STUDIES ON THE ANTIMICROBIAL EFFECT OF SOME PLANTS

Cairo University Faculty of Veterinary Medicine Department of Microbiology STUDIES ON THE ANTIMICROBIAL EFFECT OF SOME PLANTS A Thesis Presented By Ahmed Hany Mohamed Fathy (B. V.Sc., Faculty of Veterinary Medicine, Cairo University, 2003) For M. V. Sc. Degree in Veterinary Medical Science (Bacteriology, Immunology and Mycology) Under The Supervision of Professor Dr. Mahmoud Essam Hatem Ahmed Professor of Microbiology Faculty of Veterinary Medicine Cairo University Professor Dr. Atteya Hassan Ata Professor of Pharmacology Faculty of Veterinary Medicine Cairo University Professor Dr. Mohamed Samih Mahmoud Professor of Pharmacology Faculty of Veterinary Medicine Cairo University Professor Dr. Ahmed Tawfik Mohamed Animal Health Research Institute 2012

Approval Sheet The committee for the discussion has decided to nominate Ahmed Hany Mohamed Fathy for Master of Veterinary Science (Bacteriology Immunology Mycology) from Faculty of Veterinary Medicine Cairo University. The Committee: Dated on: 4/4/2012

AKNOWLEDGMENT I would like to express my sincere gratitude and thanks to my Professor Dr. Mahmoud Essam Hatem Ahmed for his valuable supervision and his great support throughout this work. I ask for mercy to Professor Dr. Mohamed Samih Mahmoud who helped me and encouraged this work. I would like to express my sincere gratitude and thanks to Professor Dr. Atteya Hassan Ata for his active and helpful guidance during work. I would like to express my sincere gratitude and thanks to Professor Dr. Ahmed Tawfik Mohamed for his encouragement and support. I would like to express my sincere gratitude and thanks to My Family for their great help and support.

Contents CONTENTS Subject Page - 1. Introduction 1. o Aim of the work 4. - 2. Review of Literature.. 5. o 2.1. Guava. 6. o 2.2. Aloe......... 14. o 2.3. Thyme....... 16. o 2.4. Basil..... 17. o 2.5. Stevia... 21. o 2.6. Peppermint....... 24. o 2.7. Miller.... 26. - 3. Material and Methods....... 27. - 4. Results...... 41. - 5. Discussion....... 51. - 6. Summary..... 57. - 7. Reference.... 59. List of photos.: Photo. Page - Guava (Photos. 1, 2&3).... 34. - Aloe (Photos. 4, 5& 6)...... 35. - Thyme (Photos. 7, 8 & 9)..... 36. - Basil (Photos. 10, 11, 12 & 13).... 37. - Stevia (Photos. 14, 15 &16). 38. - Peppermint (Photos. 17, 18& 19) 39. - Miller (Photos. 20, 21 & 22) 40. - The inhibitory zone for S. aureus at concentration of plant leaves extract 200 mg/ml (Photo. 23)..... 47. - The inhibitory zone for S. aureus at concentration of plant leaves extract 100 mg/ml (Photo. 24).... 47. - The inhibitory zone for S. agalactiae at concentration of plant leaves extract 200 mg/ml (Photo. 25).... 48. I

Contents - The inhibitory zone for P. aeruginosa at concentration of plant leaves extract 200 mg/ml (Photo. 26).... 48. List of figures: Figure (1): The antimicrobial activity for each plant leaves extract clarifying the diameter of inhibitory zone for the susceptible microorganism. Figure (2): The antimicrobial activity for Aloe vera leaves extract clarifying the diameter of inhibitory zone for the susceptible microorganisms (S. aureus and S. agalactiae) Figure (3):Showing the antimicrobial activity for Psidium guajava leaves extract clarifying the diameter of inhibitory zone for the susceptible microorganisms (S. aureus, S. agalactiae and Ps. aeruginosa) Figure (4): Showing the antimicrobial activity for Mentha piperita leaves extract clarifying the diameter of inhibitory zone for the susceptible microorganisms (S. aureus and S. agalactiae) Figure (5): Showing the antimicrobial activity for Thymus vulgaris leaves extract clarifying the diameter of inhibitory zone for the susceptible microorganisms (S. aureus) Figure (6): Showing the MIC for the different plant leaves extract on the tested microorganisms Page 44 45 45 46 46 50 II

Contents List of Tables: Page Table (1): The antimicrobial potential of the plant leaves extract 42-43 Table (2): The MIC of the plant leaves extract (R = Resistant) 50 III

INTRODUCTION 1. INTRODUCTION Finding healing powers in plants is an ancient idea. People in all continents have long applied politics and imbibed infusions of hundreds, if not thousands, of indigenous plants. Many plants were used in the past for healing and these plants are still widely used in ethnomedicine around the world. Primitive man observed and appreciated the great diversity of plants available to him. The plants provided food, clothing, shelter, and medicine. Much of the medicinal use of plants seems to have been developed through observations of wild animals, and by trial and error. Many drugs commonly used today are of herbal or plant origin. Indeed, about 25% of the prescription drugs dispensed in the United States contain at least one active ingredient derived from plant material. Some are made from plant extracts; others are synthesized to mimic a natural plant compound. It is estimated that there are 250,000 to 500,000 species of plants exist on earth (Borris, 1996). Relatively both human and animal use a small percentage (1 to 10 %) of these as food. Hippocrates (in the late fifth century B.C) recorded 300 to 400 medical plants (Schultes, 1978). For example, guava, which has many pharmacological - 1 -

INTRODUCTION activities, has analgesic, anticholinergic, anticonvulsant, antidiarrheal, antimycobacterial, anti-inflammatory, antilipolytic and antibacterial activities. Aloe also has alkalinizing effect, analgesic, local anesthetic, antibacterial, antifungal and antitumor activities (Ivan, 1996). Clinical microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts. First, it is reported that, on average, two or three antibiotics derived from micro-organisms are launched each year (Clark, 1996). After a downturn in that pace in recent decades, the pace is again quickening as scientists realize that the effective life span of any antibiotic is limited. World-wide spending on finding new anti-infective agents is expected to increase 60 % from the spending level in 1993 (Alper, 1998). Second, the public is becoming increasingly aware of problems with the over prescription and misuse of traditional antibiotics, that leads to a very important problem worldwide which is bacterial drug resistance (Eisenberg et al., 1993). Bacterial resistance becomes an especially important and controversial topic in human and veterinary medicine mainly resulted from misuse of antibiotics, the thing, which is leading to economic loses in exploring new antibiotics and researches, which are conducted to solve this problem. - 2 -

INTRODUCTION Recently, there are several plants, which have uses in a medical branch. In microbiology, clinical microbiologists are conducting many studies on the antimicrobial activity of the plants. An example for that, a fermented aqueous extract from Stevia rebaudiana Bertoni showed strong bactericidal activity towards a wide range of food-borne pathogenic bacteria including enterohemorrhagic Escherichia coli O157: H7 (Tomita et al., 1997). The uses of some herbs, their essential oils or their active compounds, as means of control of pathogens constitute an alternative to chemical additives showed in the study of activities of the thyme on Shigella. Thyme and its essential oils, thymol and carvacrol showed inhibition of Shigella species (Bagamboula et al., 2001). - 3 -

INTRODUCTION Aim of the work The present investigation aims to study the antimicrobial activity and minimal inhibitory concentration (MIC) of methanol leave extracts of seven plants, which are Psidium guajava (Guava), Aloe vera (Aloe), Thymus vulgaris (Thyme), Ocimum basillicum (basil), Stevia rebaudiana (Stevia), Mentha piperita (peppermint) and Eruca sativa (Miller) against the selected microorganisms which are 2 Gram positive bacteria Staphylococcus aureus and Streptococcus agalactiae, 2 Gram negative bacteria Pseudomonas aeruginosae and E. coli and 1 fungus which is Candida albicans. - 4 -

REVIEW OF LITERATURE 2. REVIEW OF LITERATURE Borris (1996) recorded that there are 250,000 to 500,000 species of plants exist on earth. Cichewicz and Thorpe (1996) made alcoholic extractions for some plant leaves by drying the plant leaves. These leaves were grinded to fine texture and then soaked in methanol for extended period. The slurry was then filtered and washed, after which it might be dried under reduced pressure and redissolved in the alcohol to a determined concentration. Taylor et al. (1996) used the water extract for plant leaves by soaking of plant powder in the distilled water, plotted dry, made into slurry through blending, and then strained or filtered. The filtrate could be centrifuged (approximately 20000 X g, for 30 minutes) multiple times for clarification. Marjorie (1999) classified and scheduled different plants according their antimicrobial activity and which part of the plant was active. He explained the importance of using plant extracts instead of antibiotics as antimicrobial agents. - 5 -

REVIEW OF LITERATURE 2.1. Psidium guajava (Guava): Acevedo et al. (1996) reported the taxonomic position of Psidium guajava as follows: Family: Myrtaceae Subfamily: Myrtoideae Genus: Psidium Species: Psidium guajava Ivan (1996) reported the antimicrobial activities of Psidium guajava leaves extract. It had antibacterial activity on Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes (MIC 10.0 mg/disc). Acetone extract of dried leaves was active on E.coli, Pseudomonas aeruginosa, Salmonella B, Salmonella Newport, Salmonella Typhi, Sarcina lutea, Serratia marcescens, Shigella flexneri, Staphylococcus albus and Staphylococcus aureus. The hot water extract of dried leaves at concentration of 1.0 ml in broth culture was active on Epidermophyton floccosum. The acetone extract of dried leaves of Psidium guajava at concentration of 50 mg/ disc was active on Staphylococcus aureus and was active on Streptococcus pyogenes (MIC 10mg/ disc). Ethanol extract (95%) of Psidium guajava was active on E.coli, Pseudomonas aeruginosa, Salmonella B, Salmonella Newport, Salmonella Typhi, Shigella flexneri, Staphylococcus albus. - 6 -

REVIEW OF LITERATURE Jaiarj et al. (1999) found that alcohol and chloroform extracts of leaves of Psidium guajava (Guava) were effective against Aeromonas hydrophila, Shigella species and Vibrio species. Tona et al. (1999) screened the traditional preparation of Psidium guajava leaves as antidiarrhoeal. They found that the leaves extract has pronounced antibacterial action in addition to antiamoebic and antispasmodic actions. Gislene et al. (2000) evaluated the antimicrobial activity of Psidium guajava leaves extract with antibiotic susceptible and resistant microorganisms. It had an effect on Staphylococcus aureus and Candida albicans with inhibition zone on sensitivity plate 7 mm. Arima and Danno (2002) isolated four antibacterial compounds from leaves of guava (Psidium guajava). Two new flavonoid glycosides and two known flavonoids were identified. The minimum inhibition concentration of the two new flavonoid glycosides was 200 microgram /ml for each against Salmonella Enteritidis, and 250 microgram /ml and 300 microgram /ml against Bacillus cereus. Holetz et al. (2002) screened the antibacterial and antifungal activity of Psidium guajava leaves extract. They found some activity against Staphylococcus aureus and Candida albicans. - 7 -

REVIEW OF LITERATURE Voravuthikunchai et al. (2004) reported an activity of Psidium guajava leaves extract on E. coli O 157: H7 using paper disc agar diffusion method. Neviton et al. (2005) evaluated the antibacterial activities of aqueous and ethanol: water extract from leaves of Psidium guajava against bacteria. They found that the aqueous extract was active against Staphylococcus aureus with MIC = 500 µg/ ml and Bacillus subtilis with MIC = 500 µg/ ml and inactive against E. coli and Pseudomonas aeruginosa. The ethanol: water extract showed higher antimicrobial activity as compared to aqueous extract. The leaves extract had an effect on Staphylococcus aureus with MIC 25 µg/ ml. Chan et al. (2005) evaluated the methanol extract of Psidium guajava leaves for antibacterial and wound healing properties. Psidium guajava leaves extract prevented growth of 81.8 % of different strains of Staphylococcus aureus, E. coli, Pseudomonas aeruginosa and Proteus species using the well diffusion method. Abbas (2005) tested extracts of Psidium guajava leaves for its inhibitory effect against 70 strains of Staphylococcus aureus. The extract showed inhibitory results however, significant differences were noted when 50 fusidic acid Staphylococcus aureus strains were tested. Guava leaves gave an average inhibitory zone of 15.3 mm. The - 8 -

REVIEW OF LITERATURE concentration of the extracted leaves was 83.33 mg /ml. The results suggested the possibility of using such plant extract as an alternative therapy to the ineffective antibiotics. Rattanachaikunsopon and Phumkhachorn (2007) evaluated the flavonoids isolated from the leaves of Psidium guajava against antimicrobial activity of fish bacterial pathogens. The flavonoids were shown to have bacteriostatic effect on all of the tested bacteria. Ahmed and Yagoub (2007) tested the petroleum ether, methanol and water extracts of Psidium guajava leaves to determine their possible antimicrobial activity against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (E. coli and Salmonella Typhi) and fungi (Aspergillus niger and Candida albicans). Each extract was used in concentration of 100, 50, 25 and 10%. Gram-positive and Gram-negative bacteria showed susceptibility toward all extracts, the zones of their inhibition ranged between 13-28 mm. Aspergillus niger showed complete resistance toward all extracts at all concentrations, while Candida albicans was inhibited by water extract. All concentrations of petroleum ether extract and only concentration at 10% of methanol extract, the zones of inhibition ranged between 13-20 mm. - 9 -

REVIEW OF LITERATURE Hoque et al. (2007) investigated the antibacterial properties of Psidium guajava against a number of common food borne pathogens and spoilage bacteria. Screening for antibacterial activity was determined by disc diffusion assay against 21 strains of food borne pathogens; Listeria monocytogenes (5 strains), Staphylococcus aureus (4 strains), E. coli O157:H7 (6 strains), Salmonella Enteritidis (4 strains), Vibrio parahaemolyticus and Bacillus cereus, and 5 food spoilage bacteria; Pseudomonas aeruginosa, P. putida, Enterococcus faecalis, and Aeromonas hydrophila (2 strains). The Minimum inhibitory concentration (MIC) of the extracts was determined using the micro dilution method. The results indicated that ethanol extracts of guava inhibited all L. monocytogenes and S. aureus strains, and V. parahaemolyticus (IFO 12711), B. cereus (IFO 3457), Pseudomonas aeruginosa PA 01, E. faecalis and two A. hydrophila strains, (NFRI 8282) and (NFRI 8283). On the other hand, aqueous extracts of guava inhibited all Staphylococcus aureus strains, V. parahaemolyticus (IFO 12711), E. faecalis (IFO 12669) and the two A. hydrophila strains. The MIC of ethanol extracts of guava showed the highest inhibition for L. monocytogenes (JCM 7676) (0.1 mg/ml), Staphylococcus aureus (JCM 2151) (0.1 mg/ml), Staphylococcus aureus (JCM 2179) (0.1 mg/ml) and V. parahaemolyticus (IFO 12711) (0.1 mg/ml) and the lowest inhibition for E. faecalis (IFO 12669) and the two A. hydrophila strains (4.0 mg/ml). No significant effects of temperature and ph were - 10 -

REVIEW OF LITERATURE found on guava extracts against cocktails of L. monocytogenes, and Staphylococcus aureus. Guava leaves extracts showed higher antimicrobial activity against Gram-positive bacteria compared to Gram-negative bacteria except for V. parahaemolyticus, Pseudomonas aeruginosa, and A. hydrophila. None of the extracts showed antimicrobial activity against E. coli O157:H7 and Salmonella Enteritidis. The results suggested that guava extracts possess compounds containing antibacterial properties that can potentially be useful to control food-borne pathogens and spoilage organisms. Geidam et al. (2007) studied the effect of Psidium guajava aqueous leaves extract on the swab content from the navel of the day old chicks of both broilers and layers which had revealed the presence of several Gram positive and Gram negative organisms such as E. coli, Staphylococcus species, Streptococcus species, Proteus species, Klebsiella species, and Corynebacterium. The susceptibility tests on the isolated organisms by the extract under study had showed an appreciable dose dependent zone of inhibition ranging from 13-25 mm. The activity of the extract 400 mg/ ml can be favorably compared with that of the standard antibiotic-oxytetracycline 10 mg/ ml particularly between the E. coli and Streptococcus with 25-30 mm and 20-22 mm as inhibition zone, respectively where no significant difference was observed. The extract exhibited a highest MIC of 12.5-11 -

REVIEW OF LITERATURE mg/ ml against Staphylococcus species, while concentration of 25.0 mg/ ml was noted as the MIC values against both E. coli and Streptococcus species. Gonçalves et al. (2008) screened the antimicrobial effect of essential oils and methanol, hexane, ethyl acetate extracts from Psidium guajava leaves. The extracts were tested against diarrhea causing bacteria; Staphylococcus aureus, Salmonella species and Escherichia coli. Strains that were screened included isolates from seabob shrimp, Xiphopenaeus kroyeri (Heller) and laboratory-type strains. Of the bacteria tested, Staphylococcus aureus strains were most inhibited by the extracts. The methanol extract showed greatest bacterial inhibition. No statistically significant differences were observed between the tested extract concentrations and their effect. The essential oil extract showed inhibitory activity against Staphylococcus aureus and Salmonella species. The strains isolated from the shrimp showed some resistance to commercially available antibiotics. These data support the use of guava leaf-made medicines in diarrhea cases where access to commercial antibiotics is restricted. In conclusion, guava leaves extract and essential oil are very active against Staphylococcus aureus, thus making up important potential sources of new antimicrobial compounds. - 12 -

REVIEW OF LITERATURE Buvaneswari et al. (2011) studied Psidium guajava leaf extracted in 75% methanol for their antimicrobial potentiality against 8 clinically important microbial strains S. aureus, Bacillus species, E. coli, Klebsiella species, Proteus vulgaris, Pseudomonas species, Salmonella species and Shigella species. The extract of Psidium guajava leaves were exhibited good antibacterial activity against almost all test cultures except Klebsiella species. The most antibacterial activity was against S. aureus. - 13 -

REVIEW OF LITERATURE 2.2. Aloe vera (Aloe): Duke (2002) reported the taxonomic position of Aloe vera as follows: Family: Asphodelaceae Genus: Aloe Species: Aloe vera Ivan (1996) reported that Aloe vera dried entire plant juice on agar plate was active on Proteus vulgaris and Pseudomonas aeruginosa. Fresh leaf juice at a concentration of 1:50 on agar plate was active on Streptococcus pyogenes, Corynebacterium xerosis and Staphylococcus aureus. Undiluted fresh leaf juice in broth culture was active on Bacillus subtilis, Enterobacter species, E.coli, Serratia marcescens, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus pyogenes. The anthraquinone fraction of fresh leaf juice on agar plate was active on Trichophyton mentagrophytes. Dried juice from entire plant, applied externally to human adult, was active in treating trichophytiasis. Rajurkar et al. (2001) evaluated the antibacterial activity of Aloe vera leaves extract (aqueous, ethyl acetate, methanol and chloroform) against some common pathogenic bacteria; Escherichia coli, Staphylococcus aureus and Salmonella Gallinarum in vitro. - 14 -