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.
Sabourouds dextrose slants incubated at 25C 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
Brewers 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

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~gnrs
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 Dunnetts 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

netts 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)
038 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 Dunnetts 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 Dunnetts 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. DSilva (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.
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