International Journal of Drug Development and Research

Keywords

Psuedomonas sp., Bacillus sp., Cardamom, Clove, Cinnamon, Spices

Introduction

There were three antimicrobial agents, cloves, cardamom and cinnamon used on two organisms, Bacillus and pseudomonas. Cloves are the dried flower buds of Syzygiumaromaticum belonging to the family Myrtaceae. Cloves were originally from the Maluku islands in Indonesia and are used in culinary all around the globe. Mainly harvested in India, Zanzibar, Pakistan, Srilanka and Indonesia, they have numbing effect in the mouth. They have been used in India mainly as ayurvedic medicine prominently in dentistry [1]. The oil derived from it is mainly used as a pain killer in dental surgeries and in aromatherapy when warming and stimulation is required in digestive diseases [2].Cloves increases hydrochloric acid in the gut and helps in peristalsis, thus used as a carminative. Because of the warming effect, it is advised to be avoided in cases of fire. Some other uses include its usage in impotency, morning sickness and coldness in the stomach after drowning accidents [3]. Clove tea is also taken as a beverage in many parts of the country Cinnamon, scientifically called Cinnamomumverum is a spice retrieved from the bark of trees of the genus Cinnamomum that is commonly used as flavouring agent in both salty as well as sweet food items [4]. The antibacterial activity of cinnamon maybe due to its essential oil. There are many active components present in the essential oil that contributes this activity. Major constituent of the oil is cinnamaldehyde[5]. A natural antioxidant, cinnamaldehyde from the extract of cinnamon bark if orally administered can help against the formation of stomach ulcer [6]. Also when added to food, the oil was not harmful and helped in inhibiting the growth of molds, yeast and bacteria [7].The extract also has regulatory effect on lipids and glucose levels in the blood [8].The oil can also be applied topically to provide relief against neuralgia and headache. Certain types of cancers can also be prevented by prolong usage of this oil [9].

Cardamom is the dried fruit of the tall perennial herbaceous plant, ElettariacardamomumMaton, and belonging to the family Zingiberaceae [10]. This herb is cultivated commercially in India, Sri Lanka, Guatemala and Tanzania. The leaves are lanceolate, green or dark green, glabrous on both surfaces with acuminate apex. The fruit are triocular, ovoid, oblong or greenish-brown capsules containing about 15-20 reddish brown seeds. The cardamom seeds have a warm, slightly pungent and highly aromatic flavour. Therefore, it is used as a spice in meat products such as Bologna and Frankfurter [11]. Cardamom oil is used in food, perfumery, and liquor a pharmaceutical industries as a flavour and a carminative. In medicine, it is used as a powerful aromatic, antiseptic, stimulant, carminative, stomachic, expectorant, antispasmodic and diuretic [11][12]. In studies carried out in Turkey, the antimicrobial activities of different plants and their extracts used as spices or aromatic herbs including Nigella sativa, nettle, onion, garlic, peppermint, cumin, cinnamon and thyme have been investigated [13][14][15][16][17].

There are two organisms that were used in this experiment: Bacillus and pseudomonas. Bacillus subtilis belongs to the genus Bacillus and is a rod shaped bacteria. It can form a protective endospore which can be extremely tough. This endospore has the ability to protect the organism from harsh environmental conditions. This bacteria was conventionally grouped as an obligate aerobe but recent studies have proved otherwise. Laboratory culture of this bacteria is easy and inexpensive [18][19]. The other organism used was Pseudomonas which is a gram negative aerobe of the family pseudomonadacaeae. They are able to live in a variety of niches due to its great metabolic diversity. Also, they are available in a variety of strains, thus they are used extensively in scientific research. Due to the presence of spores and polar flagella, they can survive even in harsh conditions. Thus the laboratory culture of this organism isn’t difficult or expensive either. [20][21][22].

Materials and Method

To check the anti-microbial properties of spices on bacteria, we studied the effect of two spices on the growth of two bacterial strains. The spices used were clove and asafoetida. The bacteria taken under study were - Bacillus and a Pseudomonas species. The growth media used was Nutrient agar which remains solid even at high temperatures. It is used mostly for routine cultivation of non-fastidious bacteria. Any growth is visible as colonies on the broth. As liquid broth, colonies form clumps in the soupy substance. The media contained:

Ingredients Grams/Litre

HiVeg peptone 5.00

HiVeg extract 1.50

Yeast extract 1.50

Sodium chloride 5.00

Agar 15.00

Procedure

Petri plates were sterilized along with two conical flasks, containing 5.6 g of Nutrient agar in 200 mL of distilled water each, in an autoclave for 20 minutes at 121 degrees.

After sterilization, the media was allowed to cool slightly, till a temperature of about 60 degrees.

In a laminar air flow, under sterile conditions, the media was poured into twelve petri plates and allowed to solidify.

The media in one plate was inoculated with Bacillus from a fresh culture, using a sterile inoculating loop. Then using an L-shaped glass rod, the inoculum was spread evenly on the solidified agar media. This step was repeated with six more petri plates.

The remaining six petri plates with solidified agar were spread plated with Pseudomonas from a fresh culture.

Using a well cutter, five wells were made in the inoculated media contained in the plates.

Crushed clove was measured appropriately and added into the wells in four plates plates, two inoculated with Bacillus and the other with Pseudomonas.

Similarly, the other spices were measured and added to the wells of the remaining plates.

One petri plate with an inoculum of Bacillus and another with an inoculum of Pseudomonas were sealed and marked as control for this experiment. These did not contain the spices.

All the petri plates were sealed and put in an incubator, along with the controls, for a period of 24hrs. The temperature was set at 37 degrees.

After 24hrs, the plates were observed under light and the zone of inhibition was noted and measured.

Observation

The diameters of the zones of inhibition in each plate were measured to see the effect of the spices on the growth of the organism. The values (in cm) were as follows:

Cardamom

In case of bacillus there was a completely clear plate that was found. However, in case of Pseudomonas in the concentration of 0.06, there was no zone of inhibition that was found. Higher concentration of 0.12 yielded a zone of 1cm

Result

The diameter of the zone of inhibition was found to be the maximum in petri plate initially inoculated with Bacillus and with clove in the wells cut into the agar media. This was followed by the petri plate inoculated with Bacillus and with cinnamon in the wells cut into the agar media. It was also observed that the petri plates with lower concentrations of cinnamon had a greater zone of inhibition for Bacillus but not for Psuedomonas while in petri plates with higher concentration of clove the greatest zones of inhibition were observed in both bacteria.

Discussion

Clove is shown to be more effective as seen from the above values. This may be due to the compound Eugenol which is present in the essential oil derived from clove. This has toxic properties if used in large quantities. Clove for that reason has been used since long back in Ayurveda for its medicinal properties [23]. It has an anaesthetic effect and used for numbing small regions like teeth. Thus, it has been used actively in dentistry. Also as compared to allopathic analgesic, this is natural and so does not have any side effects [24]. Clove also contains in small quantities active anti-microbial tannins like gallotannic acid and methyl salicylate. These compounds though toxic in large doses are extremely instrumental in relieving pain. Since they are not present in large quantities, their effect is not observed much in the essential oil derived from clove [25].

Acknowledgments

We would like to thank our Honourable Chancellor Dr G.Viswanathan. VIT University for his essential and constant guidance in the efficient proceeding of this project. We would also like to extend our gratitude to our family and friends for their encouragement and support in the timely completion of this project.

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12

References

1. 1) Saha R, Bhupendar K, Chandekar A, Upmanyu N. Spices as anti-microbial agents- A review.International Research Journal of Pharmacy, 2012 : 3, 2
2. 2) Balch, Phyllis and Balch, James. Prescription for Nutritional Healing. Avery Publishing, 2000: 3, 94
3. 3) Alqareer A, Alyahya A, Andersson L. The effect of clove and benzocaine versus placebo as topical anesthetics. Journal of dentistry 2012; 34
4. 4) Iqbal, Mohammed (1993). International trade in non-wood forest products: An overview.Food and Agriculture Organization of the United Nations. Retrieved November 12, 2012.
5. 5) Simic A, Sokovic MD, Ristic M, Grujic-Jovanovic S, Vukojevic J, Marin PD: The chemical composition of some Lauraceae essential oils and their antifungal activities. Phytother Res 2004, 18:713- 717
6. 6) Blumenthal M: The Complete Commission E Monographs, Therapeutic Guide Herbal Medicines. Boston, Mass: Integrative Medicine Communications; 1998:110.
7. 7) Matan N, Rimkeeree H, Mawson AJ, Chompreeda P, Haruthaithanasan V, Parker M: Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. Int J Food Microbiol 2006, 107:180-185.
8. 8) Kim SH, Hyun SH, Choung SY. Anti-diabetic effect of cinnamon extract on blood glucose indb/dbmice. JEthnopharmacol, 2006: 104,119- 123
9. 9) Nadkarni KM. Indian MeteriaMedica. Popular Prakashan, Bombay, India, 1976:228-231.
10. 10) Anonymous. Zingiberaceae.In:Encyclopoedia Britannica Macropaedia, 1977: 15, 1150.
11. 11) BaytopT.Türkiye’deBitkilerileTedavi. ?.Ü. Yay. No: 3255, Eczac?l?kFak. No: 40,?stanbul, 1984
12. 12) KorikontimathVS, Mulge R, Zachariah JT. Variations in essential oil constituents in highyielding selections of cardamom. J. Plantation Craps, 1999: 27, 230-232.
13. 13) A?ao?lu S, Berkta? M, Güdücüo?lu H. Çörekotu (Nigella sativa) tohumununantimikrobiyalaktivitesiüzerinebirara?t?r ma. Y.Y.Ü.Sa?. Bil. Derg, 1999: 5, 215-217.
14. 14) Aksu I, Kaya M. Türksucu?uüretiminde?s?rganotu (Urticadioica L.) kullan?m?n?nsucu?unmikrobiyolojiközelliklerineetkisi .Türkiye 7.G?da Kongresi, 22-24 May?s, Ankara, 2002: 847-857.
15. 15) ÇonAH,AyarA,GökalpHY. Baz?baharatuçucuya?lar?n?nçe?itlibakterilerekar??antimikrobiyaletkisi.G?daDerg., 1998: 23, 171-175.
16. 16) Karap?narM,Aktu??E.Baharatlar?nlaktikasitbakterilerininüremesivelaktikasitolu?turmas?üzerineinhibitifvestimülatifetkileri. E.Ü. Müh. Fak.Derg. Seri:BG?daMüh.,1986: 4, 79-87.
17. 17) Topal?.Sar?msakveso?an’?nantimikrobiyaletkileriüzerindeara?t?rmalar.I.Uluslararas?G?daSempozyumu , Bursa, 1989: 450-461.
18. 18) Madigan M, MartinkoJ.Brock Biology of Microorganisms. Prentice Hall. ISBN, 2005: 11, 0-13
19. 19) Nakano, Michiko M, Zuber, Peter. Anaerobic Growth of A "Strict Aerobe" (Bacillus Subtilis). Annual Review of Microbiology, 1998: 52, 165–90
20. 20) Euzéby J.P. Pseudomonas entry in LPSN, List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. Int J SystBacteriol, 1997: 47, 2, 590–2.
21. 21) Migula W. Übereinneues System der Bakterien. ArbBakteriolInst Karlsruhe, 1894: 1, 235–328.
22. 22) Migula, W. System der Bakterien, Jena, Germany: Gustav Fischer,1900: 2
23. 23) Clove (Eugenia aromatica) and Clove oil (Eugenol). National Institutes of Health, Medicine Plus. nlm.nih.gov, 2012
24. 24) Kurokawa, Masahiko. Purification and Characterization of Eugeniin as an Antiherpesvirus Compound from Geumjaponicum and Syzygiumaromaticum.JPET 1998; 284, 2, 728– 735.
25. 25) Niwano Y, Keita, Yoshizaki, Fumihiko, Kohno, Masahiro, Ozawa, Toshihiko. Extensive screening for herbal extracts with potent antioxidant properties. Journal of Clinical Biochemistry and Nutrition, 2011: 48, 1, 78–84.