Antimicrobial activity against pathogenic microorganisms by extracts ...

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31261, Saudi Arabia. 2 Department of Nutrition and Food Processing, Faculty of Agricultural Technology, Al-Balq'a Applied. University, Al-Salt 19117, Jordan.3 ...
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Journal of Food, Agriculture & Environment Vol.7 (2) : 103-106. 2009

Antimicrobial activity against pathogenic microorganisms by extracts from herbal Jordanian plants Amjad Khalil 1*, Basem F. Dababneh 2 and Ahmad H. Al-Gabbiesh

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Biotechnology Research Group, College of Sciences, King Fahd University of Petroleum and Minerals, P.O. Box 665, Dhahran 31261, Saudi Arabia. 2 Department of Nutrition and Food Processing, Faculty of Agricultural Technology, Al-Balq’a Applied University, Al-Salt 19117, Jordan.3 Department of Applied Medical Sciences, Al-Zarka College, Al-Balq’a Applied University, Al-Zarka 313, Jordan. *e-mail: [email protected], [email protected], [email protected] Received 15 January 2009, accepted 10 April 2009.

Abstract The aim was to study antimicrobial activity of a group of herbal medicinal plants, including Achillea biebersteinii, Phlomis viscosa, Ainworthia trachycarpa, Solanum elaeagnifolium, Arum hygrophilum, Varthemia iphionoides, Crupina crupinastrum, Teucrium polium, Achillea santolina, Micromeria nervosa, Chenopodium murate, Ballota philistaea, Onosma roussaei, Fagonia mollis, Marrubium vulgare, Calotropis procera, Salvia hierosolymitana, Ballota undulata, Hallogeton alopecuroides, Scrophularia hierochuntica and Nonea melanocarpa, grown in Jordan. The tested medicinal plants showed different antimicrobial activity in different extract amounts (5, 10, 15, 20, 40, 60, 80 and 100 ppm) against the tested microorganisms. Minimum inhibition concentration (MIC) and the diameter of inhibition zone (DIZ) were determined by in vitro bioassays using hole-plate diffusion method against two bacterial species, Staphylococcus aureus and Pseudomonas aeruginosa, and one fungi, Candida albicans. Extracts of most tested plants, except Arum hygrophilum and Micromeria nervosa, showed antimicrobial activity against some of the tested microorganisms. The antimicrobial activity was highest in Crupina crupinastrum extract (5, 10, 15, 20, 40 and 60 ppm) which gave the largest inhibition zone (DIZ 24 mm) at 60 ppm followed by the extracts from Achillea biebersteinii with highest effect at 60 ppm (DIZ 18 mm). This study shed the light on the antimicrobial ability of extracts from Jordanian medicinal plants, which can be used as natural antimicrobial agents in pharmaceutical and food preservation systems. Key words: Herbal medicinal plants, anti-bacterial activity, anti-fungal activity.

Introduction It is well known that infectious diseases account for high proportion of health problems, especially in the developing countries. Microorganisms have developed resistance to many antibiotics, and this has created immense clinical problem in the treatment of infectious diseases 5. This resistance has increased due to indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases. This situation forced scientists to search for new antimicrobial substances from various sources, such as medicinal plants 10. The first three significant antibiotics, namely, tyrothricin (of bacterial origin), penicillin (a rediscovery of an antibiotic of fungal origin) and actinomycin (a product of actinomycetes) were used20. Due to the widespread and often indiscriminate use of antimicrobial drugs, many microorganisms have acquired resistance to specific antibiotic treatments, and these strains are particularly evident in the hospital environment 6. This has created immense clinical problem in the treatment of infectious diseases 5, 13. In addition to this problem, antibiotics are sometimes associated with adverse effects on host which include hypersensitivity, depletion of beneficial gut and mucosal microorganisms, immuno-suppression and allergic reactions 7. Antimicrobials of plant origin are efficient in the treatment of infectious diseases mitigating simultaneously many of the side effects that are often associated with synthetic antimicrobials 8. Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009

The antimicrobial activities of plant oils and extracts have formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies 12, 16. Moreover, the increasing use of plant extracts in the food, cosmetic and pharmaceutical industries suggests that in order to find active compounds, a systematic study of medicinal plants is very important 14. Medicinal plants have been tested for biological, antimicrobial and hyperglycemia activity 3, 4, 21.They have also been tested for antiallergenic, antihelminthic, hepatoprotective, analgesic, antipyretic, antileishmania and insecticidal activities 1, 11, 19. The aim of this study was to test the activity of plant extracts of medicinal plants on inhibiting the growth of pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, and one pathogenic fungus, Candida albicans. Materials and Methods Plant material: The plant materials were collected from different areas in Jordan between February and May of 2004. These plants were identified by Dr. Maha Siyof, The National Center for Agricultural Research and Technology Transfer, Jordan. In this study, the aerial parts of plants were used for testing their antimicrobial activity. Scientific and common names of the tested plants and common medicinal uses are summarized in Table 1. 103

Preparation of extracts: Plant materials were dried in shade at room temperature and ground by using a blender. Two hundred and fifty gram of plant powder was soaked in 1.25-1.5 L of 95% ethanol for 5 days at room temperature. The mixture was mixed daily for regular infusion. After a five-day period, the extract was filtered by using Whatman filter paper No. 1. The filtrate was dried by using a rotary evaporator at 60°C. The dried extract was stored in sterile glass bottles at -20°C until use 9. Microorganisms: Two bacterial species, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, and yeast Candida albicans were used. These microorganisms were obtained from the Hospital of the University of Jordan. Screening of antimicrobial activities: Inoculums containing 106 bacterial cells/ml or 108 cells/ml for yeast were spread on nutrient agar medium. Antimicrobial activity test was carried out by using the hole-plate diffusion method. Holes were made on the media by using 8 mm cork borer. The dried plant extracts were dissolved in dimethylsulfoxide (DMSO) to a final extract amount of 500 ppm. Each hole (diameter 8 mm) was filled with 120 µl (60 ppm) of plant extract. The plant extracts were tested at different concentrations (5, 10, 15, 20, 40 and 60 ppm). The inoculated agar plates were incubated at 37ºC for 24 h. After the incubation period, the diameter of inhibition zone to each hole was measured in mm. Negative controls DMSO and 96% ethanol showed no antimicrobial activity against any of the tested microorganisms. Statistical analysis: Data of DIZ, presented as means of three replicates, were analyzed using factorial arrangement in complete random design (CRD) with SAS Version 9 software package 17. LSD analysis was used to compare means. Significant differences were defined at p•0.05.

Results and Discussion Extracts of most tested plants, except Arum hygrophilum and Micromeria nervosa, showed antimicrobial activity against some of the tested microorganisms. The antimicrobial activity was highest in Crupina crupinastrum extract (5, 10, 15, 20, 40 and 60 ppm) which gave for S. aureus the largest inhibition zone (DIZ 24 mm) at 60 ppm followed by the extracts from Achillea biebersteinii with highest effect at 60 ppm (DIZ 18 mm). P. aeruginosa was inhibited by most of used plant extracts. C. albicans was inhibited by some extracts, the inhibition being highest by Halogeton alopecuroides extract at concentrations of 40 and 60 ppm (Tables 2 and 3). Extracts from Achillea biebersteinii (collected from Turkey) showed no antimicrobial activity against P. aeruginosa but inhibited S. aureus growth at 300 ppm 15. In this study, extracts from A. biebersteinii inhibited S. aureus growth at 10 ppm which means that using higher concentration of the plant extract will increase the inhibition efficiency against S. aureus. This variation could be due to the different methods used or due to the variation in quality or composition of the same plant species or could be due to differences in the environmental conditions and genetic variation. The essential oils of Salvia fruticosa, collected from different geographic locations in the island of Crete, gave different response when all plants were cultured under the same condition for three years 18. This result was suggested to be due to variation in quality and composition of plants and might depend more on the genetic background of the plants and less on climate variation. Extracts from Achillea bierbersteinii and Achillea santolina inhibited P. aeruginosa, with the highest effect at 60 ppm (Tables 2 and 3). In another related work, alcoholic and oil extract from Achillea falcata inhibited the growth of S. aureus and P. aeruginosa 2. These results reveal that most Achillea plants from Jordan habitats show very good inhibition against Grampositive (S. aureus) and Gram-negative (P. aeruginosa) bacteria.

Table 1. Scientific and common name and medical uses of the tested plants. Scientific name

Common name

Medical uses

Achillea biebersteinii Achillea santolina Ainsworthia trachycarpa Arum hygrophilum Ballota philistaea Ballota undulata Calotropis procera

Yarrow Milfoil Ainsworthia Green arum Horehound Common ballota Mudar plant, Giant milkweed; Mudar Yercum Wall- or Nettle-leafed goosefoot; Australian spinach False seawort Fagonbush

Carminative, insect repellant Intestinal colic, dysentery, carminative, insect repellant

Chenopodium murale Crupina crupinastrum Fagonia mollis Halogeton alopecuroides Marrubium vulgare Micromeria nervosa Nonea melanocarpa Onosma roussaei Phlomis viscosa Salvia hierosolymitana Scrophularia hierochuntica Solanum elaeagnifolium Teucrium polium Varthemia iphionoides

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White horehound Savory Saltwort Shrubby phlomis Jerusalem sage Figwort Silverleaf nightshade Cat thyme Varthemia

Internal bacterial infection, cancer, poisoning and circulatory system Antioxidant, diuretic, haemostatic Antioxidant, diuretic, haemostatic Antipyretic and anti-inflammatory activity Treatment of dyspepsia and jaundice

Antimicrobial, cancer treatment Carminative, useful for cough and cold, stomochic, bronchitis, asthma

Anti-inflammatory Contraceptive and corticosteroid drugs Kidney, liver diseases, diabetes, anti-inflammatory for stomach and intestine, stimulates glands for piles Infections, kidney stones, prostate illness, impotence, testicle pain

Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009

Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009

105

5 13ghi 12ijk 0m 0m 0m 12ijk 18cd 10jkl 11kl 0m 0m 10 jkl 12 ijk 0m 0m 0m 0m 0m 0m 11kl 0m

10 14gh 13hij 0m 0m 0m 13hij 19bc 11kl 12ijk 0m 0m 10 jkl 14 gh 0m 0m 0m 0m 0m 0m 12ijk 0m

15 16ef 13hij 0m 0m 0m 12ijk 20b 12ijk 12ijk 0m 0m 12 ijk 14 gh 0m 0m 0m 0m 0m 0m 12ijk 0m

20 17de 13hij 0m 0m 0m 11kl 20b 13hij 14gh 0m 0m 12 ijk 14 gh 0m 0m 0m 0m 0m 0m 13hij 0m

40 17de 15gh 0m 0m 0m 12ijk 24a 13hij 16ef 0m 0m 12 ijk 14 gh 0m 0m 0m 0m 0m 0m 14gh 0m

60 18cd 15fg 0m 0m 0m 15fg 24a 14gh 17de 0m 0m 13 ghi 14 gh 0m 0m 0m 0m 0m 0m 14gh 0m

S. aureus* (Extracts in ppm) 80 19bcd 16ef 0m 0m 0m 15fg 25a 15fg 17de 0m 0m 14 gh 15 fg 0m 0m 0m 0m 0m 0m 15fg 0m

100 19bcd 16ef 0m 0m 0m 15fg 26a 15fg 17de 0m 0m 14 gh 15 fg 0m 0m 0m 0m 0m 0m 15fg 0m

5 10ijk 9jkl 10ijk 9 jkl 0m 0m 8 kl 9 jkl 8 kl 0m 0m 10 ijk 0m 0m 0m 0m 8 kl 8 kl 7l 7l 12 ghi

10 11hij 11hij 11hij 10 ijk 0m 0m 9 jkl 9 jkl 9 jkl 0m 0m 10 ijk 0m 0m 0m 0m 9 jkl 9 jkl 9 jkl 8 kl 13 fgh

15 11hij 11hij 11hij 11hij 0m 0m 9 jkl 9 jkl 9 jkl 0m 0m 10 ijk 0m 0m 0m 0m 11 hij 10 ijk 10 ijk 8 kl 13 fgh

S. aureus (Extracts in mm) MIC DIZ 80 19 80 16 N.A N.A N.A N.A N.A N.A 60 15 40 24 80 15 60 17 N.A N.A N.A N.A 80 14 80 15 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 80 15 N.A N.A

P. aeruginosa (Extracts in mm) MIC DIZ 80 12 10 11 80 20 80 15 N.A N.A N.A N.A 80 13 80 14 40 12 N.A N.A N.A N.A 5 10 N.A N.A N.A N.A N.A N.A N.A N.A 80 17 80 19 80 19 80 20 80 17

DIZ= Diameter of inhibition zone (mean in mm). MIC= Minimum inhibition concentration (mm) N.A= Not Active.

Achillea biebersteinii Phlomis viscosa Ainworthia trachycarpa Solanum elaeagnifolium Arum hygrophilum Varthemia iphionoides Crupina crupinastrum Teucrium polium Achillea santolina Micromeria nervosa Chenopodium murate Ballota philistaea Onosma roussaei Fagonia mollis Marrubium vulgare Calotropis procera Salvia hierosolymitana Ballota undulate Hallogeton alopecuroides Scrophularia hierochuntica Nonea melanocarpa

Scientific name of the plant used

C. albicans (Extracts in mm) MIC DIZ 80 17 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A 80 17 N.A N.A 60 18 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A

Table 3. Minimum inhibitory concentration and inhibition zone diameter of crude plant extracts against pathogenic microorganisms.

20 11hij 11hij 11hij 11hij 0m 0m 10 ijk 10 ijk 10 ijk 0m 0m 10 ijk 0m 0m 0m 0m 11 hij 10 ijk 12 ghi 12 ghi 15 cde

40 11hij 11hij 14 efg 12 ghi 0m 0m 11 hij 12 ghi 12 ghi 0m 0m 10 ijk 0m 0m 0m 0m 15def 14 efgh 13 fgh 18 abc 16 cde

60 11hij 11hij 18 abc 13 fgh 0m 0m 11 hij 13 fgh 12 ghi 0m 0m 10 ijk 0m 0m 0m 0m 16 cde 18 abc 18 abc 19 ab 16 cde

P. aeruginosa* (Extracts in ppm)

* values were means of triplicate readings . Means with different superscript letters are significantly different (P•0.05) at plant extract concentration used.

A. biebersteinii P. viscosa A. trachycarpa S. elaeagnifolium A. hygrophilum V. iphionoides C. crupinastrum T. polium A. santolina M. nervosa C. murate B. philistaea O. roussaei F. mollis M. vulgare C. procera S. hierosolymitana B. undulate H. alopecuroides S. hierochuntica N. melanocarpa

Scientific name of the plant used 80 12ghi 11hij 20 a 15 def 0m 0m 13 fgh 14 efgh 12 ghi 0m 0m 10 ijk 0m .0 m 0m 0m 17 bcd 19 ab 19 ab 20 h 17 bcd

100 12ghi 11hij 20 a 15 def 0m 0m 13 fgh 14 efgh 12 ghi 0m 0m 10 ijk 0m 0m 0m 0m 17 bcd 19 ab 19 ab 20 h 17 bcd

5 11g 0h 0h 0h 0h 0h 14 e 0h 11 g 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

10 12f 0h 0h 0h 0h 0h 14 e 0h 12 h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

15 15d 0h 0h 0h 0h 0h 15 d 0h 15 d 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

20 15d 0h 0h 0h 0h 0h 16 c 0h 16 c 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

40 15d 0h 0h 0h 0h 0h 16 c 0h 17 h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

60 16c 0h 0h 0h 0h 0h 16 c 0h 18 a 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

C. albicans* (Extracts in ppm) 80 17b 0h 0h 0h 0h 0h 17 h 0h 18 a 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

100 17b 0h 0h 0h 0h 0h 17 h 0h 18 a 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h 0h

Table 2. Antimicrobial activity (DIZ mean in mm) of medicinal plants at various concentrations against (Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans).

The antimicrobial activities of Achillea biebersteinii 15 agreed with our results in term of inhibited Staphylococcus aureus but not Pseudomonas aeruginosa. Furthermore, the oil was found to have strong antifungal activities against Candida albicans which agree with results of our extracts from Achillea biebersteinii. The results of this work suggest that extracts from tested plants possesses compounds with antimicrobial properties that can be used especially as antibacterial and antifungal agents in new drugs for treating infectious diseases in human. Acknowledgements The authors would like to thank the Higher Council for Science and Technology for their financial support. Thanks also to Abbeer Kharma for her technical assistance. References

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Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009