Chemical Composition of Volatile and Extractive Components of Canary (Tenerife) Propolis

The vegetation of the Canary Islands is characterized by a large number of endemic species confined to different altitudinal levels. It can be assumed that these circumstances determine the characteristic features of the chemical composition of local beekeeping products, including propolis. We report, for the first time, the chemical composition of propolis from Tenerife (Canary Islands). The volatile emissions of three propolis samples collected from different apiaries are represented by 162 C1–C20 compounds, of which 144 were identified using the HS-SPME/GC-MS technique. The main group of volatiles, consisting of 72 compounds, is formed by terpenoids, which account for 42–68% of the total ion current (TIC) of the chromatograms. The next most numerous groups are formed by C6–C17 alkanes and alkenes (6–32% TIC) and aliphatic C3–C11 carbonyl compounds (7–20% TIC). The volatile emissions also contain C1–C6 aliphatic acids and C2–C8 alcohols, as well as their esters. Peaks of 138 organic C3–C34 compounds were recorded in the chromatograms of the ether extracts of the studied propolis. Terpene compounds form the most numerous group, but their number and content in different samples is within very wide limits (9–63% TIC), which is probably due to the origin of the samples from apiaries located at different altitudes. A peculiarity of the chemical composition of the extractive substances is the almost complete absence of phenylcarboxylic acids and flavonoids, characteristic of Apis mellifera propolis from different regions of Eurasia and North America. Aromatic compounds of propolis from Tenerife are represented by a group of nine isomeric furofuranoid lignans, as well as alkyl- and alkenyl-substituted derivatives of salicylic acid and resorcinol.


Introduction
Propolis is one of the most valuable beekeeping products, but a honeybee colony's need for it is not very great.Therefore, only a small proportion of worker bees are involved in collecting raw plant materials for its production.Meanwhile, the role of propolis in ensuring the survival of the colony in a far-from-sterile environment cannot be overestimated: it has high activity against various pathogens [1-3] and plays a key role in the 'social immunity' of bees [4,5].
In recent years, increasing attention has been paid to propolis as an antidote to human microbial pathogens.In terms of antisepticity, propolis stands out among most other beekeeping products, second only to bee venom in terms of potency [6][7][8].It exhibits a wide range of medicinal properties, which are not limited to antimicrobial action, as reflected in a number of recent comprehensive reviews [9][10][11][12].In particular, its beneficial effects on the human digestive system, in the treatment of a number of gynecological diseases, and in the healing of wounds and burns, as well as in dermatology and a number of oncological diseases, have been shown [13][14][15][16][17].
It is generally accepted that the antibacterial activity and other medicinal properties of propolis are due primarily to phenolic compounds of plant origin, flavonoids, phenolcarboxylic acids and their esters [9,10,18].These natural compounds are found not only in plant tissues but also in their external secretions in the form of resin, exudates and leakage from wounded tissues, and perform protective functions against pathogens and parasites.It is these properties that encourage bees to collect these materials, in need of a means of combating pests and pathogens in their hives.Bees are selective and preferential in relation to plant sources of raw materials for the production of propolis [2,[19][20][21], and when searching for them, they are guided by various insufficiently studied olfactory and optical signals.A consequence of the selectivity of bees that deliver raw materials is the existence of certain regional 'types' or varieties of propolis [22].For example, the middle latitudes of Eurasia and North America are characterized by the 'poplar type', the plant precursor of which is exudates on the buds of different types of poplar containing the flavonoid aglycone, phenylcarboxylic acids and their derivatives [18,19,23].In boreal regions, outside the growing range of poplar, bees collect resin from the buds of downy birch (Betula pubescens), which are not only rich in flavonoids but also contain large amounts of sesquiterpenol esters of phenylpropenoid acids [23,24].Another source of resin are aspen (Populus tremula and P. tremuloides) buds, characterized by a high content of phenylpropenoid glycerides [23,25,26].In the boreal zone, a mixed 'birch-aspen type' of propolis is often found [23], and on the border with the mid-latitude zone, poplar markers are also found in its composition [27].In other phytogeographical zones and regions, there are local sources of resin acceptable to foraging bees, and there they produce specific types of propolis, such as green Brazilian propolis from the tropical zone [28] or Argentine Andean propolis [29].The botanical predecessor of the former is Baccharis dracunculifolia, and that of the latter is Larrea nitida.
In isolated island areas, often rich in endemic plant species, one can also expect the existence of propolis with a specific chemical composition that allows it to be considered a separate variety.This applies to diterpenoids-rich propolis from the Greek islands [30] and to Pacific propolis from Okinawa and Taiwan [31].Such isolated areas include the islands of the Canary archipelago, where beekeeping has long been practiced, but its products still remain poorly understood.In particular, there is only one report on the chemical composition of two propolis samples from the island of Gran Canaria [32].Judging by the data presented, these samples demonstrate significant qualitative differences both from propolis from the temperate zone and from its known varieties of tropical origin.This is undoubtedly due to the presence on the island of specific plant sources of resins or other types of secretions that are attractive to the bees bred on it.It would be interesting to study the chemical composition of propolis from other Canary Islands, since each of them is characterized by biogeographical differences.This work reports for the first time the chemical composition of propolis from Tenerife, the largest of the islands of the archipelago and possessing plant species endemic only to this island [33].

Composition of Volatile Components of Propolis
Determination of the volatile organic components (VOCs) of propolis was carried out using the method of solid phase microextraction combined with gas chromatography with mass spectrometric detection (HS-SPME/GC-MS).Chromatograms recorded using this method contained peaks of 162 C 1 -C 20 compounds belonging to different classes of organic substances.Table 1 shows the composition of VOCs, grouped by class of organic compounds, as well as the substances included in each group in order of increasing retention index.The largest contribution to the total ion current (TIC) of the chromatograms was made by the group of terpenoids, numbering 72 compounds.The monoterpenoid subgroup, formed by 34 individual components (11 of them were found in all three samples), accounted for 27-56% of the TIC.However, 12 monoterpenoids were recorded in only one of the three samples.An even greater difference was observed in the subgroup of sesquiterpenoids: 27 out of a total of 36 were found in only one of the samples.The second largest group of VOCs was formed by C 6 -C 17 alkanes and alkenes, the contribution of which to the TIC chromatograms varied greatly and ranged from 6% to 32%.It is interesting that even-numbered homologs predominated in terms of their contribution to the TIC.The VOCs contained 17 aliphatic carbonyl compounds, the proportion of which ranged from 7% to 20% of the TIC.The largest amounts were acetone, nonanal and heptanal.In addition, the fugitive emissions contained C 2 -C 8 aliphatic alcohols and C 1 -C 6 acids, as well as seven esters.However, the latter were found in only one of the samples (Pr-1).Of the seven aromatic compounds, toluene and p-cymene were present in the highest amounts in all three samples.

Extractive Components
Chromatograms of ether extracts of all three propolis samples were formed by peaks of 138 organic compounds, most of which contained polar groups and were recorded in the form of TMS derivatives.The identified compounds could be divided into 12 groups, as shown in Table 2.As in the case of VOCs, the largest group was formed by terpenoids, but its structure was different.It was formed by 10 sesquiterpenes, 22 diterpenes and 13 triterpenes.Sesquiterpenes were present only in small quantities (1.2-1.7%TIC) and only in extracts from two propolis samples.Without exception, all diterpenes were classified as resin acids and related C 20 compounds (totarol and neoabietal).Three compounds with retention indices of 2515, 2665 and 2596 were conditionally assigned to this group based on the presence of characteristic ions in the mass spectra and the MS pattern of ion fragmentation.A subgroup of triterpenoids was represented by tetracyclic lanosterol, dihydrolanosterol and masticadienoic acid, as well as pentacyclic compounds of the oleanane, ursane and lupane group.The largest amount (21.4% TIC) and the largest number of triterpenes were found in the extract from sample 1.
The second largest group was formed by 16 C 17 -C 33 n-alkanes and 10 C 23 -C 33 alkenes; the contribution of this group to the TIC was significant and amounted to 15-48%.The group of aliphatic acids (11-21% TIC) included 21 compounds with a number of carbon atoms from 9 (azelaic acid) to 34.Aliphatic compounds also included relatively small amounts of normal C 18 -C 34 alcohols and aldehydes, and these were not detected in one of the samples (Pr-2).
The group of aromatic compounds included four substituted alkyl and alkenyl resorcinols, five alkyl and alkenyl salicylates and nine furofuranoid lignans.If representatives of the last subgroup were present in the extracts of all three propolis samples, then resorcinol derivatives were found in two of them and substituted salicylates only in one.
The contribution of compounds not assigned to any of the 12 groups amounted to 0.1-5.5% TIC, and the share of unidentified components accounted for 3.5-10.5%TIC.

Discussion
Until now, there has been only one report on the chemical composition of Canarian (Gran Canaria) propolis [32].Although Tenerife and Gran Canaria are located close to each other, the composition of their vegetation cover is markedly different: they have many endemic species that are found only on one of them [33].Therefore, it seems interesting to compare the chemical composition of propolis originating from these islands.
The data obtained on the composition of propolis from Tenerife and Gran Canaria differ in many aspects, and in some cases, this may be explained by differences in the experimental technique.For example, the list of VOCs shown in Table 1 determined by the HS-SPME/GC-MS method includes a much larger number of compounds than those isolated from propolis by hydrodistillation, which loses the most volatile compounds.The list of extracted compounds (Table 2) does not contain sugars and related sugar acids and alcohols, while in the work [32], their share in one propolis sample accounts for approximately 56% of TIC, and in the second, approximately 10% of TIC.This is due to the fact that the weakly polar diethyl ether we used for extraction dissolves practically no polar sugars, unlike 70% aqueous ethanol.
Another difference is that the authors of the cited work note that the chemical composition of the extracts from both Gran Canaria propolis samples is very similar, while the extracts from the three Tenerife samples show significant differences.For example, in the extract from sample Pr-2, sesquiterpenes, aliphatic alcohols and carbonyl compounds were not detected even at trace levels, while extract Pr-3 did not contain phenolic compounds.These differences may be due to the fact that the Tenerife propolis samples were collected from apiaries located at different altitudinal levels (from 600 to 1400 m above sea level) with different floristic compositions.
A distinctive characteristic of the chemical composition of the studied propolis samples from Tenerife is the presence of a large number and, in two out of three samples, a high content of diterpenoids, mainly resin acids, while in the case of propolis from Gran Canaria, one compound of this class was found with a relative contribution of 0.1% TIC.The most likely plant source of resin acids in propolis from Tenerife is the secretion of an endemic pine species, Pinus canariensis.There is no information in the available literature on the chemical composition of the resinous secretions of this species, and our assumption is based on the qualitative composition of diterpene acids in propolis, characteristic of resins of all pine species [34].The lower boundary of closed pine forests in Tenerife lies at an altitude of 1000-1200 m above sea level, but individual trees are found in plantings right up to the coastline.Thus, the lowest content of resin acids in the extract of sample Pr-3, collected in the San Miguel region at an altitude of about 600 m a.s.l., can be explained by the limited availability of this source of resin for bees.
Another difference concerns the composition of phenolic compounds, represented in Tenerife propolis by alkyl derivatives of resorcinol and salicylic acid, but absent in propolis samples from Gran Canaria.A probable plant source of these lipids is Mangifera indica (Anacardiaceae), brought from Indonesia to the Canary Islands at the end of the 18th century and widely cultivated on all the islands of the archipelago.These compounds were first discovered in Indonesian propolis [35] and later in Thai propolis [36].Both publications name M. indica as the source of these compounds.Their absence in sample Pr-3, as well as in propolis samples from Gran Canaria, can be explained by the inaccessibility of mango plants to bees.On the other hand, a similar feature of the chemical composition of propolis from Gran Canaria and Tenerife is the high content of furofuranoid lignans.However, the plant precursor of these compounds, which have beneficial effects on brain function [37][38][39], including protection against Parkinson's disease, Alzheimer's disease, stroke, and other neurodegenerative diseases, remains unknown.
Thus, based on our research and earlier results [32], we can preliminarily state that Canarian propolis is significantly different in chemical composition from known types of propolis from the mid-latitude and tropical zones, and its distinctive feature is a high content of furofuran lignans. of an HP7890A gas chromatograph with a 5975C VL MSD Triple-Axis Detector (Agilent Technologies, Santa Clara, CA, USA) for 15 min.The apparatus was fitted with an HP-5ms capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) with electronic pressure control and a split/splitless injector.The latter was operated at 220 • C in splitless mode.The helium flow rate through the column was 1 mL min −1 .The initial column temperature was 40 • C and rose to 180 • C at a rate of 3 • C min −1 .The MSD detector acquisition parameters were as follows: the transfer line temperature was 280 • C, the MS source temperature was 230 • C, and the MS quad temperature was 150 • C. The EI mass spectra were obtained at 70 eV of ionization energy.Detection was performed in the full scan mode.After integrating and summing the areas of the recorded peaks, the fraction of separated components in the total ion current (TIC) was calculated.All analyses were carried out in duplicate.
In a separate experiment, the retention times of the n-alkanes used as standards in the calculation of chromatographic retention indices on the above-mentioned GC/MS equipment used were determined.From 0.5 to 10 µL of C 5 -C 17 n-alkanes were injected into a 16 mL vial for HS-SPME with a silicone membrane containing 5 mL of pure glycerol using a 10 µL microsyringe, increasing the dose for each subsequent homologue.The mixture was thoroughly mixed, and DVB/CAR/PDMS fiber was introduced into the gas phase above it for 2-3 s.The chromatogram of reference alkanes was recorded under the above conditions.

Determination of Extractive Components
After concentrating the volatile compounds on the sorption fiber for SPME, the propolis was transferred from the vial to a 50 mL conical flask, and 25 mL of diethyl ether was added and stirred on a magnetic stirrer for 30 min.The solvent was separated, and the extraction was repeated twice.The combined extracts were filtered through a paper filter, and the ether was completely removed on a rotary evaporator.Five milligrams of the viscous residue was transferred into a 2 mL vial and dissolved in 220 µL of dry pyridine, 80 µL of BSTFA was added and the resulting solution was heated for 30 min at a temperature of 60 • C.
The resulting TMS derivatives were separated on the above-mentioned GC-MS apparatus, equipped with the same HP-5ms column.The initial temperature of the column thermostat was 50 • C and increased linearly at a rate of 3 • C min −1 to 320 • C. The chromatograph injector, heated to 280 • C, operated with a division of the carrier gas flow (1:10).The helium flow rate through the column was 1 mL min −1 in constant flow mode.
Under the given conditions, a calibration mixture of C 10 -C 40 n-alkanes was separated, and the recorded retention times were used to calculate RI Exp values in the chromatograms of the extracts.

Component Identification
To identify the components, mass spectrum and chromatographic retention index (RI) were used.The mass spectrometric identification of volatiles was carried out using an automatic system for GC-MS data processing supplied by the NIST 14 library, as well as by computer search libraries containing the spectra and RI values from Adams' [40] and Tkachev's [41] collections.The RI values of TMS derivatives were compared with those in the NIST collection [42], as well as with those presented in a recently published atlas containing mass spectra and retention indices of more than 1750 organic components of various origins in the form of TMS derivatives, including bee products [43].An identification was considered reliable if the results of the computer search of the mass spectra library were confirmed by the experimental RI Exp values, i.e., if their deviation from the published database values (RI Lit ) did not exceed ±10 u.i.If the result of mass spectrometric identification was not confirmed chromatographically due to the absence of RI values in the available databases, or if the RI Exp and RI Lit values differed by more than 10 u.i., the identification was considered tentative (the names of the tentatively identified compounds in Tables 1 and 2 are followed by a question mark).