0306-4492/92$5.00+ 0.00 0 1992Pergamon Press Ltd
Camp. &o&m. Physiol. Vol. 10X, No. I, pp. 205-208, 1992 Printed in Great Britain
EFFECT OF GALLERIA MELLONELLA LARVAE PREPARATION AND HONEYBEE PRODUCTS ON CELL CULTURES N.
A. SPIRIDONOV,V. V. ARKHIPOV, A. A. NARIMANOV,S. A. SHABALINA,L. A. ZVERKOVA, E. M. SHVIRST and M. N. KONDRASHOVA Institute of Theoretical and Experimental Biophysics, Academy of Sciences, Pushchino, Moscow Region, 142292, Russia (Received 12 September 1991)
Abstract-l. The action of the extract from larvae of the great wax moth Galleria mellonellu 15. used in Russian folk medicine was studied. 2. Two active components influencing the growth and morphological differentiation of cells in vitro were
found. 3. The presence of these components in the extract was conditioned by consumption of honeybee products by Galleria mel~onella larvae. 4. Cytotoxicity of honeybee products was studied.
INTRODUCTION
and aromatic conjugated with carbohydrates tides, substances was used in the work. Samples of honey, royal jelly, beebread, propolis and dry honeycombs were obtained from beekeepers in the Moscow region. Polyfloric bee-gathered pollen was from the Latvian Republic. Honey and royal jelly were dissolved in the cultivation medium, other honeybee products were extracted with ethanol or water (1: 5, w/v) for 24 hr prior to experiments.
An extract from larvae of the great wax moth Galleria mellonella L. has been used in Russian folk medicine
to treat cardiovascular and pulmonary diseases, and as a geriatric remedy (Mukhin et al., 1991). Recently we have described the general chemical composition of the extract and some of its active components (Spiridonov et al., 1992). Pharmacological investigation of the extract revealed its stimulating influence on the oxidative metabolism of cardiac and aortic tissues (Rachkov et al., 1992), the adaptogenic, hypocoagulant and cardioprotective properties (Rachkov et al., in preparation). Substances stimulating the morphological differentiation of dissociated neurons in culture were found in the extract (Spiridonov et al., 1984). In the present work different cell cultures were used as a test system for investigation of the biological activity of the extract from G. mellonella larvae and the honeybee products consumed by the insect. The origin of the active components revealed is discussed. MATERIALS
Tests of biological activity The biological activity of G. mellonella larvae extract and honeybee products was tested on the following cell lines. Human iymphoblastoid cells Raji were grow> on RPMI-1640 and DMEM mediums (Serva). mouse mveloma x63-Ag.8.653 cells and Sp2/0-Ag.14~cells were grown-on the medium DMEM in the presence of 10% faetal calf serum (FBS) (Serva). Fibroblasts 3T3, CH3 and BHK-21 were cultured in the medium MEM (Sigma) in the presence of 10% bovine serum. The initial cell density was 180-200 thousand cells per ml in all experiments. The substances to be tested were introduced into the medium simultaneously with inoculation of cells. The influence on cell growth was judged from the final cell density, as determined in a hemocytometer after 72 hr of cultivation at 37°C. Embryonic mice liver tissue was cultured in Eagle’s medium with 10% faetal calf serum (Serva) which contained 1 g/l glucose, 2 IUjl insulin, 8 pg/l hydrocortisone and 50mg/l kanamycin sulfate. Liver tissue was cut into 0.4-0.6 mm pieces, washed on kapron sieves with cultivation medium three times, and placed in plastic Petri dishes (100 mg tissue per 100 square cm of plate). Explantates were cultured at 37°C in an atmosphere with 5% carbon dioxide. At the beginning of cultivation the level of the medium did not exceed 1 mm. Then it was raised to 34 mm by addition of new medium portions every &6 hr in the first day of cultivation. After that three-quarters of the old medium was replaced with a fresh one. Tested substances were present in all portions of the medium in the stated concentrations. Giant neurons of freshwater snail Lymnaeu stugnalis L. were isolated from ganglia and cultured in a chemically defined medium on a glass surface as described earlier (Kostenko et al., 1983). The substances to be tested were
AND METHODS
Galleria mellonella were collected in the Moscow region. The larvae were reared in laboratory conditions on their natural diet (empty honeycombs, containing beebread and the remainder of honey) and an artificial diet (Marston and Brown, 1974) which additionally containing 8% of purified beeswax and 10% of honey. Extraction of biologically active substances from the larvae was carried out with 40% ethanol as described earlier (Spiridonov et al., 1984). The extract was stored in the dark at 4°C and used during a year. The extract was concentrated on a vacuum rotary evaporator and fractioned on Sephadex LH-20, G-50 and G-10 columns (Pharmacia Fine Chemicals) eluted with 50% ethanol. Isolation of high molecular weight conjugates from the extract was performed as earlier described (Spiridonov et al., 1984, 1992). Fraction 3-G-50 which contained pep205
N. A. SPIRIDONOV et al.
206
introduced into the medium simultaneously with implantation of neurons, their influence on the morphological differentiation was judged from the percentage of neurons which formed neurites after 14 days of cultivation. RESULTS
Znfruence of extractfrom Galleria mellonella growth of cells in culture
larvae on
Extract of G. mellonella larvae, grown on the natural diet, stimulated the growth of a human lymphoblastoid Raji cell line. The data obtained in experiments with Raji cells grown in a medium with a varying serum content are presented in Fig. 1. Addition of the extract to the medium stimulated the cell growth in all series of experiments, the effect increasing with extract concentration. It was most pronounced in the medium with a low (0.3%) serum content, where the cells did not proliferate without the extract. The fact that stimulation of cell growth was also observed in the medium with a high (10%) serum content evidenced the presence in the extract of a growth stimulating factor that is absent in faetal calf serum. Similar results were obtained in experiments with a mouse x63-Ag.8.653 cell line (Fig. 2). In this case the growth stimulating effect of the extract was less pronounced and was revealed at lower concentrations. The extract did not stimulate the growth of Sp2/0, 3T3 and CH3 cell lines (data are not presented). Separation of the extract on a Sephadex LH-20 column showed that the growth stimulating activity is due to the low molecular weight fraction of the extract (fraction 3-LH-20, Fig. 3). The molecular weight of the active factor, as determined with gel chromatography on a Sephadex G-10 column. was less than 700 daltons.
d-
01--/o
5 10 20 40 80 Concentration of G.mellonella extract in the medium (pg/ml)
Fig. 2. The effect of the extract from Galleria mellonella larvae, grown on the natural diet, on the growth of mice myeloma x63-Ag.8.653 cells, cultured in DMEM medium in the presence of 3% serum.
Influence of honeybee products on growth of Raji cells
It is known that many insects are capable of accumulating biologically active substances consumed with their food. Such a possibility was suggested for the wax moth by Young (1961). To clarify the origin of the cell growth stimulating factor, we compared the activities of extracts from G. mellonellu larvae, grown on the natural and artificial diets. It was found that the extract from larvae, fed on the artificial diet, did not stimulate the growth of Raji cells (Table 1). This circumstance made us to investigate the influence of honeybee products on the growth of Raji cells. The results of these experiments are nresented in Fig. 4. It is seen that not one the
0 Concentration of Gmellonella extract in the medium (pg/ml) Fig 1. The effect of the extract from Galleria mellonella larvae, grown on the natural diet, on the final density of Raji cells, cultured in the medium RPMI-1640 with a varying serum content. Serum content in the medium: A-IO%, B-2%, CG.3%.
Volume elution (ml) Fig. 3. Gel chromatography of the extract from Galleria mellonella larvae, grown on the natural diet, on a Sephadex LH-20 column (2.6 x 40 cm). Elution was carried out with 50% ethanol. The growth stimulating activity of the fractions obtained was tested on Raji cells, cultured in the medium RPMI-1640 in the presence of 3% serum.
Effect of Galleria and Apis extracts Table I. The effect of extracts from Gderia mellonella larvae, grown on the natural and artificial diets, on the growth of Raji cells cultured in the medium RPMI-1640 with 10% serum Diet, received by the larvae Dry honeycombs Artificial diet
180
Quantity of extract in the medium (mgiml)
Final cell density, as compared with control (%)
160
0.1 1.0 0.1 1.0
121*5 l52+5 102 * 5 106k7
100
cells. Honey, royal jelly and the extracts of pollen displayed no cytotoxic action. The extracts of beebread and dry honeycombs caused a moderate inhibition of cell grows. The extracts of propolis possessed a pronounced cytotoxicity, completely suppressing the growth of the cells in concentrations of 0.05Xl.5 mg/ml. The water extract of propolis which contained mainly aromatic acids and saccharides was less toxic than the ethanol extracts which contained flavonoids. Injfuence of high molecular weight conjugates from Galleria mellonella larvae on growth of cell cultures High molecular weight conjugates, containing peptides, saccharides and aromatic substances, which stimulated the growth of neurites in culture of dissociated neurons, have been isolated from G. mellonella larvae earlier (Spiridonov et al., 1984; 1992). In this work we studied their action on different cell cultures. It was found that conjugates exerted no significant influence on the growth of the following stable cell lines: Raji, Sp2/0, 3T3, CH3 and BHK-21 (data are not shown). At the same time, the conjugates stimulated the attachment to substratum and the growth of primary mice liver tissue cultures. The period of attachment of tissue explantates to plastic substratum was 20-26 hr in the control. In the presence of the conjugates in the medium significant acceleration of attachment of explantates to substra-
207
140 120
80 60
*:A 0
5
10
10
20
40
I30
Concentration of conjugates in the medium (pg/ml) Fig. 5. The effect of high molecular weight conjugates from Galleria mellonella larvae on the growth of mice liver tissue explantates.
turn was observed. Explantates, cultured in the presence of 20 pg/ml conjugates in the medium, attached the most rapidly (in 4-6 hr). In the presence of 10 pgg/ml and 40 pg/ml conjugates in the medium the most part of the explantates attached in lo-15 hr. Introduction of the conjugates into the medium stimulated also formation of monolayer growth zones around explantates (Fig. 5). The highest growth activity exhibited explantates, cultured in the presence of 20 pg/ml conjugates. The high concentration of the conjugates (160 pg/ml) inhibited both attachment of explantates and monolayer growth zone formation. Origin oj‘ conjugated substances Extract from G. mellonella larvae, grown on an artificial diet, did not contain high molecular weight conjugates and did not stimulate neurite growth in culture of dissociated neurons. At the same time, 40% ethanol extract from dry honeycombs exerted the same stimulating action on the neurite growth,
Concentration in the medium (pg/ml) on the growth of Raji cells, cultured in DMEM medium with 10% serum. 1. Ethanol (96%) extract of pollen; 2. Water extract of pollen; 3. Royal jelly; 4. Honey; 5. Ethanol (40%) extract of dry honeycombs; 6. Ethanol (96%) extract of beebread; 7. Water extract of beebread; 8. Water extract of propolis; 9. Ethanol (96%) extract of propolis; 10. Ethanol (40%) extract of propolis. Fig. 4. The effect of honeybee
products
208
N. A.
SPIRIDONOV
(cl
I
Fig. 6. The effect of high molecular weight conjugates from Galleria mellonella larvae and 40% ethanol extract from dry honeycombs on the neurite growth in culture of dissociated neurons of Lymnaea stagnalis. A control; B 10 pg/ml conjugates in the medium; C 150 pg/ml extract of dry honeycombs in the medium.
as conjugates from G. mellonella larvae, fed on dry honeycombs did (Table 1). Hence, we conclude that substances stimulating neurite growth have exogenous origin and come to G. mellonella larvae from their natural food. DISCUSSION
The extract from Galleria mellonella larvae, used in Russian folk medicine, contains two active components, influencing the growth of cells in culture. The components revealed act selectively on cells of different types. One of them (conjugates of peptides, saccharides and aromatic substances) stimulates the growth and morphological differentiation of primary cell cultures. The other component (unidentified low molecular weight factor) stimulates the growth of anchorage-independent lymphoblastoid cells. The extract retained its growth stimulating properties during a year, which is evidence of the stability of its active components. The growth stimulating activity of the extract is conditioned by consumption of honeybee products by G. mellonella larvae. Substances stimulating the growth of neurites were found in dry honeycombs. Direct extraction of the factor stimulating the growth of lymphoblastoid cells from honeybee products gave no positive results. It is possible that the factor is a product of metabolic transformation of an inactive precursor consumed by larvae with their natural food. Another possible explanation is that the growth stimulating activity of honeybee products is masked by their toxic components. Of all the honeybee products tested, only beebread, dry honeycombs and propolis inhibited the growth of Raji cells. The cytotoxic action of propolis was most pronounced. The results of our experiments with propolis are in agreement with the data of other authors who studied the action of propolis on HeLa and KB cultures (Hladon et al., 1980; Ban et al., 1983). It is supposed that the toxicity of propolis for mammalian cells, as well as its antimicrobic
et al.
action, are due to the plant phenolic substances (Chisalberti, 1979; Ban et al., 1983). Comparison of our results with the literature data on the antimicrobial action of propolis (Chernyak, 1973; Metzner et al., 1977; Shub et al., 1981) shows that the cytotoxic action of propolis manifests itself at lower concentrations (0.0545 mg/ml) than its antimicrobial action (0.5-15 mg/ml for staphylococci, Escherichia coli, Candida and Saccharomyces). This indicates that the antimicrobial activity of propolis is not a specific one and is due to its general toxicity. Acknowledgements-We wish to thank Dr Biol. M. A. Kostenko for laboratory facilities provided and valuable directions in culturing neurons of Lymnaea stag&is.
REFERENCES Ban J., Popovic S. and Maysinger D. (1983) Cytostatic effects of propolis in vitro. Acta Pharm. Jugoslav. 33, 245-255. Chemyak N. F. (1973) On synergistic action of propolis and some antibacterial preparations. Antibiotiki 18, 259-261 (in Russian). Chisalberti E. L. (1979) Propolis: a review. The Bee World 60, 59-84. Hladon B., Bulka W., Ellnain-Wojtaszek M., Skrzypczak L., Szafarek P., Chodera A. and Kowalewski Z. (1980) In vitro studies on the cytostatic activity of propolis extracts. Arsneim.-Forsch. 30, 1847-1848. Kostenko M., Musienko V. and Smolikhina T. (1983) Ca2+ and pH affect the neurite formation in cultured mollusc isolated neurons. Brain Res. 276, 43-50. Marston N. and Brown B. (1974) Constituents in diet for Galleria mellonella. J. Econ. Entomol. 67, 497-500. Metzner J., Schneidewind E. and Friedrich E. (1977) Effect of propolis and pinocembrin on blastomyces. Pharmuzie 32, 730. Mukhin S. A., Spiridonov N. A., Rachkov A. K. and Kondrashova M. N. (1991) “Vita”--effective cardiovascular and geriatric preparation. In Fundamentalnie Nauki-Narodnomu Hoziaistvu, Nauka, Moscow. (in Russian). Rachkov A. K., Spiridonov N. A., Mukhin S. A. and Kondrashova M. N. (1991) Stimulation of oxidative metabolism in rat tissues with Galleriu mellonellu preparation. Gomeopatia i Electropunclura, Novosibirsk (in Russian). Shub T. A:, Kagramanova K. A., Voropaeva S. D., Kivman G. Ya. (1981) Effect ofnronolis on strains of Sfauhylococcus au&us resistant to antibiotics. Antibioriki 26, 2688271 (in Russian). Spiridonov N. A., Kashparova E. V. and Baskunov B. P. (1992) On chemical composition of biologically active preparation from Galleria mellonella. Comp. Biochem. Phvsiol. 102C. 198-202. Spiridonov N. .A., Kostenko M. A., Volkova S. P., Poeorelov A. G. and Kondrashova M. N. (1984) substances from I&ence of biologically active Galleria mellonella larvae on neurons of Lymnaea stagnalis in culture. Comp. Biochem. Physiol. 78C, 207-210. Spiridonov N. A., Sviridova T. A. (1985) Stable protease from GaBeriu mellonellu larvae dissolves murine zonae pellucidae. Comp. Biochem. Physiol. MA, 853-856. Young R. G. (1961) The effect of dietary beeswax and wax components on the larvae of the greater wax moth, Galleria mellonella (L.). Ann. Entomol. Sot. Amer. 54, 657659.
Camp. &o&m. Physiol. Vol. 10X, No. I, pp. 205-208, 1992 Printed in Great Britain
EFFECT OF GALLERIA MELLONELLA LARVAE PREPARATION AND HONEYBEE PRODUCTS ON CELL CULTURES N.
A. SPIRIDONOV,V. V. ARKHIPOV, A. A. NARIMANOV,S. A. SHABALINA,L. A. ZVERKOVA, E. M. SHVIRST and M. N. KONDRASHOVA Institute of Theoretical and Experimental Biophysics, Academy of Sciences, Pushchino, Moscow Region, 142292, Russia (Received 12 September 1991)
Abstract-l. The action of the extract from larvae of the great wax moth Galleria mellonellu 15. used in Russian folk medicine was studied. 2. Two active components influencing the growth and morphological differentiation of cells in vitro were
found. 3. The presence of these components in the extract was conditioned by consumption of honeybee products by Galleria mel~onella larvae. 4. Cytotoxicity of honeybee products was studied.
INTRODUCTION
and aromatic conjugated with carbohydrates tides, substances was used in the work. Samples of honey, royal jelly, beebread, propolis and dry honeycombs were obtained from beekeepers in the Moscow region. Polyfloric bee-gathered pollen was from the Latvian Republic. Honey and royal jelly were dissolved in the cultivation medium, other honeybee products were extracted with ethanol or water (1: 5, w/v) for 24 hr prior to experiments.
An extract from larvae of the great wax moth Galleria mellonella L. has been used in Russian folk medicine
to treat cardiovascular and pulmonary diseases, and as a geriatric remedy (Mukhin et al., 1991). Recently we have described the general chemical composition of the extract and some of its active components (Spiridonov et al., 1992). Pharmacological investigation of the extract revealed its stimulating influence on the oxidative metabolism of cardiac and aortic tissues (Rachkov et al., 1992), the adaptogenic, hypocoagulant and cardioprotective properties (Rachkov et al., in preparation). Substances stimulating the morphological differentiation of dissociated neurons in culture were found in the extract (Spiridonov et al., 1984). In the present work different cell cultures were used as a test system for investigation of the biological activity of the extract from G. mellonella larvae and the honeybee products consumed by the insect. The origin of the active components revealed is discussed. MATERIALS
Tests of biological activity The biological activity of G. mellonella larvae extract and honeybee products was tested on the following cell lines. Human iymphoblastoid cells Raji were grow> on RPMI-1640 and DMEM mediums (Serva). mouse mveloma x63-Ag.8.653 cells and Sp2/0-Ag.14~cells were grown-on the medium DMEM in the presence of 10% faetal calf serum (FBS) (Serva). Fibroblasts 3T3, CH3 and BHK-21 were cultured in the medium MEM (Sigma) in the presence of 10% bovine serum. The initial cell density was 180-200 thousand cells per ml in all experiments. The substances to be tested were introduced into the medium simultaneously with inoculation of cells. The influence on cell growth was judged from the final cell density, as determined in a hemocytometer after 72 hr of cultivation at 37°C. Embryonic mice liver tissue was cultured in Eagle’s medium with 10% faetal calf serum (Serva) which contained 1 g/l glucose, 2 IUjl insulin, 8 pg/l hydrocortisone and 50mg/l kanamycin sulfate. Liver tissue was cut into 0.4-0.6 mm pieces, washed on kapron sieves with cultivation medium three times, and placed in plastic Petri dishes (100 mg tissue per 100 square cm of plate). Explantates were cultured at 37°C in an atmosphere with 5% carbon dioxide. At the beginning of cultivation the level of the medium did not exceed 1 mm. Then it was raised to 34 mm by addition of new medium portions every &6 hr in the first day of cultivation. After that three-quarters of the old medium was replaced with a fresh one. Tested substances were present in all portions of the medium in the stated concentrations. Giant neurons of freshwater snail Lymnaeu stugnalis L. were isolated from ganglia and cultured in a chemically defined medium on a glass surface as described earlier (Kostenko et al., 1983). The substances to be tested were
AND METHODS
Galleria mellonella were collected in the Moscow region. The larvae were reared in laboratory conditions on their natural diet (empty honeycombs, containing beebread and the remainder of honey) and an artificial diet (Marston and Brown, 1974) which additionally containing 8% of purified beeswax and 10% of honey. Extraction of biologically active substances from the larvae was carried out with 40% ethanol as described earlier (Spiridonov et al., 1984). The extract was stored in the dark at 4°C and used during a year. The extract was concentrated on a vacuum rotary evaporator and fractioned on Sephadex LH-20, G-50 and G-10 columns (Pharmacia Fine Chemicals) eluted with 50% ethanol. Isolation of high molecular weight conjugates from the extract was performed as earlier described (Spiridonov et al., 1984, 1992). Fraction 3-G-50 which contained pep205
N. A. SPIRIDONOV et al.
206
introduced into the medium simultaneously with implantation of neurons, their influence on the morphological differentiation was judged from the percentage of neurons which formed neurites after 14 days of cultivation. RESULTS
Znfruence of extractfrom Galleria mellonella growth of cells in culture
larvae on
Extract of G. mellonella larvae, grown on the natural diet, stimulated the growth of a human lymphoblastoid Raji cell line. The data obtained in experiments with Raji cells grown in a medium with a varying serum content are presented in Fig. 1. Addition of the extract to the medium stimulated the cell growth in all series of experiments, the effect increasing with extract concentration. It was most pronounced in the medium with a low (0.3%) serum content, where the cells did not proliferate without the extract. The fact that stimulation of cell growth was also observed in the medium with a high (10%) serum content evidenced the presence in the extract of a growth stimulating factor that is absent in faetal calf serum. Similar results were obtained in experiments with a mouse x63-Ag.8.653 cell line (Fig. 2). In this case the growth stimulating effect of the extract was less pronounced and was revealed at lower concentrations. The extract did not stimulate the growth of Sp2/0, 3T3 and CH3 cell lines (data are not presented). Separation of the extract on a Sephadex LH-20 column showed that the growth stimulating activity is due to the low molecular weight fraction of the extract (fraction 3-LH-20, Fig. 3). The molecular weight of the active factor, as determined with gel chromatography on a Sephadex G-10 column. was less than 700 daltons.
d-
01--/o
5 10 20 40 80 Concentration of G.mellonella extract in the medium (pg/ml)
Fig. 2. The effect of the extract from Galleria mellonella larvae, grown on the natural diet, on the growth of mice myeloma x63-Ag.8.653 cells, cultured in DMEM medium in the presence of 3% serum.
Influence of honeybee products on growth of Raji cells
It is known that many insects are capable of accumulating biologically active substances consumed with their food. Such a possibility was suggested for the wax moth by Young (1961). To clarify the origin of the cell growth stimulating factor, we compared the activities of extracts from G. mellonellu larvae, grown on the natural and artificial diets. It was found that the extract from larvae, fed on the artificial diet, did not stimulate the growth of Raji cells (Table 1). This circumstance made us to investigate the influence of honeybee products on the growth of Raji cells. The results of these experiments are nresented in Fig. 4. It is seen that not one the
0 Concentration of Gmellonella extract in the medium (pg/ml) Fig 1. The effect of the extract from Galleria mellonella larvae, grown on the natural diet, on the final density of Raji cells, cultured in the medium RPMI-1640 with a varying serum content. Serum content in the medium: A-IO%, B-2%, CG.3%.
Volume elution (ml) Fig. 3. Gel chromatography of the extract from Galleria mellonella larvae, grown on the natural diet, on a Sephadex LH-20 column (2.6 x 40 cm). Elution was carried out with 50% ethanol. The growth stimulating activity of the fractions obtained was tested on Raji cells, cultured in the medium RPMI-1640 in the presence of 3% serum.
Effect of Galleria and Apis extracts Table I. The effect of extracts from Gderia mellonella larvae, grown on the natural and artificial diets, on the growth of Raji cells cultured in the medium RPMI-1640 with 10% serum Diet, received by the larvae Dry honeycombs Artificial diet
180
Quantity of extract in the medium (mgiml)
Final cell density, as compared with control (%)
160
0.1 1.0 0.1 1.0
121*5 l52+5 102 * 5 106k7
100
cells. Honey, royal jelly and the extracts of pollen displayed no cytotoxic action. The extracts of beebread and dry honeycombs caused a moderate inhibition of cell grows. The extracts of propolis possessed a pronounced cytotoxicity, completely suppressing the growth of the cells in concentrations of 0.05Xl.5 mg/ml. The water extract of propolis which contained mainly aromatic acids and saccharides was less toxic than the ethanol extracts which contained flavonoids. Injfuence of high molecular weight conjugates from Galleria mellonella larvae on growth of cell cultures High molecular weight conjugates, containing peptides, saccharides and aromatic substances, which stimulated the growth of neurites in culture of dissociated neurons, have been isolated from G. mellonella larvae earlier (Spiridonov et al., 1984; 1992). In this work we studied their action on different cell cultures. It was found that conjugates exerted no significant influence on the growth of the following stable cell lines: Raji, Sp2/0, 3T3, CH3 and BHK-21 (data are not shown). At the same time, the conjugates stimulated the attachment to substratum and the growth of primary mice liver tissue cultures. The period of attachment of tissue explantates to plastic substratum was 20-26 hr in the control. In the presence of the conjugates in the medium significant acceleration of attachment of explantates to substra-
207
140 120
80 60
*:A 0
5
10
10
20
40
I30
Concentration of conjugates in the medium (pg/ml) Fig. 5. The effect of high molecular weight conjugates from Galleria mellonella larvae on the growth of mice liver tissue explantates.
turn was observed. Explantates, cultured in the presence of 20 pg/ml conjugates in the medium, attached the most rapidly (in 4-6 hr). In the presence of 10 pgg/ml and 40 pg/ml conjugates in the medium the most part of the explantates attached in lo-15 hr. Introduction of the conjugates into the medium stimulated also formation of monolayer growth zones around explantates (Fig. 5). The highest growth activity exhibited explantates, cultured in the presence of 20 pg/ml conjugates. The high concentration of the conjugates (160 pg/ml) inhibited both attachment of explantates and monolayer growth zone formation. Origin oj‘ conjugated substances Extract from G. mellonella larvae, grown on an artificial diet, did not contain high molecular weight conjugates and did not stimulate neurite growth in culture of dissociated neurons. At the same time, 40% ethanol extract from dry honeycombs exerted the same stimulating action on the neurite growth,
Concentration in the medium (pg/ml) on the growth of Raji cells, cultured in DMEM medium with 10% serum. 1. Ethanol (96%) extract of pollen; 2. Water extract of pollen; 3. Royal jelly; 4. Honey; 5. Ethanol (40%) extract of dry honeycombs; 6. Ethanol (96%) extract of beebread; 7. Water extract of beebread; 8. Water extract of propolis; 9. Ethanol (96%) extract of propolis; 10. Ethanol (40%) extract of propolis. Fig. 4. The effect of honeybee
products
208
N. A.
SPIRIDONOV
(cl
I
Fig. 6. The effect of high molecular weight conjugates from Galleria mellonella larvae and 40% ethanol extract from dry honeycombs on the neurite growth in culture of dissociated neurons of Lymnaea stagnalis. A control; B 10 pg/ml conjugates in the medium; C 150 pg/ml extract of dry honeycombs in the medium.
as conjugates from G. mellonella larvae, fed on dry honeycombs did (Table 1). Hence, we conclude that substances stimulating neurite growth have exogenous origin and come to G. mellonella larvae from their natural food. DISCUSSION
The extract from Galleria mellonella larvae, used in Russian folk medicine, contains two active components, influencing the growth of cells in culture. The components revealed act selectively on cells of different types. One of them (conjugates of peptides, saccharides and aromatic substances) stimulates the growth and morphological differentiation of primary cell cultures. The other component (unidentified low molecular weight factor) stimulates the growth of anchorage-independent lymphoblastoid cells. The extract retained its growth stimulating properties during a year, which is evidence of the stability of its active components. The growth stimulating activity of the extract is conditioned by consumption of honeybee products by G. mellonella larvae. Substances stimulating the growth of neurites were found in dry honeycombs. Direct extraction of the factor stimulating the growth of lymphoblastoid cells from honeybee products gave no positive results. It is possible that the factor is a product of metabolic transformation of an inactive precursor consumed by larvae with their natural food. Another possible explanation is that the growth stimulating activity of honeybee products is masked by their toxic components. Of all the honeybee products tested, only beebread, dry honeycombs and propolis inhibited the growth of Raji cells. The cytotoxic action of propolis was most pronounced. The results of our experiments with propolis are in agreement with the data of other authors who studied the action of propolis on HeLa and KB cultures (Hladon et al., 1980; Ban et al., 1983). It is supposed that the toxicity of propolis for mammalian cells, as well as its antimicrobic
et al.
action, are due to the plant phenolic substances (Chisalberti, 1979; Ban et al., 1983). Comparison of our results with the literature data on the antimicrobial action of propolis (Chernyak, 1973; Metzner et al., 1977; Shub et al., 1981) shows that the cytotoxic action of propolis manifests itself at lower concentrations (0.0545 mg/ml) than its antimicrobial action (0.5-15 mg/ml for staphylococci, Escherichia coli, Candida and Saccharomyces). This indicates that the antimicrobial activity of propolis is not a specific one and is due to its general toxicity. Acknowledgements-We wish to thank Dr Biol. M. A. Kostenko for laboratory facilities provided and valuable directions in culturing neurons of Lymnaea stag&is.
REFERENCES Ban J., Popovic S. and Maysinger D. (1983) Cytostatic effects of propolis in vitro. Acta Pharm. Jugoslav. 33, 245-255. Chemyak N. F. (1973) On synergistic action of propolis and some antibacterial preparations. Antibiotiki 18, 259-261 (in Russian). Chisalberti E. L. (1979) Propolis: a review. The Bee World 60, 59-84. Hladon B., Bulka W., Ellnain-Wojtaszek M., Skrzypczak L., Szafarek P., Chodera A. and Kowalewski Z. (1980) In vitro studies on the cytostatic activity of propolis extracts. Arsneim.-Forsch. 30, 1847-1848. Kostenko M., Musienko V. and Smolikhina T. (1983) Ca2+ and pH affect the neurite formation in cultured mollusc isolated neurons. Brain Res. 276, 43-50. Marston N. and Brown B. (1974) Constituents in diet for Galleria mellonella. J. Econ. Entomol. 67, 497-500. Metzner J., Schneidewind E. and Friedrich E. (1977) Effect of propolis and pinocembrin on blastomyces. Pharmuzie 32, 730. Mukhin S. A., Spiridonov N. A., Rachkov A. K. and Kondrashova M. N. (1991) “Vita”--effective cardiovascular and geriatric preparation. In Fundamentalnie Nauki-Narodnomu Hoziaistvu, Nauka, Moscow. (in Russian). Rachkov A. K., Spiridonov N. A., Mukhin S. A. and Kondrashova M. N. (1991) Stimulation of oxidative metabolism in rat tissues with Galleriu mellonellu preparation. Gomeopatia i Electropunclura, Novosibirsk (in Russian). Shub T. A:, Kagramanova K. A., Voropaeva S. D., Kivman G. Ya. (1981) Effect ofnronolis on strains of Sfauhylococcus au&us resistant to antibiotics. Antibioriki 26, 2688271 (in Russian). Spiridonov N. A., Kashparova E. V. and Baskunov B. P. (1992) On chemical composition of biologically active preparation from Galleria mellonella. Comp. Biochem. Phvsiol. 102C. 198-202. Spiridonov N. .A., Kostenko M. A., Volkova S. P., Poeorelov A. G. and Kondrashova M. N. (1984) substances from I&ence of biologically active Galleria mellonella larvae on neurons of Lymnaea stagnalis in culture. Comp. Biochem. Physiol. 78C, 207-210. Spiridonov N. A., Sviridova T. A. (1985) Stable protease from GaBeriu mellonellu larvae dissolves murine zonae pellucidae. Comp. Biochem. Physiol. MA, 853-856. Young R. G. (1961) The effect of dietary beeswax and wax components on the larvae of the greater wax moth, Galleria mellonella (L.). Ann. Entomol. Sot. Amer. 54, 657659.
