Antimicrobial activity and Phytochemical investigations of some volatile oils obtained from some Sudanese medicinal plants By Egbal Hashim Mohammed {B. Sc. Agric., University of Alexandria} Athesis submitted in partial fulfillment for the degree of Master of Science (Pharmacognosy) Department of Pharmacognosy Faculty of Pharmacy University of Khartoum June 2003 DEDICATION Dedicated to the Soul of my grandmother, my mother, my father, my brothers, my sister and my friends and colleagues. Acknowledgements I would like to thank my supervisor Dr. Abdalla Mohammed Elhassan. Dean Faculty of Pharmacy, (The National Ribat University) for his great efforts and valuable advises throughout this work. I would also like to express my thanks to my co- supervisor Dr. Mohammed Elfatih Ahmed Omer. Medicinal and Aromatic Plants Research Institute, National Center for Research. [MAPRI]. for his supervision, guidance, care and help throughout this work. I would also like to thank the staff of Pharmacognosy Department (Faculty of Pharmacy. University of Khartoum) particularly Dr. Abdel El khalig Muddathir. Head of the Department of Pharmacognosy for his strong support. I would also like to express my thanks to Dr. Hassan El subki.[MAPRI] for his valuable help. My gratitude to the staff of [MAPRI], of both Departments of Microbiology and Phytochemistry for their kind help during the practical work. I wish to thank all the staff members of the National Laboratory and Khartoum Hospital for their help in collecting the samples of microorganisms. Lastly my gratitude is also extended to Prof. Saeed Ahmed El shafee (University of Sudan), for his strong support. Abstract Ten volatile oils obtained from eight Sudanese plants belonging to five families, were tested for antimicrobial activity against four standard organisms (Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus), fifty seven clinical isolates of E.coli, Pr. vulgaris, Ps. aeruginosa, S. aureus (collected randomly from Sudanese patients) and three standard fungi (Aspergillus niger, Candida albicans and Saccharomyces cervisiae). Most volatile oils tested have moderate activity against all standard organisms. All tested volatile oils showed some degree of antimicrobial activity. The volatile oil of Citrus aurantifolia obtained from leaves recorded the highest activity against S.aureus; on the other hand the volatile oil of Pimpinella anisum showed low activity against S.aureus. Volatile oils of Mentha spicata, Ocimum basilicum and Eucalyptus globulus have low activity against Ps. aeruginosa, while Pr. vulgaris was resistant to volatile oil of Eucalyptus globulus. The activity of volatile oils against clinical isolates showed that volatile oils of Mentha spicata, Cymbopogon citratus, Citrus aurantifolia (leaves, fruit peel) and Citrus sinensis (leaves, and fruit peel) scored high activity against all clinical isolates while volatile oils of Citrus paradisi, Pimpinell anisum, Ocimum basilicum and Eucalyptus globulus showed low activity compared with other volatile oils. Reference antibiotics were tested against standard organisms and the results were compared with the activity of the volatile oils. Volatile oils showed some degree of antifungal activity. Volatile oil of Citrus aurantifolia obtained from leaves showed high activity against all standard fungi tested. Chemical components of volatile oils tested were studied by thin layer chromatography. ﻡﻴﺤﺭﻟﺍ ﻥﻤﺤﺭﻟﺍ ﷲﺍ ﻡﺴﺒ ﺹﺨﻠﻤ لﺌﺍﻭﻋ ﺱﻤﺨ ﻲﻟﺇ ﻲﻤﺘﻨﺘ ﺔﻴﻨﺍﺩﻭﺴ ﺕﺎﺘﺎﺒﻨ ﺔﻴﻨﺎﻤﺜ ﻡﺍﺩﺨﺘﺴﺍ ﻡﺘ .ﺔﻴﻟﺎﺤﻟﺍ ﺔﻴﻭﻴﺤﻟﺍ ﺕﺍﺩﺎﻀﻤﻟﺍ ﻡﻅﻌﻤ ﻪﺠﺍﻭﺘ ﻲﺘﻟﺍ ﺔﻤﻭﺎﻘﻤﻟﺍ ﺞﻟﺎﻌﺘ ﺔﻴﺭﺘﻜﺒ ﺕﺍﺩﺎﻀﻤ ﻁﺎﺒﻨﺘﺴﺍ ﻭﺤﻨ ﻲﻌﺴﻤ ﻲﻓ ﺔﻴﺒﻫﺫﻟﺍ ﺔﻴ ﺩﻭﻘﻨﻌﻟﺍﻭ ﺔﻴﺭﺎﺠﻨﺯﻟﺍ ﺔﻔﺌﺍﺯﻟﺍﻭ ﺔﻴﻨﻭﻟﻭﻘﻟﺍ ﻪﻴﻜﺭﺸ ﻻﺃ :ﻰﻫﻭ ﺔﻴﺴﺎﻴﻘﻟﺍ ﺔﻴﺭﺘﻜﺒﻟﺍ ﺕﺎﺒﻭﺭﻜﻤﻟﺍ ﻥﻤ ﺔﻌﺒﺭﺍ ﺩﻀ ﺕﻭﻴﺯﻟﺍ ﻙﻠﺘ ﺭﺎﺒﺘﺨﺍﻭ ﻪﻴﺭﻁﻋ ﺕﻭﻴﺯ ﺭﺸﻋ ﺹﻼﺨﺘﺴﻻ ﻪﻴﺘﺎﺒﻨ ﺀﺎﻀﻴﺒﻟﺍ ﺔﻀﻴﺒﻤﻟﺍ :ﻰﻫﻭ ﺔﻴﺴﺎﻴﻘﻟﺍ ﺔﻴﺭﻁﻔﻟﺍ ﺕﺎﺒﻭﺭﻜﻤﻟﺍ ﻥﻤ ﺔﺜﻼﺜ ﺩﻀﻭ ﻥﻴﻨﺍﺩﻭﺴ ﻰﻀﺭﻤ ﻥﻤ ﺎﻴﺌﺍﻭﺸﻋ ﺕﻌﻤﺠ ﺔﻴﻜﻴﻨﻴﻠﻜﺇ ﺔﻨﻴﻋ ﻥﻭﺴﻤﺨﻭ ﺔﻌﺒﺴ ﺩﻀﻭ ﺱﺭﺎﺠﻠﻓ ﺱﻴﺘﻭ ﺭﺒﻟﺍﻭ .ﺔﻴﻭﻌﺠﻟﺍ ﺔﻴﺭﻁﻔﻟﺍﻭ ﺀﺍﺩﻭﺴﻟﺍ ﺔﻴﺸﺎﺸﺭﻟﺍﻭ ﺓﺭﺒﺘﺨﻤﻟﺍ ﺕﺎﺒﻭﺭﻜﻤﻟﺍ ﺩﻀ ﺔﺘﻭﺎﻔﺘﻤ ﺔﻴﻟﺎﻌﻓ ﺕﻴﻁﻋﺃ ﺔﻠﻤﻌﺘﺴﻤﻟﺍ ﺔﻴﺭﻁﻌﻟﺍ ﺕﻭﻴﺯﻟﺍ - .ﺏﻭﺭﻜﻤﻟﺍ ﺱﻔﻨ ﺩﻀ لﻗﺍ ﺔﻴﻟﺎﻌﻓ ﻰﻁﻋﺃ ﻥﻭﺴﻨﺎﻴﻟﺍ ﻥﻤ ﻱﺭﻁﻌﻟﺍ ﺕﻴﺯﻟﺍ ﺎﻤﻨﻴﺒ ﺔﻴﺒﻫﺫﻟﺍ ﺔﻴﺩﻭﻘﻨﻌﻟﺍ ﺩﻀ ﺓﺭﻴﺒﻜ ﺔﻴﻟﺎﻌﻓ ﻥﻭﻤﻴﻠﻟﺍ ﻕﺍﺭﻭﺃ ﻥﻤ ﺹﻠﺨﺘﺴﻤﻟﺍ ﺕﻴﺯﻟﺍ ﺯﺭﺤﺃ- ﺔﻴﻟﺎﻌﻓ ﺕﻁﻋﺃ ﻥﺎﺒﻟﺍﻭ ﻥﺎﺤﻴﺭﻟﺍﻭ ﻱﺩﻠﺒﻟﺍ ﻉﺎﻨﻌﻨﻟﺍ ﻥﻤ ﺔﺼﻠﺨﺘﺴﻤﻟﺍ ﺔﻴﺭﻁﻌﻟﺍ ﺕﻭﻴﺯﻟﺍ- .ﺔﻴﺭﺎﺠﻨﺯﻟﺍ ﺔﻔﺌﺍﺯﻟﺍ ﺩﻀ ﺔﻴﻨﺩﺘﻤ ﺭﺎﻤﺜ ﺭﺸﻗﻭ لﺎﻘﺘﺭﺒﻟﺍﻭ ﻥﻭﻤﻴﻠﻟﺍ ﻕﺍﺭﻭﺃ ﻥﻤ ﺔﺼﻠﺨﺘﺴﻤﻟﺍ ﺕﻭﻴﺯﻟﺍ ﻥﺇ ﺩﺠﻭ .ﻰﻀﺭﻤ ﻥﻤ ﺔﻟﻭﺯﻌﻤﻟﺍ ﺔﻀﺭﻤﻤﻟﺍ ﺕﺎﺒﻭﺭﻜﻤﻟﺍ ﺩﻀ ﺔﻴﻟﺎﻌﻓ ﺕﻁﻋﺃ ﺔﻴﺭﻁﻌﻟﺍ ﺕﻭﻴﺯﻟﺍ ﻥﺃ ﺩﺠﻭ- ﻥﻭﺴﻨﺎﻴﻟﺍﻭ ﺕﻭﺭﻓ ﺏﻴﺭﻘﻟﺍ ﻕﺭﻭ ﻥﻤ ﺔﺼﻠﺨﺘﺴﻤﻟﺍ ﺔﻴﺭﻁﻌﻟﺍ ﺕﻭﻴﺯﻟﺍﻭ .ﺔﻀﺭﻤﻤﻟﺍ ﺕﺎﺒﻭﺭﻜﻤﻟﺍ لﻜ ﺩﻀ ﺔﻴﻟﺎﻋ ﺔﻴﻟﺎﻌﻓ ﺕﻁﻋﺃ ﻱﺩﻠﺒﻟﺍ ﻉﺎﻨﻌﻨﻟﺍﻭ ﻥﻭﻤﻴﻠﻟﺍ ﺔﺸﻴﺸﺤﻭ لﺎﻘﺘﺭﺒﻟﺍﻭ ﻥﻭﻤﻴﻠﻟﺍ .ﺔﻠﻤﻌﺘﺴﻤﻟﺍ ﻯﺭﺨﻷﺍ ﺕﻭﻴﺯﻟﺍ ﻊﻤ ﺔﻨﺭﺎﻘﻤ لﻗﺍ ﺔﻴﻟﺎﻌﻓ ﺕﻁﻋﺃ ﻥﺎﺒﻟﺍﻭ ﻥﺎﺤﻴﺭﻟﺍﻭ ﺕﻭﻴﺯﻟﺍ ﺔﻴﻟﺎﻌﻓ ﻊﻤ ﺞﺌﺎﺘﻨﻟﺍ ﺔﻨﺭﺎﻘﻤ ﺕﻤﺘ ﻡﺜ ﻥﻤﻭ ﺔﻴﺭﺎﻴﻌﻤﻟﺍ ﺕﺎﻨﺌﺎﻜﻟﺍ ﺩﻀ ﺔﻴﻌﺠﺭﻤﻟﺍ ﺔﻴﻭﻴﺤﻟﺍ ﺕﺍﺩﺎﻀﻤﻟﺍ ﺽﻌﺒ ﺭﺎﺒﺘﺨﺍ ﻡﺘ- ﺔﻴﺴﺎﻴﻘﻟﺍ ﺕﺎﻴﺭﻁﻔﻟﺍ لﻜ ﺩﻀ ﺔﻴﻟﺎﻋ ﺔﻴﻟﺎﻌﻓ ﻲﻁﻋﺃ ﻥﻭﻤﻴﻠﻟﺍ ﻕﺭﻭ ﻥﻤ ﺹﻠﺨﺘﺴﻤﻟﺍ ﻱﺭﻁﻌﻟﺍ ﺕﻴﺯﻟﺍﻭ.ﺓﺭﺒﺘﺨﻤﻟﺍ ﺔﻴﺴﺎﻴﻘﻟﺍ ﺕﺎﻴﺭﻁﻔﻟﺍ ﺩﻀ ﺔﺘﻭﺎﻔﺘﻤ ﺔﻴﻟﺎﻌﻓ ﺕﻁﻋﺃ ﺔﻴﺭﻁﻌﻟﺍ ﺕﻭﻴﺯﻟﺍ- .ﺓﺭﺒﺘﺨﻤﻟﺍ .ﺔﻘﻴﻗﺭﻟﺍ ﺢﻴﺍﺭﺸﻟﺍ ﺎﻴﻓﺍﺭﻏﻭﺘﺎﻤﺭﻜ ﺔﻘﻴﺭﻁﺒ ﺔﻴﺭﻁﻌﻟﺍ ﺕﻭﻴﺯﻟﺍ لﻜ لﻴﻠﺤﺘ ﻡﺘ- CONTENTS DEDICATION………………………………………………..i Acknowledgements..……….....................................................…….ii Abstract…………....……..........................................................……..iii Arabic abstract.............……....................................................……. iv Contents……………………………………………………….v List of tables……………..…..………………………………..vi List of figures………………………………………… …… viii 1.INTRODUCTION …...………………….…………………..1 2.EXPERIMENTAL………………….……..…………44 3.RESULTS AND DISCUSSION ..……..……..…..…59 CONCLUSION ….…………...………………………………106 REFERENCES .......................................................………….108 APPENDIX ……….…………………………………………..116 List of tables (Table 1) Volatile oils reported to exert antimicrobial activity [1988-1999]…….……………………………………………….28 (Table 2) Developing solvents…..………………………..……………..58 (Table 3) Yield of volatile oils obtained from some Sudanese plants…..61 (Table 4) Antimicrobial activity of some volatile oils of Sudanese plants against standard organisms………………….. …………63 (Table 5) The activity of volatile oils against clinical isolates 5.1 Mentha spicata……………………………………………...72 5.2 Ocimum basilicum…………….………………………….. ..72 5.3 Eucalyptus globulus…………………………………………. 72 5.4 Cymbopogon citratus…………………………………….... 73 5.5 Citrus aurantifolia [leaves]……………………………….. 73 5.6 Citrus aurantifolia [fruit peel]..…………………………… 73 5.7 Citrus paradisi……………………………………………. 74 5.8 Citrus sinensis [leaves]…………………………………… 74 5.9 Citrus sinensis [fruit peel]..……………………………….. 74 5.10 Pimpinell aanisum……………………………………….. 75 (Table 6) The activity % of volatile oils obtained from Sudanese plants against clinical isolates………………………………………...82 (Table 7) Antimicrobial activity of reference drugs against standard organisms……………………………………………………….86 (Table 8) Suscepitability of standard fungi to volatile oils tested………92 (Table 9) Results of Thin layer chromatography. Solvent system: Petroleum ether +Toluene ……………………………………...98 (Table 10) Results of Thin layer chromatography. Solvent system: Petroleum ether + Hexane + Ethyl acetate ………………..100 (Table 11) Results of Thin layer chromatography. Solvent system: Hexane + Chloroform + Toluene ….……….………………102 (Table 12) Results of Thin layer chromatography. Solvent system: Toluene +Petroleum ether+Formic acid…….………………104 (Table 13) Screening for antibacterial activity of the volatile oils* obtained from Sudanese plants against the clinical isolates of Escherichia coli.……………………………….………………..116 (Table 14) Screening for antibacterial activity of the volatile oils* obtained from Sudanese plants against the clinical isolates of Proteus vulgaris……………………………………………...117 (Table 15) Screening for antibacterial activity of the volatile oils* obtained from Sudanese plants against the clinical isolates of Pseudomonas aeruginosa……………………………………….118 (Table 16) Screening for antibacterial activity of the volatile oils* obtained from Sudanese plants against the clinical isolates of Staphylococcus aureus…………………………………….119 (Table 17) Screening for antifungal activity of the volatile oils obtained from Sudanese plants against three standard organisms…...120 List of figures (Fig 1) Some common components of volatile oils……………………12 (Fig 2) Antibacterial activity of some volatile oils obtained from Sudanese plants against Escherichia coli………………………...65 (Fig 3) Antibacterial activity of some volatile oils obtained from Sudanese plants against Proteus vulgaris…………………..……65 (Fig 4) Antibacterial activity of some volatile oils obtained from Sudanese plants against Pseudomonas aeruginosa…….……….66 (Fig 5) Antibacterial activity of some volatile oils obtained from Sudanese plants against Staphylococcus aureus………………… 66 (Fig 6) The activity of volatile oil of Mentha spicata against all clinical isolates tested....………………………….…………… 76 (Fig 7) The activity of volatile oil of Ocimum basilicum against all clinical isolates tested………..………..………………………….76 (Fig 8) The activity of volatile oil of Eucalyptus globulus against all clinical isolates tested……….…………………………………….77 (Fig 9) The activity of volatile oil of Cymbopogon citratus against all clinical isolates tested……………………………………………77 (Fig 10) The activity of volatile oil of Citrus aurantifolia (Leaves) against all clinical isolates tested.………………..……..……….78 (Fig 11) The activity of volatile oil of Citrus aurantifolia (Fruit peel) against clinical isolates tested……...…………………………...78 (Fig 12)The activity of volatile oil of Citrus paradisi against all clinical isolates tested……...…..………………………………………79 (Fig 13) The activity of volatile oil of Citrus sinensis (Leaves) against all clinical isolates tested……….….………………………………79 (Fig 14) The activity of volatile oil of Citrus sinensis (Fruit peel) against all clinical isolates tested…………………………………….80 (Fig 15) The activity of volatile oil of Pimpinella anisum against all clinical isolates tested…………………..……………………….80 (Fig 16) The activity % of some volatile oils obtained from Sudanese plants against the clinical isolates of Escherichia coli….………83 (Fig 17) The activity % of some volatile oils obtained from Sudanese plants against the clinical isolates of Proteus vulgaris………….83 (Fig 18) The activity % of some volatile oils obtained from Sudanese plants against the clinical isolates of Pseudomonas aeruginosa….84 (Fig 19) The activity % of some volatile oils obtained from Sudanese plants against the clinical isolates of Staphylococcus aureus…..84 (Fig 20) Antibacterial activity of Gentamycin against standard organisms of Escherichia coli…………..………………………………… 87 (Fig 21) Antibacterial activity of Gentamycin against standard organisms of Pseudomonas aeruginosa……………………….……………88 (Fig 22) Antibacterial activity of volatile oil of Citrus aurantifolia obtained from leaves and Ampcillin against Staphylococcus aureus…………………………………………...89 (Fig 23) Antibacterial activity of volatile oil of Citrus aurantifolia obtained from leaves and Gentamycin against Staphylococcus aureus…...…………………………………….90 (Fig 24) The activity of some volatile oils obtained from Sudanese plants against Aspergillus niger………………………………………...93 (Fig 25) The activity of some volatile oils obtained from Sudanese plants against Candida albicans…………………..……………….…94 (Fig 26)The activity of some volatile oils obtained from Sudanese plants against Saccheromyces cervisiae………………...……………..95 (Fig 27)Thin Layer Chromatogram Separation of different volatile oils, solvent system: Petroleum ether +Toluene……………………99 (Fig 28)Thin Layer Chromatogram Separation of different volatile oils, solvent system: Petroleum ether + Hexane + Ethyl acetate ..…101