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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.11, pp 151-160, 2016
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A study on Antidermatophytic Potential of
Ocimum tenuiflorum Essential Oil and Chemical Composition Evaluation
Vishnu Sharma, Anima Sharma* and Ruchi Seth
Department of Biotechnology, JECRC University, Jaipur, India
Abstract : The Dermatophytes engage in an important role in the atmosphere. They invade the keratinophilic substrates with causing the superficial infections. The certain augmentation in the infection in the human, there is urgent need to search out a new therapeutics or remedies from nature. In the presented study, the chemical composition of Ocimum tenuiflorum’s volatile oil and its anti-dermatophytic potential was evaluated against isolated Dermatophyte species from Jaipur (India). The Clear pale yellowish colored oil containing 40 volatile components was extracted & analyzed using Hydro Distillation Process and GC & GC-MS methods. The main constitutions were obtained as β-Caryophyllene (38.90%) and Eugenol (19.63%)in the oil and it revealed excellent inhibition activity against test fungal organisms with presence of Maximum Inhibition Zone of 37 mm against T. mentagrophytes(KU578106)as well as 31.67 mm for M. gypsiumand 28.33mmfor M. nannum as compared to standard. The results studied in the present research, helps to look out new natural therapeutic drugs from Ocimum tenuiflorum as an anti-dermatophytic agent than the standard.
Keywords: Dermatophytes; Ocimum tenuiflorum; Volatile; Hydro Distillation; β-Caryophyllene.
Introduction:
Fungi have renowned as a unique division in nature from evolution1. In fungi divisions, the Deuteromycota Division has awide account of pathogenic fungi in which some have the ability to penetrate the natural keratin and play a role as keratinolytic agent2-3. The inventions of fungi cause infection, known as Mycoses4. During the last decades, Mycotic infections are raised up to 20–25% in world’s population5. All these factors have an attempted to search new drugs to treat mycoses.
Plants are an affluent source of organic compounds, many of those are using as drug agents against several infectious and non-infectious diseases, by the modern medicinalsystem6. They are livestock, are used by people from Homeopathy, Allopathy, Unani as well as Ayurvedic medicine from Vedic time7-8.Ayurveda is a "Science of life and longevity" from prehistoric Sanskrit that is one of the remedial systems of India depends on lifestyle, diet, and herbs9.
The plant’s Cytotoxic attitudes are formulated them as anexcellent antiseptic and antifungal agents10. There is growing attention in medicinal plants as an alternative source of synthetic molecules with inspiration in the existing marketplace11. The Ocimum tenuiflorum (Krishna Tulsi) is an aromatic herbaceous plant with 30–60 cm long hairy stems and simple phyllotaxis green or purple leaves & caudiform floral segments that contain Oleanolic acid, linalool, Eugenol, β-elemene and β-caryophyllene as the major constituent of the essential oil. The Ocimum tenuiflorum oil can be used as possible pharmaceutical products with therapeutic, perfumery properties12-14. On the Basis of remedial activity of Ocimum tenuiflorum, in the present study the detailed
chemical composition of its volatile oil and anti-dermatophytic activity was evaluated against Microsporum species and Trichophyton mentagrophytes(KU578106)species.
Material & Methodology:
The Antifungal activity of the Ocimum tenuiflorum was carried out against isolated fungal species of Dermatophytes by the agar diffusion method17. The sterile 6.0 mm Standard size Whatman No.1 filter paper discs soaked with Ocimum tenuiflorum oil were placed on agar plates containing fungal spore suspension. Respectively Ketoconazole (10mcg/disc) and Fluconazole (10mcg/disc) were also used as a positive control against isolated Dermatophytes. The plates were incubated at 30ºC for 48 to 72 hrs. Three replicates were kept in each case and average values were calculated. The diameter of the inhibition zones (including the diameter of the disk) was measured in mm and the activity index was calculated on the basis of the size of the inhibition zone with analyzing data in mean ± SE via subjected to one-way ANOVA with significant (P<0.05).
In the subsequent method, the different concentrations of essential oil were also diluted series of Pure: 1/2: 1/4: 1/5: 1/7 (Pure: 50: 25: 20: 14%) of essential oil and DMSO solutions. The discs soaked in different concentrations were placed on agar plates containing fungal spore suspension and incubated at 30ºC for 48 to 72 hrs.
The Minimum inhibitory concentration was evaluated using microdilution method in essential oils using Sabouraud Dextrose Broth Medium18-20. In eppendorfs tubes, Different concentrations of Ocimum tenuiflorum oil diluted with DMSO ranging in 5: 10: 15: 20: 25: 30: 50: 75: 100µl. Then 0.1 µl inoculums’ suspension was inserted deep into each eppendorfs containing broth with different concentration of oil as well as an oil-free control. The tubes were then incubated at 30ºC for 48-72 hours to determine the MIC. MIC was read to be the lowest concentration at which there was no visible growth of the organism by visual inspection.
Result:
The Clear pale yellowish colored oil was extracted from Ocimum tenuiflorum with an account of 40 volatile components, representing 96.51% of the total oil which were identified.
Table I: Chemical composition of leaf essential oil of Ocimum tenuiflorum
Peak Number |
Reaction Time |
Area% |
Name of Compound |
RI |
Molecular Weight |
Molecular Formula |
|
13.353 |
0.12 |
Para Cymene |
1022 |
134 |
C10H14 |
|
14.954 |
0.03 |
Gamma Terpinene |
1056 |
136 |
C10H16 |
|
15.567 |
0.02 |
1-Octanol |
1069 |
130 |
C8H18O |
|
16.358 |
0.01 |
Trans Linalool Oxide |
1086 |
170 |
C10H18O2 |
|
17.107 |
0.02 |
Nonanal |
1102 |
142 |
C9H18O |
|
20.068 |
0.48 |
Linderol |
1165 |
154 |
C10H18O |
|
20.587 |
0.17 |
4-Terpineol |
1176 |
154 |
C10H18O |
|
28.486 |
0.41 |
α- Cubebene |
1349 |
204 |
C15H24 |
|
29.227 |
19.63 |
Eugenol |
1366 |
164 |
C10H12O2 |
|
29.765 |
2.65 |
Copaene |
1378 |
204 |
C15H24 |
|
30.441 |
2.27 |
β-Elemen |
1394 |
204 |
C15H24 |
|
31.047 |
0.49 |
α-Guaiene |
1408 |
204 |
C15H24 |
|
31.925 |
38.90 |
β-Caryophyllene |
1429 |
204 |
C15H24 |
|
32.602 |
0.11 |
β-Barbatene |
1445 |
204 |
C15H24 |
|
33.058 |
2.25 |
α-Humulene |
1456 |
204 |
C15H24 |
|
33.374 |
0.05 |
Gamma Amorphene |
1464 |
204 |
C15H24 |
|
33.968 |
0.05 |
Gamma Muurolene |
1478 |
204 |
C15H24 |
|
34.196 |
2.59 |
Germacrene-D |
1483 |
204 |
C15H24 |
|
34.364 |
0.09 |
β-Selinene |
1487 |
204 |
C15H24 |
|
34.697 |
0.55 |
Cubebol |
1495 |
222 |
C15H26O |
|
35.126 |
0.24 |
β-Chamigrene |
1506 |
204 |
C15H24 |
|
35.837 |
0.83 |
Delta Cadinene |
1524 |
204 |
C15H24 |
|
36.595 |
0.04 |
β-Calacorene |
1543 |
200 |
C15H20 |
|
37.183 |
0.06 |
Spathulenol |
1558 |
220 |
C15H24O |
|
38.492 |
20.39 |
Caryophyllene Oxide |
1592 |
220 |
C15H24O |
|
38.661 |
0.50 |
Viridiflorol |
1596 |
222 |
C15H26O |
|
38.871 |
0.16 |
Cedroxyde |
1602 |
220 |
C15H24O |
|
39.286 |
0.85 |
Humulene Epoxide Ii |
1613 |
220 |
C15H24O |
|
39.951 |
0.09 |
Epicubenol |
1630 |
222 |
C15H26O |
|
40.793 |
0.12 |
β-Eudesmol |
1653 |
222 |
C15H26O |
|
40.911 |
0.21 |
Gamma Eudesmol |
1656 |
222 |
C15H26O |
|
41.074 |
0.24 |
β-Costol |
1660 |
220 |
C15H24O |
|
41.989 |
0.10 |
α-Bisabolol |
1685 |
222 |
C15H26O |
|
42.170 |
0.76 |
Sesquicineole |
1690 |
222 |
C15H26O |
|
42.931 |
0.14 |
α-Costol |
1710 |
220 |
C15 H24O |
|
43.281 |
0.09 |
Cembrene |
1720 |
272 |
C20H32 |
|
43.416 |
0.20 |
Larixol |
1724 |
306 |
C20H34O2 |
|
47.590 |
0.15 |
Phytone |
1843 |
268 |
C18H36O |
|
55.277 |
0.20 |
N-Nonadecanol-1 |
2081 |
284 |
C19H40O |
|
56.220 |
0.18 |
Phytol Isomer |
2111 |
296 |
C20H40O |
|
|
96.51 |
|
|
|
|
Figure 1: Chromatogram of Essential oil of Ocimum tenuiflorum by GC.
The percentage composition and names of the essential oil components are listed in Table-1. The major components of essential oil were β-Caryophyllene (38.90%), Eugenol (19.63%), Caryophyllene-Oxide (20.39%), Copaene (2.65), Germacrene-D (2.59%); β-Elemen (2.27%), α- Humulene (2.25%) and other components were present in trace amounts as presented in Table 1. In Disc diffusion method (Table 2), Ocimum tenuiflorum confirmed excellent antidermatophytic activity against selected test fungi. Maximum zone of inhibition was found to be 37mm against T. mentagrophytes(KU578106) (Inhibition Zone (IZ): 37mm, Activity
Index (AI): 1.23) as compared to the standard drug. Likewise, the plant oil also revealed excellent activity against M. gypsium(IZ: 31.67 mm, AI: 1.17) and M. nannum(IZ: 28.33mm, AI: 1.88) as compared to standard.
Table 2: Antifungal activity of Ocimum tenuiflorum essential oil with Activity index for Ketoconazole against pathogenic organisms
Test Strain |
Antifungal activity of Ocimum tenuiflorum |
||||
IZ of Oil Sample |
IZ of Ketoconazole |
AI |
IZ of Fluconazole |
AI |
|
T. mentagrophytes (KU578106) |
37±1.06 |
30±3.88 |
1.23 |
14±0.70 |
2.64 |
M. gypseum |
31.67±1.17 |
20±2.82 |
1.58 |
- |
- |
M. nannum |
28.33±3.06 |
15±0.70 |
1.88 |
15.5±1.5 |
1.83 |
The concentration of oil used 100%. IZ = inhibition zone (in mm) including the diameter of disc (6 mm), AI = activity index. Experiments were performed in triplicate and data analyzed are mean ± SE subjected to one-way ANOVA with significant (P<0.05).
Ketoconazole is a best antifungal standard against Dermatophytes. Fluconazole was observed in resistant as compared to Ketoconazole. It has resulted only for the T. menta agrophytes(KU578106) andM. nannumfor growth inhibition.The Minimum inhibition activity of Ocimum tenuiflorum essential oil was evaluated by modified dilution in oil concentration method against different test fungi. The results showed (Table 2 & 3) that all selected test fungi have good records of resistance activity against neat essential oil (100% concentration) with the formation of inhibition zone. In All Selected fungi, T. mentagrophytes (KU578106)shown the highest inhibitory action at 50% oil concentration mixed in DMSO.
Table 3: Inhibition zone activity of Ocimum tenuiflorum essential oil at various Dilutions against pathogenic organisms
Inhibition zone activity of Ocimum tenuiflorum essential oil at various Dilutions |
||||
Test Fungi |
Oil Dilutions |
|||
1/2 (50%) |
1/4 (25%) |
1/5 (20%) |
1/7 (14%) |
|
T. mentagrophytes (KU578106) |
22.33±1.17 |
16.66±1.24 |
21±1.41 |
10.30±0.47 |
M. gypseum |
20.9±0.63 |
15.5±1.76 |
11.53±0.45 |
8.63±0.61 |
M. nannum |
21.33±1.65 |
19±0.70 |
15.33±1.18 |
11.33±0.47 |
The concentration of oil used 100%. IZ = inhibition zone (in mm) including the diameter of disc (6 mm), AI = activity index. Experiments were performed in triplicate and data analyzed are mean ± SE subjected to one-way ANOVA with significant (P<0.05).
For further investigation, the oil was diluted up to 14% concentration and checked against same fungi and observed the zone of 10.30 mm diameter. Similarly,M. nannum revealed the results for diluted essential oil concentration of 20% and exhibited highest inhibitory action with 11.33mm inhibition zone.
Table 4: Minimum Inhibition concentration of Ocimum tenuiflorum essential oil against pathogenic organisms
Sr. No. |
Concentration of oil(in µl/ml) |
Test Fungi |
||
T. mentagrophytes (KU578106) |
M. gypseum |
M. nannum |
||
|
5 |
- |
- |
- |
|
10 |
+ |
- |
- |
|
15 |
+ |
+ |
+ |
|
20 |
++ |
+ |
+ |
|
25 |
++ |
++ |
+ |
|
30 |
++ |
++ |
+ |
|
50 |
+++ |
++ |
++ |
|
75 |
+++ |
+++ |
+++ |
|
100 |
+++ |
+++ |
+++ |
- No Inhibition; + Fair Inhibition; ++ Moderate Inhibition: +++ excellent inhibition
The zone size forT. mentagrophytes (KU578106)had the highest zone size for 50% value after pure concentration but the Minimum Inhibition activity was observed at 14% concentration with zone size of 10.30mm including thediameter of thedisc. In the present investigation, T. mentagrophytes(KU578106)and M. nannum were found to be more susceptible fungus at 100% concentration of oil whereas rests of fungi were found to be less sensitive.The MIC Value against all selected strains was observed to start from 10µl/ml concentration as aminimum of essential oil mixed in DMSO. The best observances were recorded from 50µl/ml concentration. At 5-10µl/ml concentrations were observed with fungal growth as well as in Negative control.
Discussion:
In the20th century, the natural remedies have an attraction for strong efficacy, broad spectrum as direct sources of therapeutics, Affordable by the populace, Raw base elaboration up to complex semi-synthetic chemical compounds, Taxonomic markers, Renewable source capability21-22.The allopathic systems of medicine have less popularity among people that is based on fast therapeutic actions of synthetic drugs. But the traditional route of the healthcare system is returned with herbal medicine that is renowned as “Return to Nature”23-24. Plant Essential oils are the best candidature against Dermatophytes25. The β-Caryophyllene (38.90%), Eugenol (19.63%),Caryophyllene-Oxide (20.49%), Copaene(2.65), Germacrene-D (2.59%), β-Elemen (2.27%), α- Humulene (2.25%) and other components were present in trace amounts of Ocimum tenuiflorum essential oil as presented in Table 1.
The Present study showed the similarity with Joshi and Hoti, 2014 from North West Karnataka, India. In their investigation the major constitutions of Ocimum tenuiflorum essential oil were 1, 8-eucalyptol (72.71 %), α-terpineol (2.54 %), terpinen-4-ol (0.34 %), and linalool (0.24 %) were the main methyl Eugenol (82.9%), β-Caryophyllene (4.1%), Borneol (2.4%), Germacrene D (2.3%) and α-Copaene (1.9%) 26. Similarly again In Belgaum, India, GC-MS analysis was concluded for Ocimum tenuiflorum essential oil with O. gratissimum oil and reported the major volatile constitution as methyl Eugenol (92.4%) and Eugenol (2.4%), β-Caryophyllene (1.3%). similarly again In Northern India reported the 39 constituents comprising 98% of the oil with themajority of Eugenol (46.2%), (ε)-Caryophyllene (27.6%) and β-Elemene (16.3%) in Ocimum sanctum oils27. Subsequently In South-east of Borazjan, Iran, 1, 8-Cineole, β-Bisabolen, and Eugenol was found as the main compound in O. sanctum oils28.
The present results of Antidermatophytic activity of Ocimum tenuiflorum essential oil by disc diffusion methods showed the agreement with Gupta et al., 2014 who used the plant extract for evaluation of antifungal activity29. Here we applied the same procedure to plant essential oil for evaluating the antifungal potential against dermatophytic strains. In present study, researcher observed the excellent antifungal activity as T. mentagrophytes(KU578106)(Inhibition Zone (IZ): 37 mm, Activity Index (AI): 1.23), M. gypsium(IZ: 31.67 mm, AI: 1.17) and M. nannum(IZ: 28.33 mm, AI: 1.88). For MIC Investigation, the Inhibition Activity was showed by the 15µl/ml as lowest value for Minimum inhibition concentration. While the lowest value of using concentration of essential oil as 5µl/ml and10 µl/ml was recorded as negative for MIC evaluation. The 10 µl/ml
concentration of essential oil also showed the imitative MIC Value only for T. mentagrophytes (KU578106).In this study, Minimum Inhibition activity for all fungal floras the best Minimum Inhibition activity was for T. mentagrophytes (KU578106)of 10.30mm and M nannum of 11.33mm at IZ at14% concentration including the diameter of the disc. For M. gypseum the best Minimum Inhibition activity was observed at 20% conc. with an inhibition zone of 11.53mm.
Figure 2: Chromatogram of essential oil of Ocimum tenuiflorum by GC-MS.
Figure-3: Graphical representation of the comparative Antifungal activity of Eucalyptus globulus essential oil with standards against Dermatophytes.
A similar approach was performed by Balakumar et al., 2011 against T. mentagrophytesand E. floccosum by using disc diffusion method and observed the excellent antifungal activity of O. sanctum leaves extracts against clinically isolated dermatophytes at the 200 µg/mL concentration30. Another similar study was done by the Silva et al., 2005 in Brazil on Extracts of Ocimum gratissimum leaves for in vitro antifungal activity, using agar dilution technique against dermatophytes with a positive 80% inhibition of selective dermatophytes (Microsporum canis, M. gypseum, Trichophyton rubrum and T. mentagrophytes)31. In another study Mahariya and Sharma, 2013 evaluated the in vitro antifungal activity against pathogenic fungi by disc diffusion method and microdilution method using the N sativa essential oil32.In their study, resulted in the strong antifungal activity against Microsporum gypseum, Trichophyton rubrum and Trichophyton simiiwith adiameter of inhibition zone and activity index 38 mm (AI: 1.90), 20 mm (AI: 1.33) and 35 mm (AI: 1.09). Sharma et al., 2011 also reported the Minimum inhibitory concentrations (MIC) range between 3.12 -12.5 mg/ml and above for the extracts of the Solanum melongena L., Lawsonia inermis L. and Justicia gendarussa B. against Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum gypseum and Microsporum fulvum using Agar cup diffusion. Similarly once again, they also observed certain MIC ranging between 0.156-1.25 mg/ml in case of Piper betle, Allamanda cathertica33-34. Sharma et al., 2012 also reported the positive inhibitions (IZ=7mm and AI=0.19mm) of O. tenuiflorum extracts against A. niger respectively35.There are required further studies & analysis to prove the higher antidermatophytic agent of Ocimum tenuiflorum as the results obtained higher showed the potential of Ocimum tenuiflorum than the standard.
Acknowledgements:
The authors express their sincere thanks for the kind contribution of Dr. Puneet Bhargava, Skin &VD Department, and Dr. R.K. Maheshweri, Department of Microbiology, SMS Medical College & Hospital for providing the infected skin samples from Tinea patients. Also express our acknowledgments to Dr. Ajay Kumar, Scientist, Advanced Instrumentation Research Facility, JNU, New Delhi, India for GC/GC-MS facilities.
Conflicts of interest: There are no conflicts of interest to declare for any of the authors in the study.
References:
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