Scientific world-vol9 final.pmd

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: 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 Phlomis bracteosa 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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Scientific World, Vol. 9, No. 9, July 2011



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