Studies on the antimicrobial activity of Nigella sativa seed (black cumin)

Journal of Ethnopharmacology, 34 (1991) 215-278 Elsevier Scientific Publishers Ireland Ltd.

275

Studies on the antimicrobial activity of Nigella sativa seed (black cumin) M.S.M.

Hanafy

and M.E. Hatem

Departments of Pharmacology and Microbiology, Faculty of Veterinary Medicine, Cairo Univrrsit_v, Cuiro. Gix (Accepted

(Egypt)

June 5. 1991)

Filter paper discs impregnanted with the diethyl ether extract of Nigellu sutiw seeds (25-1100 pg extract/disc) caused concentrationdependent inhibition of Gram-positive bacteria represented by Stuphy/ococcu.~ aureus. Gram-negative bacteria represented by Pseudomonas aeruginosa and Escherichia coli (but not Salmonella typhimurium) and a pathogenic yeast Cot&to ulbicuns. The extract showed antibacterial synergism with streptomycin and gentamicin and showed additive antibacterial action with spectinomycin, erythromycin, tobramycin, doxycycline, chloramphenicol, nalidixic acid, ampicillin, lincomycin and sulphamethoxyzole-trimethoprim combination. The extract successfully eradicated a non-fatal subcutaneous staphylococcal infection in mice when injected at the site of infection. Key words: NigeNa sativa: antimicrobial;

antibiotics;

bacterial

infection;

food poisoning.

Introduction

Materials and Methods

Previous work on Nigella sativa L. (black cumin; family, Ranunculaceae) has shown that seed extracts inhibit the growth of Escherichia co/i, Bacillus subtilis and Streptococcus feacalis (Saxena and Vyas, 1986). Methanolic extracts of Nigella sativa seeds have been reported to prevent adhesion of viable cells of Streptococcus mutans to smooth surfaces; therefore, it was suggested that this plant can be of value in preventing dental plaques and caries (Namba et al., 1985). The aim of the present work was to evaluate the antimicrobial activity of a N. sativa seed extract on certain pathogenic bacteria representing Gram-negative and Gram-positive bacteria and a pathogenic yeast (Candida albicans). Test organisms used in this study are known to be the cause of disease in man and animals and some are known to be involved in food poisoning.

Seed source Seeds of Nigella sativa were obtained from com-

Correspondence to: Dr. Veterinary Pharmacology, Cairo

University,

Cairo,

M. Samih M. Hanafy. Faculty of Veterinary Giza.

mercial sources and were known to be from freshly harvested crops grown in Egypt. Seeds were authenticated at the Vivi Tackholm Herbarium, Cairo University. Preparation of ether extract discs

Two hundred grams of ground seeds of Nigella sativa were soaked in 500 ml of diethyl ether for 6 h, then filtered. Ether was evaporated (60°C) leaving a clear dark brown oily liquid. The yield of this extract was 11% v/w in terms of dry starting material. Serial dilutions of the ether extract were prepared in ether so that 1 ml contained 100 times the amount of the extract required per disc. Aliquots (1 ml) of these dilutions were transferred to sterile bottles containing batches of 100 discs (6 mm in diameter) of filter paper (Whatmann No. 1). Bottles were placed in a water bath at 50°C with occasional shaking to allow an even distribu-

Dept. of Medicine,

Egypt.

0378-8741/$03.50 0 1991 Elsevier Published and Printed in Ireland

Scientific

Publishers

Ireland

Ltd.

276

tion of the extract between discs until complete evaporation of ether had been achieved. Antimicrobial sens~tivit.y testing Antimicrobial activity was semiquantitatively determined using the disc-agar diffusion techniques described by Finegold and Martin (1982). Local isolates of Staphy/ococcu.s aureus and Candida a~bicans and standard strains of Salmonella typhimurium (ATCC 14028), Escherichia coEi (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) (Bactrol Discs, Difco) were used. Overnight cultures of these strains were prepared in Mueller-Hinton broth, standardized to match McFarland No. 0.5 barium sulphate tubes and used to inoculate Mueller-Hinton agar plates. Discs impregnated with different amounts of the ether extract were placed on the surface of these plates and incubated at 37°C for 18 h. Diameters of the zones of inhibition were recorded. Commercial diagnostic antibiotic sensitivity testing discs (Oxoid) were also used for comparison. Interaction between extract and antibacterial drugs Disc diffusion t~hniques were used for this study as described by Krogstad and Moellering (1979). Two discs, one impregnated with the ether extract of Nigeila sativa (400 &disc) and the other disc containing an antibiotic (Oxoid, U.K.) were placed on the surface of an inoculated MuellerHinton agar plate. The distance between the two discs was slightly greater than the sum of the radii of the zones of inhibition when tested separately. Plates were incubated at 37°C for 18 h. Bacterial growth at the area between the two discs was examined and results were interpreted as: (i) antibacterial synergism when bridging between the zones of inhibition was observed, (ii) antibacterial antagonism when truncation was observed or (iii) additive antibacterial activity when two independent circular zones of inhibition were observed (Krogstad and M~llering, 1979). Effects of the extract on non-fatal staphylococcal infection in mice The experimental design was as described by Cleeland and Grunberg (1979). Fifteen male

albino mice (weight range: 22-25 g) were divided into three equal groups. Each mouse was given a subcutaneous 0.2 ml injection of an overnight culture of Staph_v~o~occusaureus in the loose skin between the shoulders. The site of staphylococcal infection was then infiltrated with 1 ml sterile saline containing either 2 mg of Nigella saliva extract (suspended with the aid of a few drops of glycerine) (group I) or 0.5 mg gentamicin (Garamycin, Schering) (group 2, positive control) or a few drops of glycerine (the extract vehicle) (group 3, negative control). Mice were left for 24 h, then killed. A swab was taken from the site of infection in each mouse and cultured on a plate of nutrient agar, incubated at 37’C for 24 h, then examined for bacterial growth. Plates showing no bacterial growth or the presence of no more than 10 colonies indicated successful treatment of the staphylococcal infection (Cleeland and Grunberg, 1979).

15 -

5 *

lo-

!!! 5-

if! !I

0

25

% 20

25

50

X00 xl0

400Eso?l Stph

15 10

Fig. I. The relationship between the logarithm disc content of the ether extract of Nigella saliva and the zone of inhibition of plates of Mueller-Hinton agar inoculated with Staphylococcus aureus (Stph), Escheriehia coli (Esch), Pseudomonas aeruginosa (Psdmf or Candida olbicvns (Cand).

271

TABLE

I

MEAN DIAMETER OF ZONES OF INHlBlTlON AROUND 6-mm DISCS CONTAINING EITHER THE ETHER EXTRACT OF NIGELLA SATIVA OR COMMERCIAL DIAGNOSTIC ANTIBIOTIC SENSITIVITY TESTING DISCS ON MUELLERHINTON AGAR PLATES INOCULATED WITH STAPHYLOCOCCUS AUREUS (Stph), ESCHERlCHlA COLl (Esch), SALMONELLA

TYPHIMURIUM

(Salm).

Drug

N. surivrr extract

Lincomycin Streptomycin Gentamicin Spectinomycin Erythromycin Tobramycin Doxycychne Chloramphenicol Nahdixic acid Ampicillin Sulphamethoxyzole

+ trimethoprim

PSEUDOMONAS

AERUGINOSA

(Psdm) OR CANDIDA

ALBICANS

(Cand)

N = 3

Disc content

Zone of inhibition

(cm)

(wg)

Stph

Esch

Salm

Psdm

Cand

400 200 100 50 25 2 IO IO 100 I5 IO 30 30 30

2.1 1.5 I.0 0.8 0.7 2.2 1.8 1.7 1.2 2.1 2.1 2.3 2.7 2.0

2.5 1.2 0.7 0.7 0.7 0.0 2.5 I.8 2.0 I.0 2.0 2.6 2.0 2.2

0.0 0.0 0.0 0.0 0.0 0.0 2.2 1.7 2.5 0.9 1.8 2.2 2.5 1.5

2.5 1.3 I.0 0.0 0.0 0.0 I.5 1.9 I.0 0.7 1.7 0.8 2.1 1.3

2.1 1.6 1.2 8.0 0.0 NT NT NT NT NT NT NT NT NT

10 23.75 + 1.25

3.0 2.0

I.8 2.7

1.6 2.3

0.9 0.8

NT NT

NT = not tested.

TABLE

2

INTERACTIONS

BETWEEN

THE ETHER

Drugs

Spectinomycin Erythromycin

OF NIGELLA

Ampicillin Lincomycin Streptomycin Gentamicin + trimethoprim

antibacterial

action;

AND ANTIBACTERIAL

Organism

(ag)

Stph

Esch

Psdm

100 15

A A A A A A A A S S A

A A

A A

A A A A A A S S A

A A A A A A S S A

1

Sulphamethoxyzole

SAT/VA (400 &disc)

Disc content

IO 30 30 30 IO

Tobramycin Doxycycline Chloramphenicol Nalidixic acid

A = additive

EXTRACT

1; IO 23.75 + 1.25 S = synergistic

antibacterial

action.

DRUGS

278

ReHlhS In vitro.antibacterial activity Discs containing the ether extract of Nigella sativa caused inhibition zones in plates inoculated with Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans but not Salmonella typhimurium. The diameter of zone of inhibition was proportional to the logarithm of disc drug content. This is shown in Fig. 1 and Table 1. Interaction between extract and antibacterial

drugs

Bridging (antibacterial synergism) was observed between the inhibition zones caused by extractimpregnanted discs and streptomycinand gentamicin-impregnated discs (Table 2). Two circular zones of inhibition without bridging or truncation (additive antibacterial action) were observed when the extract was tested with other drugs (Table 2). Antibacterial antagonism was not seen. Effect of the extract on non-fatal staphyloccocal infection in mice All nutrient agar plates inoculated with swabs taken from the site of the staphylococcal infection in mice in group 1 (treated with Nigella sativa extract) and group 2 (treated with gentamicin) showed either no growth or no more than one colony. Plates inoculated with swabs from mice in group 3 (treated with saline) showed ateas of heavy bacterial growth forming a continuous layer not separated into colonies.

extract used data suggest of the extract positive than

in this study (25400 pg/disc). The (Fig. 1) that the antibacterial action may be more pronounced on Gramon Gram-negative bacteria. Nigella sativa extract showed antibacterial synergism with streptomycin and gentamicin. These findings suggest that preparations from that plant, if given with these antibacterial drugs, would enhance their efficacy. Results of in vivo studies show that the ether extract successfully eradicated localized infection with Staphylococcus aureus in mice. Thus, Nigella sativa seeds can possibly provide the basis for a for the successful antibacterial preparation chemotherapy of localized infections. Other possible applications of Nigella sativa is in the preservation of food and prevention of food poisoning since some of the bacterial species inhibited by Nigella sativa extract are known to be involved in food poisoning. The odour and taste of the ether extract of Nigella sativa are weak and not disagreable, if not pleasant, which would favour its use in food technology. The possible use of extracts of Nigella sativa seed in the prevention of dental plaques and carries has been previously suggested (Namba et al., 1985).

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532-557. Krogstad,

Results of this work show that microgram concentrations (25400 &disc) of the ether extract of Nigella sativa seeds inhibited growth of several species of pathogenic bacteria representing Grampositive bacteria (Staphylococcus aureus), Gramcoli and bacteria (Escherichia negative Pseudomonas aeruginosa) and a pathogenic yeast (Candida albicans). Salmonella typhimurium was non-sensitive to the range of concentrations of the

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