INVESTIGATION OF ACTIVE COMPOUND IN CLOVE (Syzygium aromaticum) EXTRAC AND COMPARED WITH INHIBITORS OF GROWTH OF SOME TYPES OF BACTERIA CAUSING FOOD POISONING

Antimicrobial properties of Syzygium aromaticum were investigated and phytochemical active groups were done, clove oil contain a unique active compound known as eugenol was extracted and isolated to be tested. Four different pathogenic bacterial strains (Staphylococcus auras, Listeria monocyogenes, Salmonella typhimurium and Escherichia coli) were obtained and isolated from contaminated food, three concentrations for the plant (eugenol extract) were prepared to be a treatments, T1, T2 and T3 with percentage of 100%, 50%, 25% respectively. Antimicrobial activity was measured by using disc diffusion method and the best treatment was T1, which tested for eugenol qualitative and quantitative activity that was determined by using (HPLC). The results showed that the inhibition zones were increased with the increasing of concentrations of eugenol extract thus it was very clearly that the results encourage the use of natural Sources like some plants or some parts of plants to solve some problems done by bacteria activities that cause food poisoning. . key words: volatile oil, eugenol, hplc, clevenger. ةيقارعلا ةيعارزلا مولعلا ةلجم 2019 : 50 ) 6 ) : 1645 1651 مرك نع يرحتلا بكرملا يئاذغلا ممستلل ةببسملا ايرتكبلا عاونا ضعب ومن تاطبثم عم هتنراقمو لفنرقلا صلختسم يف لاعفلا مرك يلع ديرف ماستبا سردم زكرم دادغب ةعماج /كلهتسملا ةيامحو قوسلا ثوحب Ifak_77@yahoo.com صلختسملا ربتخا ت لا صئاصخ لا داضم ة لل تابوركيمل لفنرقلا يف Syzygium aromaticum. عيماجملل ةيئايميكلا تا رابتخلاا لمع مت و هرابتخلا هلزعو هصلاختسا مت لونيجويلا ىعدي ديرف لاعف بكرم يوتحي لفنرقلا تيز .ةلاعفلا يريتكب تلالاس عبرأ ةيضرم ا ( ةفلتخم Staphylococcus auras, Listeria monocyogenes, Salmonella typhimurium and Escherichia coli ) تلزع ةثولملا ةيذغلأا نم و ترضح صلختسمل تا زيكرت ةثلاث لونيجويلا ، تاجلاعم نوكتل T1 و T2 و T3 و زيكا رت بسنب 100 ٪ و 50 ٪ و 25 سايق مت .يلاوتلا ىلع ٪ ل ةداضملا ةيلاعفلا ةقيرط مادختساب تابوركيمل لاا شن ا ر ب صرقلا تناك ةجلاعم لضفاو T1 ةينقت لامعتساب تددح يتلاو لونيجويلل ةيعونلاو ةيمكلا اهتيلاعف تربتخا يتلاو ايفارجوتامورك لا ءادلأا يلاع لئاس (HPLC) . قطانم نأ جئاتنلا ترهظأ لا طيبثت دادزت زيكرت ةدايز عم صلختسم اذل لونيجويلا احضاو ناك ادج لا مادختسا عجشت جئاتنلا نأ رداصم ابنلا ضعب لثم ةيعيبطلا ءا زجأ وأ تات اه ةطشنأ اهثدحت يتلا لكاشملا ضعب لحل يئاذغلا ممستلا ببست يتلا ايريتكبلا . ةيحاتفملا تاملكلا ,رايط تيز : لونيجوي , hplc , .رجنفلاكلا *Received:11/1/2019, Accepted:17/4/2019 Iraqi Journal of Agricultural Sciences –2019:50(6):1645-1651 Karm 1646 INTRODUCTION Food spoilage is a process in which foods to be undesirable, unacceptable and un useful for human consumption due to changes in almost many characteristics, also it is clear that spoiled foods may be un safe to eat specially when its contain pathogens or a toxin present(18). Many problems were reported that we cannot use chemical preservatives products safely any more due to carcinogenic effects of these chemicals, thus the requirement has been increased for natural preservatives (27, 28). Bacteria are an expected component of decomposing animals and plants (7). A group of Gram-positive bacteria, including Lactobacillus, Pediococcus, Leuconostoc and Oenococcus, under low oxygen, low temperature, and acidic conditions, turn to be spoilage organisms on foods. Listeria spp., Staph sp. diseases that can be serious and cause damages to human and animals. (3, 4). Many pathogenic microbial organisims such as Enterobacteriaceae they are gram-negative, facultative anaerobic bacteria that include a number of human pathogens (Salmonella sp., E. coli, Shigella, Yersinia). Bacillus and Geobacillus spp. Clostridium spp. cause spoilage of canned foods. These bacteria are widespread in nature in soil, on plant surfaces and in digestive tracts of animals and are therefore present in many foods. (10, 26). In many parts of the word clove used for malaria, cholera, tuberculosis and parasitic that cause illnesses for human. Cloves also may use to alleviate muscle spasms, skin ulcers and sties in the eyes. Clove oil is also a potent insecticide, repelling disease-causing mosquitoes and other insects. Clove oil is an essential oil extracted from clove plants especially from its flowers, stems and leaves. The quality of clove oil is normally indicated by its eugenol and carryophyllene contents (25, 31). Methyl eugenol (ME), as a constituent in leaves, fruits, stems, and/or roots, may be released when that part of a plant is damaged. Methyl eugenol is a component of several essential oils that are sold for use in aromatherapy, oils that used for massage treatment and alternative medicines (25). Many pathogenic organisims had become resistant nowadays for many manufactured antibiotics (9). So various researches have been done to improve and focus on the pharmatical characteristics of plants and their parts as alternative sources for many synthetic medicinal drugs, for example clove Syzygium aromaticum. (29). MATERIALS AND METHODS Sample preparation and extraction Dried clove samples were collected from the local markets of Baghdad\ Iraq. Grinding was done for clove bud with grinder in the laboratory of Market Researches and Consumer Protection Center. The samples were kept in closed containers after being chopped into small pieces (1 mm). Preparation extracts (oil Extract) Clevenger is used for oil extraction to extract cloves volatile oil from S. aromaticum plant. 10g of ground cloves and 150 mL of distilled water. The cloves were allowed to be wetted in the water for about 15 min, then the mixture was distilled after that transferring the distillate quantitatively to the separator funnel, the distillate was extracted twicly with 2.0 mL of CH2Cl2 Dichloromethane (DCM), the DCM extracts were combined, adding Na2SO4 to be dried, genteelly evaporated to get eugenol as a pale yellow oil (15, 20). Tree different concentration were prepared to be tested as treatments (T1 = 25% extract +75% DW), (T2 = 50% extract + 50% DW), (T3 = 100% extract) Then keep the oil in the refrigerator until use. Determination of active compounds The determination of some active groups, like {Flavonoids, Phenols, Tannins, Terpenes, Alkaloids, Coumarins}(13). HPLC (Shimadzu\Japan) used for purification and quantification of phenolic compounds in volatile oil of cloves buds plant, the mobile phase solution is consists of (Acetonitrile: Deionized distilled water with the ratio of 40:60) respectively, then the column C18 was used as stationary phase, flow rate 1.25 ml / min and the wavelength 210 nm, and injection was 25 μl of sample (15). Bacterial isolation Four types of bacteria were isolated from contaminated food: Salmonella sp. bacteria was isolated from several samples of meat and meat products, Escherichia coli bacteria was isolated from several vegetables samples, Lesteria sp. and Staph sp. were the gram Iraqi Journal of Agricultural Sciences –2019:50(6):1645-1651 Karm 1647 positive bacteria that were isolated from ice cream and vegetable fresh foods, These bacteria were identified according to several tests were depended first the Gram stain, second microscopic examination for morphology exam and some biochemical tests (5, 11), all these tests were done in Center of Market Researches and Consumer Protection. Antimicrobial activity assay Antibacterial activity was confirmed by use of disc diffusion method (27). The clove extract and the antibiotic drug Oflaxacin, stocks were also made for it at 30 μg \ml the Oflaxacin served as positive controls. The nutrient agar plates were spread with 100 μL of respective culture of pathogenic bacteria and the loaded discs were placed onto the surface of agar. The plates were left for 5 min for drying then plates were incubating at 37°C for 24 h. The results shows the inhibition zone which were measured in millimeters was determined (19). All tests in this study were done with 5 replications. RESULTS AND DISCUSSION Determination of active compound All treatments of oil extraction from powder samples of S. aromaticum plant were detected for their quality of active groups, so some active substances such as Flavonoids, Phenols, Tannins, Terpenes and Alkaloids were optioned as it found in table (1). Table 1. Active compounds of studied treatments in oil extraction for Syzygium aromaticum Extractions Flavonoids Phenols Tannins Terpenes Alkaloids


INTRODUCTION
Food spoilage is a process in which foods to be undesirable, unacceptable and un useful for human consumption due to changes in almost many characteristics, also it is clear that spoiled foods may be un safe to eat specially when its contain pathogens or a toxin present (18). Many problems were reported that we cannot use chemical preservatives products safely any more due to carcinogenic effects of these chemicals, thus the requirement has been increased for natural preservatives (27,28). Bacteria are an expected component of decomposing animals and plants (7). A group of Gram-positive bacteria, including Lactobacillus, Pediococcus, Leuconostoc and Oenococcus, under low oxygen, low temperature, and acidic conditions, turn to be spoilage organisms on foods. Listeria spp., Staph sp. diseases that can be serious and cause damages to human and animals. (3, 4). Many pathogenic microbial organisims such as Enterobacteriaceae they are gram-negative, facultative anaerobic bacteria that include a number of human pathogens (Salmonella sp., E. coli, Shigella, Yersinia). Bacillus and Geobacillus spp. Clostridium spp. cause spoilage of canned foods. These bacteria are widespread in nature in soil, on plant surfaces and in digestive tracts of animals and are therefore present in many foods. (10,26). In many parts of the word clove used for malaria, cholera, tuberculosis and parasitic that cause illnesses for human. Cloves also may use to alleviate muscle spasms, skin ulcers and sties in the eyes. Clove oil is also a potent insecticide, repelling disease-causing mosquitoes and other insects. Clove oil is an essential oil extracted from clove plants especially from its flowers, stems and leaves. The quality of clove oil is normally indicated by its eugenol and carryophyllene contents (25,31). Methyl eugenol (ME), as a constituent in leaves, fruits, stems, and/or roots, may be released when that part of a plant is damaged. Methyl eugenol is a component of several essential oils that are sold for use in aromatherapy, oils that used for massage treatment and alternative medicines (25). Many pathogenic organisims had become resistant nowadays for many manufactured antibiotics (9). So various researches have been done to improve and focus on the pharmatical characteristics of plants and their parts as alternative sources for many synthetic medicinal drugs, for example clove Syzygium aromaticum. (29).

MATERIALS AND METHODS Sample preparation and extraction
Dried clove samples were collected from the local markets of Baghdad\ Iraq. Grinding was done for clove bud with grinder in the laboratory of Market Researches and Consumer Protection Center. The samples were kept in closed containers after being chopped into small pieces (1 mm).

Preparation extracts (oil Extract)
Clevenger is used for oil extraction to extract cloves volatile oil from S. aromaticum plant. 10g of ground cloves and 150 mL of distilled water. The cloves were allowed to be wetted in the water for about 15 min, then the mixture was distilled after that transferring the distillate quantitatively to the separator funnel, the distillate was extracted twicly with 2.0 mL of CH2Cl2 Dichloromethane (DCM), the DCM extracts were combined, adding Na2SO4 to be dried, genteelly evaporated to get eugenol as a pale yellow oil (15,20). Tree different concentration were prepared to be tested as treatments (T1 = 25% extract +75% DW), (T2 = 50% extract + 50% DW), (T3 = 100% extract) Then keep the oil in the refrigerator until use.

Determination of active compounds
The determination of some active groups, like {Flavonoids, Phenols, Tannins, Terpenes, Alkaloids, Coumarins} (13). HPLC (Shimadzu\Japan) used for purification and quantification of phenolic compounds in volatile oil of cloves buds plant, the mobile phase solution is consists of (Acetonitrile: Deionized distilled water with the ratio of 40:60) respectively, then the column C18 was used as stationary phase, flow rate 1.25 ml / min and the wavelength 210 nm, and injection was 25 µl of sample (15).

Bacterial isolation
Four types of bacteria were isolated from contaminated food: Salmonella sp. bacteria was isolated from several samples of meat and meat products, Escherichia coli bacteria was isolated from several vegetables samples, Lesteria sp. and Staph sp. were the gram positive bacteria that were isolated from ice cream and vegetable fresh foods, These bacteria were identified according to several tests were depended first the Gram stain, second microscopic examination for morphology exam and some biochemical tests (5, 11), all these tests were done in Center of Market Researches and Consumer Protection. Antimicrobial activity assay Antibacterial activity was confirmed by use of disc diffusion method (27). The clove extract and the antibiotic drug Oflaxacin, stocks were also made for it at 30 μg \ml the Oflaxacin served as positive controls. The nutrient agar plates were spread with 100 μL of respective culture of pathogenic bacteria and the loaded discs were placed onto the surface of agar. The plates were left for 5 min for drying then plates were incubating at 37°C for 24 h. The results shows the inhibition zone which were measured in millimeters was determined (19). All tests in this study were done with 5 replications.

RESULTS AND DISCUSSION Determination of active compound
All treatments of oil extraction from powder samples of S. aromaticum plant were detected for their quality of active groups, so some active substances such as Flavonoids, Phenols, Tannins, Terpenes and Alkaloids were optioned as it found in table (1). It was clearly that the active compounds were in positive relation due to the rising of extract concentration and that results is compatible with the results of a study by Abo El-Maati, et al 2016, that concluded cloves extracts may be an effective antioxidant and antimicrobial and bioactive agents to promote health(1) and the these quality test shows that flavonoid and phenols were the highest results. The most suitable explanation were applied in a results study by Adaramola and Onigbinde (2), they found that clove bud has more flavonoids and phenols than Tannins and Terpenes or the extraction solvents were able to extract more flavonoids and phenols than other compounds. Positive correlation among all active compounds of clove bud and their antimicrobial and antioxidant capacity which may be due the presence of many phytochemicals and synergistic effect of all the phytochemicals that may be present. (6, 14) as shown in table (1).

Antimicrobial activity
Four pathogenic bacteria were collected, isolated and identified according to their morphological and chemical tests and gram stain test. Tow were positive gram, Lesteria sp. and Staph sp., others were gram negative,

Escherichia coli and Salmonella typhimurium.
These bacteria were chosen for their high resisted towards many chemical antibiotics also they had a high ferociousness. Disc diffusion method was confirmed and inhibition zones were measured in millimeters. The inhibition zones for tow positive tested bacteria Lesteria sp. and Staph sp. increased with the increasing of the concentration of the extract, these results were in agreement with results of many researches (8,23,27). It is found that the negative gram tested bacteria, Escherichia coli and Salmonella typhimurium were proportional in their response toward the treatments and shows a gradual increasing in inhibition zones with the rising of extract concentrations, these facts were in accord with some studies (21,32,33) as it shown in table (2). All tests in this study were done with 5 replications. All bacterial strains were sensitive to the activity of clove extract which damages the cells and inhibits the growth of both gram positive and gram negative bacteria this was explained and found that the primary mechanism of action of clove extract is membrane damage, which leads to cell death. (12,17,30).
Table2. Growth inhibition zone (mm) by clove extract T1, T2 and T3 against pathogenic bacteria and standard antibiotic (control treatment). Each sample result is a mean for five replications.
It was clearly that there is a positive relation between the concentration of the extract and the inhibition zones, after using a method to extract and isolate the eugenol so it is clear that the major phytochemical in clove is eugenol, that was in agreement with a study by Mittal, et al 2014, they recorded that major phytochemicals found in clove oil is mainly eugenol that is about (70-85%) followed by eugenyl acetate (15%) and finely βcarryophyllene which is less than 5% (22). It was obvious the best result is at the treatment T3 so the HPLC test was done and the results shows that the most important active compound which was detected is the aromatic oil called eugenol, the retention time (RT) of the extract compared with retention time (RT) of standard compound (15). RT of eugenol stander was 12.20 as showed in figer (1), and the RT for T3 was tested and it was 12.19 as showed in figer (2) and this result shows the major area for eugenol which is a prove that this active component was responsible for the greet activity of this plant and that was coinciding with a study by some researchers that identificated and analyzed the chemicals of clove extract, (16,24). The results of this study encourage the use of natural sours like some plants or some parts of plants and the present tests improve the bioactivity including antibacterial action thus related to active compounds in the clover (Syzygium aromaticum) especially in the buds, actually this could be one of many promised treat to solve some series problems done by bacteria activities that cause food poisoning and harmful for humans health.