Essential Oil Composition and Antibacterial Studies of Vitex negundo Linn . Extracts

Lessne M, Clerckx-braun F, Dduhoux P, Van Ypersele de Strihou C. 1. Pharmacokinetic study of torsemide in human: An overview of its diuretic effects. Int J Clin Pharmacol Ther Toxicol 1982;20:382-7. March C, Farthing D, Wells B, Besenfelder E, Karnes HT. Solid-phase 2. extraction and liquid chromatography of torsemide and metabolites from plasma and urine. J Pharm Sci 1990;79:453-7. Ghys A, Denef J, De Suray J, Gerin M, George A, Delarge J, 3. et al. Pharmacological properties of the new potent diuretic torsemide in rats and dogs. Arzneimittelforschung 1985;35:1520-6. Ventura R, Nadal T, Alcalde P, Pascual JA. Fast screening method 4. for diuretics, probenacid and other compounds of doping interest. J Chromatogr A 1993;655:233-42. Barroso MB, Alonso RM, Jimenez RM. Simultaneous determination 5. of torsemide and its major metabolite M5 in human urine by highperformance liquid chromatography-electrochemical detection. J Chromatogr Sci 2001;39:491-6. Neugebauer G, Besenfelder E, Von Mollendorff E. Pharmacokinetic 6. and metabolism of torsemide in man. Arzneimittelforschung 1988;38:164-8. Barroso MB, Meiring HD, De Jong, RM Alonso A, Jimenez RM. 7. Gas chromatographic-mass spectrometric analysis of the loop diuretic torsemide in human urine. J Chromatogr B Biomed Sci Appl 1997;690:105-13. Engelhardt S, Meineke I, Brockmoller J. Improved solid-phase 8. extraction and HPLC measurement of torsemide and its important metabolites. J Chromatogr B Analyt Technol Biomed Life Sci 2006;831:31-5.

Vitex negundo (family: Verbenaceae), is an important medicinal plant found throughout India.Though almost all of its parts are used in Ayurvedic and Unani systems of medicine, the extracts from its leaves and roots are the most important in the Þ eld of medicine and drug 1 .Its leaves 2 and seeds 3 are widely used externally for rheumatism and inflammations of joints and are also reported to have insecticidal properties.Internally, decoction of its leaves is taken as diuretic, expectorant, vermifuge, tonic and febrifuge 4 .The chemical components of the essential oil of leaf isolated from V. negundo [5][6][7][8][9] and other Vitex species 10 have been reported by several researchers in the past.Its essential oil is found to be useful for sloughing wounds and ulcers.The leaves of V. negundo are reported to possess pesticidal, antifungal and antibacterial properties 11 so in the present study, besides GC/MS analysis of fresh leaves, ß owers and dried fruits, the antibacterial potential of all three different oils and Þ ve successive extracts was studied.This study will be useful to identify the bioactive compounds of the oil, which may be responsible for the various medicinal properties of the plant.
The fresh leaves and flowers of V. negundo were collected in the month of August and September 2005 while fruits were collected in the month of October and November in the same year, from local areas of Kurukshetra, Haryana (India).The plant got identiÞ ed and authenticated by FRI, Dehradun and a voucher specimen of the sample (Sr.No. 160/Flora of Haryana) has deposited in the NWFP Herbarium collection at Forest Research Institute and College, Dehradun, India.
The freshly collected leaves, ß owers and dried fruits of V. negundo were washed twice with water to remove dust, just before hydrodistillation.Each of the leaves, ß owers and fruits were then subjected to hydrodistillation separately for 8 h using a closed type Clevenger apparatus for extraction of oils lighter than water.Yellowish oil so obtained was separated from the distillate with the help of hexane and dried over anhydrous MgSO 4 .Percentage yield of essential oil was calculated and it was stored in sealed glass bottles in a refrigerator until analysis.In case of leaves oil extraction, before subjecting to hydrodistillation, the leaves were treated with a 10% KOH solution for 3 h and then washed twice with fresh water.Shade dried leaves were pulverized and extracted successively with petroleum ether + ethyl acetate + CH 3 OH + C 2 H 5 OH + H 2 O in a Soxhlet extractor for 18 h.The solvents were evaporated under vacuum and percentage yield of each extract was calculated.Each of the extract was stored in a sealed glass bottle in a refrigerator until analysis.
GC analysis was carried out on Shimadzu GC17A GC V3 system, equipped with FID and fitted with a column (30 m×0.25 mm, film thickness 0.2 µm).Temperature parameters: column oven-100º, injection port-250º, detector-300º.Time programming: 100º for 2 min, temperature raised from 20º to 300º for 10 min, total time 22 min.Carrier gas used was He at 600 kPa primary pressure with 3 ml/min purge ß ow, column pressure 100 kPa.GC/MS analysis was performed on Shimadzu GC/ MS QP 5000 with GC17A GC system fitted with DB-6 chrompack capillary column (30 m × 0.25 mm, film thickness 0.2 µm).Temperature programming: 50º -300º at 20º/min.Carrier gas used was He at 55 kPa pressure, 53 ml/min ß ow rate.Electron impact mode of ionization with ionization energy 70 eV and ion source temperature 170º.Peaks were identified by comparison of relative GC retention times with standards from literature, retention indices on BP -1 column [12][13] , peak enrichment on co-injection with authentic standard wherever possible and comparison of mass spectra with literature data [14][15][16] .Relative percentage amounts were computed from GC peak areas without FID response factor correction.
The bacteria used for antibacterial tests were Gram (+) Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 0121) and Gram (-) Escherichia coli (MTCC 0051), Pseudomonas aeruginosa (MTCC 0741).All the strains used for these studies were procured from MTCC, IMTECH, Chandigarh, India.Antibacterial potential of all three samples of essential oils and successive extracts was evaluated by agar well diffusion method.Nutrient agar plates were swabbed with the broth culture of the respective microorganisms (diluted to 0.5 McFarland Standard) and were kept at room temperature for 15 min for absorption to take place.Wells of 8 mm diameter were punched into the agar medium and Þ lled with 100 µl each of the essential oils and extracts.DMSO, DMF and hexane were taken as solvent blank and Ciprofloxacin was used as positive control.The inoculated agar plates were incubated for 24 h at 37°.All the tests were made in triplicate and diameter of the inhibition zones was calculated in mm.The average of diameter of the inhibition zones of each sample was taken called clearing zone (CZ) and the antimicrobial index (AI) was computed as the clearing zone (CZ) minus the diameter of the hole divided by the diameter of the hole.
The results of present investigation matches with the results of earlier findings except in that one constituent namely viridiflorol was not found in these studies.The major constituents named ethyl-9-hexadecenoate, δ-guaiene, caryophyllene epoxide, valencene, α-selinene and germacren-4-ol were found first time along with thirteen minor constituents in essential oil of leaf of V. negundo.Limonene, 1,8cineole, citral, cinnamic aldehyde, eugenol, terpinen-4ol 5,10 , sabinene and viridiß orol 6,9 , which were reported as major constituents of the oil of leaves could not be detected in the present studies.Fig. 1 shows the results of well diffusion test and zone of inhibition of tested  samples against blank.The blank solvents DMSO, DMF and hexane did not show any zone of inhibition.
All the extracts and essential oils were found to be highly effective in inhibiting the growth of bacteria at a minimum concentration of 30 and 60 μg/100 μl, respectively (Table 2).Each of the essential oil and extracts were found to be active against B. subtilis and E. coli with antimicrobial index (AI) ranging from 0.3 to 1.8.Leaf essential oil inhibited S. aureus with maximum AI of 1.5 while fruit essential oil showed its inhibition against E. coli and B. subtilis with AI of 1.3 and 1.0, respectively.Flower oil did not show any activity against S. aureus while leaf and fruit oils were ineffective against P. aeruginosa.Ethyl acetate extract was found to be most potent among all the extracts tested.Petroleum ether and aqueous extracts did not show any activity against P. aeruginosa while all the extracts were found potent against S. aureus.Ciprofloxacin was used as positive standard control and the results of tested samples were very promising in comparision to standard drug ciproß oxacin.The oral route of administration is considered as the most widely accepted route.But the most evident drawback of the commonly used oral dosage forms like tablets and capsules is difÞ culty in swallowing, leading to patients incompliance particularly in case of pediatric and geriatric patients 1 .Thus, a new delivery system known as oral fast dissolving/disintegrating (FDDS)/melt-in-mouth tablets gaining importance.These oral dosage forms dissolve rapidly in saliva and can be swallowed without the need of drinking water 2 .Elimination of bitterness is an important criterion in product formulation of mouth dissolving tablets 3 .

Development of Mouth
Superdisintegrants added in the formulation increase the dissolution characteristics thus increasing the bioavailability of drug 4 .Mouth dissolving tablet disintegrate in mouth and are useful for potent drugs
Dissolving Tablets of Clozapine Using Two Different Techniques R. S. MASAREDDY*, R. V. KADIA AND F. V. MANVI Department of Pharmaceutics, K. L. E. S's College of Pharmacy, Nehrunagar, Belgaum-590 010, India Masareddy, et al.: Mouth Dissolving Tablets of ClozapineMouth dissolving tablets constitute an innovative dosage form that overcomes the problems of swallowing and provides a quick onset of action.In view of enhancing bioavailability an attempt has been made to study two different methods direct compression and sublimation in formulation of mouth dissolving tablets of clozapine.Total four formulations using various superdisintegrants and subliming agents were prepared.All prepared formulations were evaluated for physico-chemical parameters.The formulations exhibited good disintegration properties with total disintegration time in the range of 25 to 35 s.Comparative evaluation of two methods showed direct compression method is a better alternative to sublimation method as its formulations rapidly disintegrate in oral cavity.In vitro cumulative percentage drug release for formulations prepared by direct compression with explotab superdisintegrants shows 99.79 while sublimation method using camphor 93.58 release in 12 min.Kinetic studies indicated that all the formulations followed fi rst order release with diffusion mechanism.Key words: Clozapine, direct compression, sublimation, mouth dissolving *For correspondence E-mail: rsmasareddy@rediffmail.com

TABLE 1 : CHEMICAL COMPOSITION OF LEAVES, FLOWER AND DRIED FRUIT ESSENTIAL OIL OF VITEX NEGUNDO Leaf oil Retention Peak Flower oil Retention Peak Dried Fruit oil Retention Peak Constituents index Area (%) Constituents index Area (%) Constituents index Area (%)
t -trace (<0.1%), *Newly reported in this oil