ANTIBACTERIAL ACTIVITIES OF TRIGONELLA FOENUM-GRAECUM AND ZINGIBER OFFICINALE

ANTIBACTERIAL ACTIVITIES OF TRIGONELLA FOENUM-GRAECUM AND ZINGIBER OFFICINALE

Mamta Bhatia* and Alka Sharma

Department of Food Technology, Guru Jambheshwar University of Science and Technology, Hisar-125021, Haryana, India

ABSTRACT

An in vitro study was conducted for screening antibacterial activities of seeds of Trigonella foenum-graecum (fenugreek)and rhizomes of Zingiber officinale (ginger), in their different forms viz.  aqueous extracts, essential oils and powders against some common food borne pathogens. Bacterial strains involved in the study were Bacillus cereus (MTCC 430), Enterococcus feacalis (MTCC 439), Escherichia coli (MTCC 1687), Psuedomonas aeruginosa (MTCC 424) and Staphylococcus aureus (MTCC 5021).  Results revealed that powdered forms of T. foenum-graecum and Z. officinale remained ineffective in arresting the growth of all the bacterial strains under investigation. During preliminary screening, aqueous extracts and essential oils of test spices did not exhibit any growth inhibitory zone towards any test bacteria. In broth dilution technique, essential oil of Z. officinale arrested all the bacterial strains under observation while essential oil of T. foenum-graecum and aqueous extracts of T. foenum-graecum and Z. officinale remained ineffective.

Aqueous extracts

INTRODUCTION: Current economic and biological assessment upon withdrawal of most of the conventional synthetic preservatives registered for control of microorganisms  implicated in food spoilage have elicited widespread interest in providing new perspectives for the development of future antimicrobials based on natural substances, those are socially more acceptable ¹. According to the council for Agriculture Science and Technology, an international consortium of 36 scientific and professional societies, many biologically derived substances from plant and animal sources exhibit antimicrobial properties in foods in which normally they are found or may be developed for commercial use as preservative to other food products. Biologicals, because of their natural origin, are biodegradable and mostly do not leave toxic residues or byproducts to contaminate the environment and spices are one among them.

T. foenum-graecum (fenugreek) is an important spice in India, Egypt, Saudi Arabia, Iran, Armenia and Turkey. Trigonella seeds are bitter, mucilaginous, aromatic, tonic,  emollient, anticarcinogenic ², anti inflammatory ³, hypoglycaemic ⁴, hepatoprotective ⁵, immuno- modulatory ⁶, larvicidal ⁷ and wound healing activities ⁸. These are rich source of phytochemicals, especially the steroidal saponins (chiefly composed of diosgenin), which are  the starting compounds for the manufacture of over 60% of the total steroid drugs by the pharmaceutical industry .

Z. officinale (ginger) is generally consumed as fresh paste and dried powder in culinary practices and for flavoring tea. The pungent compounds of Z. officinale include gingerols, shogaols, paradols and zingerone ^9,.  Rhizomes of Z. officinale and its constituents have been proven for their antioxidant ¹⁰  and  antiplatelet efficacies ¹¹.

Present study focuses on the growth inhibitory activities  of  T. foenum-graecum  and Z. officinale towards some common  food borne pathogens.

MATERIALS AND METHODS:

Procurement of  Spice Samples: Dried seeds of T. foenum graecum were procured in a single lot, in the amounts of  500 g, from a  wholesaler spice-seller, local market, Hisar, India. Procured seeds were cleaned manually for extraneous material and were ground to powdered form in laboratory grinder. Fresh rhizomes of Z. officinale  (rhizomes) were purchased in the amounts of  1 kg, from grocery shop, local market, Hisar, India. Z. officinale rhizomes were washed with distilled water  to remove extraneous matter followed by their  peeling and drying in shade for 5 days. Dried  rhizomes thus obtained were ground to powdered form. Commercial preparations of essential oils of T.foenum graecum  and Z. officinalewere used.

Chemicals and Culture Media: Ethyl violet azide dextrose agar, Ethyl violet azide dextrose broth, MacConkey agar, MacConkey agar, MacConkey broth, Nutrient agar and Nutrient broth were obtained from Hi-Media Pvt. Ltd, India.  Dimethylsulphoxide (DMSO) and  Sodium chloride (NaCl) were purchaged from Central drug house Pvt. Limited, India.

Bacterial cultures: Pure cultures of Bacillus cereus (MTCC 430), Enterococcus feacalis (MTCC439), Escherichia coli (MTCC 1687), Psuedomonas aeruginosa (MTCC 424) and Staphylococcus aureus (MTCC 5021) were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. The reference bacterial strains were maintained on respective media  slants, subcultured bimonthly to maintain their viability and were stored at 4±1°C.Bacterial strains were adjusted with sterilized normal saline to contain approximately 1×10⁷  cfu/ml (used as inoculum for experiments). Incubation temperatures, incubation periods and media used for test microbes are mentioned in Table 1.

TABLE 1:  BACTERIAL STRAINS TESTED

 Prepearation of Aqueous Spice Extracts: Aqueous extracts were prepared in the laboratory ¹². Powdered spice samples of T. foenum-graecum and Z. officinale  were steeped overnight (temperature: 24-27°C) in sterilized distilled water in a ratio of 1:1 (w:v), followed by their  homogenization in a blender at high speed for 2 min. The homogenized spice mixtures were filtered through Whatman No. 1 filter paper. Filtrates thus obtained, were sterilized by passing through syringe filters containing 0.45 um pore size membrane filters and  were collected in sterilized glass vials.

Determination of antibacterial activities of test spice samples: Spice agar method ¹³ was opted for  investigating  antibacterial activities  of powdered forms of test spices at their different concentration levels (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0%, (w/v)). The petriplates supplemented with abovestated spice concentrations were examined for bacterial growth at an interval of 12 hours, for an incubation period of 30 days.

Similar sets of experiments were conducted without any spice sample that served as negative control. Agar well assay ¹⁴ was used for the preliminary screening  of  aqueous extracts (50, 80, 100 (ul/well)) and essential oils  (30ul/well) of  reference spices against bacterial strains under observation. Dimethylsulphoxide (DMSO) served as negative control.

Results were expressed as net zones of inhibition. Broth dilution technique ¹⁵ was followed for the better insight into the antibacterial activities of  test spice samples (aqueous extacts and essential oils). Test substances were used at various concentration levels viz. 2000, 1000, 500, 250, 125, 62.50, 31.25, 15.62, 7.81, 3.90, 1.95, 0.97, 0.48, 0.24, 0.12, 0.06 ul/ml; and the concentrations, at which bacterial growth was fully inhibited, were recorded. Dimethylsulphoxide (DMSO) served as negative control. All the abovementioned experiments were conducted in triplicates results obtained were highly reproducible.

RESULTS AND DISCUSSION: The results revealed that powdered forms of T. foenum-graecum and Z. officinale  upto 6.0% level (w/v), remained ineffective in arresting the growth of  bacterial strains under observation, and visible growth of all the bacterial strains was noticed on 2^nd day of  incubation as in control set of petriplates containing no spice sample (Table 2).  The potential of spices in the culinary, non culinary and medicinal fields is based on the chemistry and composition of  their volatile aromatic secretions generally known as essential oils. Seeds of T. foenum-graecum contain 0.02-0.05% essential oil ¹⁶, while rhizomes  of Z. officinale  are composed of 2-3 % essential oil ¹⁷. The inertness of powdered forms   may be attributed to very high volatility and subsequent losses of antimicrobial components during grinding and drying.  Composition of fresh and dried rhizomes of Z. officinale was determined by Gas chromatography (GC) and GC-MS techniques and it was observed that fresh rhizomes usually contain a greater proportion of the lower boiling components and  up to 80% of the volatile components can be lost during drying ¹⁸.  During preliminary screening aqueous extracts (50, 80, 100 (ul/well)) and essential oils (30 ul/well) of  both the test spices did not exhibit any growth inhibitory zone towards any reference bacterial strain (Table 3).

However, from the results of broth dilution technique, it was found that essential oil of Z. officinale at  different concentrations viz. 250 (ul/ml), 1000 (ul/ml), 2000 (ul/ml), 1000 (ul/ml), 250 ( ul/ml) inhibited B. cereus, E. faecalis, E. coli, P. aeruginosa, S. aureus respectively, while essential oil of T. foenum-graecum and   aqueous extracts of  T. foenum-graecum and Z . officinale  could not arrest test bacteria (Table 4). It was also noticed that g+ve bacterial strains were inhibited at lower concentration levels of essential oil of Z. officinale. This differential behaviour of test microbes may be attributed to their different membrane structures  and their relative permeability towards substrate components ^{19, 20}. The sensitivity of  microbes towards essential oil of  Z. officinale followed the following order : B. cereus = S. aureus > E. faecalis = P. aeruginosa > E.coli.

TABLE 2: INHIBITORY EFFECTS OF POWDERED FORMS OF T. FOENUM-GRAECUM AND Z. OFFICINALE ON THE GROWTH OF BACTERIAL STRAINS

T. f-g : Trigonella foenum-graecum; Z. o.: Zingiber officinale

TABLE 3: INHIBITORY EFFECTS OF AQUEOUS EXTRACTS AND ESSENTIAL OILS OF REFERENCE SPICES ON BACTERIAL STRAINS (AGAR-WELL ASSAY)

T.f-g : Trigonella foenum-graecum, Z.o.: Zingiber officinale, DMSO: Dimethylsulphoxide

TABLE 4: INHIBITORY EFFECTS OF AQUEOUS EXTRACTS AND ESSENTIAL OILS OF REFERENCE SPICES ON BACTERIAL STRAINS (BROTH DILUTION TECHNIQUE)

T.f-g : Trigonella foenum-graecum, Z.o.: Zingiber officinale, DMSO: Dimethylsulphoxide, ND: Not Detected

 CONCLUSION: The results of this in vitro study indicate that among all the spice forms tested, only essential oil of Zingiber officinale  arrested  food borne pathogens effectively and hence, it may be considered for further studies (in vitro and  in vivo ), against other microbes of spoilage and health significance.

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    How to cite this article:

    Bhatia M and Sharma A: Antibacterial Activities of Trigonella foenum-graecum and Zingiber officinale. Int J Pharm Sci Res, 2012; Vol. 3(9): 3228-3232

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