Introduction
Plant extracts can be very significant in the therapeutic treatment of microbial infections and extracts are often purified and incorporated into commercially available remedies7. These chemical compounds, referred to as secondary metabolites, are produced by plants as part of their defence mechanism in response to microbial infection7. Plants such as lemon (Citrus limon)2, mint (Mentha piperita)8, garlic (Allium sativum)3, and roots such as ginger (Zingiber officinale)3 produce active compounds which have demonstrated antimicrobial activity and are commonly used in commercially available remedies to treat microbial infections of the upper respiratory tract7. These compounds include nitrogenous alkaloids, flavonoides5 and polypeptides7. Common causative agents of bacterial upper respiratory tract infections (URT) include Streptococcus pneumoniae, Streptococcus pyogenes and Klebsiella pneumoniae 4. This study aims to look at the antimicrobial effect of commercially altered natural products: a capsule containing allium sativum oil 1 mg (equiv. to 3 grams fresh bulb ) and a peppermint dried leaf infusion to determine their efficacy against the test bacteria. Antimicrobial activity of freshly prepared substances (garlic, ginger, mint and lemon) was compared to the commercially prepared substances using disc diffusion assays. Minimum inhibitory concentrations were then determined from products that displayed antimicrobial activity.
Fig 3. Streptococcus pyogenes
Fig 2. Streptococcus pneumoniae

Fig 1. Klebsiella pneumoniae

Materials and Method
Initial Screening Each substance was prepared to desired consistency appropriate for disc diffusion assay Two horse blood agar (HBA) and two nutrient agar plates were inoculated with bacteria to grow a lawn culture. HBA agar was used to grow Streptococcus species, NA for K. pneumoniae. A diffusion disc was impregnated with each test substance preparations and placed on the agar plate. Plates were then incubated at 37o C for 24 – 48 hours. Antimicrobial activity was determined by the zone of inhibition. Substances that did not show anti-microbial activity were omitted from further testing. MIC was determined by performing a two fold serial dilution with [2 X] test substance and combining it with molten NA. Calf serum was added to mixture to support the growth of the Streptococcus species

Materials and Method
Minimum Inhibitory Concentration NA Plates Aliquot test substance and combine with molten nutrient agar to achieve a two fold serial dilution from 50 % to12.5 % concentration.
10ml

NA + calf serum Plates
5ml
5.75ml 2.8ml

1.25ml [2 X] Test substance

11.5ml

50% 1.5 ml of calf serum was combined with agar for the

25%
10ml

12.5%
15ml 18.75ml 10ml

50%
15.75ml

25%
18.7ml

12.5%

Streptococci sp. plates. Fig 4. Stage 2 schematic protocol

1.5ml calf serum in all NA Agar

Results
Initial Screen of Antimicrobial Activity
Table 1. Compounds which displayed antimicrobial activity against S. pyogenes, S. pneumoniae & K. pneumoniae.
Microorganism Test Substance Mint Pure Mint extract Peppermint tea Raw garlic Censovis garlic capsule Ginger Lemon juice
• •

• The disc diffusion assay
Klebsiella pneumoniae
R R R S R R S

Streptococcus pyogenes
R R R S R R R

Streptococcus pneumoniae
R R R S R R S

suggested that S. pyogenes, • S. pneumoniae & K. pneumoniae were sensitive to raw garlic.

• S. pyogenes & K. pneumoniae were sensitive to pure lemon juice. • Mint, pure mint extract, ginger,

peppermint tea and the contents of the garlic capsule had no effect on any of the three test microbes.

S = Sensitive (Zone of inhibition > 6 mm) R = Resistant (Zone of inhibition ≤ 6 mm)

Results
Minimum Inhibitory concentrations for Garlic Infusion & Lemon juice
• The garlic infusion inhibited growth of both Streptococci sp. therefore the MIC is likely to be below 12.5 %.

Table 2. Minimum Inhibitory concentrations for Garlic Infusion and Lemon juice
Microorganism Test Substance

Streptococcus pyogenes
< 12.5 % > 50 %

Streptococcus pneumoniae
< 12.5 % > 50 %

Klebsiella pneumoniae
< 25 % < 12.5 %

• K. pneumoniae growth occurred at the 12.5 % concentration for the garlic infusion but was inhibited on the 25% plate indicating the MIC is between 25 % and 12.5 %. • The lemon juice had no inhibitory effect for both Streptococci sp. suggesting that the MIC is greater than 50 %.
• However, the lemon juice inhibited the growth of K. pneumoniae at all concentrations indicative of an MIC less than 12.5 %.

Garlic Infusion Lemon juice

Discussion
Garlic contains many antimicrobial compounds, however it is most known for containing an organosulfur compound allicin (diallyl thiosulfinate)6. As outlined in Table 1, the commercially prepared capsule containing purified allicin had minimal impact on the growth of the three test microbes. In comparison, raw garlic that was freshly minced was able to inhibit growth on all three test species. Allicin is not present in garlic cloves, instead exists as a precursor alliin within the cell wall of the clove6. When the clove is damaged (minced), the cell wall ruptures, and alliin is transformed into allicin by the enzyme, allinase6. Over time, antimicrobial activity of allicin decreases4, offering a possible explanation for the capsules inactivity as opposed to the fresh preparations activity. Allicin inhibits the acetyl-CoA- forming system in bacteria as well as other thiol containing enzymes by reacting with the thiol groups within the microbe6.

Fig 5. Target site of Allicin within the acetyl – CoA forming system in the Citric acid cycle.

Discussion
As shown in Table 1, pure lemon juice had an inhibitory effect on the growth of S. pneumoniae and K. pneumoniae. Diluted lemon juice had minimal effect on the growth of both streptococci sp. but inhibited the growth of K. pneumoniae at all concentrations indicative of an MIC less than 12.5%. Alkaloids and flavonoids derived from lemon have been shown to have a range of antimicrobial activities including inhibiting cell proliferation5, causing cell membrane damage5, inhibiting nucleic acid synthesis5 and inhibiting energy metabolism5. Variances in susceptibility between the test substances as observed in Table 1 can be explained by substance permeability throughout the agar1,8, the cell wall and the microbial colony. The extent of damage to the cell wall could also explain variances.

Fig 6. Example of how flavonoids can reduce bacterial colonies on agar

Conclusion: According to this study, certain raw products had an inhibitory effect on the growth of microbes that are commonly associated with upper respiratory tract infections more than their commercial counterparts. Further studies are necessary to explore a broader range of products and extraction methods.

References
1. Ahmed, S. A., Jabbar, I. I., & Abdul, H.E., (2012). Study the Antimicrobial Activity of Zingiber officinale roots against some of the Pathogenic Bacteria. Al- Mustansiriya. J Sci 23, 63-70. 2. Al-Ani, W., Al-Haliem, S., & Tawfik, N., (2010). Evaluation of the antibacterial activity of citrus juices: An in vitro study.

Al-Rafidain Dent J 10(2), 376-382.
3. Ankri, S., & Mirelman, D., (1999). Antimicrobial properties of Allicin from garlic. Microbes and Infection 1(2) 125-129. 4. Bakri, I., & Douglas, C., (2005). Inhibitory effect of garlic extract on oral bacteria. Archives of Oral Biology 50, 645-651. 5. Cushnie, T., & Lamb, A., (2011). Recent advances in understanding the antibacterial properties of flavonoids. Inter J

of Antimicrobial Agents 38 (2) 99–107
6. Dhanavade, M.J., Jalkute, C. B., Ghosh, J.S. & Sonawane, K.D.(2011). Study Antimicrobial Activity of Lemon (Citrus

lemon L.) Peel Extract. Brit J of Pharm and Toxicol 2(3) 119-122.
7. Lanzotti, V., Bonanomi, G., & Scala, F., (2013). What makes Allium species effective against pathogenic microbes?

Phytochem Rev 12 751-772.
8. Rios, J.L., & Recio, M. C., (2005). Medicinal Plants and antimicrobial activity. J. Ethnopharmocology 100 80-84. 9. Saeed, S., Naim, A., & Tariq, P., (2006). In vitro antibacterial activity of peppermint. Pak. J. Bot. 38(3) 869- 872.