru, Picos, Pariquera-Açu, Ponta Grossa, Lavras, and Mira Bela showed low contents of total flavonoids: 1.97%, 1.243%, 0.884%, 0.859%, 0.685%, and 0.311%, respectively. It is interesting to note that all samples, except from Bauru, present flavone C-glycosides among their constituents. However, only the mentioned sample exhibited high content of caffeic acid ethyl phenyl ester derivatives. Among the samples studied in the present work, there seems to be a direct correlation between the contents of total phenols and total flavonoids (Table 1).
Glycosides are not frequent in propolis. Glycosides of caffeic acid (54, 58, and 59) have rarely (if at all) been reported as propolis constituents. C-glycosyl and O-glycosyl flavonoids were found in stingless bee honeys [47]. In the honey of Apis mellifera the contents of aglycone flavonoids typical of propolis (galangin, pinocembrin, quercetin) were found to be much higher than the contents of flavonoid glycosides. This has been attributed to a contamination of the honey by plant resins and propolis [48].
For O-glycosylated flavonoids, fragmentation pathway starts with the cleavage of the glycosidic bonds and elimination of the sugar moieties, with charge retention on the aglycone [52]. Tentative identification was based mainly on the MS data, UV-DAD spectra, and literature data.   compound for propolis, having previously been identified from Citrus genotypes [54]. Among the constituents identified stand out the preny- lated flavonols 109, 125, 126, and 128-133. Prenylated phenylpropanoids typical of Brazilian green propolis were also detected, such as compounds 95, 96, 117, and 119 (artepillin C).
The chemical affinities among the analyzed propolis samples are shown in Figure 2. One of the clusters of the dendrogram combines the samples from Bauru (SP, southeast), Lavras (MG, southeast), and Cabo Verde (BA, northeast). It is somewhat surprising the emergence in this cluster of the sample from the latter locality, which is much up north from the commonly admitted distribution of green propolis. The three samples share several prenylated phenylpropanoids (e. g., 9, 14, 24, 25, and 35) typical of green propolis ( Table 4). The sample from Cabo Verde stands apart in the clade for the more frequent possession of flavonoid glycosides, for example, 88-91, 98, 99, and 101 (Table 4). Flavonoids are major compounds in propolis from temperate   [19,55]. However, flavonoids commonly detected in propolis are aglycones, and only rare flavonoid glycosides have been reported as propolis constituents. The samples from Pariquera-Açu (SP, southeast) and Ponta Grossa (PR, south) contain also some of these green propolis constituents but emerge at the base of the clustering analysis tree (Figure 2). Both contain substances rarely or never reported for green propolis, such as flavonoid glycosides (e.g., 81, 88, and 97; Table 4). They also contain or share diterpenes, such as 17 and 23 (Table 4), a class of substances known to occur in Brazilian propolis [43,56,57] and abundant in Mediterranean propolis [20,58].