Antimicrobial activity of Commercial essential oils on human pathogens

 

Dr. B. R. Malathy1, Sweetlin Ajitha P2, Sangeetha K. S3, Swetha Thampy3, Kamala G3

1Reader, Department of Microbiology, Sathyabama Dental College and Hospital, Chennai – 600119.

2Research Scholar, Department of Microbiology, Sathyabama Dental College and Hospital, Chennai – 600119.

3IVth Year BDS Students, Sathyabama Dental College and Hospital, Chennai – 600119.

*Corresponding Author E-mail: maluravi_71@yahoo.co.in

 

ABSTRACT:

Essential oils (EOs) are natural extracts from the seeds, stems, roots, flowers, bark and other parts of the plant prepared by steam distillation. They are complex, volatile, natural compounds formed by aromatic plants as secondary metabolites. They are known for their bactericidal, virucidal, fungicidal, sedative, anti-inflammatory, analgesic, spasmolytic and locally anesthetic properties. They are generally composed of a combination of substances like terpenes, phenolics, aldehydes or alcohols.  The complex composition and different mechanisms of action of EOs may be an advantage over other antimicrobials to prevent the development of resistance of pathogenic bacteria. With this background, the aim of this study was to evaluate the antimicrobial activity of five essential oils like basil, lime, rosemary, thyme and canada balsam against 14 microbes. The effects of essential oil on the selected microbes were determined by agar well diffusion method. The zone of inhibition was observed and measured in millimeter. Essential oils which showed inhibitory diameter >15 mm were further tested to determine the minimum inhibitory concentration (MIC). S. aureus, E. coli, S. mutans, S. sanguinis, C. albicans and M. furfur were inhibited by all essential oils. K. pneumoniae, P. aeruginosa and E .faecalis were inhibited only by thyme and not by other essential oils. The MIC values ranged from 50% to 0.10%. The least MIC value of 0.10% was shown by thyme and basil to S. aureus, thyme to E.coli and all essential oils against C. albicans except lime.

 

KEYWORDS: Essential oils, antimicrobial activity, antibiotics, minimum inhibitory concentration, microbes.

 

 


INTRODUCTION:

Resistance of bacteria to antibiotics is a public health concern throughout the world. The number of bacteria developing resistance to antibiotics keep increasing. As a result, treatable diseases become untreatable, morbidity and mortality among patient’s increases and exert huge economic and emotional burden on the families and on the health care system of the country. Alternatives to antibiotics have been explored to overcome the burden of antibiotic resistance and its side effects. Plant based products offer a promising solution to this and has been increasingly investigated for the past one decade.

 

One such product obtained from medicinal and aromatic plants are essential oils. They are aromatic in nature since they contain mixture of chemical substances like terpenes, aldehydes, alcohols, esters, phenolic, ethers and ketones1.

 

Essential oils are volatile liquids, soluble in lipid and organic solvents, possess strong odor and are composed of complex compounds. They can be synthesized from plant parts such as flowers, buds, leaves, stems, twigs, seeds, fruits, roots, wood or bark etc. They are stored in secretary cells, cavities, canals and epidermic cells or glandular trichomes2. They are extracted by various methods such as simultaneous distillation extraction, microwave assisted hydrodistillation, ultrasound assisted extraction, cold pressing, hot maceration etc3. They can be administered in small quantities by inhalation, as topical massaging oil on skin, rarely they are taken orally.

 

Some essential oils possess antibacterial, antifungal and antiviral activity4-7. Anticancer properties of essential oils have been studied against various cancers - prostate cancer and glioblastoma8, melanoma9, breast cancer10, colon cancer11, liver cancer12, cervix cancer13, lung cancer14, oral cancer15, pancreas cancer16 using cancer cell lines. They are also used in aromatherapy and for the treatment of diseases such as alzheimer, cardiovascular disorders, cancer and to alleviate labor pain in pregnancy17-22. Since they are hydrophobic they can easily penetrate the cell membrane of bacteria and kill them due to leakage of molecules and ions from the bacterial cell23.

 

The major components present in these essential oils determine their biological properties. Thyme oil which is extracted from Thymus vulgaris constitute thymol (10-64%), carvacrol (2-11%), γ -terpinene (2-31%), β -cymene (10-56%)24. Rosemary oil extracted from Rosmarinus officinalis constitute α -pinene (2–25%), bornyl acetate (0-17%), camphor (2-14%) and 1,8-cineole (3-89%)25. Basil oil is extracted from Ocimum basilicum constitutes methyl eugenol (78.02%), α-cubebene (6.17%), nerol (0.83%) and ε-muurolene (0.74%)26. Canada balsam extracted from Balsam fir tree constitute β-Pinene (38%), δ-3-carene (12%), α- and β –terpineol (1.5%), bornyl acetate (9%)27. Lime oil extracted from Citrus aurantifolia constitutes limonene (39.74%) and β-pinene (25.44%)28.

 

The aim of this study was therefore to investigate the antimicrobial properties of five commercial essential oils such as basil (Ocimum basilicum), thyme (Thymus vulgaris), canada balsam (Balsam fir tree), lime (Citrus aurantifolia) and rosemary (Rosmarinus Officinalis)

 

MATERIALS AND METHODS:

Totally 5 essential oils basil, Canada balsam, lime, rosemary and thyme were selected to study their antimicrobial activity against the following bacterial and fungal strains maintained in the laboratory - Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis, Salmonella paratyphi A, Salmonella typhimurium, Salmonella typhi, Streptococcus mutans, Streptococcus sanguinis, Candida albicans and Malassezia furfur MTCC-1374.

 

Determination of antimicrobial activity:

Agar well diffusion method was used to study the antimicrobial activity. Muller Hinton agar (MHA) was used for non-fastidious bacteria, Mueller Hinton blood agar (MHBA) was used for fastidious bacteria, for C. albicans Sabouraud's dextrose agar and for M. furfur Dixon’s agar were used29. Fresh cultures grown on agar medium was inoculated in peptone water and incubated for 2 hours, turbidity was adjusted using 0.5 MacFarland standard (2 × 108 colony forming units/µL). Sterile swabs were used for confluent spreading of the inoculums on agar plates. For S. mutans and S. sanguinis, 2 to 3 colonies on blood agar was touched with wire loop and it was streaked confluently on MHBA. Wells were punched on agar plates using sterile cork borer and were loaded with 100µL of essential oils. Plates were incubated at 37şC for 24 hours. Zone of inhibition around the wells were measured in millimeter and recorded30.

 

Minimum inhibitory concentration (MIC):

MIC was done for those organisms which showed zone of inhibition of more than 15mm for undiluted essential oils. Serial doubling dilution of the essential oil was done in methanol and 100µL from each dilution was loaded in wells punched on agar. Undiluted essential oil was considered as 100% and serial dilutions had 50%, 25 %, 12.50%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.20 %, 0.10% in concentration. MIC was considered as the lowest percentage of essential which showed zone of inhibition31. Methanol was used as negative control.

 

RESULTS:

Five essential oils - basil, lime, rosemary, canada balsam and thyme were tested for antimicrobial activity against 14 microbial strains by well diffusion method. S. aureus, E. coli, S. typhimurium, S. sanguinis, S. mutans, C. albicans and M. furfur MTCC-1374 were inhibited by all the five essential oils tested. K. pneumoniae, P. aeruginosa, and E. faecalis were inhibited only by thyme oil. S. epidermidis was not inhibited by lime oil and P. mirabilis by rosemary oil. Similarly, S. paratyphi A and S. typhi were not inhibited by canada balsam (Table 1).

 

Fig: 1 MIC by well diffusion method for thyme oil against K. pneumoniae

 

Fig 2: MIC by well diffusion method for rosemary oil against S. aureus

 

 

MIC of essential oils ranged from 0. 10% to 50%. v/v. S. aureus showed lowest MIC value of 0.10% to thyme, basil and lime oil, 0.39% for canada balsam and highest MIC 25% for rosemary oil. S. epidermidis showed highest MIC of 50% to thyme oil and lowest MIC value of 0.78% for basil. The highest MIC for E.coli was obtained for rosemary oil (3.13%) and lowest MIC of 0.10% for thyme, basil and lime. K. pneumoniae, P. aeruginosa, E. faecalis and P. mirabilis, K. pneumoniae, P. aeruginosa, E. faecalis and P. mirabilis were inhibited by only thyme oil and showed MIC of 6.25%, 12.5%, 6.25% and 25% respectively (Fig 1). were inhibited by only thyme oil and showed MIC of 6.25%, 12.5%, 6.25% and 25% respectively (Fig 1).

 

The MIC values for S. typhimurium ranged from 50% to rosemary and canada balsam to 0.78% for basil. The MIC values of S. mutans were 0.20% for rosemary, thyme and lime, 1.56% for canada balsam and 0.39% for basil oil. The MIC values for S. sanguinis were 0.10% to lime, 0.20% for thyme and basil, 0.39% for canada balsam and 0.78 for rosemary oil. C. albicans showed MIC value of 0.10% for all except lime whose MIC was 3.13%. M. furfur showed MIC of 0.78% for thyme and basil and for the rest the MIC was 0.39%. (Fig 2, Table 2).


 

Table 1: Antimicrobial activity of essential oils by well diffusion method

Organisms

Rosemary

Canada balsam

Thyme

Basil

Lime

Zone of inhibition in millimeter (mm)

S. aureus

33

19

26

20

33

S. epidermidis

33

19

16

21

-

E. coli

24

30

20

19

32

K. pneumoniae

-

-

18

-

-

P.aeruginosa

-

-

18

-

-

E. faecalis

-

-

24

-

-

P. mirabilis

-

14

16

14

20

S. paratyphi A

12

-

20

28

30

S. typhimurium

23

22

22

25

34

S. typhi

20

-

20

16

25

S. mutans

20

25

23

22

28

S. sanguinis

Big zone

Big zone

Big zone

Big zone

Big zone

C. albicans

30

20

33

Big zone

28

M. furfur MTCC-1374

Big zone

Big zone

Big zone

Big zone

Big zone

 

Table 2: MIC values of essential oils against microbes in % (v/v)

S. No

Organism

Rosemary

Canada balsam

Thyme

Basil

Lime

1

S. aureus

25

0.39

0.10

0.10

0.10

2

S. epidermidis

6.25

12.5

50

0.78

-

3

E. coli

3.13

0.39

0.10

0.10

0.10

4

K. pneumoniae

-

-

6.25

-

-

5

P. aeruginosa

-

-

12.5

-

-

6

E. faecalis

-

-

6.25

-

-

7

P. mirabilis

-

-

25

-

25

8

S. paratyphi A

-

-

12.5

0.39

3.13

9

S. typhimurium

50

50

25

0.78

25

10

S. typhi

6.25

-

25

0.39

25

11

S. mutans

0.20

1.56

0.20

0.39

0.20

12

S. sanguinis

0.78

0.39

0.20

0.20

0.10

13

C. albicans

0.10

0.10

0.10

0.10

3.13

14

M. furfur MTCC-1374

0.39

0.39

0.78

0.78

0.39

 


DISCUSSION:

Essential oils are known for their antibacterial, antifungal and insecticidal activities. At present, 300 among the 3000 known essential oils are used commercially in pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries. Lagha R et al, studied antibacterial activity of five essential oils against E.coli and reported, it was inhibited by O. majorana, T. zygis and R. officinalis and was not inhibited by J. communis and Z. officinale. Highest antibacterial effect was obtained for T. zygis which is similar to our study where thyme oil showed least MIC value of 0.10% for E.coli32. In a study done by Man A et al, thyme oil showed MIC of 6.3% for E.faecalis, 1.6% for E.coli, 3.1% for K. pneumonia and 50% for P.aeruginosa33 whereas in our study MIC values were 6.25%, 0.10%, 3.13% and 12.5% respectively for the above mentioned organisms. All the 60 clinical isolates of E.coli were sensitive to basil and rosemary essential oils and basil was more effective than rosemary oil in their MIC values was reported in a study done by Sienkiewicz M et al34. Rosemary showed MIC of 0.03% for MRSA, 0.3% for E.coli, 0.1% for P.aeruginosa and C. albicans ATCC strains35, whereas in ours study rosemary didn’t have activity on P.aeruginosa, but showed same MIC of 0.1% for C.albicans and increased MIC of 3.13 for E.coli. The MIC for rosemary against S.aureus was 15µL/mL, against E.faecalis was 30µL/mL, against E.coli and P.aeruginosa was > 30µL/mL. Thyme showed MIC of 0.5µL/mL for S.aureus, < 0.5µL/mL for E.faecalis, 1.0 µL/mL for E.coli and 10µL/mL for P.aeruginosa36. When rosemary was tested against oral microbes, it was found that Streptococcus mitis was found to be most susceptible and E. faecalis was most resistant37. Lime oil showed no activity on S. mutans whereas in our study lime showed an MIC of 0.20%38. Thyme showed MIC of < 0.005% and basil showed MIC of < 0.048% against 114 isolates of C.albicans39, in our study the MIC values were 0.10% for both thyme and basil. Thyme oil was more inhibitory to P. mirabilis than penicillin G40, in our study thyme showed high MIC value of 25 % against the same bacteria. Highest antimicrobial activity for thyme oil similar to our results were also reported by Carvalho M et al46.

 

Malassezia genus includes lipid dependent yeast which appears as normal flora of human skin and other homoeotherms41. Under certain conditions, it causes Pityriasis versicolor and folliculitis, seborrheic dermatitis or exacerbate several skin diseases such as atopic dermatitis and dandruff42,43. Clinical isolates of fluconazole resistant M. furfur were inhibited by Thymus vulgaris essential oil44. Standard strain of M.furfur used in our study showed MIC value of 0.78% for thyme oil. M furfur isolated from dog showed MIC of 260µg/mL against Rosemary officinalis oil45. In our study rosemary oil showed MIC of 0.39% against the standard strain of M furfur. Further canada balsam and lime also showed similar MIC values, basil and thyme showed MIC of 0.78%. This shows that all the five oils tested in our study may serve as potential antifungal agent against M furfur and can act as a therapeutic alternative in future. To conclude, among the five essential oils tested against 14 microbes, S.aureus, E.coli, S.mutans, S.sanguinis, C.albicans and M.furfur showed inhibition for all essential oils whereas K.pneumoniae, P.aeruginosa and E. faecalis were inhibited only by thyme. The MIC values ranged from 50% to 0.10%. The least MIC value of 0.10% was shown by thyme and basil to S.aureus, thyme to E.coli and all essential oils against C.albicans except lime. Essential oil may play a huge role in aroma therapy, cosmetics, topical ointments, mouth washes, tooth pastes, food preservatives, hand sanitizers, insecticides, fragrant etc.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

ETHICAL CLEARANCE:

Since the study involved laboratory maintined strains, no ethical was obtained.

 

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Received on 04.04.2020           Modified on 15.06.2020

Accepted on 20.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(8):4440-4444.

DOI: 10.52711/0974-360X.2021.00771