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Antibacterial, antifungal, antiamoebic, antiinflammatory and antipyretic studies on propolis bee products
Journal of Ethn~phurmac~logy,
35 ( 199
I)
7-l
R-82
Elsevier Scientific Publishers Ireland Ltd.
Antibacterial, antifungal, antiamoebic, antiinflammatory antipyretic studies on propolis bee products
and
Jan W. Dobrowolski”, S.B. Vohorab, Kalpana Sharmab, Shaukat A. Shahb, S.A.H. Naqvib and P.C. Dandiyab UInsfitute of Management
and Protection
~icro~~~~~gy,
of Environment,
~~mdard
University,
Krakow P.O.
{Poland)
~~rndard
and ‘departments
Nugar,
New
of Pharmacolag~,
Lk#zi-I IO W2
Biochemistry
and
{India)
(Accepted June 5. 1991) Propolis bee preparations revealed good antibacterial (particularly against Gram-positive bacteria), antifungal (against those responsible for superficial and dermatomycoses) and antiinflammatory (against acute and chronic models of inflammation) effects but no antiamoebic or antipyretic capacity. Key wwds:
bee products; propolis: antimicrobial; antiinflammatory;
Introduction Apitherapy or therapy with bee products (e.g. honey, pollen, propolis, fortified honey, herb honey, etc.) is an old tradition which has been revived by recent researches (International Symposium on Apitherapy, 1985). These products, which are used both as health foods and medicine, are receiving renewed attention worldwide because of their beneficial effects and a general ‘back to nature’ trend. Propolis is a resinous wax-like substance which bees collect from plants and use as glue or putty to line their hives and fill up cracks. It invariably contains pollen grains which are a rich source of essential elements, e.g. Ca, Mg, Fe, Cu, Zn, Mn, Ni, etc. (Dobrowolski, 1987). Propolis products are claimed to be useful in a variety of ailments including infectious diseases (Grochowski et al., 1987) and arthritis (Zielonka et al., 1987). Considering these claims, antibacterial, antifungal, antiinflammatory and antipyretic studies on three propolis preparations were, therefore, undertaken,
Correspondence to: Prof. P.C. Dandiya. Department of Pharmacology, Hamdard University, P.O. Hamdard Nagar. New Delhi I IO 062. India.
0378-g74l/SO3.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
antipyretic: antiamoebic
Since the Rheumatic Diseases Foundation (De Fabio, 1982) has indicated that certain rheumatoid diseases may be the result of infection with an amoeba, we decided to include antiamoebic studies in the present investigation. Materials and Meth~ Natural materials
The following products were procured from APIPOL, the Polish Bee Keepers Association Laboratories at Krakow (Poland): (i) propolis granules (PG) containing 300 mg propolis per gram; (ii) drazteki propo~~sowe (red coloured tablets, PR) containing 350 mg propolis per tablet with a mean tablet weight of 1.2 g; and (iii) naturalany pszczeli pylek kwaiatowy (yellow coloured tablets, PY) containing 350 mg pollen grains per tablet of mean weight 1.2 g. Aqueous solutions or suspensions of these materials in 1% gum acacia were used for all investigations. Penicillin (IDPL, Rishikesh), streptomycin (Hindustan Antibiotics, Pune), tetracycline (Pfizer, Bombay), griseofulvin (Glaxo, Bombay), metronidazole (May and Baker, Bombay), phenylbutazone (Alembic, Baroda), flurbiprofen (FDC, Bombay) and hydrocortisone acetate (Wyeth, Bombay) were used as standards for comparison.
Animal stock Antiinflammatory and antipyretic studies were carried out in Wistar albino rats (150-250 g) of either sex, housed in groups of 5-6 animals in polypropylene cages kept in air-conditioned rooms (25--28”(Z) and maintained on a standard pellet diet (Gold Mohar, Lipton India Ltd., Calcutta) and clean drinking water ad libitum. Toxicity studies were carried out in Swiss mice (20-35 maintained in a similar manner.
g)
Microorganisms used Five Gram-positive (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus vir~dan~~. Diplococcus pneumoniae and Corynebacterium diphtheriae) and five Gram-negative (Escherichia co/i, Salmonella typhi, Salmonella puratyphi-A, Salmonella paratyphi-B and Shigella jlexneri) organisms were procured from the Public Health Serivces Board, London. Pure cultures of 10 fungi (Crypt0~o~~u.s neojbr~adure~Ia eapsulatum, mans, ~istoplasma Microsporum canis, Microsporum mycetomi, gypseum, Phialophora jeanselmei, Piedra hortae, Trichophyton mentagrophytes, Trichophyton rubrum and Trichosporon cutaneum) were obtained from the Central Drug Research Institute, The fungi were cultured on Lucknow. Sabouroud’s dextrose slants incubated at 25°C for 7 days. A virulent strain of Enlamoeba histolytica was procured from the All India Institute of Medical Sciences, New Delhi. Screening methods The filter-paper disk method (Cruickshank, 1965) was employed for the study of the in vitro effects of the five Gram-positive and five Gramnegative organisms grown on nutrient agar. The diameter of the filter paper disk was 6 mm, and inhibition zones greater than 10 mm were considered significant. Antifungal studies were conducted in vitro by the method of Aytoun et al. (1960) using Sabourouds’ dextrose agar. The slants were observed daily for 7 days. If no growth was observed during this period, the test drug was considered active.
Antiamoebic studies were carried out in vitro by the method of Vinayak and Prakash (1969). The fungal cultures were maintained on diphasic egg medium with an over layer of Ringer and buffalo calf serum. Effects were tested using liver marmite liquid medium. The test material was dissolved in triple-distilled water and tested at concentrations of 5-20 mg/ml. The experiments were repeated 6 times and the results were expressed as modal values. A~tii~~ammatory studies Five different techniques in rats were employed: (i) formaldehyde-induced arthritis (Brownlee, 1950); (ii) carrageenan-induced paw oedema (Winter et al., 1962); PGE, (100 mg/ml) induced paw oedema (Ghosh and Singh, 1974); (iv) cotton pellet granuloma (Meier et al., 1950), slightly modified by using 10 mg cotton pellets implanted on both sides of the abdomen instead of in the axillae; and (v) adjuvant-induced arthritis (Wakesman et al., 1960). Antipyrefic studies Brewer’s yeast-induced pyrexia in rats (Gujral et al., 1956) was used to test the antipyretic capacity. Rectal temperature was recorded using a Teletherm electronic thermometer with liquid para~n-lubricated probes inserted to a uniform depth of 5 cm and kept in situ for 1 min. Biochemical studies Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AP) were measured by the method of Basu et al. f 1986) on day-10 in formaldehyde-induced arthritic rats. Adrenal ascorbic acid content was estimated in normal and formaldehyde-induced arthritic rats on day-10 in PG-, PR- and PYtreated groups by the method described by Oser ( 1965). General toxicify PC, PR and PY were administered to groups of 10 mice (5 male, 5 female) in doses up to 2 g/kg p.o. The animals were observed continuously for 1 h, intermittently for 3 h and at 24 h and 48 h for all gross symptoms and any mortality.
79
Cryptococcus
neoformans
Yadurella
Yycetomi
q Fig. 1. Antifungal
NO
SLIGHT
GROWTH
FULL
preparations
relative
to griseofulvin
GROWTH
activity
of propolis
GROWTH
I
TABLE
ANTIBACTERIAL Tabular
I B
capsulatum
Histoplasma
ACTIVITY
values represent
OF PROPOLIS
mean zone of inhibition
insignificant. Filter paper discs were soaked controls were inactive. Organisms
PREPARATIONS (mm). Diameter
in aqueous
Penicillin
Streptomycin
Tetracyclin
(100 I.U.)
(1 w)
(100Irg)
24
I8
21 24 21 22
I8 12 14 12
23.6
E. coli S. Iyphi
Gram-positive s. uureus s. p.Vc~g“nrs S. viridun.s D. pneumoniue C. diphlheriue
Collective
mean
of the tilter paper disc = 6 mm. Values
solutions/suspensions
of the concentration
I IO mm were considered
in parenthesis
PG (IO mg)
PR (IO mg)
PY (IO mg)
28 28 27 30 18
16
16 17 16 14
II
14.8
26.2
13.8
15.0
13
26
16
10 12 12 I2
23
16
20
16
20
14
23
16
13 12 12 II I3
I3 II II II I2
20.4
15.6
12.2
11.6
16 13 13 II
12
I2 IO IO 6 9.8
Gram-negative
S. parutyphi-A S. purutyphi-B S. ,jlesneri
Collective
mean
for 30 min. Vehicle
I I.8
IO 6 6 6 12 8.0
80
(data not shown) and carrageenan-induced paw oedema. Only PY exhibited antiinflammatory effect in the cotton pellet granuloma test (Table 4). Generally, for all propolis preparations, the peak effects against PGEz were discernible at +4 h (Table 3).
Statistics
Mean, S.E.M., analysis of variance and Dunnett’s t-test were used for the statistical analysis of data. Results Antimicrobial
Antipyretic
studies
PR and PG exhibited some antibacterial activity, particularly against Gram-positive orgnisms (Table 1). PY exhibited mild antibacterial effects against four Gram-positive (S. aureus, S. pyogenes, S. viridans, D. pneumoniae) and two Gramnegative (E. cofi, Sh. flexneri) bacteria. PG and PR exhibited definite antifungal activity against the superficial and dermatomycoses groups of fungi (Fig. 1). No activity was observed for PG and PR against the subcutaneous and systemic mycoses. PY did not elicit effects against any of the fungal cultures used in these investigations. None of the propolis preparations exhibited antiamoebic activity. Antiinflammatory
Biochemical
EFFECT
studies
Levels of ALT and AST were significantly elevated in formaldehyde-induced arthritic rats, but treatments with PG, PR and PY only partly reversed this elevation and these decreases were not found to be statistically significant (data not shown). AP levels remained unaltered both in animals with inflammation only and those receiving the various treatments. Adrenal ascorbic acid content was significantly depressed (P < 0.05) in arthritic rats (166.5 f 18.9 pmol/l per min) as compared to that in normal rats (224.5 f 10.2 pmolll per min). The test treatments (PG, PR and PY) failed to restore these levels to normal.
studies
All the propolis preparations under study elicited moderate to marked antiinflammatory activity against formaldehyde-induced arthritis (Table 2) and PGEz-induced paw oedema (Table 3) but no action against adjuvant-induced arthritis
TABLE
studies
Yeast-induced stable pyrexia was seen in all animals 18 h after injection. None of the propolis preparations exhibited significant antipyretic action at doses of 100-200 mg/kg p.o. although a transient fall in rectal temperature (30-60 min in duration) was seen just after administration of the propolis preparations (data not shown).
2 OF PROPOLIS
PREPARATIONS
Treatment (dose/kg, route)
ON FORMALDEHYDE-INDUCED
Paw volume Initial 1.04 f 0.96 f
PC (100 mg. p.o.) PR (200 mg, p.0.) PY (200 mg. p.0.)
0.93 f 0.02 1.01 f 0.04 0.98 zt 0.06
Tabular values represent nett’s I-test: Significance
0.06 0.06
IN ALBINO
RATS
Antiinflammatory activity (‘%I)
(ml) Ten-day
Normal saline (10 ml. p.o.) Hydrocortisone (25 mg, i.p.)
ARTHRITIS
average
1.57 * 0.05 1.23 zt 0.06 1.26 f 1.34 f 1.34 f
0.02 0.04 0.05
the mean f S.E.M. of 7-10 animals. Statistical relative to the saline control: *P < 0.05, **P
analysis < 0.01.
49.1** 36.7** 37.7** 32. I * by one-way
analysis
of variance
followed
by Dun-
81
TABLE 3 EFFECT OF PROPOLIS PREPARATIONS RATS Phlogistic agent (cont.. vol.)
PGE, (100 @ml, 0.1 ml)
Carrageenan (1% suspension in sterile saline, 0.1 ml)
ON PGE,- AND CARRAGEENAN-INDUCED
PAW OEDEMA IN ALBINO
Treatment (dose/kg, p-0.. -30 min) Time interval (h)
Normal saline (10 ml)
Flubiprofen (1.7 mg)
PG (100mg)
PR (200 mg)
PY (200 mg)
+l
0.50 f 0.05
+2
0.63 f 0.03
+3
0.60 f 0.02
+4
0.65 f 0.02
+I
0.31 f 0.03
+2
0.70 f 0.06
+3
0.55 f 0.05
0.38 zt (24.0) 0.16 f (74.6) 0.33 l (45.0) 0.32 f (50.8) 0.16 f (48.4) 0.17 f (75.7) 0.1 I f (80.0)
0.30 f (40.0) 0.42 f (33.3) 0.38 f (36.7) 0.32 f (50.8) 0.41 f (--_) 0.61 i (12.8) 0.56 zt (-)
0.30 zt (40.0) 0‘38 f (39.7) 0.42 zt (30.1) 0.28 f (56.9) 0.38 f (--) 0.53 zt (24.3) 0.48 f (12.7)
0.31 f (38.0) 0.41 * (34.9) 0.40 f (33.3) 0.31 f (52.3) 0.23 i (25.8) 0.48 f (31.4) 0.46 f (16.4)
0.06 0.05* ox&** 0.04* 0.04** 0.04** 0.04**
0.04* 0.06’ 0.05** O.OS** 0.03 0.06 0.05
0.03* 0.04** 0.02* 0.07** 0.05 0.06 0.06
0.05* 0.04** 0.04** 0.04** 0.05 0.08 0.05
Tabular values represent mean paw ,volume (ml) t S.E.M. of 67 animals. Tabular tigures in parentheses indicate ‘%Iantiinflammatory effect. Statistical an&y& by one-way analysis of variance followed by Dunnett’s r-test. Significance relative to saline controls: *P c 0.05, **P < 0.01.
TABLE 4
General toxicity The propolis preparations were well tolerated in mice up to a dose of 2 g/kg p.o. None of the treated mice died during the 48-h period of observation,
EFFECT OF PROPOLIS PREPARATlONS ON COTTON PELLET GRANULOMA IN ALBINO RATS
Discussion
Treatment (dose/kg per day. route)
Dry weight of granuloma (mg)
Inhibition (‘X)
Normal Saline (10 ml, p.o.) Hydrocortisone acetate (5 mg, i.p.1 PG (100 mg, p.0.) PR (200 mg. p.0.) PY (200 mg, p.0.)
54.40 zt 2.67
-
27.51 54.88 51.11 40386
49.4 6.0 24.9
zt zt tt f
1.13* 2.45 1.31 1.42*
Tabular values represent the mean + S.E.M. of 69 animals/group. Statistical analysis by one-way analysis of varianee followed by Dunnett’s f-test. Significant relative to saline control. *P < 0.01.
A study of the findings here shows that the propolis preparations exhibited definite antibacterial (particularly against Gram-positive bacteria), antifungal (except against subcutaneous and systemic mycoses and with PY) and antiinflammatory effects but no antiamoebic or antipyretic actions. Antiinflammatory effects were observed against an acute (endogenous mediator PGE+nduced paw oedema) and a chronic (formaldehyde-induced arthritis) model of inflammation but not against carrageenan and immunological (adjuvant arthritis) models. The effects against PGEz oedema, though discernible even at +I h, were maximum at
82
+4 h. The preparations failed to restore stressinduced depressed adrenal ascorbic acid content and could only partly reverse the elevated levels of serum ALT and AST of arthritic rats. The mechanisms of action of the propotis preparations, therefore, remain obscure and warrant further investigation. The effects of PG, PR and PY were observed at 5-25 mg/ml in vitro and 100-200 mg/kg, p.o. to rats in vivo. No untoward effects or mortality were noted in mice at doses up to 2 g/kg, p.o. This suggests a wide therapeutic index for these preparations. Arthritis, being a chronic distressing and disabling ailment, requires therapy for prolonged periods. Natural food additives like prop&is, unlike the nonsteroidal and steroidai ant~in~ammatories~ could, therefore, constitute a more ideal approach towards its ~ngement. The present study should stimulate further research on these preparations.
Brownlee, G. f 1950) Effect of deoxycortone and ascorbic acid on formaldehyde-induced arthritis in normal and adrenaiectomised rats. Luncet 1, 157-I 59. Cruickshank, R. (1965) ~~~j~~/ ~~er~~j~~f~~~,1Ith Edn. E. and S. Livingstone Ltd.. Edinburgh, p. 894. Defabio, A. fi982) Rheumuraid iXspasrs Cuwd rrt tusr, Rheumatoid Disease Foundatj~n, Franktin. Tennessee, pp. I-16. Dobrowoiski, J.W., Tomaszewski, R., Druzga, M. and Zawodny. Z. (1987) Herb-honeys with pollens as natural sources of deficient and bialogically essential elements. In: M. Said. M.A. Rahman and L.A. D’Silva (Eds.), Ekmenrs in Heulrh and fXxwe, Hamdard University Press, Karachi, pp, 63-73. Ghosh, M.N. and Singh, N. 11974) fnhibitory effects of a pyroiizidine atkaloid crotalburinine in rat paw oedema. Bririslt Journui ef P/Iurrnue~t~#g~ 5 I, 503-508. Grochowski. J., Bilinska, M. and Stankiewicz.. D. (1987) Propolis in treatment of intradermal bacterial infection in guinea pigs. Biufelyn Ap@t& 71%. 25-26. Gujrat, ML.. Kohl;, R.P., Bhargava, K.P. and Saxena. P.N. (1956) An experimental study ofthe effect of some drugs on temperature in normal and pyretic rats. fndiun Journul u/‘ Medicul
Reseurch 43. 89-94.
Symposium on Apitherupy ( 1985) Krakow. Poland, March 23-26. Meier, R.. Schuler. W. and Desaniler, P. (1950) Zur frage des mechan~smus der hemmung des bindgewe~w~hst~ms durch cortisone. &prrietriicr 6, 469472. Oser, B.L. (1965) Wus&S P/l~sju/itgj~ul Ckw~Lsrry 14th Edn. McGraw-Hill Book Co,, New York, pp, 703-704. Vinayak, V.K. and Prakash. 0. f 1969) A comparative evaluation of metronidazole and other amoebicidal drugs on the strains of ~ntu~~~~h~t ~ti~t#~~t~~u. It&m Jm~rtwi of ~~~~~~~~I~ Intwwtioturl
Acknowledgements The authors are grateful to Hakeem Abduf Hameed, Chancellor, and Prof. M. Amin, Vice Chancellor of Jamia Hamdard, New Delhi, for facitities and encouragement. Thanks are due to Mr. E.A. Khan for the statistical analyses and to Mr. T.A. Farooqui, Mr. Pradeep Roy, Mrs. Rosamma Phillip and Mr. Muneer Khan for technical assistance. References Aytoun, R.S.C., (3ampbel1, A.H.. Napier, E.J. and kiter, D.A.L. (f96Q) Mycological aspects of action of grieseofulvin against dermatophytes. Artkives qf Dertnctr&g~ 8 1. 650-656. Basu, B.N., Ray, K.. jch~upu~ui. R.L. and Khanna, K.K. ( 1986) Mrrnucilo~He&Ii: Luhorutor,r Prorprlurcs_&r D&trier tr& ~~~/z~~~j~~.r,Narionai Institute of Communicable Diseases, Delhi, fndia, pp. I15---1IS.
R~~~nr~~~ 57, 84 t-847.
Wakesman, B.H.. Pearson, C.M. and Sharp, LT. (1960) Studies of arthritis and other lesions induced in rats by injection of myocardial adjuvant. II: Evidence that disease is a discriminated immunological response to exogenous antigen. Journul of Immunology 85, 403-417. Winter, C.A.. Risley. EA. and Nuss, C.W. (1964) Carrageenininduced oedema in hind paw of the rat as an assay for antiinflammatory drugs. Pr<~e&q.r ($ r/~ .Socicq ,j&r E.vp~rit~7~~tttftl Biolog)’ 11I. 544-547. Zielonka, E., Zaborski. W., Dymarczyk. M. and Kuziemska. G. f 1987) Role of propalis in the tr~tment of rheumatic diseases. B&&j~~ Api+/ 718. 29-32.
35 ( 199
I)
7-l
R-82
Elsevier Scientific Publishers Ireland Ltd.
Antibacterial, antifungal, antiamoebic, antiinflammatory antipyretic studies on propolis bee products
and
Jan W. Dobrowolski”, S.B. Vohorab, Kalpana Sharmab, Shaukat A. Shahb, S.A.H. Naqvib and P.C. Dandiyab UInsfitute of Management
and Protection
~icro~~~~~gy,
of Environment,
~~mdard
University,
Krakow P.O.
{Poland)
~~rndard
and ‘departments
Nugar,
New
of Pharmacolag~,
Lk#zi-I IO W2
Biochemistry
and
{India)
(Accepted June 5. 1991) Propolis bee preparations revealed good antibacterial (particularly against Gram-positive bacteria), antifungal (against those responsible for superficial and dermatomycoses) and antiinflammatory (against acute and chronic models of inflammation) effects but no antiamoebic or antipyretic capacity. Key wwds:
bee products; propolis: antimicrobial; antiinflammatory;
Introduction Apitherapy or therapy with bee products (e.g. honey, pollen, propolis, fortified honey, herb honey, etc.) is an old tradition which has been revived by recent researches (International Symposium on Apitherapy, 1985). These products, which are used both as health foods and medicine, are receiving renewed attention worldwide because of their beneficial effects and a general ‘back to nature’ trend. Propolis is a resinous wax-like substance which bees collect from plants and use as glue or putty to line their hives and fill up cracks. It invariably contains pollen grains which are a rich source of essential elements, e.g. Ca, Mg, Fe, Cu, Zn, Mn, Ni, etc. (Dobrowolski, 1987). Propolis products are claimed to be useful in a variety of ailments including infectious diseases (Grochowski et al., 1987) and arthritis (Zielonka et al., 1987). Considering these claims, antibacterial, antifungal, antiinflammatory and antipyretic studies on three propolis preparations were, therefore, undertaken,
Correspondence to: Prof. P.C. Dandiya. Department of Pharmacology, Hamdard University, P.O. Hamdard Nagar. New Delhi I IO 062. India.
0378-g74l/SO3.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
antipyretic: antiamoebic
Since the Rheumatic Diseases Foundation (De Fabio, 1982) has indicated that certain rheumatoid diseases may be the result of infection with an amoeba, we decided to include antiamoebic studies in the present investigation. Materials and Meth~ Natural materials
The following products were procured from APIPOL, the Polish Bee Keepers Association Laboratories at Krakow (Poland): (i) propolis granules (PG) containing 300 mg propolis per gram; (ii) drazteki propo~~sowe (red coloured tablets, PR) containing 350 mg propolis per tablet with a mean tablet weight of 1.2 g; and (iii) naturalany pszczeli pylek kwaiatowy (yellow coloured tablets, PY) containing 350 mg pollen grains per tablet of mean weight 1.2 g. Aqueous solutions or suspensions of these materials in 1% gum acacia were used for all investigations. Penicillin (IDPL, Rishikesh), streptomycin (Hindustan Antibiotics, Pune), tetracycline (Pfizer, Bombay), griseofulvin (Glaxo, Bombay), metronidazole (May and Baker, Bombay), phenylbutazone (Alembic, Baroda), flurbiprofen (FDC, Bombay) and hydrocortisone acetate (Wyeth, Bombay) were used as standards for comparison.
Animal stock Antiinflammatory and antipyretic studies were carried out in Wistar albino rats (150-250 g) of either sex, housed in groups of 5-6 animals in polypropylene cages kept in air-conditioned rooms (25--28”(Z) and maintained on a standard pellet diet (Gold Mohar, Lipton India Ltd., Calcutta) and clean drinking water ad libitum. Toxicity studies were carried out in Swiss mice (20-35 maintained in a similar manner.
g)
Microorganisms used Five Gram-positive (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus vir~dan~~. Diplococcus pneumoniae and Corynebacterium diphtheriae) and five Gram-negative (Escherichia co/i, Salmonella typhi, Salmonella puratyphi-A, Salmonella paratyphi-B and Shigella jlexneri) organisms were procured from the Public Health Serivces Board, London. Pure cultures of 10 fungi (Crypt0~o~~u.s neojbr~adure~Ia eapsulatum, mans, ~istoplasma Microsporum canis, Microsporum mycetomi, gypseum, Phialophora jeanselmei, Piedra hortae, Trichophyton mentagrophytes, Trichophyton rubrum and Trichosporon cutaneum) were obtained from the Central Drug Research Institute, The fungi were cultured on Lucknow. Sabouroud’s dextrose slants incubated at 25°C for 7 days. A virulent strain of Enlamoeba histolytica was procured from the All India Institute of Medical Sciences, New Delhi. Screening methods The filter-paper disk method (Cruickshank, 1965) was employed for the study of the in vitro effects of the five Gram-positive and five Gramnegative organisms grown on nutrient agar. The diameter of the filter paper disk was 6 mm, and inhibition zones greater than 10 mm were considered significant. Antifungal studies were conducted in vitro by the method of Aytoun et al. (1960) using Sabourouds’ dextrose agar. The slants were observed daily for 7 days. If no growth was observed during this period, the test drug was considered active.
Antiamoebic studies were carried out in vitro by the method of Vinayak and Prakash (1969). The fungal cultures were maintained on diphasic egg medium with an over layer of Ringer and buffalo calf serum. Effects were tested using liver marmite liquid medium. The test material was dissolved in triple-distilled water and tested at concentrations of 5-20 mg/ml. The experiments were repeated 6 times and the results were expressed as modal values. A~tii~~ammatory studies Five different techniques in rats were employed: (i) formaldehyde-induced arthritis (Brownlee, 1950); (ii) carrageenan-induced paw oedema (Winter et al., 1962); PGE, (100 mg/ml) induced paw oedema (Ghosh and Singh, 1974); (iv) cotton pellet granuloma (Meier et al., 1950), slightly modified by using 10 mg cotton pellets implanted on both sides of the abdomen instead of in the axillae; and (v) adjuvant-induced arthritis (Wakesman et al., 1960). Antipyrefic studies Brewer’s yeast-induced pyrexia in rats (Gujral et al., 1956) was used to test the antipyretic capacity. Rectal temperature was recorded using a Teletherm electronic thermometer with liquid para~n-lubricated probes inserted to a uniform depth of 5 cm and kept in situ for 1 min. Biochemical studies Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AP) were measured by the method of Basu et al. f 1986) on day-10 in formaldehyde-induced arthritic rats. Adrenal ascorbic acid content was estimated in normal and formaldehyde-induced arthritic rats on day-10 in PG-, PR- and PYtreated groups by the method described by Oser ( 1965). General toxicify PC, PR and PY were administered to groups of 10 mice (5 male, 5 female) in doses up to 2 g/kg p.o. The animals were observed continuously for 1 h, intermittently for 3 h and at 24 h and 48 h for all gross symptoms and any mortality.
79
Cryptococcus
neoformans
Yadurella
Yycetomi
q Fig. 1. Antifungal
NO
SLIGHT
GROWTH
FULL
preparations
relative
to griseofulvin
GROWTH
activity
of propolis
GROWTH
I
TABLE
ANTIBACTERIAL Tabular
I B
capsulatum
Histoplasma
ACTIVITY
values represent
OF PROPOLIS
mean zone of inhibition
insignificant. Filter paper discs were soaked controls were inactive. Organisms
PREPARATIONS (mm). Diameter
in aqueous
Penicillin
Streptomycin
Tetracyclin
(100 I.U.)
(1 w)
(100Irg)
24
I8
21 24 21 22
I8 12 14 12
23.6
E. coli S. Iyphi
Gram-positive s. uureus s. p.Vc~g“nrs S. viridun.s D. pneumoniue C. diphlheriue
Collective
mean
of the tilter paper disc = 6 mm. Values
solutions/suspensions
of the concentration
I IO mm were considered
in parenthesis
PG (IO mg)
PR (IO mg)
PY (IO mg)
28 28 27 30 18
16
16 17 16 14
II
14.8
26.2
13.8
15.0
13
26
16
10 12 12 I2
23
16
20
16
20
14
23
16
13 12 12 II I3
I3 II II II I2
20.4
15.6
12.2
11.6
16 13 13 II
12
I2 IO IO 6 9.8
Gram-negative
S. parutyphi-A S. purutyphi-B S. ,jlesneri
Collective
mean
for 30 min. Vehicle
I I.8
IO 6 6 6 12 8.0
80
(data not shown) and carrageenan-induced paw oedema. Only PY exhibited antiinflammatory effect in the cotton pellet granuloma test (Table 4). Generally, for all propolis preparations, the peak effects against PGEz were discernible at +4 h (Table 3).
Statistics
Mean, S.E.M., analysis of variance and Dunnett’s t-test were used for the statistical analysis of data. Results Antimicrobial
Antipyretic
studies
PR and PG exhibited some antibacterial activity, particularly against Gram-positive orgnisms (Table 1). PY exhibited mild antibacterial effects against four Gram-positive (S. aureus, S. pyogenes, S. viridans, D. pneumoniae) and two Gramnegative (E. cofi, Sh. flexneri) bacteria. PG and PR exhibited definite antifungal activity against the superficial and dermatomycoses groups of fungi (Fig. 1). No activity was observed for PG and PR against the subcutaneous and systemic mycoses. PY did not elicit effects against any of the fungal cultures used in these investigations. None of the propolis preparations exhibited antiamoebic activity. Antiinflammatory
Biochemical
EFFECT
studies
Levels of ALT and AST were significantly elevated in formaldehyde-induced arthritic rats, but treatments with PG, PR and PY only partly reversed this elevation and these decreases were not found to be statistically significant (data not shown). AP levels remained unaltered both in animals with inflammation only and those receiving the various treatments. Adrenal ascorbic acid content was significantly depressed (P < 0.05) in arthritic rats (166.5 f 18.9 pmol/l per min) as compared to that in normal rats (224.5 f 10.2 pmolll per min). The test treatments (PG, PR and PY) failed to restore these levels to normal.
studies
All the propolis preparations under study elicited moderate to marked antiinflammatory activity against formaldehyde-induced arthritis (Table 2) and PGEz-induced paw oedema (Table 3) but no action against adjuvant-induced arthritis
TABLE
studies
Yeast-induced stable pyrexia was seen in all animals 18 h after injection. None of the propolis preparations exhibited significant antipyretic action at doses of 100-200 mg/kg p.o. although a transient fall in rectal temperature (30-60 min in duration) was seen just after administration of the propolis preparations (data not shown).
2 OF PROPOLIS
PREPARATIONS
Treatment (dose/kg, route)
ON FORMALDEHYDE-INDUCED
Paw volume Initial 1.04 f 0.96 f
PC (100 mg. p.o.) PR (200 mg, p.0.) PY (200 mg. p.0.)
0.93 f 0.02 1.01 f 0.04 0.98 zt 0.06
Tabular values represent nett’s I-test: Significance
0.06 0.06
IN ALBINO
RATS
Antiinflammatory activity (‘%I)
(ml) Ten-day
Normal saline (10 ml. p.o.) Hydrocortisone (25 mg, i.p.)
ARTHRITIS
average
1.57 * 0.05 1.23 zt 0.06 1.26 f 1.34 f 1.34 f
0.02 0.04 0.05
the mean f S.E.M. of 7-10 animals. Statistical relative to the saline control: *P < 0.05, **P
analysis < 0.01.
49.1** 36.7** 37.7** 32. I * by one-way
analysis
of variance
followed
by Dun-
81
TABLE 3 EFFECT OF PROPOLIS PREPARATIONS RATS Phlogistic agent (cont.. vol.)
PGE, (100 @ml, 0.1 ml)
Carrageenan (1% suspension in sterile saline, 0.1 ml)
ON PGE,- AND CARRAGEENAN-INDUCED
PAW OEDEMA IN ALBINO
Treatment (dose/kg, p-0.. -30 min) Time interval (h)
Normal saline (10 ml)
Flubiprofen (1.7 mg)
PG (100mg)
PR (200 mg)
PY (200 mg)
+l
0.50 f 0.05
+2
0.63 f 0.03
+3
0.60 f 0.02
+4
0.65 f 0.02
+I
0.31 f 0.03
+2
0.70 f 0.06
+3
0.55 f 0.05
0.38 zt (24.0) 0.16 f (74.6) 0.33 l (45.0) 0.32 f (50.8) 0.16 f (48.4) 0.17 f (75.7) 0.1 I f (80.0)
0.30 f (40.0) 0.42 f (33.3) 0.38 f (36.7) 0.32 f (50.8) 0.41 f (--_) 0.61 i (12.8) 0.56 zt (-)
0.30 zt (40.0) 0‘38 f (39.7) 0.42 zt (30.1) 0.28 f (56.9) 0.38 f (--) 0.53 zt (24.3) 0.48 f (12.7)
0.31 f (38.0) 0.41 * (34.9) 0.40 f (33.3) 0.31 f (52.3) 0.23 i (25.8) 0.48 f (31.4) 0.46 f (16.4)
0.06 0.05* ox&** 0.04* 0.04** 0.04** 0.04**
0.04* 0.06’ 0.05** O.OS** 0.03 0.06 0.05
0.03* 0.04** 0.02* 0.07** 0.05 0.06 0.06
0.05* 0.04** 0.04** 0.04** 0.05 0.08 0.05
Tabular values represent mean paw ,volume (ml) t S.E.M. of 67 animals. Tabular tigures in parentheses indicate ‘%Iantiinflammatory effect. Statistical an&y& by one-way analysis of variance followed by Dunnett’s r-test. Significance relative to saline controls: *P c 0.05, **P < 0.01.
TABLE 4
General toxicity The propolis preparations were well tolerated in mice up to a dose of 2 g/kg p.o. None of the treated mice died during the 48-h period of observation,
EFFECT OF PROPOLIS PREPARATlONS ON COTTON PELLET GRANULOMA IN ALBINO RATS
Discussion
Treatment (dose/kg per day. route)
Dry weight of granuloma (mg)
Inhibition (‘X)
Normal Saline (10 ml, p.o.) Hydrocortisone acetate (5 mg, i.p.1 PG (100 mg, p.0.) PR (200 mg. p.0.) PY (200 mg, p.0.)
54.40 zt 2.67
-
27.51 54.88 51.11 40386
49.4 6.0 24.9
zt zt tt f
1.13* 2.45 1.31 1.42*
Tabular values represent the mean + S.E.M. of 69 animals/group. Statistical analysis by one-way analysis of varianee followed by Dunnett’s f-test. Significant relative to saline control. *P < 0.01.
A study of the findings here shows that the propolis preparations exhibited definite antibacterial (particularly against Gram-positive bacteria), antifungal (except against subcutaneous and systemic mycoses and with PY) and antiinflammatory effects but no antiamoebic or antipyretic actions. Antiinflammatory effects were observed against an acute (endogenous mediator PGE+nduced paw oedema) and a chronic (formaldehyde-induced arthritis) model of inflammation but not against carrageenan and immunological (adjuvant arthritis) models. The effects against PGEz oedema, though discernible even at +I h, were maximum at
82
+4 h. The preparations failed to restore stressinduced depressed adrenal ascorbic acid content and could only partly reverse the elevated levels of serum ALT and AST of arthritic rats. The mechanisms of action of the propotis preparations, therefore, remain obscure and warrant further investigation. The effects of PG, PR and PY were observed at 5-25 mg/ml in vitro and 100-200 mg/kg, p.o. to rats in vivo. No untoward effects or mortality were noted in mice at doses up to 2 g/kg, p.o. This suggests a wide therapeutic index for these preparations. Arthritis, being a chronic distressing and disabling ailment, requires therapy for prolonged periods. Natural food additives like prop&is, unlike the nonsteroidal and steroidai ant~in~ammatories~ could, therefore, constitute a more ideal approach towards its ~ngement. The present study should stimulate further research on these preparations.
Brownlee, G. f 1950) Effect of deoxycortone and ascorbic acid on formaldehyde-induced arthritis in normal and adrenaiectomised rats. Luncet 1, 157-I 59. Cruickshank, R. (1965) ~~~j~~/ ~~er~~j~~f~~~,1Ith Edn. E. and S. Livingstone Ltd.. Edinburgh, p. 894. Defabio, A. fi982) Rheumuraid iXspasrs Cuwd rrt tusr, Rheumatoid Disease Foundatj~n, Franktin. Tennessee, pp. I-16. Dobrowoiski, J.W., Tomaszewski, R., Druzga, M. and Zawodny. Z. (1987) Herb-honeys with pollens as natural sources of deficient and bialogically essential elements. In: M. Said. M.A. Rahman and L.A. D’Silva (Eds.), Ekmenrs in Heulrh and fXxwe, Hamdard University Press, Karachi, pp, 63-73. Ghosh, M.N. and Singh, N. 11974) fnhibitory effects of a pyroiizidine atkaloid crotalburinine in rat paw oedema. Bririslt Journui ef P/Iurrnue~t~#g~ 5 I, 503-508. Grochowski. J., Bilinska, M. and Stankiewicz.. D. (1987) Propolis in treatment of intradermal bacterial infection in guinea pigs. Biufelyn Ap@t& 71%. 25-26. Gujrat, ML.. Kohl;, R.P., Bhargava, K.P. and Saxena. P.N. (1956) An experimental study ofthe effect of some drugs on temperature in normal and pyretic rats. fndiun Journul u/‘ Medicul
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Symposium on Apitherupy ( 1985) Krakow. Poland, March 23-26. Meier, R.. Schuler. W. and Desaniler, P. (1950) Zur frage des mechan~smus der hemmung des bindgewe~w~hst~ms durch cortisone. &prrietriicr 6, 469472. Oser, B.L. (1965) Wus&S P/l~sju/itgj~ul Ckw~Lsrry 14th Edn. McGraw-Hill Book Co,, New York, pp, 703-704. Vinayak, V.K. and Prakash. 0. f 1969) A comparative evaluation of metronidazole and other amoebicidal drugs on the strains of ~ntu~~~~h~t ~ti~t#~~t~~u. It&m Jm~rtwi of ~~~~~~~~I~ Intwwtioturl
Acknowledgements The authors are grateful to Hakeem Abduf Hameed, Chancellor, and Prof. M. Amin, Vice Chancellor of Jamia Hamdard, New Delhi, for facitities and encouragement. Thanks are due to Mr. E.A. Khan for the statistical analyses and to Mr. T.A. Farooqui, Mr. Pradeep Roy, Mrs. Rosamma Phillip and Mr. Muneer Khan for technical assistance. References Aytoun, R.S.C., (3ampbel1, A.H.. Napier, E.J. and kiter, D.A.L. (f96Q) Mycological aspects of action of grieseofulvin against dermatophytes. Artkives qf Dertnctr&g~ 8 1. 650-656. Basu, B.N., Ray, K.. jch~upu~ui. R.L. and Khanna, K.K. ( 1986) Mrrnucilo~He&Ii: Luhorutor,r Prorprlurcs_&r D&trier tr& ~~~/z~~~j~~.r,Narionai Institute of Communicable Diseases, Delhi, fndia, pp. I15---1IS.
R~~~nr~~~ 57, 84 t-847.
Wakesman, B.H.. Pearson, C.M. and Sharp, LT. (1960) Studies of arthritis and other lesions induced in rats by injection of myocardial adjuvant. II: Evidence that disease is a discriminated immunological response to exogenous antigen. Journul of Immunology 85, 403-417. Winter, C.A.. Risley. EA. and Nuss, C.W. (1964) Carrageenininduced oedema in hind paw of the rat as an assay for antiinflammatory drugs. Pr<~e&q.r ($ r/~ .Socicq ,j&r E.vp~rit~7~~tttftl Biolog)’ 11I. 544-547. Zielonka, E., Zaborski. W., Dymarczyk. M. and Kuziemska. G. f 1987) Role of propalis in the tr~tment of rheumatic diseases. B&&j~~ Api+/ 718. 29-32.