Screening of Antimicrobial Activity of Fenugreek Seeds

Plants have formed the basis for traditional medicine systems in most societies and have been used for thousands of years in countries like China and India. Plant-based systems still play a vital role in health care, and WHO has estimated about 80% of the world’s inhabitants rely mainly on traditional medicine for their primary health care. Various plants (whole or some parts) are known to be useful use for human benefit and welfare. Of these benefits, is the capacity of some plant seeds, fruits, leaves and other parts exerting pharmacological activities such as, analgesics, diuretics, antispasmodics, antimicrobial activity.. etc. Fenugreek (Trigonella foenum graecum Linn.) is an annual herb which has widely been consumed throughout the world as a food, a food additive and in the traditional remedies science civilizations. Fenugreek, being rich in photochemicals, has traditionally been used as a food, forage and medicinal plant [1]. The active principles of many drugs found in plants are secondary metabolites. The antimicrobial activities of plant extracts may reside in a variety of different components [2]. Fenugreek seeds contain lysine and L-tryptophan rich proteins, mucilaginous fibre and other rare chemical constituents such as saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline, which are thought to account for many of its presumed therapeutic effects [3]. The steroidal saponins (diosgenin, yamogenin, tigogenin and neotigogenin) are thought to inhibit cholesterol absorption and synthesis and hence its potential role in arteriosclerosis.


Introduction
Plants have formed the basis for traditional medicine systems in most societies and have been used for thousands of years in countries like China and India. Plant-based systems still play a vital role in health care, and WHO has estimated about 80% of the world's inhabitants rely mainly on traditional medicine for their primary health care. Various plants (whole or some parts) are known to be useful use for human benefit and welfare. Of these benefits, is the capacity of some plant seeds, fruits, leaves and other parts exerting pharmacological activities such as, analgesics, diuretics, antispasmodics, antimicrobial activity.. etc. Fenugreek (Trigonella foenum -graecum Linn.) is an annual herb which has widely been consumed throughout the world as a food, a food additive and in the traditional remedies science civilizations. Fenugreek, being rich in photochemicals, has traditionally been used as a food, forage and medicinal plant [1]. The active principles of many drugs found in plants are secondary metabolites. The antimicrobial activities of plant extracts may reside in a variety of different components [2].
Fenugreek seeds contain lysine and L-tryptophan rich proteins, mucilaginous fibre and other rare chemical constituents such as saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline, which are thought to account for many of its presumed therapeutic effects [3]. The steroidal saponins (diosgenin, yamogenin, tigogenin and neotigogenin) are thought to inhibit cholesterol absorption and synthesis and hence its potential role in arteriosclerosis.
Locally, fenugreek seeds have traditionally and commonly been used to treat diabetes, coughs, congestion, bronchitis, fever, high blood pressure, headache, migraines, diarrhea, flatulence, anemia, irregular menstrual cycles and arthritis, to ease labor pains and menstruation pain, and as an appetite stimulant [4]. Fenugreek has also been used as an external poultice to control inflammation and dandruff. Modern medicine is beginning to provide confirmation of many of the traditional medicinal applications of fenugreek seeds [5,6]. To the best of our knowledge, no previous studies have been reported for antimicrobial activity of fenugreek. Thus, this study was aimed to evaluate the antimicrobial activity of fenugreek seeds in order to be used in some infectious diseases.

Materials and Methods
Fenugreek was procured from the local market. Sixty gram of fenugreek (seeds and powdered seeds) was extracted by maceration in 200 ml of cold, hot and boiling water. Different concentrations of fenugreek powder (20, 40, and 100 gm) were extracted in methanol as organic solvent. In vitro, antimicrobial activities of the extractions of different concentrations were tested for the antimicrobial activity using both Gram positive and Gram negative bacteria (six microorganisms including E. coli, C. albicans, P. vulgaris, St. aureus St. epidermis and St. saprophyticus). Antimicrobial activity evaluations were performed using two different methods: agar disc diffusion and agar-well diffusion method [7,8]. Gentamicin (10 µg) was used as a positive control [9,10]; pure methanol and distilled water were used as a negative control in both methods. In addition, the extraction of fenugreek seeds in boiling water was used to test its effect on some staphylococcus species (St. epidermis, St. saprophyticus and St. aureus) using disc diffusion method.

Abstract
The pharmaceutical industry has produced a large number of new antibiotics over the last three decades, but resistance to these antibiotics by microorganisms has increased. Statistically, WHO estimates that medicinal plants are the primary medicine for 2/3 of the world's population. Recent studies focus on plant research all over the world to extract appropriate and efficient antimicrobial drugs. Fenugreek (Trigonella foenum-graecum Linn.) is an annual herb which has widely been consumed throughout the world as a food, a food additive and in the traditional remedies science civilizations. This study was aimed preliminary to investigate in vitro antimicrobial activity of fenugreek seeds against Gram-negative and Gram-positive bacteria and other microorganisms such as Samples were incubated at 37 o C. The diameter of clear zone of inhibition surrounding each disc was measured in millimeters. The antimicrobial activity was determined by measuring the diameter of zone of inhibition that is the mean of triplicates ± SD of two replicates. Table 2 show that the extraction of fenugreek seeds by boiling water has some inhibitory effect on the growth of St. Aureus bacteria in disc diffusion method and agar well diffusion method with a zone inhibition of diameters of 22 mm and 10 mm respectively. To verify this finding; other species of staphyloccus were used to test this effect. Table 3 indicates that fenugreek has some antimicrobial effect on all the species of staphyloccus and it also shows that the inhibition is directly proportion to the extract concentration. The other microorganisms (E. coli, C. albicans, and P. vulgaris) were found to be resistance to all aqueous extractions including the boiling water. The effects of different methanol fenugreek powder extract concentrations by disc diffusion method and by agar well diffusion method did not show any inhibitory effect on the growth of all the microorganisms used (concentrations: 20, 40 and 100 gm per 200 ml, data not shown).

Discussion
The potential for developing antibacterial into medicine appears rewarding from the perspective of drug development and phytomedicine. There are several studies of antibiotics resistance of human pathogens to available antibiotics [11,12]. Biomolecules of plant origin appear to be one of the alternatives for the control of these antibiotic resistant human pathogens. In North Africa, fenugreek is one of the medicinal plants that are traditionally used for therapy. In the present study, crude extracts of the plant materials (seeds and powder) obtained in aqueous and alcoholic solvents were tested against six different microorganisms including E. coli, C. albicans, P. vulgaris, St. aureus, St. epidermis and St. saprophyticus. The results show that only the boiling water extraction of fenugreek seeds has an inhibitory effect on the growth of St. aureus by the two different testing methods, agar disc diffusion and agar-well diffusion (Tables 1 &  2). This may be explained by that only the boiling water extract contains the antimicrobial active ingredients of fenugreek that are responsible for the antimicrobial effect, while both cold water extract and methanol are not suitable for such purposes. This antimicrobial effect is confirmed by testing the boiling water extract of fenugreek seeds on other three different species of staphyloccus (St. epidermis, St. aureus, and St. saprophyticus). All the species show sensitivity to this extraction type ( Table  3). The antimicrobial activity of the boiling water extraction of fenugreek seeds is affected by its concentration, as changing the seed concentrations from 60 gm per 200 ml of water to 100 gm of seed per 200 ml of water results to about 2-fold in zone of growth inhibition from 7mm to 12mm for St. aureus ( Table  3). The finding of this preliminary screening of antimicrobial activity of fenugreek seeds suggest that the minimum inhibitory concentration (MIC, MIC50) is determined in future studies when a quantitative sensitivity result is required. Furthermore, the two antimicrobials (antimicrobial-like effect of fenugreek seeds and gentamicin) interactions are suggested to be studied to identify the nature of interaction (additive/synergistic using summed fractional inhibitory concentration). However, before such studies carried out, mode of action and active ingredients of the fenugreek seeds are needed to be identified (see below).
All these preliminary indications dedicate further studies for the effect of fenugreek seed extraction on staphylococcus growth since infections caused by St. aureus are difficult to treat with conventional antibiotics [13]. In vitro inactivity of other extractions on staphylococcus and other microorganisms may not necessary translate to their in vivo inactivity. The determination of the exact compound which is responsible for this effect may pave the way for new potential drugs. Previously, it has also been shown that scopoletin, a coumarin derivative of coumaric acid, responsible for the antimicrobial activity of fenugreek against prokaryotes [14].

Conclusion
This study suggests that extracts of fenugreek may have antibacterial activity against some human pathogens. Therefore, it paves the way for further research to identify the active compounds responsible for the plant biological activity and its mechanism.