ANTIMICROBIAL ACTIVITY OF THE ESSENTIAL OIL OF PHLOMIS BRACTEOSA R. K. Joshi*, Veena Pande**, B. C. Thakuri***
*Regional Medical Research Centre, Indian Council of Medical Research, Belgaum, Karnataka-590 010, India.
**Department of Biotechnology, Kumaun University Nainital, Uttarakhand-263002, India.
***Siddhanath Science Campus, Mahendranagar, Kanchanpur, Nepal. Abstract: The essential oil was isolated from the aerial parts of Phlomis bracteosa Royle ex Benth., a hairy erect herb. The leaves and flowers of this species are used as carminative, stimulant and locally known as ‘Ballikotu, Calba, Salba’ in Turkish traditional medicine. The antimicrobial activity of Phlomis bracteosa was studied using well diffusion method. The activity was tested against human pathogenic bacteria and fungi at different concentrations (0.25 µg/ml, 0.125 µg/ml and 0.062 µg/ml) of the essential oil. Keywords: Phlomis bracteosa; Lamiaceae; Antimicrobial activity.
different parts of the world6-16. The major constituent of theessential oil of Phlomis bracteosa was reported as
Essential oils are known to possess antimicrobial activity,
germacrene D (34.3%). Other minor constituents were
which has been evaluated mainly in liquid medium. Several
sabinene (6.2%), germacrene D-4-ol (4.9%), linalool (4.8%)
essential oils and their isolates have been found to exhibit
strong antibacterial and antifungal activity. The essential oilare supposed to interfere with intermediary metabolism of
The essential oils of Phlomis lanata,Phlomis fruticosa,
microorganisms by changing the rate of an enzyme reaction
Phlomis cretica, Phlomis samia, Phlomis ferruginea and the
influencing nutrient uptake from the medium affecting enzyme
ethanol extract of Phlomis fruticosa showed antimicrobial
synthesis at nuclear or ribosomal level or changing membrane
activities15, 18-20, while the essential oil of Phlomis linearis has
also been reported anti-angiogenic and anti-inflammatoryactivity21. The iridoids, like lamiide isolated from the
Phlomis bracteosa Royle ex Benth. of the family Lamiaceae is
dichloromethane and methanol extracts of Phlomis crinita
an erect hairy plant 20-80 cm, with heart-shaped toothed leaves,
showed the topical anti-inflammatory activity22. The
and pink-purple flowers crowded into a few large whorls and
compounds, luteolin 7-O-â-D-glucopyranoside and chrysoeriol
forming an interrupted spike. The whorls are 2.5-4 cm across,
7-O-â-D-glucopyranoside were isolated from Phlomis
corolla 1.5-2 cm, the tube shorter than the calyx, upper lip larger
brunneogaleata and have anti-malarial property23. Phlomis
hooded, very hairy and with a fringe of white hairs, the lower
grandiflora used in folk remedy as gastroprotective crude drug
lip smaller, 3-lobed, calyx hairy, with five narrow awl-shaped
and possess antiulcerogenic activity24. To the best of our
teeth, much shorter than the calyx tube. The bracts linear-
knowledge this is the first report on the antimicrobial activity
lanceolate, bristly-haired, without a spiny tip. The leaves are
of the essential oil of Phlomis bracteosa.
5-10 cm, stalked, hairy. The plant is distributed in temperateregion from Kashmir to Kumaun and Afghanistan to South
West China from an elevation of 1200-4000 m1,2. Plant Material Phlomis species are recorded as herbal drugs being usedethno pharmacologically, tonic and as stimulant3. The leaves
The aerial parts of Phlomis bracteosa were collected before
and flowers of Phlomis species are used as carminative,
flowering phase from the Pindari glacier area, district Bageswer
stimulant and locally known as ‘Ballikotu, Calba, Salba’ in
of North-West Himalaya of Uttarakhand, India, at a height of
Turkish traditional medicine4,5. Several essential oil
3200 m. The plant was identified by Prof. Y. P. S. Pangtey,
constituents of the Phlomis species have been reported from
Botany Department, Kumaun University, Nainital. The
Author for Correspondence: R. K. Joshi, Regional Medical Research Centre, Indian Council of Medical Research, Belgaum, Karnataka-590 010, India. Email: firstname.lastname@example.org
Scientific World, Vol. 9, No. 9, July 2011
voucher specimen (No. 3318) was confirmed and deposited
Antibacterial Activity of the Essential Oil
in the herbarium of the Botany Department, Forest Research
The essential oil of 0.25 µg/ml, 0.125 µg/ml and 0.062 µg/ml
were prepared with some modification in 50% ethanol25-27 and
Isolation of Essential Oil
tested against human pathogens Staphylococcus aureus(Gram-positive), Pseudomonas aeruginosa, Escherichia coli
The aerial parts (1 kg) of Phlomis bracteosa were steam
and Proteus vulgaris ((Gram-negative) bacteria. It was
distilled for 3 hours using a copper still fitted with spiral
demonstrated by well diffusion method.
glass condensers for 3 hours and extracted with n-hexaneand dichloromethane. The organic phase was dried over
Antifungal Activity of the Essential Oil
anhydrous sodium sulphate and the solvent was recovered
The essential oil of 0.25µg/ml, 0.12µg/ml and 0.062µg/ml were
using a thin film rotary vacuum evaporator at 25o-30oC. The
prepared with some modification in 50% ethanol25-27 and tested
against Microsporum canis, Trichophyton rubrum and
Antimicrobial Assay Candida albicans fungi. It was demonstrated by welldiffusion method. Media Preparation Well Diffusion Method
Antibacterial and antifungal activities of the essential oil of
Nutrient agar (NA) was used for the screening of antibacterial
Phlomisbracteosa were tested using well diffusion method28.
activity of Gram-positive and Gram-negative bacteria. NA was
The autoclaved media was poured in the sterilized petri plates.
weighed as per instructions provided by the manufacturer
These plates were dried for a period of 20 minutes under
and dissolved in distilled water. After proper plugging, it was
aseptic condition before its use. Freshly grown cultures of
autoclaved at 120oC and 15 lbs for 20 minutes. Autoclaved
the tested bacteria and fungi in their media were streaked
nutrient agar when cooled at 45oC was poured into sterilized
over the plates using a platinum wire inoculation loop. On
petri dishes containing nearly 20 ml agar medium under
sterile media plates, well of 6.0 mm diameter were punched
aseptic condition and kept undisturbed as such till solidify.
with the help of a sterile gel cutter. Wells were sealed with the
After solidification, these petri plates were incubated at
molten media to prevent the escape of essential oil through
37oC±1oC overnight for sterility testing.
bottom. In the well of separate Petri plates 15 µl of different
concentrations (0.25µg/ml, 0.125µg/ml and 0.062µg/ml) ofessential oil were delivered. The positive control were used
Preparations of potato dextrose agar (PDA), sabouraud’s agar
gentamicin 1% (w/v) (Fulford (India) Limited, Hyderabad)
(SA) and yeast extract potato dextrose agar (YEPDA) media
and fluconazole 1% (w/v) (Lark Laboratories (India) Ltd.,
were used as per instruction provided by the manufacturer,
New Delhi) for antibacterial and antifungal activity,
for different fungal strains viz., Micrococus canis,
respectively. The plates were incubated at 37oC ± 1oC for 24
trichophyton rubrum and Candida albicans, respectively.
hours for Gram-positive and Gram-negative bacteria and 25oC
After proper plugging, the media were autoclaved at 120oC
± 1oC, 30oC ± 1oC and 30oC ± 1oC for Microsporum canis,
and 15 lbs for 20 minutes. Autoclaved PDA, SA and YEPD
Trichophyton rubrum and Candida albicans fungi for ten
media when cooled at 45oC was poured into sterilized petri
days, seven days and 48 hours respectively. The plates were
plates containing nearly 20 ml growth media under aseptic
observed for the zone clearance around the wells. The zones
condition till solidified. After solidification these petri plates
of inhibition were calculated by measuring the diameter of
were incubated for sterility testing. The PDA plates incubated
the inhibition zone around the well in millimeter including the
at 25oC± 1oC while SA and YEPD plates at 30oC± 1oC for ten
well diameter. The readings were taken in three different
days, seven days and 48 hours, respectively.
replicates and the average values were tabulated (Table 1). Table 1: Antimicrobial activity of the essential oil of Phlomis bracteosa Royle ex Benth.
a Dilution of essential oil in 50 % ethanol, V/V: applied dose: 15 µlb Standard: applied dose: 15 µlDiameter of well = 6 mmNA= No activity
Scientific World, Vol. 9, No. 9, July 2011
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