FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 61–63

Antimycotic activity of the essential oil of

Artemisia nilagirica
N. Kishore,1 N. K. Dubey1∗ and J. P. N. Chansouria2
1 Department of Botany Banaras Hindu University, Varanasi, India
2 Department of Experimental Medicine and Surgery, Banaras Hindu University, Varanasi, India
Received 7 March 2000
Revised 20 June 2000
Accepted 15 August 2000

ABSTRACT: During screening of essential oils isolated from leaves of 10 angiospermic taxa against dermato-
phytes Epidermophyton floccosum and Trichophyton violaceum, the essential oil of Artemisia nilagirica was found
to possess complete antidermatophytic activity by the poisoned food technique. The minimum inhibitory concen-
tration of the oil was found to be 200 ppm. It was fungistatic in nature and had a broad fungitoxic spectrum. An
ointment of the essential oil prepared in polyethylene glycol showed pronounced efficacy as a herbal antifungal
agent against dermatomycosis induced in guinea-pigs within 14 days of application. The oil was standardized on
the basis of its different physico chemical properties, such as specific gravity, specific rotation, refractive index,
acid value, saponification value, carbonyl percentage and phenolic content. Copyright  2001 John Wiley & Sons,
Ltd.
KEY WORDS: Artemisia nilagirica; Epodermophyton floccosum; Trichophyton violaceum; essential oil; antimy-
cotic

Introduction
Cutaneous infections of vertebrates include a wide vari-
ety of diseases. The majority of these infections are
caused by a homogeneous group of keratinophylic fungi
known as dermatophytes or ring-worm fungi.1 Some
synthetic antifungal drugs have been discovered for the
cure of dermatomycosis. However, because of differ-
ent side effects on human systems, the use of most of
the synthetic drugs has remained restricted.2,3 Such side
effects are nil or atleast much less with drugs of herbal
origin.4,5 The present research comprises testing of some
essential oils for their antidermatophytic activity, and
animal trials with ointment prepared from the effective
essential oil of Artemisia nilagirica in curing induced
dermatomycosis of guinea-pigs caused by Epidermophy-
ton floccosum and Trichophyton violaceum. The oil of
A. nilagirica has been standardized on the basis of its
physico chemical properties.
oils. The pure oils were tested for their antidermatophytic
activity by the poisoned food technique6 at 1000 ppm.
Sabouraud dextrose agar medium was prepared, auto-
claved and cooled to 40 ° C. The oils (0.01 ml) were
dissolved separately in 0.01% Tween-80 (0.5 ml) in pre-
sterilized Petri plates (7.5 cm diameter). Sabouraud dex-
trose agar medium (9.5 ml) was pipetted into each Petri
plate and was mixed thoroughly to obtain a final concen-
tration of 1000 ppm (v/v). For control sets, the requisite
amount of sterilized water in place of the oils was added
to the medium. The experiment was run in triplicate
and was repeated twice. Discs of the test dermatophytes
(5 mm diameter) were cut from the periphery of 7 day
old cultures using a sterilized cork borer and were asepti-
cally inoculated upside-down on the centre of each Petri
plate of the treatment and control sets. The Petri plates
were incubated at 37 š 1 ° C in an incubation chamber.
The percentage inhibition of growth of the test dermato-
phytes was calculated by the mean values of colony
diameters, using the following formula:

Material and Methods dc dt

Fresh leaves of 10 angiospermic taxa of the locality were
collected and were subjected separately to hydrodistilla-
tion in a Clevenger’s apparatus to extract their essential
*Correspondence to: N.K. Dubey, Department of Botany Banaras
Hindu University, Varanasi, India.
Inhibition of growth.%/ D ð 100
dc
where dc D average fungal colony diameter in control
sets and dt D average fungal colony diameter in treat-
ment sets.
The essential oil of A. nilagirica, which showed
absolute antidermatophytic activity against both the test

Copyright  2001 John Wiley & Sons, Ltd.

62 N. KISHORE, N.K. DUBEY AND J. P. N CHANSOURIA

dermatophytes, was tested for its minimum inhibitory Results

concentration (MIC) using graded concentrations, viz.
200, 300, 400, 500, 700 and 800 ppm using the usual During antidermatophytic testing, the essential oil of
poisoned food technique. The fungistatic/fungicidal A. nilagirica showed complete inhibition of growth of
nature of the oil was tested at its MIC using the both test dermatophytes (Table 1), while the rest of the
technique proposed by Thompson.7 The antidermato- oils showed moderate antidermatophytic activity. The
phytic spectrum of the oil was determined against eight oil showed complete inhibition of growth of both the
human pathogenic fungi, also using the poisoned food test dermatophytes at 200 ppm, therefore, its minimum
technique. inhibitory concentration was recorded as 200 ppm, at
The potency of the A. nilagirica oil was determined which it exhibited a fungistatic nature. The oil showed a
in the cure of induced dermatomycosis of guinea-pigs by broad antidermatophytic spectrum (Table 2), inhibiting
applying the ointment prepared in polyethylene glycol. most of the human pathogenic fungi tested. However,
Guinea-pigs weighing 300–400 g were inoculated sepa- the oil could not completely inhibit Candida albicans.
rately with Epidermophyton floccosum and Trichophy- During the culture positivity test, the ointment of the
ton violaceum using the method of Srivastava et al.8 oil prepared in polyethylene glycol started to cure the
The hair in an area (approximately 1.5 cm) on either induced mycosis of guinea-pigs caused by E. floccosum
side of the dorsal midline position of the test animals and T. violaceum on the 14th and 10th days, respec-
was shaved off using a safety razor. Each test dermato- tively (Table 3). The physico chemical properties of the
phyte was grown separately on Sabourand dextrose agar essential oil of A nilagirica are recorded in Table 4.
medium. The mycelium mat removed from the medium
was macerated separately with 500 mg sterilized white
sand and a paste was prepared with honey. The paste Discussion
was applied on the shaved area of the guinea-pigs. The
freshly inoculated areas were covered with a piece of Although some products of higher plant origin have been
sterilized plastic sheet with the help of adhesive tape tested for their antimycotic activity, most of them have
for 2 h. Isolation of the dermatophytes from the infected not been subjected to chemotherapeutic and pharma-
areas was done 2 days after inoculation, i.e. on the third cological investigations. However, determination of the
day, following the method of Gray and Douglas9 to curative potency of products for the cure of mycosis need
determine establishment of infection. Eight animals were
taken for each treatment and control set. Two concentra- Table 1. Screening of some essential oils against Epider-
tions of polyethylene glycol (1500 and 200 m.w.) were mophyton floccosum and Trichophyton violaceum Data
are mean š SD
mixed and a creamy base was prepared. The requisite
amount of the oil was added to the base to make the Inhibition of growth
concentration of the paste 500 ppm. Treatment of the Plant species Family E. floccosum T. violaceum
animals was started from the sixth day after inocula-
Aegle marmelos Rutaceae 75 š 3.4 70 š 1.4
tion and was continued until complete recovery from the Ageratum conyzoides Asteraceae 80 š 3.16 85 š 3.6
infection was achieved. The ointments (400–500 mg) Ammomum subulatum Zigiberaceae 69 š 3.8 73 š 3.8
were applied twice a day, in the morning and in the Artemisia nilagirica Asteraceae 100 š 0 100 š 0
Callistemon
evening. For control animals, only polyethylene glycol lanceolatus Myrtaceae 75 š 1.4 80 š 3.6
was applied similarly. The results were recorded in terms Foeniculum vulgare Apiaceae 65 š 3.16 60 š 2.82
of percentage culture recovery. Cultures for examina- Juniperus communis Cupressaceae 75 š 1.4 70 š 3.4
Lantana camara Verbinaceae 85 š 2.82 90 š 3.8
tion were made by placing the scales and hair portions Leucas aspera Lamiaceae 80 š 3.16 83 š 3.4
collected from the active borders of the infection sites Santalum album Santalaceae 70 š 1.4 65 š 3.4
on alternate days by the method of Gray and Douglas.9
The results were recorded in terms of percentage culture
Table 2. Antidermatophytic spectrum of Artemisia nila-
recovery, following Wahab et al.,10 using the formula: girica oil against some human pathogenic fungi
Total no. of sites
positive for culture Inhibition of growth (%)
in each set Fungi 200 ppm 500 ppm
Culture recovery.%/ D ð 100
Total no. of sites Aspergillus flavus 80 š 1.15 100 š 0
in each set Aspergillus fumigatus 100 š 0 100 š 0
The physico chemical properties of the essential oil of A. Candida albicans 60 š 2.64 85 š 1.122
Microsporum fulvum 100 š 0 100 š 0
nilagirica, e.g. specific gravity, specific rotation, refrac- Microsporum gypseum 100 š 0 100 š 0
tive index, acid value, saponification value, carbonyl per- Trichophyton mentagrophytes 80 š 2.112 100 š 0
centage and phenolic contents, were recorded following Trichophyton rubrum 77 š 2.85 100 š 0
Trichophyton simmii 85 š 1.112 100 š 0
Guenther11 and Mishra.12

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 61–63
 ESSENTIAL OIL OF ARIEMISIA NILAGIRICA 63

Table 3. Efficacy of the ointment of Artemesia nilagirica Table 4. Physico chemical properties of
oil against lesions on guinea pigs induced by Epidermo- Artimisia nilagirica oil
phyton floccosum and Trichophyton violaceum
Parameters Values
Culture recovery (%)
Colour Yellow
Treatment Treatment set Odour Pungent smell
(days) Control setŁ E. floccosum T. violaceum Specific gravity 0.9592105
Specific rotation .C/17.412766 °
2 100 100 100 Refractive index 1.3962
4 100 100 100 Solubility Soluble in:
6 100 100 100 Alcohol 1:1
8 100 100 100 Acetone 1:2
10 100 100 100 Acid number 5.6
12 100 100 80 Saponification value 21.03750
14 100 100 35 Ester value 15.43750
16 100 75 10 Carbonyl percentage 9.2
18 100 35 0 Phenolic content Absent
20 100 0 0
Ł
Polyethylene glycol was used in control set.
2. Stevens DA, Levine MB, Derisinaky SC. Am. J. Med. 1976; 60:
191.
to be established for a clear idea of their antimycotic 3. Brass C, Calgiani JN, Campbell SC. Rev. Infect 1980; 2: 656.
4. Franz C. Plant Res. Dev. 1989; 37: 111.
activity. The ointments of the oil were prepared in 5. Pratibha, Yadav, Dubey NK. J. Mycopathol. Res. 1996; 34:
polyethylene glycol because of its compatibility with 21–27.
many dermatological medicaments.13 The findings indi- 6. Perrucci S, Manciatiti F, Ciont PL, Flamini G, Morelli I,
Machioni G. Planta Med. 1994; 60: 184–187.
cate the possibility of A. nilagirica oil as an ideal herbal 7. Thompson DP. Mycologia 1989; 81: 151–153.
antimycotic agent. Unlike synthetic chemotherapeutics, 8. Srivastava OP, Chandra B, Mathur IS, Gupta HP. Ind. J. Exp.
it would constitute a renewable, easily available and Biol. 1978; 16: 700–701.
9. Gray SM, Douglas. Mycology for the Clinical Laboratories.
herbal antidermatophytic agent. Reston: Reston, VA, 1979; 157–182.
10. Wahab S, Tondon RN, Jacob Z, Chandra B, Srivastava OP. Ind.
J. Med. Res. 1982; 76: 77–82.
11. Guenther E. In The Essential Oils, vol. 1. Robert E (ed.). Krieger:
References Huntington, NY, 1972; 85–226.
12. Mishra AK. PhD Thesis, Banaras Hindu University, Varanasi,
1. Rippon JW. Medical Mycology—The Pathogenic Fungi and India 1993; 78.
Pathogenic Actinomycetes. W. B. Saunders: Philadelphia, PA, 13. Robinson J, Gibaldi M, Weiner ND, Kauig JL. J. Pharm. Sci.
1974; 35–40. 1964; 53: 1245–1247.

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 61–63