ANTIBACTERIAL ACTIVITY OF PROPOLIS AS AFFECTED BY THE CONDITIONS OF ITS COLLECTION

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1 PSZCZELNICZE ZESZYTY NAUKOWE Rok }.XXV 1991 ANTIBACTERIAL ACTIVITY OF PROPOLIS AS AFFECTED BY THE CONDITIONS OF ITS COLLECTION H e l e n a R y b a k - C h m i e l e w s k a, J a n i n a M u s z y ń s Zofia Konopacka Apiculture Section, Institute of Pomology and Floriculture Puławy, Poland k a, Summary A total of 129 propolis sampies were collected over the years ,. 1982, 1983 and from the apiaries of the Apiculture Section, Inst. of Pomology and Floriculture in Pulawy. The collected propolis showed substantial variation from sample to sarnple both in the eontent of contaminants and in antibacterial act ivity. In the majority of cases high activ ity was associated with high yields of the produet and with the site of the apiary (decidou) trees including poplars and birches), The activity of propolis was not directly related tocontamination rate andcotony strength. INTRODUCTION An increased demand for propolis makes it necessary to deterrnine as comprehensively and accurately as possible the factors that promote the collection of high amounts of the contamination-free and active produet. The aim of the study was to deterrnine the relationship between the antibacterial activity of propolis and the conditions in which it arises. The study was a Iollow-up of earlier investigations (Muszyńska, Konopacka, Rybak 1983) and are jointly meant to improve the methods for stimulating propolis production. MATERIAL AND METHODS The investigations were conducted in the years and in the year 1982, 1983 and 1985 in the apiaries of the Apiculture Section in Puławy. In the years a relationship was investigated between propolis aetivity and the timing of its production and between propolis 47

2 activity and the strength of the colonies of ongm. In the remammg years a close attention was paid to the comparison of propolis sampies coming from apiaries located at different natural environments and to the relationship between propolis activity and its contamination rate. In the years propolis was yielded by colonies described in detail (Muszyńska, Konopacka, Rybak 1983). Later on, detailed descriptions of individual colonies were discontinued to allow a simultane- GUS study of a greater number of propolis - producing apiaries. Those apiaries were located at different sites: woodless farmland, Vistula - si de terrain bordering on farmland, Vistula - side mixed groves, riverside parkiand and meadow groves, spruce forest. In the present study the propolis was collected oniy from the escape boards made of perforated zinc as described earlier (Muszyńska, Konopacka, Rybak 1983). The boards we re fitted in the hives after the bars were removed and were allowed to stay there for at least a month. Three collection periods were recognized each year: from 24th April to 21th June (spring), from 7th June to 10th August (summer) and from ] 1th August to 1st October (autumn). Board a piacement date in a given season and in a given apiary was determined both by climatic and nectar flow patterns affecting plant growth and bee coiony strength and by organizational considerations. 'The boeards were fitted on different dates in diffe-ent years but always within the time limits as given above. In order to obtain more unified results, the amount of propolis deposited on the board by a single coiony over one study period was converted to 10 days collections. The propolis was analysed for contamination rate and antibacterial activity. The total amount of propolis collected over a given study period from a colony was subjected to analysis. To measure the contamination rate all propolis in a sample was dissolved in 96% ethanol to make a 10% propolis solution. The solution was kept in a tightly stoppered flask for 4 days and the liquid was stirred every day. Subsequently, the flask content was chilled to +4 C and passed through soft quantitative filter paper. The sediment was washed with chloroform to measure its wax content. Since the content of impurities was known for each sample the sampies could be accurately compared for their antibacterial properties. The 10% solutions of propolis fractions in 96% ethanol we re taken for antibacterial studies. The antibacterial activity of propolis was tested microbiologically using the standard strain of Bacillus subtilis (Rybak-Chmielewska, Szczęsna 1990). The concentration of propolis in the medium that resulted in the complete kill of the bacterial culture was taken as an index. The range of concentrations was tentatively put at ug/cm". Propolis was assumed to have a high activity when it killed all bacteria in a culture at flg/cm 3, medium - active propolis killed bacteria at flg/cm 3 and low-active propolis required concentrations of more than 600 ug/cm" to kill bacteria. 48

3 RESULTS AND DISCUSSION Description of the propolis sampies. The percentag of impurities was deterrnined in 180 propolis samples collected over the study period (Table 1). Table Distr ibution of investigated propolis samples over contamination ranges Udział badanych próbek propolisu w określonych przedziałach zanieczyszczeń l Trait Cecha together ogółem Total contamination Ievel (Ofa) Poziom zanieczyszczeń ogólnych w procentach 40 and do , , , ,1-40 more No. of samples Liczba próbek Per cent samples 7,8 19,4 20,0 24,4 13,4 9,4 5,6 100 Procent próbek The high total eontent of impurities exceeding /0 in more than a half of the samples arises from treating wax as a constant element of propolis contamination. The eontent of wax alone in the studied samples was from several to mo'e than 10 percent and frequently reached 35%. The samples varied for their size which was due to unequal lengths of collection periods as explained in the material and methods section and also to colcny-to-colony differences in.propolis production capacity. Some of the samples were too small to be analysed for antibacterial activity. The characteristics was measured in 129 propolis samples. Ca. 70% of the samples were found to show medium-strong activity (Table 2). High and low-activity propolis accounted for less than 20% of a11samples. Antibacterial activity of propolis vs. year and season of collection. The averagę activity of propolis collected from the same site accomodating colonies originating from queens of the same bread differed over the years. Likewise, within the same apiary propolis collected in the same year and season but from different colonies may show different activity (Table 3). The substantial differences in propolis activity suggest that there are celony-to-colony differences in the way the same plant species are utilized for ipropolis production (Ghisalberti ). We were unable to deterrnine unequivocally which yielded propolis of especially high activity. It is noteworthy, however, that samples wit h the highest activity level came from locations where deciduous teres were abundant: Vistula-side meadow groves and parklands, 4 - Pszczelnicze Zeszyty Naukowe XXXV 49

4 Table 2 Distribution ofinvcstigated propolis sarnplos over ant.ibacte ri al activity ranges Udział badanych próbek propolisu w określonych przedziałlach aktywności antybakterynej Antibacterial propol.is activity (ft.g/cm 3 of medium) Antybakteryj na aktywność propolisu (f.1g/cm3 Trait pożywki) Together Cecha over ponad 600 Razem No. of samples Liczba próbek - D/O sampies 13,2 20,2 28,7 20,1 17,8 100 % próbek Table 3 Antibacterial activity of propolis (f.1g/cm 3 0f medium) as affceted by season and year of harvest Antybakteryjna aktywność propolisu (f.1g/cm3poży'wki) w zależności od pory sezonu i roku zbioru 1980 year 1981 year 1980 mk 1981 rok Apiary colcny colony sum- sumno. spr ing autumn no. spring Pasieka mer mer autumn Nr wiosna jesień Nr wiosna lato jesień lato rozdz. rozdz Stationary * Stacjonarna Itinerant * Wędrowna *' - au year the same area; stałe stanowisko pasieki *' - in each season different area; w każdym sezonie inne stanowisko Vistula-side deciduous woodland, orchards and mixed groves wit h a high percentage ofpoplars and birches. The results support earlier opinions on the occurrence of resins from birches and poplars in propolis 50

5 and their corrtribution to the antibacterialproperties of the produet (d'albore 1979, Konig 1985, Warakomska 1985, Szalai, Szalay 1987). It is also of interest that propolis produced by the same bee colony showed little variation in activity over the entire producing season (Table 3) which may be explained by worker bees' consistent interest in the same plant species. There are departures from the above regularity (Table 3). AntihacteriaI activity of propolis vs. colony strength. The comparison of propolis activity and the strength of the producer colonies (Table 4) showed that both strong and weak colonies can produce propolis of either high or low activity. Thus there is no relationship between the two factors. Antihacterial activity of propolis vs. rate of its contamination. There is no relationship between the antibacterial properties of contamination- -free propolis solutions and the percentages of impurities in the crude material from which the solutions have been prepared (Table 5). Altho- Antabaetertal activity of propolis VS. bee colcny strerigth Antybakteryjna aktywność propolisu a siła rodzm.pszczelich Table 4 Activity Aktywność Number of colonies Liczba rodzin Estirnated num ber of wo'rker bees in a celony Szacunkowa liczba pszczół robotnic w rodzinach more, więcej ( ) ( ) (lo ) ( ) ( ) Table :I Antibacterial ad ivit y of propolis (,ug/om3 of medium) ViS. percentagę of i:m,purities in investigated samples Antybakteryjna aktywność propolisu (,ug/cm3 pożywki),a udział zanieczyszczeń w badanych próbkach Activity Aktywność No. of colotlliesr Liczba rodzin mechanical mechaniczne Irnpurities (l/o) Z_a_n_i_ec_z_y_S_z_czen_.ia_w_p_r_oc_e_n_ta_c_h _ wax woskowe more, więcej ,6 (3,1~13,5) 6,3 (1,5-15,4) 6,2 (2,~17,5) 7,6 (1,7-13,9) 9,5 (4,3-17,0) 15,9 (7,9-28,5) 16,8 (6,1-39,6) 17,8 (3,7-35,2) 17,0 (6,2-31,7) 17,8 (9,1-29,2) fo 51

6 ugh the average percentage of impurities rises slightly with decreasing propolis activity to each level of activity corresponds a similar range between the extreme values. Antibacterial activity of propolis vs. propolis producingpotential of the colonies. Among the high-activity sampies those from good producers i.e. colonies showing an output of more than 2 g over 10 days eonstitute a majority. However, it should be stressed that highly active propolis may, though less irequently, come from colonieś that are poor propolis producers (Table 6). Table 6 Antibacter+al activity of propolis vs. productivity of bee colonies Antyba!kteryjna aktywność propolisu a produkcyjność rodzin PlSiilCzelich Average amount of propolis Percenbage of colonies Activity NO.of produced by a celony producing more than Aktywność colonies (an a 10 day basis) 2g!10 days (u.gjcm3 Liczba Średnia ilość propolisu OJo-owyudział rodzin medium) rodzin wg wytworzonego przez produkujących więcej rodz. (10 dni) niż 2g!10 dni ,597 (0,733-4,839) 71,4 200 u 2,983 (0,2.17-4,839) 54, ,988 (0,012-5,634) 47, ,364 (0,2.17-3,578) 25,0 more, więcej ,480 (0,490-3,438) 28,6 CONCLUSIONS An average activity of propolis collected from a single site and from colonies of the same breed varies from year to year. The colonies of the same breed and from the same locations may yield propolis that differs in activity. Locations where deciduous species prevail especially those of poplars and birches seem to be especially suited to the production of highly active propolis. The activity of propolis produced by a single colony as a rul e remains stable over the entire producing season (spring, summer and autumn). In the majority of studied cases high activity of propolis was associated with high yield of this product. Propolis activity is in no direct relationship to the eontent of impurities nor does it depend on the strength of the producer colony. REFERENCES d'a l b o r e, R. (1979) L'origine geographique de la propolis. Apidalagie, 10(3): G h i s a l b e r t i, E. L. (1979) 'Propolis: A review. Bee Warld, 60(2):59-84 K a fi i g, B. (1985) Plant sources of propolis. Bee Warld, 66(4):

7 Muszyńska, J.,. Konopacka, Z., Rybak, H. (1983) Badania nad propolisem. I. Próba określenia warunków sprzyjających pozyskiwaniu propolisu. Pszczelno Zesz. nauk. 27:59-70 R y b a k-c h m i e I e w s 'k a, H., S z c z ę s n a, T. (1991) Wpływ różnych rozpuszczalników na aktywność biologiczną propolisu. Pszczelno Zesz. nauk. 35:59-69 S z a l a i, Z." S z a l a y, L. (1987) Examinatlon of polar species (Populus sp. L.) regarding propolis produetlon. XXXI International Congress of Apiculture, programme and abstracts of papers, August 19-25, Warsaw, Poland :242 War a k o m s k a, Z. (1985) Microscopic analysis of propolis. International symposium on ajpitherapy May, Kraków, Poland ANTYBAKTERYJNE WŁASNOSCI PROPOLISU A WARUNKI POZYSKIWANIA TEGO SUROWCA Helena Rybak-Chmielewska, J aninam u s zy ń ska, Zofia Konopacka Streszczenie Scharakteryzowano 129 prób propolisu zebranego w latach '1 oraz 1982, 1983 i 1985 w pasiekach Oddziału Pszczelnictwa ISK w Puławach, stosując powałki z kraty odgrodowej. Zbiór propolisu prowadzono w trzech okresach sezonu: (wiosna) propolis gromadzony w okresie od 1.05 do 15.06, (latem) w okresie od do i (jesienią) w okresie od 1.09 do 15.10). Jedną próbę stanowił propolis uzyskany od jednej (szczegółowo 'opisanej) rodziny pszczelej w danym okresie sezonu. Każdą próbę charakteryzowano określając: ogólną ilość uzyskanego surowca, zawartość zanieczyszczeń mechanicznych i woskowych (w drodze estrakcji 96% etanolem i chloroformem) oraz stopień aktywności anty bakteryjnej. Aktywność antybakteryjną oznaczano testem na przeżywalność Bacillus subtilis w statycznych hodowlach płynnych z dodatkiem 100/0 etanolowego (etanol 96%) roztworu propolisu w stężeniach od f-tg/cm3 pożywki. Przeprowadzone badania wykazały że: Srednia aktywność propolisu uzyskanego na danym stanowisku w rodzinach tej samej rasy bywa w kolejnych latach różna. W jednym roku i tym samym okresie sezonu rodziny tej samej rasy i z tego samego stanowiska dostarczały propolis o różnej aktywności. Najbardz iej aktywny pro.polis pochodził jednak ze.stanowisk zasobnych 'W drzewa liściaste. wśród których znaczny udział miały topole i brzozy, co może sugerować, iż takie stanowiska są szczególnie przydatne do intensywnej produkcji tego surowca. Aktywność propolisu wytworzonego przez określoną rodzinę pszczelą przeważnie utrzymywała się w ciągu całego sezonu produkcyjnego (wiosna, lato, jesień) na zbliżonym poziomie. Wysoka aktywność propolisu w większości badanych przypadków była związana z wysokim zbiorem tego surowca. Aktywność propolisu nie pozostawała w prostej zależności od stopnia jego zanieczyszczenia i nie zależała od siły rodziny, z której go pozyskano, 53