FABAD J. Pharm. Sci., 31, 192-197, 2006 RESEARCH ARTICLE
Microscopic Analysis of Propolis Samples Collected from East Anatolia (Kemaliye-Erzincan)
Summary
Microscopic Analysis of Propolis Samples Collected from East Anatolia (Kemaliye-Erzincan)
The plant choices of honey bees for collecting propolis were investigated by pollen analysis. A total of 30 different propolis samples were collected in the Kemaliye-Erzincan region and examined under microscope to determine pollen contents, which reflect the flora of the region.
By determining pollen intensity of propolis pollen slides, the botanical origins of propolis samples were determined. Thirty- two different plant families were identified by pollen analysis of the 30 propolis samples. Species belonging to Apiaceae, Asteraceae, Campanulaceae, Fabaceae, Fagaceae, Lamiaceae, Liliaceae, Pinaceae, Rosaceae, Salicaceae, Rhamnaceae, and Scrophulariaceae families were primarily found as the botanical origins of propolis.
The plant choices of honey bees for collecting propolis according to season were also investigated by using Shannon Index. It was observed that Corylus spp., Onobrychis spp., Quercus spp., Teucrium spp., and Linaria spp. were the most preferred in spring and Eryngium spp., Carduus spp., Corylus spp., Medicago spp., Quercus spp., Nepeta spp., Teucrium spp., Zizyphus spp., Linaria spp., and Veronica spp. were the most preferred in fall.
Key Words: Propolis, pollen, pollen analysis, plant family, Kemaliye-Erzincan
Received : 03.03.2008 Revised : 27.03.2008 Accepted : 29.05.2008
*° Hacettepe University, Faculty of Science, Department of Biology, 06532 Ankara, Turkey Correspondng author e-mail: gencay@hacettepe.edu.tr
INTRODUCTION
Propolis (bee glue) is a sticky dark-colored material that honey bees collect from living plants, mix with wax and use in construction and adaptation of their nests [1]. Bees use propolis in a thin layer on the internal walls of their hive or other cavity they inhabit.
It is used to block holes and cracks, to repair combs,
to strengthen the thin borders of the comb, and for making the entrance of the hive weather-tight or easier to defend. Propolis is also used as an
“embalming substance” to cover the carcass of a hive invader that the bees have killed but cannot transport out of the hive. Honey bees collect the resinous exu-
for its pharmaceutical properties and it is now known as a natural medicine for its anti-bacterial, anti-fungal, anti-tumoral, anti-oxidative, immunomodulator and many other beneficial properties [3].
The presence of pollen in propolis has been reported by Junkunz [4], who found Lupinus, Robinia and Onobrychis sativa pollen grains in the insoluble por- tion of propolis. Vanhaelen and Vanhaelen-Fastre [5]
presented microphotographs of propolis originating from different countries in the world, with pollen grains clearly visible in some.
Pollen analysis of propolis was studied extensively in 1979 by d’Albore [6]. In the following years, pollen analysis of Polish propolis was made by Warakomska and Maciejewicz [7]. In Turkey, the first scientific research was published by Sorkun and Bozcuk [8].
Subsequently, many studies related to propolis were published in Turkey, among them those by Sorkun et al. [9-10], Keskin et al. [11], Kolankaya et al. [12], Kartal et al. [13], and Erdem and Ölmez [14].
The aim of the present work was to determine the plants that form the botanical origin of propolis in East Anatolia by characterizing the pollen. Although many studies have been conducted in Anatolia about the microscopic and chemical composition of propolis, this work is one of the first detailed studies on the pollen content of Turkish propolis.
MATERIALS and METHODS
In 2003, a microscopic study was made of 30 propolis samples collected from 10 bee hives in Kemaliye- Erzincan. During the study period, samples were gathered from each research hive on two occasions in different periods, and a total of 30 propolis samples were collected. The plant species chosen by the bees in this region were determined using the Shannon Index [15].
Ni= pollen amount belongs to i plant (i=related plant species)
N= total amount of investigated plant taxa
Collecting of propolis samples from hives
Some of the samples were collected in May from the edges of frames and bottom board of the hive by scraping with a spatula, and these are described as the spring samples. Other samples were collected from traps put into hives in May and left until Sep- tember, described as fall trap samples. The fall season propolis samples were collected using the same meth- od as with spring samples (from the edges of frames and bottom board of the hive by scraping with a spatula). Thus, from each research hive (total 10 hives), three samples were collected (one from edges of the frames and deep board in spring, one using the same method in fall and one scraped from the trap in September. Thus, during the study period, one spring (from the edges of frames and bottom board of the hive by scraping with a spatula) and two fall samples were collected from each bee farm.
The sites of the bee farms from which propolis samples were collected and symbols are given in Table 1.
Location of the Kemaliye-Erzincan region where the samples were collected is given in Figure 1.
Preparation of propolis samples for microscopic analysis
The materials were prepared for examination under the microscope according to the method of Warako- mska and Maciejewicz [7].
Samples were ground to a powder. After grinding, powder was mixed with ethanol-ether-acetone (1:1:1) and shaken. Mixture was filtered through a strainer with 0.3 mm holes. The suspension was then centri- fuged at 3500-4000 rpm for 20 min. After centrifuga- tion, the supernatant was poured and from the resid- ual sediment two slides were prepared for each sample using glycerin gelatin. The two slides prepared for each sample were investigated simultaneously and the pollen count was made.
FABAD J. Pharm. Sci., 31, 192-197, 2006
RESULTS
At the end of the microscopic analysis, both the variety of plant taxa and pollen intensity of samples were defined. When we investigated the microscopic analysis results for the variety of plant taxa as the botanical origin of propolis, from 30 propolis samples collected from 10 bee farms belonging to spring (10
samples) and fall (20 samples) periods, plant taxa of 32 different plant families were determined.
When we evaluated the results for the pollen intensi- ties of plant taxa in the pollen slides, the pollen of Asteraceae, Fabaceae, Fagaceae, Lamiaceae, Liliaceae, Salicaceae and Scrophulariaceae families were found in high amounts in the researched samples. Pollen of Salix spp. belonging to Salicaceae family was found in all samples in various amounts. The pollen inten- sities of samples are shown in Table 2. Some micro- photographs of these pollens can be seen in Figure 2.
Shannon Index was used to determine any effect on results of the season in which propolis was collected.
The plant choices of honey bees according to the seasons are given in Figure 3, as calculated by Shannon Index. Using Shannon Diversity Index, it was observed that propolis was primarily collected from Corylus spp., Onobrychis spp., Quercus spp., Teucrium spp., and Linaria spp. plants in spring and from Eryngium spp., Carduus spp., Corylus spp., Medicago spp., Quercus spp., Teucrium spp., Nepeta spp., Linaria spp., and Veronica spp. plants in fall. There wer e no significant differences in plant choices of honey bees for collecting propolis according to the season. According to the index results, Linaria spp. was the most preferred in spring, with a percentage of 2.178%. After Linaria spp., Teucrium spp. (2.15%), Corylus spp. (2.15%), Onobychis spp. (2.06%), Quercus spp (2.04%) were preferred at similar rates. A Rosaceae species (1.99%) and Nepeta spp. (1.93%) also demonstrated similar rates. In the fall, Teucrium spp. was the most preferred, with a percentage of 2.78%, followed by Teucrium spp., Veronica spp. (2.76%), Linaria spp. (2.73%), Quer- cus spp. (2.70%), Eryngium spp. (2.66%), Nepeta spp.
(2.64%), Zizyphus spp. (2.57%), and Carduus spp.
(2.56%). Salix spp. was found most often in all samples, with a percentage of 1.76% in spring and 2.34% in fall.
DISCUSSION
In this study, we investigated the botanical origin of Kemaliye-Erzincan propolis, examined the pollen intensity of plant taxa in propolis, and determined Table 1. The sites of bee farms from which propolis
samples were collected and symbols
Sample name Site Symbol
First spring sample Yeflilyurt village-Köybafl› 1/1 First fall sample Yeflilyurt village-Köybafl› 1/2 First fall trap sample Yeflilyurt village-Köybafl› 1/3
Second spring sample Dutbeli 2/1
Second fall sample Dutbeli 2/2
Second fall trap sample Dutbeli 2/3
Third spring sample Çit village-Pazarard› 3/1 Third fall sample Çit village-Pazarard› 3/2 Third fall trap sample Çit village-Pazarard› 3/3 Fourth spring sample Kekik P›nar›-Köybafl› 4/1 Fourth fall sample Kekik P›nar›-Köybafl› 4/2 Fourth fall trap sample Kekik P›nar›-Köybafl› 4/3 Fifth spring sample Kekik P›nar›- Gözlük 5/1
Fifth fall sample Kekik P›nar›- Gözlük 5/2
Fifth fall trap sample Kekik P›nar›- Gözlük 5/3 Sixth spring sample S›rakonak village- Avukat›n Çeflmesi 6/1 Sixth fall sample S›rakonak village- Avukat›n Çeflmesi 6/2 Sixth fall trap sample S›rakonak village- Avukat›n Çeflmesi 6/3
Seventh spring sample Seyit Ali quarter 7/1
Seventh fall sample Seyit Ali quarter 7/2
Seventh fall trap sample Seyit Ali quarter 7/3
Eighth spring sample Ariki quarter 8/1
Eighth fall sample Ariki quarter 8/2
Eighth fall trap sample Ariki quarter 8/3
Ninth spring sample Apça¤a village-Poser 9/1
Ninth fall sample Apça¤a village-Poser 9/2
Ninth fall trap sample Apça¤a village-Poser 9/3 Tenth spring sample Ergü village-Yukar› Ba¤lar 10/1 Tenth fall sample Ergü village -Yukar› Ba¤lar 10/2 Tenth fall trap sample Ergü village -Yukar› Ba¤lar 10/3
Figure 1. Location of the Kemaliye-Erzincan region in Turkey.
Apiaceae - R R R M R R R M R R R R R R R R R R R R R R R R R R R R R
Asteraceae M M M R S S S M S M M S R M M M R R R M S R R R M R M M R M
Betulaceae R R R R R R R R R R - R R R R R R R R R R R R R R R R M R R
Boraginaceae M R R - - - - - - - - R - - - R - R R R R - - - R - - R - -
Brassicaceae - - - - - - - - - - - - R R R R R - R - R - - - - - - R - -
Campanulaceae M R M R - - R - R - - - R R R R R R R R - R R R R R R R -
Caprifoliaceae - - - - - - - - - - R - - - - - - - - - - - - - - - - - - -
Caryophyllaceae - - R R R R - R R R R - R R R - R - - R R R R R R R - - - -
Chenopodiaceae - R - - - - - - - R R R - R - R - R R - R R R R - - - - M R
Cistaceae - - - - - - - - - - - - - - - - - - R - - - - - - - - - - R
Cupressaceae - - - - - - R R R - R R R R R R R R - R R R R R R - R R R -
Cyperaceae - - - - - - - - - - - - - - - - - - R R - - - - - - - - - -
Dipsacaceae - - - - - - - - - R - - - - - - R - -R R - R - - - R R - - -
Eleagnaceae - - - - - - - - - - - - - R - - - - - - - - - - - - - - - -
Ericaceae - - - - - - - R R - - - - R - - - - - - - - - R R - - R R -
Euphorbiaceae - - - - - - - R - - - R - - - - - - - - - - - - - - - - - -
Fabaceae S M S M R M M S M S S M M M M M M M M M M R M M M M M M M M
Fagaceae M M M M R R R R R R R R R R R - R R R R R R R R R R R R R
Gramineae - - - - - - - - - - - - R R R R - R R R R R R R R R R R - -
Junglandaceae - - - - - - - - - R R R - - R - - - R R R R R - R - R - - R
Lamiaceae M M R R M M M M M M M M M M M R M M M M R M R M M M R M R M
Liliaceae - R R R - - R - R R - - - R R R R R R R R R R R R R R R M R
Oleaceae - - - - - - - - R R - R - R - R R - R R - - - R M - R - R -
Pinaceae - - - R - - - R R - - R - R R R R M R R R R R R M R R - R
Plantaginaceae - - - - - - - - - - - R - - - - - - - - - - M M R - R R - -
Polygonaceae - R - - - - - - - - - - - - - - - R R R R R R R - R M - R
Resedaceae - - - R - - - - - - - - - - - - - - - - - - - - - - - - - -
Rhamnaceae - R R R - R R R R R R - R R R R R M M R R S R M R M R R M
Rosaceae R R R R - - - R R R R R R R R M M M M M R M R R R R R R M R
Rubiaceae - - - - - - - - - R R - R R M R R R R R - R - R - R - R R -
Salicaceae M S M M R R R R M S M S D S S S S M S S M S M M M S M M S M
Scrophulariaceae R M M S S S S M M M M M R R R M M M M M M M R R M M S M R M
Unidentified - - - - - - - - - - - - - - - - - - R R R M M M R R R R R R
A) Asteraceae B) Teucrium spp.
C) Liliaceae D) a) Asteraceae b) Salix spp.
Figure 2. Microphotographs of some identified pollens from propolis pollen slides from:
A) second bee farm propolis samples (X40) B) second bee farm propolis samples (X40) C) second bee farm propolis samples (X40) D) fifth bee farm propolis samples (X40)
FABAD J. Pharm. Sci., 31, 192-197, 2006
Figure 3. Seasonal plant choices of honey bees for collecting propolis.
Figure 3 (continue). Seasonal plant choices of honey bees for collecting propolis.
the genera preferred by honey bees for collecting propolis according to spring and fall seasons.
When we evaluated the microscopic analysis results
region. When we compare the plant species variety of the region with the microscopic results of propolis, we can easily see the harmony.
species were found more intensive. In 30 propolis samples, Asteraceae, Fabaceae, Lamiaceae, Salicaceae and Scrophulariaceae species were found in higher amounts than the other species. The pollens of these species were observed in all 30 propolis samples in dominant, secondary and minor amounts. As shown in Table 2, the intensity of pollen in the fall sample slides was much greater than in the spring samples.
According to the Shannon Index, there were no sig- nificant differences in the plant species preferred by honey bees for collecting propolis according to the season. When the results of the index were examined, only some Asteraceae species were determined to be preferred more in fall samples than in the spring (Figure 3). Thus, it can be suggested that honey bees collect propolis from similar plants in both spring and fall.
In d’Albore’s study [6], 56 samples from different regions were investigated and in each sample different plant species were identified. With respect to pollen analysis, samples from Italy were the most similar to our propolis samples. In these samples, mostly Trifo- lium pratense, Onobrychis spp., Erica arborea, Olea spp., Vitis spp, Quercus spp, Hedysarum spp. pollens were found in ratios similar to our results. In one of the samples collected from Switzerland, they found Salix spp. pollen in dominant ratios, which were also found to be dominant in the Kemaliye samples. In another study on microscopic analysis of Polish pro- polis by Warakomska and Maciejewicz [7], they found Cruciferae pollen and Salix spp. pollen, which was found in the Kemaliye-Erzincan samples.
In future researches, we aim to increase the investi- gations regarding pollen analysis of Turkish propolis from different regions to facilitate making a chemical standard for propolis in Turkey and to increase the uses of propolis in this country.
ACKNOWLEDGEMENTS
The authors thank the bee farmers of Kemaliye and Assoc. Prof. Dr. A. Murat Aytekin for statistical anal-
Project No: TOGTAG 3131.
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