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Antibacterial effects of oregano (Origanum vulgare) against gram negative bacilli



The oil, aqueous infusion and decoction of oregano (Origanum vulgare), of the family Limiaceae, were asessed for antibacterial activity against 11 different genera of Gram–ve bacilli viz., Aeromonas hydrophila, Citrobacter sp., Enterobacter aerogenese, Escherichia coli, Flavobacterium sp., Klebsiella ozaenae, K. pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, S. paratyphi B, Serratia marcescens and Shigella dysenteriae, by disc diffusion method. Oregano oil exhibited the highest activity against Citrobacter species with mean zone of inhibition of 24.0 mm ± 0.5. The aqueous infusion also showed significant inhibitory activity against Klebsiella pneumoniae (20.1 mm ± 6.1 SD), Klebsiella ozaenae (19.5 mm ± 0.5 SD) and Enterobacter aerogenes (18.0 mm). Besides, all isolates were found resistant to the aqueous decoction of oregano seeds.
Pak. J. Bot., 39((2): 609-613, 2007.
Department of Microbiology,
University of Karachi, Karachi-75270, Pakistan
The oil, aqueous infusion and decoction of oregano (Origanum vulgare), of the family
Limiaceae, were asessed for antibacterial activity against 11 different genera of Gram–ve bacilli
viz., Aeromonas hydrophila, Citrobacter sp., Enterobacter aerogenese, Escherichia coli,
Flavobacterium sp., Klebsiella ozaenae, K. pneumoniae, Proteus mirabilis, Pseudomonas
aeruginosa, Salmonella typhi, S. paratyphi B, Serratia marcescens and Shigella dysenteriae, by
disc diffusion method. Oregano oil exhibited the highest activity against Citrobacter species with
mean zone of inhibition of 24.0 mm ± 0.5. The aqueous infusion also showed significant inhibitory
activity against Klebsiella pneumoniae (20.1 mm ± 6.1 SD), Klebsiella ozaenae (19.5 mm ± 0.5
SD) and Enterobacter aerogenes (18.0 mm). Besides, all isolates were found resistant to the
aqueous decoction of oregano seeds.
Microorganisms occur nearly everywhere in nature and affect the well being of
people in a great many ways. Many different microbial species normally inhabit various
parts of our bodies, such as the oral cavity, skin and intestinal tract. A variety of Gram
negative bacilli (GNB) are members of the intestinal microbiota of most animals and
humans as normal commensals or pathogens. They are mostly members of the family
Enterobacteriaceae but members of other taxonomical groups of Vibrionaceae are also
considered in this category. The GNB emerged rapidly, caused a large outbreak of
serious infections, and contributed to mortality (Herbert et al., 2007). The GNB vary in
the frequencies that they cause either intestinal and extra-intestinal infections. The
example of intestinal disorders are enteric fever, inflammatory and non-Inflammatory
enteritis. Among the extra-intestinal diseases, such as nosocomial urinary tract infections
(Yu et al., 2006), chronic obstructive pulmonary disease (Lin et al., 2007), neonatal
septicaemia (Iregbu et al., 2006), wound infections, meningitis, pneumonia, bloodstream
infection and bacteremia with septic shock are the most prevalent diseases (Donskey,
2006). During the past 20 years, changes in health care, infection-control practices, and
antimicrobial use and resistance may have influenced the frequency that these GNB are
associated with hospital-acquired infection (Gaynes & Edwards, 2005).
Previous research studies have documented increasing rates of antimicrobial
resistance of GNB (Gupta et al., 2006). Misuse of antibiotics and ineffective infection
control have been implicated in the development and spread of resistant GNB pathogens
which are associated with increased mortality and morbidity, prolonged hospitalization
and increased costs. It is evident from literature that new multi-drug-resistant GNB
strains have emerged and proliferated in the world. For example, the occurrence of
fluoroquinolone-resistant GNB colonizing community-dwelling people with spinal cord
dysfunction is common (Roghmann et al., 2006). It has also been reported that
ciprofloxacin-resistant GNB are becoming increasingly important and that they may
cause serious infections in children (Qin et al., 2006).
Problematic organisms including non-fermentative Pseudomonas aeruginosa
(Wroblewska, 2006), multi-drug resistant Salmonella typhi (Akinyemi et al., 2000) and
other GNB producing extended-spectrum beta-lactamases (GNB-ESBL), pose a
particular difficulty for the healthcare community because they represent the problem of
multi-drug resistance to the maximum (Wroblewska et al., 2006). These organisms are
niche pathogens that primarily cause opportunistic healthcare-associated infections in
patients who are critically ill or immunocompromised. Multi-drug resistance is common
and increasing among GNB, and a number of strains have now been identified that
exhibit resistance to essentially all commonly used antibiotics, including
antipseudomonal penicillins, cephalosporins, aminoglycosides, tetracyclines,
fluoroquinolones, trimethoprim-sulfamethoxazole and carbapenems (McGawan, 2006).
The impact of antibiotic resistance on the outcome of infections due to GNB remains
highly controversial. Therapeutic options for multi-drug resistant GNB strains are
limited; for this reason, there are a continuous need for alternative new chemical entities
with such activities may be identified through a variety of approaches (Combes et al.,
Screening of natural medicinal plants is common because many infectious diseases
are known to have been treated with herbal remedies throughout the history of mankind.
Even today, plant materials continue to play a major role in primary health care as
therapeutic remedies in many developing countries (Naim & Tariq, 2006). In search for
alternative ways of infectious disease control; essential oil, aqueous infusion and aqueous
decoction from oregano were used in the present study to check their antibacterial
properties against GNB using standard disc diffusion method In vitro.
Materials and Method
Maintainance of isolates: A total of 100 bacterial isolates belonging to 11 different
genera of GNB viz., Aeromonas hydrophila (2), Citrobacter sp. (3), Enterobacter
aerogenes (2), Escherichia coli (22), Flavobacterium sp. (4), Klebsiella ozaenae (12),
Klebsiella pneumoniae (20), Proteus mirabilis (7), Pseudomonas aeruginosa (15),
Salmonella typhi (5), S. paratyphi B (2), Serratia marcescens (1) and Shigella
dysenteriae (5); were maintained on Nutrient Agar medium (Oxoid).
Preparation of aqueous infusion: Aqueous infusion of oregano seeds was prepared by
soaking 20g in 100ml sterile distilled water in sterile flask. The flask was kept for two
days with occasional shaking. The contents of flasks were filtered.
Preparation of aqueous decoction: Aqueous decoction of oregano seeds was prepared
by boiling 20g in 100ml sterile distilled water for 15minutes. The flask was then plugged
and removed from heat and allowed to cool. After cooling the contents of flask were
Essential oil of oregano: Essential oil of oregano (Planter) was purchased from the local
market of Saddar, Karachi, Pakistan.
Screening of antibacterial activity: Screening of antibacterial activity was performed by
standard disc diffusion method (Saeed et al., 2006). Hundred sterilized discs of filter
paper (6 mm diameter) were soaked in 1 ml of infusion, decoction and oil, seperately, for
1-2 minutes and then used for screening. Thus potency of each disc was 10 μl. Mueller-
Hinton agar (MHA) (Merck) was used as base medium and Mueller-Hinton broth (MHB)
was used for the preparation of inoculum. Four to five isolated colonies of tested
organisms were picked by sterile inoculating loop and inoculated in tubes of MHB (5 ml
in each). The inoculated tubes were incubated at 35-37oC for 24 hours and matched with
0.5 McFarland nephelometer turbidity standard (Saeed & Tariq, 2005). A sterile cotton
swab was dipped into the standardized bacterial test suspension to inoculate entire surface
of a MHA plate. Discs of infusion, decoction and oil were placed on the surface of
inoculated plates with the help of sterile forcep. The inoculated plates were incubated at
35-37oC for 24 hours. After incubation inhibition zone diameters were measured to the
nearest millimeter (mm).
Statistical analysis: Mean zone of inhibition and standard deviations were calculated.
Results and Discussion
Oregano has been a valuable source of natural products for maintaining human
health for a long period of time, especially in last decade, with more intensive studies for
natural therapies (Force et al., 2000). The volatile oil of oregano has been used
traditionally for respiratory disorders, indigestion, dental caries, rheumatoid arthritis, and
urinary tract disorders. As a medicinal plant, oregano has been used as antifungal,
anticoccidial, antispazmolytic, antibacterial (Ertas et al., 2005), antioxidant (Lamaison et
al., 1991), antiaggregant (Okazaki et al., 2002) and antiinflammatory (Kelm et al., 2000)
agent. In addition, it has stimulating effect of digestion and antiseptic (Cabuk et al.,
2003). The most important components of oregano are the limonene, gamma-cariofilene,
rho-cymenene, canfor, linalol. Alpha-pinene, carvacol and thymol (Arcila-Lozano et al.,
2004). Among them thymol and carvacrol are the main components of the essential oil of
oregano (Tian & Lai, 2006), which are responsible for its antioxidative, antimicrobial and
antifungal effects (Proestos et al., 2005).
Antibacterial activity of aqueous infusion, aqueous decoction and oil of oregano
seeds was evaluated against 11 different GNB that are known to cause infections in
human. In the present study, the oregano oil revealed the highest antibacterial activity. It
was followed by aqueous infusion while aqueous decoction of oregano did not show
inhibitory potential against tested bacterial isolates (Table 1).
The oregano oil exhibited significant inhibitory activity against Citrobacter spp.,
(24.0 mm ± 0.5 SD), Salmonella typhi (22.4 mm ± 1.5 SD) and Escherichia coli (19.0
mm ± 2.2 SD) (Table 1). It has long been acknowledged that oregano oil is among the
most active against strains of E. coli and also presents antimicrobial activity against
pathogenic microorganisms like Salmonella. choleraesuis, S. typhi, S. typhimurium and
many others related GNB strains of Enterobacteriaceae family (Penalver et al., 2005)
and H. pylori (Stamatis et al., 2003). Similarly, the results of present study are in
accordance with the reports on oregano oil against GNB viz., Proteus vulgaris,
Aeromonas hydrophila, Klebsiella pneumoniae and Escherichia coli (Baydar et al.,
In the present study, the antibacterial effect of aqueous infusion of oregano was next
to oil and exhibited significant inhibitory activity against Klebsiella pneumoniae (20.1 ±
6.1SD), Klebsiella ozaenae (19.5 ± 0.5SD) and Enterobacter aerogenes (18.0 ± 00SD).
It is interesting to note that aqueous infusion of oregano inhibited all type of tested
bacterial strains. The present study also described the antibacterial activity of aqueous
decoction of oregano seeds. It was found that the aqueous decoction of oregano seeds did
not posses any antibacterial effect against tested GNB.
Table 1. Antibacterial activities of oil, aqueous infusion and aqueous decoction of
oregano seeds against Gram negative bacteria
Mean zone of inhibition in mm ±
standard deviation
Organisms No. of
isolates Oil Infusion Decoction
Aeromonas hydrophila 2 12.8 ± 3.3 10.5 ± 1.5 ---
Citrobacter sp. 3 24.0 ± 0.5 10.0 ---
Enterobacter aerogenes 2 --- 18.0 ± 0.0 ---
Escherichia coli 22 19.0 ± 2.2 15.8 ± 1.5 ---
Flavobacterium sp. 4 11.6 ± 0.9 8.8 ± 1.5 ---
Klebsiella ozaenae 12 --- 19.5 ± 0.5 ---
Klebsiella pneumoniae 20 11.7 ± 1.6 20.1 ± 6.1 ---
Proteus mirabilis 7 --- 10.2 ± 1.3 ---
Pseudomonas aeruginosa 15 --- 10.8 ± 1.0 ---
Salmonella typhi 5 22.4 ± 1.5 16.1 ± 3.8 ---
Salmonella para typhi B 2 12.0 ± 1.0 13.0 ± 1.0 ---
Serratia marcescens 1 12.0 13.0 ---
Shigella dysenteriae 5 9.5 ± 0.5 11.0 ± 0.7 ---
The study demonstrated that oregano represents an economic source of natural
mixtures of antibacterial compounds that can be as effective as modern medicine to
combat pathogenic microorganisms and safe alternative to treat infectious diseases.
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(Received for publication 19 February 2007)
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Human exposure to mold species often results in infections, ranging from common responses to damaging, life-threatening conditions. The use of synthetic alternatives has been the primary resort to keep products in good conditions. The preservatives, however, were also seen to increase the risks of diseases. In this premise, this study assessed the mold inhibitory property of Oregano Tincture and Orange Peel Powder for its potential to be used in food and materials application. It also determined the level of acceptability of the finished product to evaluate its prospective marketing value. Product development and descriptive methods were employed. In evaluating the product's acceptability, questionnaires were utilized and answered by homeowners and experts in the fields of Chemistry and Biology. With regards to its mold inhibitory property, bread, cheese, and wood were used as media for direct observation using visible mold growth and mold smell as indicators. The survey revealed that the product was accepted by the respondents, in terms of its sensory attributes. No significant difference was seen in the ratings of the homeowners and experts. Observation results exhibited strong mold inhibitory property of the product. This study provides significant impression for the potential of plant substances to be utilized against problems brought about by molds.
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Ruminant productivity across the world and particularly in developing countries is becoming increasingly challenging due to human-animal competition for the food and animal nutritionists have been hunting for alternate resources and harvesting maximal efficiency for a targeted gain in livestock production. Increasing efficiency of utilization of different types of feeds by ruminants depends generally on nature of rumen fermentation and thus manipulation directed at maximizing fibre degradation, improving N use efficiency, minimizing gaseous (methane) losses and overall enhancement in energy efficiency has got significant bearing in present-day nutritional research. Plant secondary metabolites or phytochemicals that have in the past been considered as a source of anti-nutritional factors is now being considered as a source of exploitable rumen as well as host metabolic modifiers. In the hind-sight, the growing problem of antibiotic and anthelmintic resistance is an increasingly serios problem in human health care and livestock husbandry around the world, which warrants inadvertent use of chemicals or synthetics and this has stimulated interest and research in the use and effects of phytochemicals in the diets of farmed animals. Increasing concern on green house gas (GHG) emission including methane that partly emanated from ruminal methanogenesis, is often considered wastage of feed gross energy. There has been growing interest in use of plant parts and plant extracts to mitigate enteric ruminal CH4 emissions. It is imperative to Identify and evaluate plant secondary metabolites that could inhibit rumen methanogens/ methane synthesis and/or improve cell wall degradation to improve rumen energetic efficiency so as to improve productivity and reduce methane emission.
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Origanum vulgare L. (O. vulgare) is an important medicinal herb of the family Lamiaceae. In the current study, we explained the critical evaluation of traditional uses, the phytochemistry and the antimicrobial properties of O. vulgare and its subspecies, with a focus on the mechanisms of actions of the most important phytochemicals from O. vulgare subspecies. The most important phytochemicals of O. vulgare are volatile (essential oil) and non-volatile phenolic compounds (phenolic acids & flavonoids). The constituents of the O. vulgare essential oil (EO) include high percentages of thymol and carvacrol with excellent antimicrobial activity alone or in combination with other antibiotics. Interesting results have been reported the remarkable antimicrobial activities of infusion or tea products of O. vulgare with a high amount of EO against multidrug‐resistant bacterial and fungal microorganism (such as Escherichia coli, Staphylococcus aureus, Candida albicans and Pseudomonas aeruginosa). The most important antibacterial mechanisms of O. vulgare are enzyme inhibition, efflux pump inhibition, ATP depletion, biofilm formation inhibition and cytoplasmic membrane damage. The antimicrobial activity of the hirtum subspecies has been confirmed in different in-vitro and in-vivo studies. The present review confirms the clinical and preclinical research showing the O. vulgare and its subspecies antimicrobial effects.
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Antibacterial activities of different forms of peppermint (Mentha piperita) viz., aqueous infusion, decoction, juice and essential oil were investigated against 100 isolates belonging to 11 different species of Gram –ve bacilli viz., Escherichia coli (30), Klebsiella pneumoniae (25), Pseudomonas aeruginosa (15), Salmonella typhi (5), S. paratyphi A (1), S. paratyphi B (1), Proteus mirabilis (10), P. vulgaris (2), Shigella dysentriae (5), Yersinia enterocolitica (1), and Enterobacter aerogenes (5). The screening was performed by standard disc diffusion method. Essential oil of peppermint exhibited highest antibacterial activity with 11.78 mm mean zone of inhibition. The juice of peppermint also possessed antibacterial activity with 10.41 mm mean zone of inhibition, while all isolates were totally resistant to aqueous infusion and decoction of peppermint.
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The antibacterial activity of essential oils and their derivatives has been recognized for a long time. In the present study, the chemical composition and the antibacterial properties of the essential oils obtained from the aerial parts of the four Lamiaceae species, wild oregano (Origanum minutiflorum) (endemic in Turkey), oregano (Origanum onites), black thyme (Thymbra spicata) and wild savory (Satureja cuneifolia), with commercial importance in Turkey, were evaluated.The major constituent of the oils determined by GC was cavracrol (86.9% in O. onites, 84.6% in O. minutiflorum, 75.5% in T. spicata and 53.3% in S. cuneifolia). Four essential oils were investigated for activity against Aeromonas hydrophila, Bacillus amyloliquefaciens, B. brevis, B. cereus, B. subtilis, Corynebacterium xerosis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Micrococcus luteus, Mycobacterium smegmatis, Proteus vulgaris, Staphylococcus aureus and Yersinia enterocolitica, using a paper disc diffusion method. All essential oils inhibited all bacteria at concentrations of <1/100 (v/v). The essential oil of T. spicata was the most active. B. amyloliquefaciens was the most sensitive. The results of this study confirmed the possibility of using these four essential oils in food systems to prevent the growth of foodborne bacteria and extend the shelf life of processed foods.
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This study was conducted to explore the usage of essential oil mix (EOM) in broiler nutrition as a natural growth promoting substance instead of antibiotics. Different levels of EOM were added to a standard diet, to determine its effect on feed intake, daily live weight gain and feed conversion ratio compared to control and antibiotic groups. Two hundred and fifty five day-old broilers (Ross-308, n = 250) were divided into five equal groups were as follow: Control group (no EOM or antibiotic), 100 ppm EOM group , 200 ppm EOM group, 400 ppm EOM group with and an Antibiotic group (0.1% Avilamycin). The diets were prepared freshly each day. Experiment carried out 35 days. The feed intake was similar between the groups (p>0.05), but the highest daily live weight gain was observed in the 200 ppm EOM group (71.31 g) followed by Antibiotic group (65.84 g), 100 ppm EOM oil group (63.40 g), control group (61.30 g) and 400 ppm EOM group (61.17 g). Thus, daily live weight gain increased in 200 ppm EOM group by approximately 16 % over the control group and approximately 8 % over the antibiotic group. Feed conversion ratio was improved in 200 ppm EOM group by approximately 12 % over the control group and approximately 6 % over the antibiotic group. The results show that, EOM could be considered as a potential natural growth promoter for poultry.
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Oil of Mediterranean oregano Oreganum vulgare was orally administered to 14 adult patients whose stools tested positive for enteric parasites, Blastocystis hominis, Entamoeba hartmanni and Endolimax nana. After 6 weeks of supplementation with 600 mg emulsified oil of oregano daily, there was complete disappearance of Entamoeba hartmanni (four cases), Endolimax nana (one case), and Blastocystis hominis in eight cases. Also, Blastocystis hominis scores declined in three additional cases. Gastrointestinal symptoms improved in seven of the 11 patients who had tested positive for Blastocystis hominis.
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A total of 635 clinically diagnosed typhoid fever patients were bled from three different health institutions in the metropolis of Lagos, Nigeria over a period of 15 months, May 1997 to July 1998. Out of the total blood cultured, 101 (15.9% ) isolates of Salmonella species were isolated of which 68 (67.3% ) were S. typhi, 17 (16.8% ) and 16 (15.8% ) were S. paratyphi A and S. arizonae respectively. The overall isolation rate of S. typhi among patients is 10.7% , with most isolates 45.9% found among the severely-ill young adults, age group 1 6 -30 years. All isolates were subjected to anti-microbial susceptibility testing using 12 different antibiotics: chloramphenicol, ampicillin, cotrimoxazole, gentamicin, colistin sulfate, nalidixic acid, nitrofurantoin, cefotaxime, tetracycline, streptomycin, ofloxacin and ciprofloxacin. All the S. typhi and S. paratyphi A isolates showed resistance to two or more of the 10 of 12 antibiotics tested particularly the 3-first-line antibiotics commonly used (chloramphenicol, ampicillin and cotrim oxazole) in the treatment of typhoid fever in Nigeria. No isolate showed resistance to ofloxacin and ciprofloxacin, however, nalidixic acid and gentamicin showed a moderate and appreciable inhibition to most of our isolates.
The juices of leaves and stem of Mentha piperita (peppermint), skin and seeds of Pisum sativum (garden pea), skin and pulp of Momordica charantia (bitter melon) were screened for antibacterial activities against 56 isolates belonging to 11 different species of Gram-negative bacilli: Escherichia coli (19), Klebsiella pneumoniae (11), Pseudomonas aeruginosa (9), Salmonella typhi (3), Salmonella paratyphi A (1), Salmonella paratyphi B (1), Proteus mirabilis (5), Proteus vulgaris (1), Enterobacter aerogenes (4), Shigella dysenteriae (1), and Yersinia enterocolitica (1). The screening was performed by well diffusion method. Leaves of M. piperita exhibited highest antibacterial activity (average zone of inhibition 17.24 mm ± 0.87 SD) while stem of M. piperita exhibited least antibacterial activity (average zone of inhibition 15.82 mm ± 3.56 SD). The skin and seeds of P. sativum, skin and pulp of M. charantia exhibited good antibacterial activity with average zone of inhibition of 16.30 mm ± 2.02 SD, 16.39 mm ± 3.16 SD, 16.16 mm ± 2.17 SD and 15.88 mm ± 2.24 SD respectively.
Hydroalcholic extracts from four native medicinal Lamiaceae, Lycopus europaeus L., Melissa officinalis L., Origanum vulgare L. and Prunella vulgaris L. have shown significant antioxidative activities, by free radical scavenger effect on DPPH, compared with those of Rosmarinus officinalis L. and Salvia officinalis L. extracts. The antioxidative activity was partly in relation to the rosmarinic acid content. The major hydroxycinnamic compound, quantitatively determinated by HPLC, was present in large amount. The content in Prunella vulgaris L. spikes average 6.1%, based on dry weight.
Anti-oxidant bioassay-directed extraction of the fresh leaves and stems of Ocimum sanctum and purification of the extract yielded the following compounds; cirsilineol [1], cirsimaritin [2], isothymusin [3], isothymonin [4], apigenin [5], rosmarinic acid [6], and appreciable quantities of eugenol. The structures of compounds 1-6 were established using spectroscopic methods. Compounds 1 and 5 were isolated previously from O. sanctum whereas compounds 2 and 3 are here identified for the first time from O. sanctum. Eugenol, a major component of the volatile oil, and compounds 1, 3, 4, and 6 demonstrated good antioxidant activity at 10-microM concentrations. Anti-inflammatory activity or cyclooxygenase inhibitory activity of these compounds were observed. Eugenol demonstrated 97% cyclooxygenase-1 inhibitory activity when assayed at 1000-microM concentrations. Compounds 1, 2, and 4-6 displayed 37, 50, 37, 65, and 58% cyclooxygenase-1 inhibitory activity, respectively, when assayed at 1000-microM concentrations. Eugenol and compounds 1, 2, 5, and 6 demonstrated cyclooxygenase-2 inhibitory activity at slightly higher levels when assayed at 1000-microM concentrations. The activities of compounds 1-6 were comparable to ibuprofen, naproxen, and aspirin at 10-, 10-, and 1000-microM concentrations, respectively. These results support traditional uses of O. sanctum and identify the compounds responsible.
Two antiaggregant compounds, thymol (compound 1) and 3,4,3',4'-tetrahydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (compound 2) were isolated from the leaves of thyme (Thymus vulgaris L.). The structures were determined by (1)H-, (13)C-NMR and mass spectra (MS) studies. These compounds inhibited platelet aggregation induced by collagen, ADP, arachidonic acid (AA) and thrombin except that compound 2 did not inhibit platelet aggregation induced by thrombin.