<4D6963726F736F667420576F7264202D20CF2EDAE3C7CF20E3CDE3CF20D1D4EDCF> Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 ANTIMICROBIAL ACTIVITY AND THE MEDIAN LETHAL DOSE OF DILL (Anethum Graveolens ) EXTRACT . Emad Mohammad Rasheed* Madeeha Hamudi* **Kreem . M . R. * Foundation of Technical Education\ Institute of Medical Technical Technology Almansur ** Hort . Dept . College of Agric . Baghdad University . ABSTRACT Antimicrobial activities of dill (Anethum graveolens) extract were studied by agar well diffusion technique against tested organisms (Staphylococcus aureus, Esherichia coli and Candida albicans), which were streaking onto agar plates and incubated for 16-20 hours at 37°C. The results were showed that the extract did not show antibacterial activity, while the extract of dill seeds exhibited growth inhibition of C. albicans. The results were also showed that the median lethal dose (LD50) of the ethanolic extract of dill in laboratory white mice was about (1486 mg/kg Bodyweight B.W ). The clinical signs during 24 hours after subcutaneous injection of the extract were rapid breathing, dullness, then death. INTRODUCTION Plants remain the basis for development of modern drugs ; medical plants have used for years in daily life to treat diseases all over the world and all the researchers are looking for them. (Jimene Z-Medina etal., 2006). Herbs or herbal extracts contain different phytochemicals with biological activity that can provide therapeutic effects. Research interest has focused on herbs that possess hypolipidemic, antiplatelets, antitumor or immune-stimulating properties that ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Dec. 15 , 2009 . Accepted for publication May 6 , 2010 . Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 may be useful in helping reduce the risk of cardiovascular diseases and cancer (Abuharfeil et al., 2000). The extracts from aromatic plants have long been used for medicinal purposes. The antimicrobial properties of essential oils obtained from aerial parts and seeds of aromatic plants, such as rosemary (Rosmerinus officinalis), eucalyptus (Eucalyptus dives) and dill (Anethum graveolens) are well documented and the antimicrobial efficiency of different plants essential oils has been studied previously, (Soylu, etal. 2006; Elgayyar, etal., 2001; Delaquis, etal., 2002;Aurelli,etal., 1992; and Piccaglia, etal., 1993). Dill (Anethum graveolens), also known as shapt is from the family umbelliferae, The major compounds found in the essential oil of dill includes furanocoumarin, oxypeucedanin, xypeucedanin hydrate and falcarindiol, These compounds are reported to have various degrees of antimycobacterium activity (Stavri and Gibbons, 2005). D- carvone in dill extract, have also been shown to possess high antifungal activity against Candida albicans (Jirovetz, etal., 2003). Little work has been published on the antimicrobial properties of dill utilizing bacterial and fungal isolates. This study was conducted to evaluate the antimicrobial activity of extracted dill and its seeds on the growth of Staph.aureus, E. coli and C. albicans with determination of median lethal dose (LD50) of dill extract in laboratory white mice. MATERIALS AND METHODS 1. The plant: Mature and fresh dill and its seeds were purchased from local vegetable market in Baghdad city and they classified by Botany department, college Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 of science, university of Baghdad as Anethum graveolens L., family umbelliferae. 2. Plant extraction: According to the Harborne, etal., 1975, ethanolic extract of dill and its seeds have been prepared as follow, fifty grams of fresh plant leaves and stem have been put in flask with 250ml of 70% ethanol and stirred on magnatic stirrer at room temperature for 72hrs, after that, the sediments have been filtered through cotton gauze then by filter paper. The solvent was evaporated by air convection oven at 38°C. the weight of resulted extract was measured by grams and kept at 4°C until use. One hundred grams of dill seeds were macerated in a mortar and pestle and then saturated with ethanol 70% in separated flask with frequent shaking. The solvent with dissolved seeds were filtered and the filtrate was allowed at room temperature for 24 hrs for ethanol evaporation. The extracted volatile oils were stored refrigerated at (4 C) . 3. The test microorganism:- Staph. aurens, E.coli and C. albicans which were isolated from clinical cases in Baghdad hospital and identified in their laboratories. The organisms were cultured on maintenance media. 4. The antimicrobial activity test: The antimicrobial activity was performed with the agar diffusion method (Deans and Ritchie, 1987). A test culture of each bacterial strain was prepared in Mueller Hinton broth to a concentration of 1×106 organism per ml. The inoculated agar plates were prepared by mixing 1 ml of test culture in 25 ml of Mueller- Hinton agar. After the agar was solidified, Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 four wells of 4 mm-diameter were punched into agar, of each Petri dish. A volume of 100 microliter of dissolved extract in ethylene glycol and oil were inoculated into three wells and a pure ethylene glycol was inoculated into the fourth well as a control. To allow the dissolved extract and oil to diffuse into agar plates, they kept at 20°C for 30 minutes before they were transferred to incubator. The plates were incubated at 37°C for 48 h. The anticandidal activity of dill extract and oil were conducted by dispensing 15 ml of sterile sabouraud dextrose agar. The inocula were prepared by addition of 1 ml overnight Candida culture to 9 ml of Mueller- Hinton broth to yield 104 colony forming unit (CFU) per µl of the inoculum. 100µl of dissolved dill extract and seeds oil were inoculated into the wells as above using a dimethyl sulfoxide as a control. Antimicrobial activity was recorded as the width (mm) of the clear zone of inhibition surrounding the agar well. The results were reported as positive (+) if there is inhibition of growth and negative (-) if there is no growth inhibition. Triplicates sets of plates were prepared, the mean of three readings was calculated. 5. Estimation of median lethal dose (LD50) of extract:- A- Experimental animals: Adult swiss albino balb/C mice (25-30 gm B.W) have been used to estimate the subcutaneous median lethal dose of ethanolic extract of dill. The animals were kept in well air conditioned rooms at the animal house of physiology and pharmacology department, college of veterinary medicine, university of Baghdad, given pellets of balanced specially prepared animal feed and water. Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 a. Determination of (LD50) Pilot Study: Four mice have been used for determination the ranges of lethal doses that used in acute toxicity study, two mice for each selected dose of extract. The selected doses were 1400 mg/kg B.W, and 1800 mg/Kg B.W according to the lethal outcome (death one or two mice) . The range of acute toxicity doses have been selected. b. Estimation of (LD50) Thirty mice have been divide into five groups six mice each . the extract was subcutaneously administered by use of the following lethal doses: Group I= 1400 mg/kg B.W Group 2= 1500 mg/kg B.W Group 3= 1600 mg/kg B.W Group 4= 1700 mg/kg B.W Group 5= 1800 mg/kg B.W The animals have been watched for 24 hours for the development of toxicity symptoms and lethality. the log dose- probit response curve was done from which the (LD50) has been determined (Katzung, 2003). RESULTS 1) The antimicrobial activity of dill extract:- Table (1) shows the invitro activity of dill extract and oil on the growth of S. aureus, E. coli and C. albicans. The leaves and stem extract of the dill did not show any inhibition of growth of test organisms. The extracted seed oil showed that no growth inhibition in Staph. aureus and E.coli Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 except C.albicans where considerable growth inhibition zone (mm) ranged from 18-20 mm. Table (1) and figure (1). Table 1. The antimicrobial activity of dill extract and its seed on the growth of test microorganisms . Dill Test microorganisms The mean diameter of inhibition zone (mm) Leaves stem extract Seed oil − − Staph. aureus − − − E.col: − − + C. albicans 19 +: growth inhibition −: no growth inhibition Figure 1. The inhibitory effect of dill seed oil on the growth of C. albicans. A,B,C: 100 µl of dill seeds oil D: dimethel sulfoxide as acontrol . Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 2) The median lethal dose (LD50): a. Apilot study: The result of this study revealed that the dose which caused the half of death of the mice was 1486 mg/kg body weight, so the doses which are used for the experiment ranges between 1400 and 1800 mg/kg B.W. b. Determination of (LD50) (probit method): The mortality percent and conversion to probit number according to the acute toxic doses of dill extract in group 1, 2, 3, 4, and 5 were listed in table (2). Table 2. Acute toxicity effect of different lethal doses of dill extract in mice. Group number Dose mg/ml Log dose Total number Number of dead animals Mortality percent % Probit number I 1400 3.146 6 2 33.3 4.56 2 1500 3.176 6 3 50.0 5.00 3 1600 3.204 6 4 66.6 5.44 4 1700 3.230 6 5 83.3 5.95 5 1800 3.255 6 6 100.0 7.40 The median lethal dose was measured after logarithm of doses against probit response were plotted from which (LD50) was determined by vertical cross link from probit response to the log number dose figure (2). The calculated (LD50) has been 1486 mg/kg B.W. Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 Figure 2. Median lethal dose of Anethum graveolens in mice by probit method. DISCUSSION The essential oils are natural plant products and their formation and accumulation in plants have been reviewed by Croteau, 1986. The essential oils from aromatic plants are for the most volatile part and thus lead themselves to several methods of extraction such as water and steam distillation and solvent extraction (Guenther, 1972). The specific extraction method depends upon the plant materials to be distilled and the desired end product. The extract of dill showed no inhibitory activity to Gram negative and Gram positive bacteria, this may be due to the high volatibility of the oil leading to the escape or evaporation of its major antibacterial constituents during boiling or insufficient release of oil during extraction. The lack of antibacterial activity in dill extract may also be due to absence or denaturation of some active components of the essential oil which are responsible for the bacteriostatic or bacteriocidal activities. Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 Lember kovics etal., (2003) has recently shown that the composition of essential oils in aromatic plants is greatly affected by the method of extraction mainly the distribution of monoterpenes and azulenogene sesquiterpene. It has been reported that the antimicrobial properties can vary within the same plant species because the chemical composition and relative proportions of the individual constituents in the essential oils of the plants are influenced by genotype, growth stage, drying, climate, and geographical location (Rhyu, 1979 and Venskutonis, 1996). A few studies have been reported the anticandidal effect of essential oil of dill seeds (Soylu, etal., 2006 and Elgayyer, etal., 2001). The dill extracts were consistently found to be effective on fungal growth by inhibition of sporangial production. The (LD50) of dill extract was usually taken as one hundredth to one tenth to calculate the treatment dose. Such values provide a convenient way of comparing the potencies of drugs in experimental and clinical setting (Katzung, 2003). The study also showed that the (LD50) of dill extract was about 1486 mg/kg B.W and this considered that the extract is safe when injected subcutaneously. There were no previous toxicological studies that focused on the (LD50) of dill extract for comparision. The estimation of (LD50) may differ in its value among other studies which were achieved, This is due to the differences in dill sourses and consequently differences in chemicals composition, the differences in lab . animals used, their species and number, the method of (LD50) calculation and other circumstances that related to the researchers. Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 Further studies will be done to investigate the best extraction method for obtaining the pure essential oils with determination of their antimicrobial activity against other pathogenic microorganism. In conclusion , this study shows that the dill seeds oil can inhibit the growth of C.albicans and this oil can be used in same pharmaceutical preparation as anti candidal agients. REFERENCES Abu Harfeil, N.M., A. Maroqa,.and S. Vonkleist 2000. Augmentation of natural killer cell activity invitro against tumor cells by wild plants from Jordan. J. Ethnopharmacol., 71:55-63. Aurelli, P., A Constantini, and S Zolea,. 1992. Antimicrobial activity of some plant essential oils against listeria monocytogenes. J. Food prot. 5:344-348. Croteau, R. 1986. Biochemistry of monoterpenes and sesquiterpenes of the essential oils. Herbs, spices and medicinal plants: recent advances in botany, horticulture and pharmacology. 1: 81-135. Oryx press, phoenix, Az. Deans, S. and G. Ritchic. 1987. Antibacterial properties of plant essential oils. Int. J. food microbial. 5: 165-180. Delaquis P., K. stanich , B.Girard, and G.Mazza. 2002. Antimicrobial activity of individual and mix fractions of dill, cilantro, coriander and eucalyptus essential oils. Int. J. Food Microbial. 74 (1-2):101- 109. Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 Elgayyar, M., F.Draughon, D.Golden, and J.Mount, 2001. Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J. Food prot. 64(7): 1019-1024. Guenther, E. 1972. The production of essential oils: method of distillation, effenrage, maceration, and extraction with volatile solvents. In: Guenther, E.(ed.). the essential oils. History-origin in plants. Production Analysis. 1:85-188. Krieger publ. Co Malabar, FL. Harborne, J.B., J.J. Marbay, and H.Mabaray. 1975. Physiology and function of flavonoids. Academic press, New York: 970. Jimenez-Medina, E., A.Garcia-lora, , L.Paco, , I.Algarra., A. collado, and F.Garrido, 2006. A new extract of the plant calendula officinalis produces a dual invitro effect: cyto & toxic anti-tumor activity and lymphocyte activiation. BMC. Cancer, 6: 119-132. Jirovetz, L., G.Buchbauer,, A. Stoyanova, and E.Georgiev, 2003. Damianova ST. Composition, quality control and antimicrobial activity of the essential oil of long time stored dill (Anethum qraveolens L.) seeds from Bulgaria. J.Agric.Food Chem.. 18(51): 3854-3857. Katzung,G. 2003. Basic and clinical pharmacology Ninth Edition. 5: 94. Lember kovics, E., A.Kery, E.Kakasy, and B.Szoke, 2003. Effect of extraction method on the composition of essential oils. ISHS Acta Horticulture 597: International Conference on medical and Aromatic plants. (part II). Piccaglia, R., N Morotti., , E.Giovanelli,, S.Deans, and E.Eaglesham, 1993. Antibacterial and antioxidant properties of Mediterranean aromatic plants. Ind. Crop. Prod. 2: 47-50. Rasheed et al. Diyala Agricultural Sciences Journal, 2( 1 ) 16 – 27 ,2010 Rhyu, H. 1979. Gas chromatographic characterization of sages of various geographic origins. J. Food Sci. 44: 758. Soylu , E., S.Soylu, and S.Kurt, 2006. Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent phytopthoro infestans. Mycopathologia. 161 (2): 119-128. Stavri , M. and S. Gibbons, 2005. The antimycobacterial constituents of dill Anethum graveolens. Phytother. Res. 19 (11): 938-941. Venskutonis, P. 1996. Effect of dring on the volatile constituents of thyme (Thymus vulgaris L.) and Sage (salvia officinalis L.). Food Chem.. 2:219-227. . الفعالية المضادة للميكروبات والجرعة المميتة النصفية لمستخلص نبات الشبت **كريم معيان ربيع *مديحه حمودي *عماد محمد رشيد .هيئة التعليم التقني –المنصور –المعهد الطبي التقني –قسم الصيدلة * .جامعة بغداد –كلية الزراعة –قسم البستنة ** الخالصة لمستخلص )Candida albicans( البيضاء لمبيضاتاو للميكروباتدراسة الفعالية المضادة تمت نبات الشبت بتقنية االنتشار بالحفر، واختبرت جراثيم المكورات العنقودية الذهبية واالشيريشيا ساعة عند درجة 20- 16ار وحضنها لمدة كالقولونية والمبيضات البيضاء وذلك بتخطيطها على اال .درجة مئوية 37حرارة لمستخلص نبات الشبت في تثبيط نمو الجراثيم المستخدمة في تأثيرالنتائج عدم وجود أي أظهرت الشبت تأثيراً واضحاً في تثبيط نمو المبيضات البيضاء كما الدراسة في حين كان لمستخلص بذور تخلص الكحولي لنبات الشبت في الفئرانالجرعة المميتة الوسطية للمس إن إلى أيضاالنتائج أوضحت الفئران عالمات أظهرتكغم من وزن الجسم، حيث /ملغم 1486رب المختبرية البيضاء بما يقا وعشرين ساعة بعد حقن المستخلص تحت الجلد وتمثلت بالتنفس السريع األربعسريرية خالل .والخمول ثم الموت