Exploring Indian Spices As Promising Antimicrobial Agents

Background: Infectious diseases caused by pathogens, and food contamination caused by microorganisms, are compromising human health. The ecacies of antimicrobials have weakened by microbial resistance, while antibiotic toxicity is another challenge, arising the need of natural antimicrobial agents. Objective: Spices have been long used to enhance avor and aroma of food, and for their antimicrobial and antioxidant activities. In this study, antimicrobial activity of aqueous and ethanolic extracts of ve Indian spices i.e. Black pepper, Carom, Cinnamon, Clove and Cumin, was explored against Escherichia coli and Staphylococcus aureus, by agar dilution method and disk diffusion method. Methods: For agar dilution, aqueous and ethanolic extracts, with concentrations ranging from 0.5mg/ml – 8mg/ml, were used. For disc diffusion method, varying concentrations of the ethanolic extracts (50%, 75% and 100%) were used. Results: The results showed inhibitory effect on the growth of the microbes at higher concentrations of extracts. Clove’s bud showed the best antimicrobial effect, having minimum inhibitory concentration (MIC) less than 0.5mg/ml for aqueous extract and 6mg/ml for ethanolic extract against both bacteria, and had the biggest zone of inhibition i.e. 21mm, against E. coli, while Black pepper had a zone of inhibition of 20mm against S. aureus. Conclusion: It was noted that the spice extracts, in general, were more effective against S. aureus than E. coli. Therefore, spices and particularly Clove and Black pepper extracts have great potential to be further tested and developed as novel safe antimicrobial agents.


Background
Microbial diseases are proving to be the serious health issue throughout the world these days. The use of antibiotics has revolutionized the treatment of various bacterial infections. However, their indiscriminate use has led to an alarming increase in antibiotic resistance, among microorganisms, considered as one of the greatest threats to human health today. 1 This has necessitated the need for development of novel antimicrobials. Many evidences suggest that consumption of fruit and vegetables is bene cial for human health and may help in the prevention of chronic diseases. 2 Traditional healers have long been using plants to treat a wide range of diseases, but these methods of treatment are still not accepted in many parts of the world. Plants contain secondary metabolites like tannins, terpenoids, alkaloids and avonoids. These are called phytochemicals. 3 Phytochemicals are originally meant to act as the plant defense mechanism but other than this they also exert potent antimicrobial properties against sensitive and pathogenic bacteria. Likewise, some natural substances have effective antimicrobial properties where they have been used as seasonings for centuries. 4 Spices and aromatic vegetable materials have long been used in food not only for their avor and fragrance qualities but also for their preservative and medicinal properties, for instance, carom seeds have been commonly used to treat gastrointestinal disorders while clove is used for toothache due to its local anesthetic activity. Since the ancient times, they have been used for preventing food spoilage and deterioration and for extending the shelf life of food. In addition, they show other bene cial biological properties, such as antimicrobial and antioxidant activities. 5 Although, the antimicrobial properties of spices may differ depending on the form of spices added, such as fresh, dried, or extracted forms and might also differ depending on the harvesting seasons and between geographical sources. 6 Over the time, with the usage of spices, it has been hypothesized that they exert antimicrobial properties but there is no core evidence of which spice is most promising and to what extent. 7 In the present study, we have evaluated the antimicrobial activity of the extracts of ve widely used spices in India, Syzygium aromaticum (Clove), Cuminum cyminum (Cumin), Cinnamomum verum (Cinnamon), Trachyspermum ammi (Carom seeds) and Piper nigrum (Black pepper) against bacterial food borne pathogens such as Esterichia coli and Staphylococcus aureus, using two methods i.e. Agar dilution and Disk diffusion method, the results are discussed.

Material And Methods
Media used nutrient agar, LB broth Extracts spices were collected from the market. Sample was grinded, by mortar and pestle, and sieved to obtain ne powder. Names of the Indian spices used in the study are mentioned in Table 1, along with their few details. Lowering the particle size increases surface contact between samples and extraction solvents. So, smaller the particle size, better it will soak into the solvent. 8 Table 1 Names of all the spices chosen for the study with their scienti c name, name of the family, English name, common name and the plant part which is used in the research SCIENTIFIC NAME FAMILY NAME ENGLISH NAME COMMON NAME PLANT PART USED Extracts of these ve spices were prepared by soaking a small amount of sample in the selected solvent overnight and later ltered with the help of Whatman paper.
Since fresh samples are fragile and tend to deteriorate faster, dried samples of spices are taken. 8 Preparation of extracts: Aqueous extract-2 g of nely powdered sample added to 20 ml sterilized distilled water (10% concentration) and left overnight.
Ethanol extract-2 g of nely powdered sample added to 10 ml ethanol (20% concentration) and left overnight. Figure 1 shows all the spice extracts prepared. First, these mixtures were ltered with the help of funnel and Whatman paper and then using micron-lters, to remove all the residue. After measuring the volumes of the extracts obtained, they are stored in respective glass bottles. 8 Bacterial Strains used: Two very common food borne pathogens were used to test the antimicrobial effect of spices, a) Staphylococcus aureus and b) Esterichia coli. The effect of spices was also tested on the environmental microbes.
E. coli is an enteric gram-negative, multitalented Bacillus. E. coli assuredly breeds in humans and in animal gut lumen in mass culture.
Staphylococcus aureus is a Gram-positive, round-shaped bacterium that is a member of the Firmicutes, and it is a usual member of the microbiota of the body, Inoculum was prepared by adding 500 microlitres of strain to 50 ml LB broth. Inoculated broth was incubated at 37 O C for 24hr.
Each petri plate was divided into half, with a marker and inoculated with 200 microlitres of Staphylococcus aureus on one half and 200 microlitres E. coli on the other half with help of micropipette.
These plates were then left in the incubator at 37 degree Celsius for 24 hrs.

Results
The inhibitory effect increases with increasing concentration of extracts. Different spices show different effects in ethanol and water.

A-MIC determination
Minimum inhibitory concentrations (MICs) is the lowest concentration of extract which prevents the visible growth of bacteria. MIC (in mg/ml) of all the extracts are recorded in Table 2 and the results are depicted in Fig. 3. Clove extracts, both ethanolic and aqueous, are most effective against both bacteria.

B-Zone of inhibition
The zone of inhibition is the zone encircling the disk, in which no growth of bacteria is seen. It is simply measured with a metric scale. The zone of inhibition for all the extracts are recorded in Table 3. However, Fig. 4 shows the picture of the formation of zone of inhibitions for the ethanolic extracts. Clove is seen to be more effective for E. coli than Staphylococcus aureus while cumin, carom, cinnamon and black pepper are more effective for Staphylococcus aureus than E. coli. For clove, maximum inhibition was seen at 50% concentration. Cinnamon and cumin are not much effective for E. coli. Cinnamon For E. coli, no effect is seen while for S. aureus, zone of inhibition increases with increase in concentration. Major antimicrobial components in clove, cumin and cinnamon have been reported to be eugenol, cuminic aldehyde and cinnamaldehyde, respectively. 12 Eugenol has been reported to inhibit the growth of E. coli. Cinnamaldehyde has been reported to inhibit the growth of S. aureus, E. coli, and Salmonella typhimurium. The alkaloids like piperine, piperidine and resins are responsible for the antibacterial activity of black pepper. While the antimicrobial potential of carom seeds lies in its oil which mostly constitutes thymol.
In this study, the antimicrobial activity of spices, with the aqueous and ethanolic extracts of clove, cumin, carom, cinnamon and black pepper was evaluated with gram-negative bacteria, E. coli and gram-positive bacteria, S. aureus. They were tested with both agar dilution and disk diffusion method to determine the minimum inhibitory concentrations and zone of inhibition of the extracts at different concentrations. The hypothesis, that spices are quite promising antimicrobial agents has proved correct, while Clove and black pepper have showed the great antimicrobial activities against the test organisms. Clove showed the biggest zone of inhibition which is 21 mm. It was also noted that most of the spice extracts were effective against gram-positive bacteria i.e. Staphylococcus aureus that the gram-negative bacteria. Disks places on agar places inoculated with E. Coli.

Figure 3
The results for the clove extracts, for MIC determination. Supplementary Files