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134 Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008

Antimicrobial activity of selected Jordanian medicinal plant extracts against

pathogenic microorganisms

Basem F. DababnehDepartment of Nutrition and Food Processing, Faculty of Agricultural Technology, Al-Balqaa Applied University, P.O.Box 7474,

Al-Salt 19117, Jordan. *e-mail: [email protected], [email protected]

Received 14 December 2007, accepted 25 March 2008.

AbstractAntimicrobial activity of crude extracts from five commonly used medicinal plants in Jordan, Teucrium polium, Dianthus caryophyllus, Carthamus

tinctorium, Ammi visnaga and Artemisia herbaalba were evaluated against four pathogenic microorganisms over a wide range of concentrations

(50-5000 ppm). Minimum inhibition concentration (MIC) and the diameter of inhibition zone (DIZ) were determined by in vitrobioassays using

hole-plate diffusion method against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. All tested crude

plant extracts significantly exhibited antimicrobial activity and inhibited the growth ofGram-positive and Gram-negative bacteria as well as C.

albicans. The only exception was T. polium which showed no activity against the tested fungus at all tested concentrations. Antimicrobial activity

was directly proportional to the tested concentrations. The most active antimicrobial effect was recorded forD. caryophyllus extract against C.

albicans at MIC 800 ppm (DIZ = 25 mm), S. aureus at MIC 2000 ppm (DIZ = 20 mm) and P. aeruginosa at MIC 2000 ppm (DIZ = 10 mm).

A. visnaga extract possessed the highest inhibitory effect on E. coli at MIC 800 ppm (DIZ = 9 mm). This study shed the light on the ability of

extracts from Jordanian medicinal plants to combat pathogens which will help as natural antimicrobial agents as well as can be used in pharmaceutical

and food preservation systems. .

Key words:Natural antimicrobial, Jordanian medicinal plants, antibacterial activity, antifungal activity, MIC, DIZ.

www.world-food.netJournal of Food, Agriculture & Environment Vol.6(2) : 134-139. 2008

WFLPublisherScience and Technology

Meri-Rastilantie 3 B, FI-00980

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e-mail: [email protected]

Introduction

Medicinal plants have been prescribed and used with a strong

belief in their ability to cure diseases forcenturies48

. Over thepast 20 years, there has been a lot of interest in the investigation

of natural materials as sources of new antibacterial agents 6, 50,

insecticidal, acaricidal and cytotoxic activity 16. Plants used in

traditional medicine contain wide range of substances to treat

chronic as well as acute diseases. The substances that can either

inhibit the growth of microorganisms or kill them are commonly

considered for developing new drugs for treatment of various

infectious diseases 25. Many plant species are considered as

potential resource for treating diabetes and various diseases in

skin, liver, digestive and the urinary system 44. Herbal medicine in

the developing countries has evolved as an alternative solution

to health problems as a cheap source 25, 36. Therefore, medicinal

plants are intensely screened and tested for a wide range of

applications including pharmacology, pharmaceutical botany,

medical and clinical microbiology, phytopathology and food

preservation 12. Research on plants used as remedies in traditional

folk medicine can lead to identification of several biologically

active molecules from the 250,000 documented higher plant

species 33. The success achieved using medicinal plants and

herbal formulations therapeutically based on ethnomedicinal and

traditional use against a number of bacterial infections, raises

optimism about the future of phytoantibiotics. Based on the

indigenous and local knowledge, plants represent a rewarding

untapped source with a significant potential for developing

antimicrobial agents43

.Jordan is very rich in botanical diversity with more than 2000

wild plant species belonging to about 700 genera. As many as 485

species from approximately 99 plant families are categorized as

medicinal plants 37. Biologically active compounds and extracts

isolated from many plants species used in traditional herbal

medicine in Jordan have been the center of interest 2. However,

few studies on the antimicrobial activities of the Jordanian

medicinal plants were carried out. Therefore, there is still a potential

need to screen their effects on various pathogenic microorganisms.

The aim of this study was to investigate the antimicrobial inhibitory

effect of ethanolic crude extracts obtained from five medicinal

plants commonly used in traditional medicine in Jordan. Extracts

of Teucrium polium, Dianthus caryophy llus , Carthamus

tinctorius,Ammi visnaga andArtemisia herbaalba were usedagainst four microorganisms (Staphylococcus aureus,

Pseudomonas aeruginosa, Escherichia coli and Candida

albicans).

Materials and Methods

Plant materials: Five medicinal plants namely Teucrium polium,

Dianthus caryophyllus, Carthamus tinctorius, Ammi visnaga

and Artemisia herbaalba were either collected from the field or

purchased from local market. Scientific name, English common


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Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008 135

name and parts used regarding each plant are summarized in

Table 1. Authentication and identification of plant material was

confirmed by Dr. Ihab H. Al-Gabbeish, Al-Balqa Applied

University, Al-Salt College-Head Department of Biological

Sciences. The common medicinal uses and active constituents

of the plants under this study are reviewed and well documented

(Table 1).

Preparation of plant extracts: Plant material was dried in theshade at room temperature and powdered using an electric mill.

Two hundred and fifty grams of each plant powder were soaked

in 1.25-1.5 L of 95% ethanol for 5 days at room temperature. The

mixture was shaken daily for regular infusion. On the sixth day,

the extract was filtered using Whatman filter paper No. 1. Dry

crude extract was produced by evaporating ethanol under low

pressure using a rotary vacuum evaporator at 60C. The final

crude extracts were stored in labeled sterile glass vials at -20C

until used for the antimicrobial activity test 23.

Microbial test suspensions: Test organisms used in this study

are Gram-positive bacteria (Staphylococcus aureus), Gram-

negative bacteria (Pseudomonas aeruginosa and Escherichia

coli) and yeast (Candida albicans). The identified clinicallyresistant strains were obtained from the Jordan University

Hospital. The microorganisms were maintained on slants of

nutrient agar (NA) at 4C. The inoculums were incubated

overnight in nutrient broth at 37C to produce dense microbial

suspension of approximately 106cfu/ml.

Screening for antimicrobial activity:Hole-plate diffusion method

was used for studying the antimicrobial activity and determining

the minimum inhibition concentrations (MICs) 8. Each inoculum

from dense bacterial suspension containing 106 bacterial cells/ml

was spread on the surface of nutrient agar medium (NA) while the

C. albicans was spread on the surface of potato dextrose agar

(PDA). Three holes were made on the media using 6 mm diameter

sterile cork-borer. The dried plant extracts were dissolved in

dimethylsulfoxide (DMSO) to provide a stock solution with the

final concentration of 5000 ppm. A range of serial dilutions were

prepared from the stock solution to provide 4000, 3000, 2000, 1000,

800, 600, 400, 200, 150, 100 and 50 ppm. Each hole (diameter 6 mm)

was filled with 50 l from each dilution of plant extract.The inoculated agar plates were incubated at 37C for 24 hr. After

the incubation period, bioactivity was determined by the

measurement of the diameter of inhibition zone (DIZ) around each

hole in mm. The inhibition zone was recognized as the area

surrounding the hole with no growth of the tested pathogens.

Control plates received only DMSO in NA and PDA without plant

extracts and were run following the same procedure as above.

The values reported for DIZs were the average of three replicates.

Minimum inhibition concentrations (MICs) were taken as the

lowest concentration at which observable growth was inhibited

with no significant differences at higher concentrations used.

Statistical analysis: Data of DIZ are presented as means of three

replicates and analyzed using factorial arrangement in completerandom design (CRD) with SAS version 9 software package 46.

LSD analysis was used to compare means. Significant differences

were defined at p0.05.

Results and Discussion

Extracts obtained from T. polium, D. caryophyllus, C. tinctorium,

A. visnaga andA. herba-albawere tested against S. aureus (Table

2),P. aeruginosa (Table 3), E. coli (Table 4) andC. albicans (Table

5). Antimicrobial activity was determined by measuring the diameter

of inhibition zones (DIZ in mm) and the minimum inhibitory

Table 1. Ethnobotanical data and active constituents of studied plants.

Botanical source Common medicinal uses Active constituentsFamily: Labiatae (Lamiaceae)

Scientific name: Teucrium poliumUsed part: Aerial parts

English name: Mountain germander

Diuretic, diaphoretic, tonic, anti-spasmodic and cholagogic20. Antipyretic and anti-bacterial 34. Anti-inflammatory andanti-rheumatic 49. Hypoglycemic 14. Hypolipidemic 40. Anti-

oxidant 10. Anti-nociceptive 4.

Diterpenoids9, neoclerodane

diterpenes 5, sesquiterpenoids 24.Flavonoids, iridoids, crisiol 26.

Family: Caryophyllaceae

Scientific name: Dianthus caryophyllusUsed part: Petals

English name: carnation, clove pink

Skin toner38, antifungal 11, relief of acute dermatitis, tooth

pain, vomiting and gasteritis, digestive function stimulant,antispasmodic.

Flavonoids 11, anthocyanins 35.

Dianthramides 39. Antiretroviralproteins 31 and phenols 11.

Family: Compositae (Asteraceae)

Scientific name: Carthamus tinctoriusUsed part: Flowers

English name: Safflower, bastard; falsesaffron, saffron thistle.

Anti-inflammatory 22, treatment of hyberlipemia,

arteriosclerosis, osteoporoses and bone resorption,antimicrobial immunomodulating, and anti-allergic 18.

Haemostatic agent, promoting blood coagulation, cardiactonic and diuretic 28. Treatment of yin deficiency of liver

and kidney, fever, night sweat and dizziness 17.

Flavonoid glycosides 54.

Carthamine 29, fatty acids 27,annexin 19 and placenta

protein 4 42.

Family: Umbelliferae (Apiaceae)Scientific name: Ammi visnaga

Used part: FruitsEnglish name: Khella, kellin, tooth pick,

bishops weed.

Hypoglycemic 51. Bronchodilator 15. Vasodilator andmuscle relaxant 13.

Coumarins 13.

Family: Compositae (Asteraceae)Scientific name: Artemisia herba-alba

Used part: Aerial partsEnglish name: Herba-alba, wormwood,

wormseeds, shih, santonica, white

mugwort.

Treatment of neurological and cardiac disorders and sexualweakness 45. Phytotoxic and antimicrobial 53.

Hypoglycemic 1 and in the treatment of jaundice 32.Antispasmodic, anti-pyretic, eye diseases, hair pomades,

antibacterial and anti-inflammatory 33, anti-tumor, anti

ulcerogenic, diuretic33

, antihelminthic7

Falconoid 45. Alkaloids 33.Monoterpenes 21, Sesquiterpene

lactones 7.


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concentration (MIC in ppm) over a range of

concentrations 5000, 4000, 3000, 2000, 1000, 800, 600,

400, 200, 150, 100 and 50 ppm. The interaction effect of

microorganisms, plants and extract concentrations were

highly significant at p0.05. Plant extracts showed

different antimicrobial activity against the tested

pathogens and the diameter of inhibition zone was

directly proportional to the increase in plant extract

concentration reaching a plateau.Table 2 shows the screening test of plant extracts on

S. aureus. All tested plant extracts were active against

tested bacteria with variable inhibitory effects. D.

caryophyllus exhibited significantly the highest

antibacterial activity at MIC 2000 ppm with DIZ 20 mm.

T. polium, C. tinctorium and A. herba-alba caused

inhibitory effect at MIC 3000 ppm with DIZ ranging

from 14-16 mm.A. visnaga required higher MIC (4000

ppm) to reach similar inhibition effect.

P. aeruginosa (Table 3) had a marked significant

sensitivity towards both D. caryophyllus and C.

tinctorium which inhibited at MIC 2000 ppm with DIZ

10 and 13 mm, respectively. A. visnaga inhibitedP.

aeruginosa at MIC 3000 ppm with DIZ 11 mm. T. poliumandA. herba-alba required higher concentration (MIC

4000 ppm) to cause similar effect (Tables 3 and 6).

The extract ofA. visnaga significantly revealed the

highest activity (MIC 800 ppm; DIZ 9 mm) againstE.

coli (Tables 4 and 6).D. caryophyllushad a remarkable

inhibitory effect at MIC 1000 ppm with DIZ 15 mm, while

T. polium possessed moderate activity at 2000 ppm with

DIZ 10 mm. C. tinctorium andA. herba-albaboth had

the lowest inhibition activity in comparison to plant

extracts tested onE. coli.

The growth ofC. albicans was considerably inhibited

by D. cary ophy llus extract at MIC 800 ppm

(DIZ = 25 mm), followed byC. tinctorium at MIC 1000

ppm (DIZ = 9 mm), A. visnaga at MIC 2000 ppm

(DIZ = 25 mm) andA. herba-albaat MIC 4000 ppm (DIZ

= 13 mm). T. polium had no anticandidal effect at all

tested concentrations (Tables 5 and 6).

Overall MICs of active plant extracts and diameter of

inhibition zones are shown in Table 6. The inhibition

activity of plant extracts on test strains was in

decreasing order according to the minimum inhibition

concentration as follows: T. polium:E. coli > S. aureus

> P. aeruginosa > C. albicans; D. caryophyllus:

C. albicans >E. coli >P. aeruginosa and S. aureus;

C. tinctorium: C. albicans >P. aeruginosa > S. aureus

> E. col i; A. visnaga: E. col i > C. albicans >

P. aeruginosa > S .aureus; A. herba-alba: S. aureus >C. albicans,P. aureus andE. coli.

All tested plant extracts demonstrated broad spectrum

antimicrobial activity against tested microorganisms

with variable inhibitory effect, except T. polium had no

antifungal effect at all tested concentrations. This could

be related to the variations in the quality and quantity

of active compounds in the plant extracts (Table 1).

Previous papers indicated that antimicrobial activity

of botanical extracts is related to the presence of

A.

herba- alba

A.

visnaga

C.

tinctorium

D.

caryophyllus

T.

polium

Concentration

of plantextract (ppm)

0 f0 f0 f0 f0 f0

0 f0 f0 f0 f0 f500 f0 f0 f9 cde0 f100

0 f0 f0 f10bcde0 f150

0 f0 f0 f10bcde0 f2000 f7 e7 e13 abcd0 f400

0 f7 e8 de14 abc8 de6000 f9 cde8 de14 abc9 cde8000 f9 cde10bcde15 ab9 cde1000

0 f9 cde12 abcde15 ab10 bcde200013 abcd9 cde12 abcde15 ab10 bcde3000

17 a9 cde13 abcd15 ab10 bcde400017 a9 cde13 abcd15 ab10 bcde50003.5410.911.534.767.25LSD

Table 4.Antimicrobial activity (DIZ mean in mm) of medicinal

plants at various concentrations against Escherichia coli*.

* Values were mean of triplicate readings.

Means with different superscript letters are significantly different (P0.05) at plant extract concentrations used.

A.herba- alba

A.visnaga

C.tinctorium

D.caryophyllus

T.polium

Concentrationof plant

extract (ppm)

0 f0 f0 f0 f0 f0

0 f0 f0 f0 f0 f50

0 f0 f0 f0 f0 f100

0 f0 f7 e7 e0 f150

0 f0 f7 e7 e7 e200

0 f0 f7 e8 de7 e400

0 f0 f7 e8 de8 de600

0 f10 bcde8 de9 cde8 de800

0 f10 bcde8 de9 cde8 de1000

8 de10 bcde13 abc10 bcde8 de2000

14 ab11 abcd13 abc10 bcde9 cde3000

16

a

11

abcd

13

abc

10

bcde

10

bcde

400016 a12 abcd13 abc10 bcde10 bcde5000

5.654.145.485.634.53LSD

Table 3.Antimicrobial activity (DIZ mean in mm) of medicinal plants

at various concentrations against Pseudomonas aeruginosa.

Means with different superscript letters are significantly different (P0.05) at plant extract concentrations used.

* Values were mean of triplicate readings.

A.

herba- albaA.visnaga

C.

tinctorium

D.

caryophyllusT. polium

Concentration

of plantextract (ppm)

0 f0 f0 f0 f0 f0

0f

0f

0f

0f

0f

50

0f

0f

0f

8ij

0f

100 0 f0 f0 f8 ij0 f150

0 f7 j0 f9 hij7 j2000 f7 j0 f13 defgh7 j400

0 f7 j11 fghij14 cdefg9 hij6000 f8 ij11 fghij17 abcd10 ghij800

0 f12 efghi13 defgh18 abc11 fghij10008 ij12 efghi14 cdefg20 a13 defgh2000

16 abcde13 defgh15 bcdef20 a14 cdefg300016 abcde15bcdef15 bcdef20 a14 cdefg4000

16abcde

15bcdef

15bcdef

20a

14cdefg

50003.74.371.615.955.1LSD

* Values were mean of triplicate readings. Means with different superscript letters are significantly different (P0.05) at plant extract concentrations used.

Table 2.Antimicrobial activity (DIZ mean in mm) of medicinal

plants at various concentrations against Staphylo-

coccus aureus*.


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Plantspecies

A.herba-alba

A.v

isnaga

C.

tinctorium

D.caryophyllus

T.polium

Microorganism

DIZ

MIC

DIZ

MIC

DIZ

MIC

DIZ

MIC

DIZ

MIC

16

3000

15

4000

15

3000

20

2000

14

3000

S.aureus

14

4000

11

3000

13

2000

10

2000

10

4000

P.aeruginosa

17

4000

9

800

13

4000

15

1000

10

2000

E.coli

13

4000

25

2000

9

1000

25

800

N.A

N.A

C.albicans

Table6.

Minimuminhibitoryco

ncentrationanddiameterinhibitionzoneofcrudeplantextracts

againstpathogenicmicroorganisms.

DIZ=Diameterofinhibitionzone(meaninmm).

MIC=Minimuminhibitionconcentration(ppm).

N.A=Notactive.

A.herba-alba

A.visnaga

C.tinctorium

D.caryophy

llus

T.polium

Concentration

ofplant

extract(ppm)

0f

0f

0f

0f

0f

0

0f

0f

0f

0f

0f

50

0f

0f

0f

0f

0f

100

0f

0f

0f

9de

0f

150

0f

0f

0f

11de

0f

200

0f

10de

7e

11de

0f

400

0f

10de

7e

21bc

0f

600

0f

11de

7e

25b

0f

800

0f

18c

9de

25b

0f

1000

0f

25b

9de

25b

0f

2000

0f

25b

9de

25b

0f

3000

13d

25b

10de

26b

0f

4000

13d

25b

10de

26b

0f

5000

2

6.67

2.43

8.76

-

LSD

Table5.

Antimicrobialactivity

(DIZmeaninmm)ofmedicinalplants

atvariousconcentrat

ionsagainstCandidaalbicans*.

*Valuesweremeanoftriplicatereadings.

Meanswithdifferentsuperscriptlettersaresignificantlydifferent(P0.0

5)atplantextractconcentrationsused.

different chemical agents including essential oils, flavonoids,

anthocyanins and terpenoids in addition to other compounds of

phenolic nature or containing free hydroxyl groups 41. Fungitoxic

property ofD. caryophyllus againstFusarium oxysporum was

reported to be due to a group of phenolic constituents such as

kaempferide triglycoside 11. The absence of antifungal effect

against C. albicans by the range of tested concentrations of

T. polium ethanolic extract might be related to absence of active

constituents or needed to use higher concentrations. Suchpostulation requires further investigations.

Results from this study indicate that tested extracts possessed

variable antimicrobial effects against both Gram-positive and Gram-

negative bacteria as well as C. albicans. However, the plants

differed significantly in the activity against tested microorganisms.

These differences could be attributed to structural nature of the

microorganisms 52 and plant constituents. The optimal

effectiveness of medicinal plants may not be due to one active

constituent, but to the combined actions of different plant

constituents 3. Moreover, the differences observed in antimicrobial

activities of the investigated plant extracts suggest the

susceptibility variations of microorganisms to various chemical

components. Earlier results 30, 47 support our findings that the

composition of essential oil depends on the plant species, thechemotypes and the climatic conditions, which lead to variation

of their antimicrobial activities.

From the above findings it could be concluded that the tested

plant extracts exhibit a broad spectrum of activity against various

microorganisms. Further investigations to determine bactericidal,

bacteriostatic, fungicidal or fungistatic effects are recommended

with emphasis on the identification of the active antimicrobial

chemical constituents of these commonly used Jordanian medicinal

plants. Results of the present study should be considered for the

possible application of plant extracts as natural bacteriostatic and

bactericidal component in various products and as natural

preservatives extending the pharmaceutical and dietary products

shelf life, as we believe is of great importance.

Acknowledgements

The author wishes to express his appreciation and thanks to Etekal

Z. Khalaf for her laborious work, Reem M. Mohsen for technical

assistance and Ahmad Al-Gabbiesh for his continuous support.

Special thanks to Dr. Mayyada B. Shehadeh for insightful

discussion and revising the manuscript.

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