Antibacterial properties of propolis and products containing propolis from Brazil

Original

article

Antibacterial

_properties

of

_propolis

and

_products

containing propolis

from Brazil

H Menezes* M

Bacci Jr SD

_Oliveira,

FC

_Pagnocca

Centro de Estudos de Insetos Sociais, Instituto de Biociências,

Unesp, Rio Claro, São Paulo, Brazil

(Received 21 _August 1996; accepted 10 March 1997)

Summary —

Crude

propolis

and commercial

_{products containing propolis,}

such as _{ethanolic extracts,}

tablets,

capsules

and

_{powders acquired}

in São Paulo

_City

(Brazil) were

_analyzed.

The resins of the solid

products

were extracted with ethanol and found to be _present at various concentrations,

indepen-dently

of the

_propolis

concentration

_specified

on the label of the commercial

_products.

The in vitro

activity

of these resins

_against

S aureus, B cereus and B subtilis was also determined. The results showed that the antibacterial

_activity

rather than the

propolis

concentration itself should be consid-ered for

_quality

control and that some resins are

likely

to

_display

a

species-specific

action.

propolis / resin /

_Apis

_mellifera

/

_quality

control / antibacterial

_activity

/

_{Staphylococcus}

aureus

/ Bacillus cereus / Bacillus subtilis

INTRODUCTION

Propolis

(bee

glue)

has been used in folk medicine for centuries

_{(Ghisalberti, 1979).}

This is

_{supported by}

recent

_findings

about its

biological properties

such as antibacterial

(Grange

and

_Davey,

1990),

antiviral

(Amoros

et

_{al, 1992)}

or

anti-protozoan

(Starzyk

et

_{al, 1977)}

_activity,

inhibition of

tumor _processes

_(Frenkel

et

_{al, 1993),}

anti-inflammatory

action

_(Dobowolski

et

al,

1991;

_Volpert

and

Elstner, 1996),

induction

of both bone

_(Stojko

et al,

1979)

and

carti-laginous

tissue

_regeneration

_(Scheller

et _al,

1977),

free radical

_scavenging

(Scheller

et al,

1989;

_Volpert

and

Elstner, 1993),

and

aller-genic

effect

(Hausen

et

_{al, 1987a, b;}

Ginan-neschi et

_{al, 1989).}

The

_{biological properties}

and chemical

_{composition of propolis}

have been

_recently

reviewed

(Marcucci, 1995).

The characterization of these

_biological

properties together

with the current trend towards

_utilizing

natural

_products

have resulted in an increased demand _for

propo-*

Corresponding

author:

_IB-Unesp,

av 24 "A" n° 1515, PO Box 199, Rio Claro, SP, Brazil, CEP: 13506-900.

lis and

_products

_containing

_propolis,

such

as ethanol extracts,

tablets,

_capsules,

_sprays,

or

powders.

The

_expansion

of the

_{propolis industry}

requires

standardization of these

_products,

and should take into account

_regional

veg-etation

_diversity

(Vanhaelen

and

Vanhae-len-Fastré, 1979),

the

_harvesting

_activity

of distinct races of

honeybees (Singh,

_1972),

and establish the

_efficacy

of the

_products.

In

_addition,

such a standardization needs to

be

_simple

and

_inexpensive

to be accessible

to manufacturers.

In this context, the

_objective

of the

pre-sent

_study

was the evaluation of the in vitro antibacterial

_activity

of the different for-mulations

_{of propolis.}

_Samples

were

_bought

in

_pharmacies,

_drugstores

and from

bee-keepers.

MATERIALS AND METHODS

Propolis

and commercial

_products

containing propolis

Three groups of

products

were studied in this work: ten different commercial ethanolic extracts

referred to as CE1 to CE10; commercial

prepa-rations of tablets,

_capsules

and

powders

con-taining propolis;

two other ethanolic extracts

pre-pared

in our

_{laboratory using}

crude

_propolis

from

one _{site reforested with Pinus sp and another}

reforested with

Eucalyptus

sp. All these

propolis

samples

were from A

_mellifera

and the extracts

were

_prepared

with 95% pa ethanol _(Merck).

Determination of resin concentration in commercial alcoholic extracts

Aliquots

of 5.0 mL of each extract were

trans-ferred to

_{previously weighted}

_{flasks and}

evapo-rated at 50 °C until constant

_weight

was reached.

Determination of resin content in crude

propolis

and in solid commercial

_product

One hundred grams of crude

propolis

of Pinus sp

or

_Eucalyptus

_sp were mixed with 200 mL of

95% ethanol and maintained in a _rotatory water

bath shaker

_during

1 week at 25 °C. The ethano-lic extracts were then filtered in Whatmann no 1

filter paper and

designated

as E-Pinus or

E-Euca-lyptus.

The resin extraction

_procedure

for the

solid commercial

_products

was similar but

_using

200 g of every

product/200

mL of ethanol. These

extracts were

_designated

as _E-tablet,

_E-capsule

and

_E-powder.

The resin content of these five

extracts was determinated

_by

the same

proce-dure as that used with the commercial alcoholic

extract. After

_{determinating}

the resin content, all 15

_samples

were diluted with 95% ethanol to

10

_mg/mL

(stock-solution) which was used for the antibacterial

_activity

assay. The resin

con-tent _{(% resin)} was

_expressed

_{in grams of resin}

per 100 mL.

Antibacterial

_activity

determination

This determination was

adapted

from Waksman

and

_Reilly

_(1945). The

_propolis

sensitive bacte-ria

_(Grange

and

Davey,

1990)

Staphylococcus

aureus ATCC 6538, Bacillus cereus CCT 1436,

and Bacillus subtilis CCT 2471 were cultivated

(24 h, 35 _°C) on Nutrient

_Agar

medium _(Difco)

and then

_suspended

_(one unit on the MacFarland

scale) in sterile saline 0.85%. The

_suspensions

(0,1 mL) were

_sprayed

with a cotton swab onto

the surface of solid media in Petri dishes. The culture medium had been

_{previously prepared}

by mixing

9.0 mL of melted _{(47 °C)} NA medium with 1 mL of 95% ethanol _(control) or with

1 mL of dilutions of resin stock solutions. The results were

expressed

in ’limit concentration’

(LC), ie, the minimum concentration of resin

_(μg

of resin per mL of culture medium) which

com-pletely

inhibited bacterial

_growth.

The LC values

represented

the average of at least two _assays.

RESULTS

Table I shows the

_propolis

concentration

specified

on the label of the commercial

products by

manufacturers,

and the resin concentration of these

_products

determined in the _present

_{investigation.}

The ethanolic

extracts from Pinus

_(E-Pinus)

or

_Eucalyptus

(E-Eucalyptus)

crude

_propolis,

_prepared

to

contain 33%

_(w/v)

of

_propolis

were found to

respec-tively.

Similar

_analysis

showed that

com-mercial

_products

contained a minimum of

1.5 to 14.2 maximum of %

(w/v)

resin. Results of antibacterial

_activity

of the different resins

(table II)

showed that B

cereus was

_always

the most sensitive

organ-ism,

followed

_by

B

_subtilis,

and S aureus.

The LC values obtained with these organ-isms characterized the low

_activity

of CE10,

tablet,

capsule

and

_powder

resins when

com-pared

to the

_higher

_activity

of resins from

CE1, CE7,

and

_E-Eucalyptus

and E-Pinus. In

_addition,

resins from

_capsuled

and pow-dered

_propolis

were effective

against

B

cereus, but

presented

low

activity against

B subtilis and S aureus.

DISCUSSION

The

_products

_analyzed

in the current

inves-tigation

showed different resin

concentra-tions

_{(table I),}

_regardless

of the

_propolis

concentration

_specified

on their labels. This

finding probably

reflects variable crude

propolis composition including

wax, wood

fragments,

debris,

and water

_(Ackermann,

1991).

The results of antibacterial

_activity

showed differences in the

_quality

of the resins

_{investigated.}

For

_instance,

CE10 was

found to have a

_high

resin content

_(12.3

_g

_%)

(table I),

but

_presented

a

high

LC value for

all

_{microorganisms}

tested

_{(table II).}

Other evidence

_showing

that resin source and

probably

their

_composition

can, at least for antibacterial _{purposes, be} more

important

than the concentration

itself,

was obtained

different chemical

_composition

of their resin is

_responsible

for the observed effects. Some

Eucalyptus species

are known for the antibacterial

_activity

of some

compounds

in their essential oils

_(Haiji

and

Fkih-Tetouani, 1993).

In

_addition,

_inadequate

storage

could also result in a

_propolis

with

high

resin content, but low antibacterial

activity.

Inactivation of antibacterial

_compounds

during

the

_{manufacturing}

process and uti-lization of low

_{quality propolis}

are some of

the

_possible

reasons for the low antibacterial

activity

of the

_capsule,

tablet and

_powder

but our data is too limited to form a

defini-tive conclusion. Since we had no

informa-tion on the industrial

_procedures,

nor on the

phytogeographic origin

of the

_propolis

con-tained in the commercial

_products,

we were

unable to determine which of these

possi-bilities was the most

_significant.

On the other

hand,

the

_{high activity}

of

_powdered

propolis

resin

_against

_only

B cereus

(table II)

indicates that this resin _{may have}

species-specific

action.

The _present

_findings

indicate that the antibacterial

_activity,

and

_perhaps

other

bio-logical properties

of

_propolis,

could not be correlated with their resin concentration but

mostly

with their chemical

_composition,

which can be variable

according

to the col-lection site

(Tomas-Barberán

et

_{al, 1993)}

and the

_quality

of the resin. Therefore the

methodology

used was effective in the

eval-uation of the

_quality

of antibacterial

_activity

from both

_propolis

and commercial

_products

containing

propolis.

Resin

_quality

and resin concentration can

be used to have access to the in vitro

activ-ity

of a

given

commercial

product.

This and

the in vivo

_activity

may be considered to

establish the

_posology

of each

_product.

Cur-rently

resin

_quality

and

_dosage

are not

ACKNOWLEDGMENTS

We thank

Fundação Tropical

de

_Pesquisa

e

Tec-nologia

André Tosello

_(Campinas,

SP, Brazil),

for

_supplying

the

_{microorganisms}

_(CCT) utilized in this

_{investigation.}

We are

_grateful

to J

_Burgess

for revision of the

_manuscript

and to L Orlando Rosário for his technical assistance.

Résumé —

_Propriétés

antibactériennes de la

_propolis

et des

_produits

renfermant de la

_propolis

du Brésil. Des

_produits

ren-fermant de la

_{propolis disponibles}

dans le

commerce au Brésil ont été

_analysés

du

point

de vue de leur concentration en résine

et _{de l’activité antibactérienne. Ils}

compre-naient dix extraits

_alcooliques

(CE1

à

_CE10)

et des

_produits

du commerce sous forme

solide. Les résines des

_produits

solides ont

été extraites à l’éthanol et nommées

E-tablette,

E-capsule

et

_E-poudre

selon leur formulation. On a

fait,

en outre, des extraits de deux

_propolis

brutes,

nommés E-Pinus

et

_E-Eucalyptus

_(propolis

_provenant de sites reboisés

_{respectivement}

en Pinus _{sp et}

Euca-lyptus sp).

La concentration en résine a varié

de

1,5

à

14,2

%

_{(tableau I),}

_{alors que les}

éti-quettes des

_produits

du commerce

annon-çaient

une teneur

_comprise

entre 10 et

33 %. La détermination de la « concentration

limite

_{» (LC),}

c’est-à-dire la concentration minimale en résine

_(μg

de résine _{par mL} du

milieu de

_culture)

_qui

inhibe

_{complètement}

la croissance

bactérienne,

a servi à évaluer

l’activité bactérienne

(tableau II).

Les valeurs de la LC ont montré que

l’organisme

le

_plus

sensible était

_toujours

Bacillus

cereus, suivi par B subtilis et

Staphylococ-cus aureus. Certains

produits,

comme

_{CE 10,}

ont

_présenté

une concentration en résine

éle-vée mais une faible activité bactérienne.

Cela montre _{que des résines} différentes ont

des

_qualités

différentes. La

_qualité

de la résine

_{dépend peut-être}

de son

_origine,

puisque

les deux

_{préparations}

de

_propolis

faites au laboratoire

(E-Pinus

et

E-Euca-lyptus)

avaient une activité contre S aureus

et B subtilis

_légèrement

différente. _La qua-lité de la résine _peut être aussi influencée

par le

procédé

de fabrication ou les condi-tions de

_stockage.

La résine de la

_propolis

en

poudre

avait une activité élevée contre B

cereus

uniquement,

ce

qui

montre _que son

action est

_spécifique

à

_l’espèce.

La méthode

présentée

ici

_peut

être utilisée pour évaluer la concentration en résine et l’activité anti-bactérienne in vitro des

_produits

du com-merce renfermant de la

_propolis.

Il faudrait

prendre

en _compte ces deux

_{caractéristiques,}

ainsi que l’activité in

vivo,

pour établir la

posologie

de chacun de ces

_produits.

propolis

/ contrôle

_qualité

/ activité anti-bactérienne /

_{Staphylococcus}

aureus /

bacillus subtilis / Bacillus cereus

Zusammenfassung —

Antibakterielle

Wirkung

von

_Propolis

und von

propolis-haltigen

Produkten in Brasilien. Der

Harz-gehalt

und die antibakterielle Aktivität von

propolishaltigen Handelsprodukten

aus

Bra-silien wurden untersucht. Die Proben setzten

sich aus 10 Alkolholextrakten

(CE

1 bis CE

10)

und aus festen Substanzen zusammen.

Aus den festen

_{Handelsprodukten}

wurde das Harz mit Ethanol

_extrahiert,

die Pro-dukte wurden

_entsprechend

als

E-Tablette,

E-Kapsel

und E-Pulver bezeichnet. Zusätz-lich wurden noch 2 andere rohe

Propolis-proben

extrahiert und als E-Pinus

(Propo-lis aus einer mit _{Pinus spec}

_{aufgeforsteten}

Region)

und

_{E-Eukalyptus (Propolis}

aus

einer mit

_Eucaliptus

spec

aufgeforsteten

Region)

bezeichnet. Die Harzkonzentration variierte in allen

_{Aufarbeitungen}

von

1,5

-14,2% (Tabelle I),

obwohl auf den Etiketten der

_{Handelsprodukte}

ein

_Harzgehalt

von 10

- 33%

angegeben

war. Die

_Bewertung

der

bakteriziden Aktivität

(Tabelle II)

erfolgte

durch die

_Bestimmung

der

_"limit

concen-tration"

_(LC),

also der

_geringsten

Konzen-tration

_(μg

_{Harz per ml}

_{Kulturmedium),}

bei der eine totale

_Hemmung

des Bakterien-wachstums

_erfolgte.

Die _LC-Werte

erga-ben,

daß Bacillus cereus immer der

emp-findlichste

_Organismus

war,

_gefolgt

von

der Produkte wie CE 10 hatte eine hohe

Harzkonzentration,

aber eine

_niedrige

bak-terizide

_Wirkung,

wodurch eine

unter-schiedliche

_Qualität

der verschiedenen Harze

_belegt

wird. Die

_{Harzqualität}

dürfte

von seiner Herkunft

_abhängen,

da die

Pro-polis -

Laborextrakte von Pinus und

E-Eucalytus

leicht unterschiedliche

Aktivitä-ten

_gegenüber

S aureus und B subtilis

zeigten.

Zusätzlich kann die

_{Harzqualität}

auch durch den

_{Herstellungsprozess}

oder die Art der

_Lagerung

beeinflußt werden. Das Harz vom

pulverisierten Propolis

hatte

nur _gegen B cereus eine hohe

Aktivität,

demnach ist dieses Harz

_{artspezifisch}

wirk-sam.Die hier beschriebene Methode ist

geeignet,

die Harzkonzentration und Akti-vität von Harz in vitro bei

Handelsproduk-ten mit

_Propolis

zu

überpüfen.

In

Verbin-dung

mit der in vivo Aktivität sollten diese Versuche die

_Möglichkeit

eröffnen,

eine

Dosiologie

für

_jedes

dieser Produkte

fest-zusetzen.

Apis

mellifera

/

_Propolis

/ Harz /

Qua-litätsprüfung

/antibakterielle

_Wirkung

/

Staphylococcus

aureus / Bacillus cereus / Bacillus subtilis

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