Karuna Shanker, Madan M. Gupta, Santosh K ... - CIMAP Staff

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ARTICLE IN PRESS2 K. Shanker et al. / Food Chemistry xxx (2007) xxx–xxxreported from the leaves (Murakami, Kitazono, Jiwajinda,Koshimzu, & Ohigashi, 1998). Recently, a bioenhancingproperty of M. oleifera pods extract was reported andfound that niaziridin rich fraction of M. oleifera podsenhances the bioactivity of commonly used antibiotics suchas rifampicin, tetracycline and ampicillin against gram(+)and ( ) bacteria and also facilitate the absorption of drugs,vitamins and nutrients through the gastro-intestinal membranethus increasing their bio-availability (Khanujaet al., 2005). Therefore, niaziridin can be used in combinationtherapy with drugs and nutrients resulting in reduceddrug associated toxicity, reduced cost and duration ofchemotherapy.In view of the potential bioenhancing properties of thenovel nitrile glycoside niaziridin, optimization of itsextraction and chromatographic conditions for rapidand precise screening of niaziridin and niazirin fromM. oleifera, was performed. Niazirin is another bioactivenitrile glycoside belonging to M. oleifera (Faizi et al.,1994a, 1994b). To the best of our knowledge no validatedHPLC method is available for the quantitative analysis ofboth niaziridin and niazirin in drumstick (M. oleifera).The present method is robust for the quantitative determinationof niaziridin and niazirin in various tissue of drumstick.It can also be used to monitor the batch-to-batchvariation of bioenhancer content in the standardizedextracts of M. oleifera.2. Methodology2.1. Plant materials and chemicalsDrumstick (M. oleifera) leaves, pods and bark were collectedfrom CIMAP research field, suburban market ofLucknow. The plant parts were air-dried and ground topowder using a mixer grinder (Philips, India).The solvents used for extraction and chromatographicisolation were of analytical grade. Gradient grade HPLC–UV–Vis quality from Lichrosolv Ò , Merck, India and waterpurified locally on Milli-Q equipment were used. Sodiumdihydrogen phosphate from Merck, India and acetic acidof HPLC quality (Spectrochem, India) and was used forpH adjustment of the eluent.2.2. StandardsReference marker compounds, Niaziridin and Niazirinwere isolated and characterized in our lab. The puritywas determined by HPLC. Spectral details of niaziridinwere confirmed with our patent (Khanuja et al., 2005),while, the authenticity of niazirin was confirmed by thespectral data from the literature (Faizi et al., 1994a,1994b). The stock solutions were prepared in methanol.The chemical structures of bioactive nitrile glycosides ofM. oleifera are depicted in Fig. 1.A. Reference compoundsB. 1. UV Spectra of NiaziridinB. 2. UV Spectra of NiazirinNiaziridin (1)0.30220.50.10220.5AU0.200.00Niazirin (2)0.200.10AU0.00-0.10277.0377.70.00AU-0.10271.0370.5-0.20-0.205.00 10.00MinutesC. Bioactive fraction of pod extractD. Iso-absorbance Map2.00AU1.006 547 8O1CH 2 CN6' 5' 2 3H 3 C 1'4' O OHHO 3' 2'HOHO 6 547 8O1CH 2 CNNiaziridin (1) 6' 5' 2 3H 3 C 1'4' OHO 3' 2'HOHONiazirin (2)350.00nm300.00250.000.0010.00 20.00 30.00Minutes200.0010.00 20.00 30.00MinutesFig. 1. Typical HPLC chromatogram of: (A) standard mixture of (1) niaziridin and (2) niazirin, (B) UV spectra of the respective peaks (C) bioactivefraction of M. oleifera pods analyzed by absorbance at 220 nm. UV pattern of the respective peaks and (D) iso-absorbance map (contour plot) of bioactivepod extract were indicated alongside the chromatogram.Please cite this article in press as: Shanker, K. et al., Determination of bioactive nitrile glycoside(s) in drumstick ..., Food Chemistry(2007), doi:10.1016/j.foodchem.2006.12.034
ARTICLE IN PRESSK. Shanker et al. / Food Chemistry xxx (2007) xxx–xxx 32.3. Equipment and chromatographic conditionsThe HPLC analysis have been carried out on Watersequipment: pump 600E; auto-injector-717plus; columnoven; detector-996 PDA. Data acquisition and computationwere carried out with Waters Melenium Ò software.Analyses were carried out on Merck Chromolith RP18ecolumn, 5 lm (100 4.6 mm i.d.). Typical chromatogramsare shown in Figs. 1 and 2.All of the standards and extracts were filtered through a0.45 lm syringe membrane filter (Type Millipore) and analyzedby HPLC. Analysis was carried out at 25 °C onaChromolith RP18e (Merck) column. Prior to use, solventswere filtered with 0.45 lm, 50 mm diameter membrane filter(Millipore) and sonicated for 15 min in a Micro clean109 bath (Oscar, India). Chromatography was carriedout using mobile phase of methanol–sodium dihydrogenphosphate–acetic acid buffer (0.1 M, pH 3.8) (20:80) (%v/v). The flow rate during the analysis was 0.7 ml/minand peaks were detected at 220 nm. Although detectionat 275 nm was more selective but detection at 220 nmwas more sensitive and did not pose a problem in termsof co-elution with impurities. Therefore, quantitation ofniaziridin and niazirin was performed at 220 nm peak areadata.2.4. Procedure2.4.1. Extraction of M. oleifera tissuesThe dried powdered leaves, pods and bark (2 g each) ofM. oleifera were extracted with 25 ml of various combinationsof ethanol–aqueous solution (0%, 20%, 40%, 60%,80%, 100%, v/v) by incubation at 30 °C for 24 h. Eachextract was filtered and solvent removed under vacuumat 40 °C. The residue was re-dissolved in 3 ml of HPLCgrade methanol. Niaziridin and niazirin in various extractswere analyzed by an analytical HPLC system. It wasobserved that the extractability of niaziridin was greatlyinfluenced by the ratio of extracting solvent mixtures i.e.ethanol–water. The most effective solvent for the maximumextraction of niaziridin and niazirin was 40% ethanol inwater for pods and 60% ethanol in water for leaves(Fig. 3). Both the markers were not extracted from barksample under experiment conditions studied. Thus weA. Pods (40% ethanol) Extract B. Leaves (60% ethanol) Extract C. Bark (20% ethanol) Extractat 220nm220nm220nm1.50AU 1.000.50Niaziridin (1)Niazirin (2)1.501.00AU0.50Niaziridin (1)Niazirin (2)0.800.60AU0.400.200.0010.00 20.00 30.00Minutes0.0010.00 20.00 30.00Minutes0.0010.00 20.00 30.00Minutes275nm275nm275nm2.00AU1.000.800.60AU0.400.20AU0.100.200.0010.00 20.00 30.00Minutes0.0010.00 20.00 30.00Minutes0.0010.00 20.00 30.00MinutesContour plot of A.Contour plot of B.Contour plot of C.350.00nm 300.00350.00nm300.00350.00nm300.00250.00250.00250.00200.005.00 30.00Minutes200.005.00 30.00Minutes200.005.00 30.00MinutesFig. 2. HPLC profile of: (A) pods, (B) leaves and (C) bark of M. oleifera analyzed by absorbence at 220 nm and 275 nm, contour plot of HPLC pattern of:(A) pods, (B) leaves and (C) bark of M. oleifera UV absorbance at 190–400 nm, respectively.Please cite this article in press as: Shanker, K. et al., Determination of bioactive nitrile glycoside(s) in drumstick ..., Food Chemistry(2007), doi:10.1016/j.foodchem.2006.12.034
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