Antimicrobial activity of some Essential oils and Extracts from Natural sources on Skin and Soft tissue infection causing microbes:

An In-vitro Study

 

Saumya Subramanian1, Padmaja Ananth Shenoy1*, Vasudev Pai2

1Department of Microbiology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India.

2Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal, Karnataka India.

*Corresponding Author E-mail: padmaja.shenoy@manipal.edu

 

ABSTRACT:

Introduction: Skin and soft tissue infections (SSTIs) constitute the diverse group of infections with varied clinical presentation and severity. SSTI’s are the most common infections treated in hospital today posing diagnostic and therapeutic challenges. Natural herbs have been widely used as a potential source of therapeutic substances all over the world. A study was conducted to determine the antimicrobial effect of some natural extracts on important SSTI causing pathogens. Materials and methods: In this study the antimicrobial potential of essential oil of Cinnamomum zeylanicum, Eugenia caryophyllata, oil of Cymbopogon citratus, Melaleuca alternifolia, Mentha piperita, Ocimum sanctum and extracts of Curcuma longa, Azadirachta indica and Cassia fistula were analysed against common skin and soft tissue infections (SSTIs) causing microorganisms. The organisms include methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA), Streptococcus pyogenes, Streptococcus agalactiae, Cutibacterium acnes, E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Bacteroides fragilis. The antimicrobial assay was performed by agar punch well method with different concentrations of essential oil and extracts. High performance liquid chromatography (HPLC) was performed in order to identify the active constituents present in the extracts. Further a formulation was made, with the powdered extract (neem, turmeric, bark of golden shower) and cinnamon oil as the chief component to look for any probable synergetic activity. Results and discussion: In this study all the essential oils showed good antimicrobial activity than the crude extracts. Significant activity against the Gram negative organisms was exhibited by all the products used in the study. Cinnamon and Clove oil had an excellent activity against all the organism whereas tea tree, mint and Tulsi oil showed better results against the Gram positive organisms including acne vulgaris causing organisms. HPLC results emphasised the presence of eugenol and cinnamaldehyde in clove and cinnamon oil respectively. A significant zone of inhibition was observed from the formulation prepared. Hence we indicate that the natural products used in this study have therapeutic properties and therefore could be considered as an alternative medication in treatment of SSTIs.

 

KEYWORDS: Skin and soft tissue infections, natural herbs, Cinnamomum zeylanicum oil, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Cutibacterium acnes etc. HPLC.

 

 


INTRODUCTION:

Skin and soft tissue infections (SSTIs) are one of the commonly presenting problems in hospitals today. SSTIs involves suppurative microbial invasions of the epidermis, dermis and subcutaneous tissues which result in induction of host immune responses1 and causation of wide variety of clinical manifestations ranging from mild (cellulitis, erysipelas and simple abscess) to severe/life threatening infections (necrotizing fasciitis).2 The skin being the largest organ of the body colonized with variety of indigenous species of microbial flora and also can act as a source of infection during trauma, surgical site infection or burn wound infection. Microbial infection of the skin also occurs due to the haematogenous spread of the bacteria.

 

Imprudent usage of antimicrobials in the recent days have led to the emergence of multi drug resistant/extensive drug resistant/pan drug resistant microbes which are perilous and require prolonged hospitalization. Natural herbal extracts have been used widely as a potential source of therapeutic substances all over the world. The herbal preparations are widely described in the Indian medicinal science- Ayurveda.3 The presence of bioactive compounds such as alkaloids, carotenoids, phenolics, flavonoids, anthocyanins and thiols in many spices result in the antibacterial properties.4

 

Lamiaceae family or the Mint family is known to possess antimicrobial activity and cytotoxic properties showing activity against Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes and Lactobacillus acidophilus.5 The leaves of tulsi plant is used in India as a remedy to cough and expectorant from ages. An extensive study on the antibacterial, anti-cancerous and anti-oxidant properties of tulsi (Ocimum sanctum) has also been done. Indian vedic scriptures quote Cinnamon (Cinnamomum zeylanicum), as a potent antiemetic, anti-diarrheal and anti-inflammatory substance.6 The therapeutic potential of lemon grass (Cymbopogon citratus) and Clove (Eugenia caryophyllata) has also been well documented.7 An extensive study on the antibacterial, antifungal and insecticidal properties of neem (Azadirachta indica) is done. Twigs of the stem were used in the rural India from brushing teeth and leaves were crushed and applied on the wounds. Turmeric (Curcuma longa), commonly found in Asia and used in the daily food, is known for its medicinal and therapeutic uses. Tea tree oil is used mainly in topical treatments of skin in treating acne bacteria involved in acne. Studies show that 5% concentration of Tree-tea oil in the treatment of acne vulgaris is effective against C. acnes.8,9 As studied in Indian literature Cassia fistula is active against skin infections and also used in rheumatism treatment. Inhibitory effect of certain microorganisms by C. fistula is also observed.10 The present study aims at analysing the antimicrobial activity of few essential oils, extracts and their synergy against common organisms causing SSTIs obtained from a tertiary care centre.

 

MATERIALS AND METHODS:

Plant material used:

The bark of Cinnamon (Cinnamomum zeylanicum), flower bud of Clove (Eugenia caryophyllata), Lemon grass (Cymbopogon citratus), Mint leaves (Mentha piperita) and rhizome of turmeric (Curcuma longa) were obtained from the local market, Udupi. Tea tree oil was procured from Department of Pharmacognosy, Manipal college of Pharmaceutical Sciences, Manipal. Leaves of Tulsi (Ocimum sanctum) and Neem (Azadirachta indica) and bark of Golden shower (Cassia fistula) were collected from the plant grown locally in the coastal area in the month of March 2019. Before initiation of the study, all the plant materials used in the study were authenticated by the Department of Pharmacognosy, Manipal College of Pharmaceutical Science, Manipal.

 

Work carried out:

The present in-vitro experimental study was conducted in Department of Microbiology Kasturba Medical College, Manipal. The ethical clearance was obtained from Institutional ethics committee- Kasturba Medical College with registration number IEC- 22/2019.

 

Extraction of essential oils and preparation of extracts:

Clevenger apparatus was used for the extraction of essential oil. The collected essential oil was stored in the suitable container away from sunlight. Soxhlet extraction was used for preparation of extracts. Dried and pulverized drug materials were extracted with ethanol using a soxhlet apparatus. The extract was concentrated in a rotary vacuum evaporator to remove all the solvent and dried extract was stored in a desiccator with dehydrating agent.

 

Test organisms:

A total of nine bacterial strains obtained from clinical samples of SSTI’s were included in this study. These bacterial isolates included five Gram positive bacteria viz. Methicillin resistant Staphylococcus aureus (MRSA), Methicillin sensitive Staphylococcus aureus (MSSA), Streptococcus pyogenes, Streptococcus agalactiae, Cutibacterium acnes and four Gram negative bacteria viz. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Bacteroides fragilis. Matrix‑assisted laser desorption/ionization‑time of flight mass spectrometry (Vitek MS bioMerieux Inc., France) was used for the identification of bacterial isolates.

 

Antimicrobial Activity:

1. Inoculum Preparation for antimicrobial assay:

For inoculum preparation, the anaerobic organisms such as C. acnes and B. fragilis, isolates were cultured on 5% sheep blood agar and incubated in anaerobic workstation (A 35 Don Whitley, Yorkshire, UK) for 72 hours to obtain purity. Fastidious organisms like S. pyogenes and S. agalactiae were cultured on 5% sheep blood agar and incubated at 37℃ in CO2 incubator (NuAire, Inc) for 24 hours. All the other aerobic Gram-negative organisms were cultured on MacConkey’s agar and incubated for 24 hours at 37℃. After obtaining purity, to perform antimicrobial analysis, C. acnes was inoculated in thioglycollate broth and then incubated in anaerobic workstation (A 35 Don Whitley, Yorkshire, UK), for 48 hours to obtain turbidity standard of 1 McFarland, whereas direct colony suspension in normal saline was done for B. fragilis.11 Similarly direct colony suspension was also done for S. pyogenes and S. agalactiae and lawn culture was done on 5% sheep blood agar plate. Other aerobic isolates were inoculated into peptone water and incubated at 37℃ for 4 hours and the turbidity was adjusted to 0.5 McFarland.12-14

 

2. Agar well diffusion method:

The MRSA, MSSA, E. coli, K. pneumoniae and P. aeruginosa isolates were lawn cultured on Muller Hinton agar and 5% sheep blood agar plates were used for S. pyogenes, S. agalactiae, C. acnes and B. fragilis. Wells of 9mm size were made with sterile borer on the agar plates and 100μl of different concentration of each extract were dispensed. DMSO was used as diluents to make different concentration of 10%, 25%, 50%, 75% and 100% oil. The different concentration of turmeric and neem powder, 0.05g/ml, 0.1g/ml and 0.2g/ml, were made with methanol and that of bark of Golden shower powder 0.05g/ml, 0.1g/ml, 0.2g/ml, 0.4g/ml, 0.6g/ml was made with sterile distilled water. Respective DMSO negative control was put for each extract. Plates were incubated in respective incubation atmosphere. For aerobic organisms the results were read after 24 hours of incubation, while for anaerobic organisms the results were read after 48-72 hours. The procedure was performed aseptically in duplicates and mean value was calculated.

 

Vancomycin (30μg) and ceftriaxone (30μg) (HiMedia Laboratories Pvt. Ltd, Mumbai) were used as procedural control for aerobic Gram positive and Gram negative isolates respectively. Metronidazole (5μg) and clindamycin (2μg) (HiMedia Laboratories Pvt. Ltd, Mumbai) were used as procedural controls for B. fragilis and C. acnes respectively.

 

Test for active components of two best extract using HPLC:

Based on the results of antimicrobial activity, the two extracts which showed best activity were used for HPLC analysis. The HPLC run conditions was done using reversed phase C-18 column, length 3.5μm, 4.6 x 50mm. The mobile phase used was a mixture of HPLC grade methanol-acetonitrile-water in the ratio of 35:20:45. The injection volume was 10μl, the flow rate was 1.0cm3/min and the detection was done at 221nm for 20 minutes at 28oC.15

 

Preparation of unguent (Lepa):

Having known the antimicrobial activity of these herbal extracts, an unguent was prepared in Department of Pharmacognosy using virgin coconut oil as base. 5ml of coconut oil was taken and 1.25gm of the powdered extract was added (0.5gm of turmeric, 0.5gm of neem and 0.25gm of bark of golden shower). Further, 1ml of essential oil of cinnamon was added to the formulation and mixed thoroughly until the unguent was uniform. The prepared unguent was stored in refrigerator until use. The antimicrobial activity of this unguent was tested, using punch well method, against all the SSTI isolates. Virgin coconut oil was used as negative control.

 

RESULTS:

The study measured antimicrobial potential of different concentrations of herbal extracts against pathogens causing skin and soft tissue infections using agar punch well method. The experiment was repeated in duplicates under aseptic environment. The mean zone size was calculated for each extract.

 

Essential oil:

Among the six different essential oil, clove and cinnamon oil showed significant antimicrobial activity against all the nine organisms at different concentrations. At 75% and 100% concentration of clove and cinnamon oil, the highest antimicrobial activity was observed against all the organisms. Therefore, these oils were further analysed. Lemon grass oil, at 100% concentration showed an excellent activity against the anaerobes. Surprisingly very less antimicrobial activity was appreciated against the Gram negative bacteria especially E.coli and P. aeruginosa. Tea tree oil on the other hand has produced a good activity against Gram positive bacteria when compared with Gram negative bacteria. As expected, tea tree oil has had a good activity on the acne causing organism viz. MSSA and C. acnes. Moving forward to Tulsi oil, inhibitory potential of the oil is seen against all the organisms except that for P. aeruginosa. The best activity was against the C. acnes especially at 50% concentration. Mint oil has showed a positive effect on the inhibition of all the organisms to a small extent. It was observed that it had a good constant effect against Streptococcus spp. at various concentrations. Results are given in Table 1. and Figure 1.


 

Table 1: Best antimicrobial activity of the Oil extracts (Zone of inhibition diameter in mm)

Organisms

Clove

(mm)

Cinnamon

(mm)

Lemon grass

(mm)

Tea tree

(mm)

Tulsi

(mm)

Mint

(mm)

Methicillin sensitive Staphylococcus aureus

24.5

35.5

29.5

42.5

26

26

Methicillin resistant Staphylococcus aureus

23

34.5

30

45

23.5

29

Escherichia coli

25.5

32.5

9

28.5

23.5

14

Klebsiella pneumoniae

26.5

35.5

15.5

54

23.5

13

Pseudomonas aeruginosa

32.5

36.5

12.5

13

10

9

Streptococcus pyogenes

32

38.5

45.5

31.5

26.5

26.5

Streptococcus agalactiae

26.5

33

35.5

28.5

25

30.5

Cutibacterium acnes

45.5

62

59

29

42

50

Bacteroides fragilis

35.5

69

68

33

25.5

50.5

 

Table 2: Best antimicrobial activity of powdered extracts (Zone of inhibition diameter in mm)

Organisms

Turmeric (mm)

Neem (mm)

Bark of Golden shower (mm)

Methicillin sensitive Staphylococcus aureus

15.5

20

24.5

Methicillin resistant Staphylococcus aureus

16

21

26.5

Escherichia coli

9

9.5

14.5

Klebsiella pneumoniae

9

9.5

16.5

Pseudomonas aeruginosa

9.5

9

15

Streptococcus pyogenes

14

20

19.5

Streptococcus agalactiae

15

17

20

Cutibacterium acnes

17

20

31

Bacteroides fragilis

15.5

17

15

 

Bacteroides fragilis

 

C. acnes

 
Figure 1. Plates showing the best antimicrobial activity of different natural essential oils

 


For powdered extract:

Among the powdered extract used in this study, turmeric and neem did not show remarkable antimicrobial activity against the organisms. Though some effect was shown against the Gram positive organisms, none was observed against the Gram negative organisms. Both turmeric and neem extracts showed the best activity at the highest concentration viz. 0.2g/ml. Among the three powdered extract bark of golden shower showed the best results amongst all the organisms. Clearly there was a mutual relation between the activity and concentration of the powdered extract used. Among all the organisms, the best activity was seen against C. acnes at the 0.6g/ml concentration. Results are given in Table 2. and Figure 2.

 


Figure 2. Plates showing the best antimicrobial activity of different powdered extracts

 


 

HPLC analysis for Extracts:

High performance liquid chromatography (HPLC) was performed in order to identify the active constituents present in the extracts. In our study, cinnamon essential oil and clove essential oil showed good in-vitro antimicrobial activity against all the SSTI causing isolates and hence their active component was determined. According to literature study; cinnamaldehyde is known to be the chief component of the cinnamon essential oil also having a wide variety of antimicrobial property. Eugenol was another major component present in cinnamon oil. The retention time of the standard peak for cinnamaldehyde was found to be 6.475 and that of eugenol was 1.68. Figure 3. shows the chromatogram of the cinnamon essential oil used in this study. Comparing their retention times, we can identify the active components of cinnamon oil to be cinnamaldehyde and eugenol. Further we can also predict the high antimicrobial activity of cinnamon oil is due to these two components present.

 

Figure 3: Chromatogram of cinnamon essential oil by HPLC

 

Another natural compound that showed excellent antimicrobial activity was clove essential oil. According to the literature, the chief component of clove essential oil is found to be eugenol which is also found to have antimicrobial properties. Figure 4. shows the chromatogram of the clove oil used in this study. Comparing the retention time for eugenol standard and the clove oil, it is seen that the active component of clove essential oil is eugenol which gives the clove oil its antimicrobial property.

 

Figure 4: Chromatogram of essential clove oil by HPLC

 

Antimicrobial assay of unguent (Lepa):

The antimicrobial activities of individual extracts were identified and were looked for probable synergetic activity of the extract. Hence a formulation was made. The activity of this formulation was studied on various SSTI isolates and a good zone of inhibition was obtained. A negative control containing coconut oil showed no zone and therefore the inhibition was purely due of the extracts used in the formulation and not because of the virgin coconut oil. The zone sizes for each isolate were measured and compared with the individual extract. Figure 4. Shows the antimicrobial properties of formulation. 

 

Figure 5: Formulation and its antimicrobial properties

 

DISCUSSION:

Plants and their products have been used as a source of therapeutic agents extensively in the past few decades.16-19 Out of all the natural products studied; it was observed that essential oils possessed a better antimicrobial activity than the powdered extracts. While all the extracts used showed considerable activity against Gram positive organisms such as S. aureus and S. pyogenes, many showed poor activity against aerobic Gram negative organisms’ especially P. aeruginosa indicating their mechanism of action of these products on bacterial cell wall. Literature survey indicates that major components of tulsi oil, cinnamon and clove are eugenol and comparing their inhibitory profiles it is evident that eugenol has a major role in the inhibitory action of these oils.20 While looking at the potential activity of lemon grass, it shows considerable inhibitory effects on S. aureus (both MSSA and MRSA), S. pyogenes and S. agalactiae. A study conducted had showed citral, the chief component of lemon grass oil, damages the cell membrane of the bacteria thereby showing an inhibitory effect.21 The present study showed good antimicrobial activity of tea tree oil (TTO) against the anaerobic organism especially C. acnes is seen. Hence TTO can be used as an alternative drug in treatment of acne vulgaris. Considering the crude powdered extracts of turmeric, neem and golden shower, it is observed that golden shower has shown excellent activity against all the organisms including P. aeruginosa. The effect of bark of golden shower has not been studied extensively against the SSTI isolates before. It is known for the presence of tannins which have showed good antimicrobial activity. Major activity was seen against S. aureus and C. acnes which makes it a good remedial product against mild skin infections. Turmeric and neem powdered extract showed very less activity than expected. No activity was seen against Gram negative aerobic isolates for both extracts. High performance liquid chromatography (HPLC) of the best two extracts showed the presence of cinnamaldehyde and eugenol also indicating the antimicrobial activity of cinnamon and clove essential oil is contributed by these components. In addition to the herbal extracts studied, a formulation of these extracts was prepared and tested for its anti-microbial activity.

 

SUMMARY AND CONCLUSION:

Essential oil extracts showed excellent antimicrobial activity than the crude powdered extract especially against the common SSTI Gram positive pathogens such as S. aureus and S. pyogenes. The antimicrobial activity of cinnamon essential oil and clove essential oil has shown greater zone size than that of the routinely used antibiotics and hence can be considered as an alternative source or in combination with the routinely used antibiotic to treat the SSTIs for the rapid recovery. Essential oils such as tea tree oil, tulsi oil and mint oil has very good inhibitory effect on C. acnes and hence can be used as a substitute in the treatment of acne vulgaris. Further, there is a scope of development in this area like In-vivo experiments such as animal studies and randomized control trials need to be performed to check for any adverse effects on the skin and the scope of other microorganisms such as fungi and parasites can also be studied with these extracts.

 

ACKNOWLEDGEMENT:

The authors are thankful to Department of Microbiology Kasturba Medical College, Manipal, Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences and Manipal Academy of Higher Education for providing laboratory facility and other infrastructure to carry out the work.

 

FINANCIAL SUPPORT AND SPONSORSHIP:

Authors are thankful to Manipal Academy of Higher Education for funding the project under student research fund.

 

CONFLICTS OF INTEREST:

There are no conflicts of interest.

 

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Received on 16.04.2020           Modified on 10.08.2020

Accepted on 05.10.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(7):3603-3609.

DOI: 10.52711/0974-360X.2021.00623