A spectrophotometric method for simultaneously estimating 11Î²,17Î±-dihydroxy-4-pregnene-3,20-dione 21-succinate and its 1(2)-dehydrogenated product has been developed using sulphuric acid chromogens. The method was used to follow the progress of bioconversion during C-1(2)-dehydrogenation by Comamonas acidovorans MTCC 3364. Keywords: Steroid 1(2)-dehydrogenation; Simultaneous estimation; Steroid bioconversion; Hydrocortisone; Prednisolone estimatio

Neelam Lad1 and S. P. Pawar2, Kirti Pawar1*,

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Recent Research in Science and Technology 2011, 3(1): 01-04 ISSN: 2076-5061 www.recent-science.com  MICROBIOLOGY  SIMULTANEOUS SPECTROPHOTOMETRIC ESTIMATION OF 11β,17α-DIHYDROXY -4-PREGNENE-3,20-DIONE- 21-SUCCINATE AND ITS 1(2)-DEHYDROGENATED PRODUCT DURING BIOCONVERSION BY COMAMONAS ACIDOVORANS MTCC 3364 Kirti Pawar1∗, Neelam Lad1 and S. P. Pawar2 1Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences, New Vallabh Vidya Nagar, Anand- 388 121, Gujarat, India 2A R College of Pharmacy, Vallabh Vidya Nagar, Anand- 388 121, Gujarat, India  Abstract A spectrophotometric method for simultaneously estimating 11β,17α-dihydroxy-4-pregnene-3,20-dione 21-succinate and its 1(2)-dehydrogenated product has been developed using sulphuric acid chromogens. The method was used to follow the progress of bioconversion during C-1(2)-dehydrogenation by Comamonas acidovorans MTCC 3364.  Keywords: Steroid 1(2)-dehydrogenation; Simultaneous estimation; Steroid bioconversion; Hydrocortisone; Prednisolone estimation                                                           ∗ Corresponding Author, Email: drkirtipawar@rediffmail.com Introduction Two corticosteroids, 11β,17α-dihydroxy-4-pregnene-3,20-dione-21-succinate (hydrocortisone succinate) and its dehydrogenated product 11β,17α-dihydroxy-1,4-pregnadiene-3,20-dione-21-succinate (prednisolone succinate) are important anti-inflammatory agents used in variety of dosage forms. Simultaneous estimation of precursor and products during steroid 1(2)-dehydrogenation poses a difficult task. Most of the described methods utilize sophisticated, sensitive but expensive techniques like High Performance Liquid Chromatography and Gas Liquid Chromatography (Kaul & Mattiasson, 1986, Pinhero and Cabral, 1990).  The present work reports a rapid and cost effective spectrophotometric method for routine estimation of precursor and product during C-1(2)-dehydrogenation of hydrocortisone succinate by Comamonas acidovorans MTCC 3364. The method is based on characteristic chromogen formation by the steroids which show concentration dependent difference in absorption at the selected wavelength.   Materials and Methods Ethyl acetate solutions of hydrocortisone succinate and prednisolone succinate containing 25 μg of compound were vacuum dried in separate tubes. The residue was dissolved in 3.0 ml 40%, 60% and 98% sulphuric acid, tubes kept in boiling water bath for different time intervals and cooled to room temperature. Absorption spectra of the developed chromogen were recorded between 250 and 650 nm. To select the best wavelength for simultaneous determination of the compounds, a series of tubes containing 10 to 100 μg compounds were taken, chromogens prepared and absorbance determined at selected wavelengths.  Comamonas acidovorans MTCC 3364 was grown in a medium containing (g l-1): (NH4)2SO4, 3; CaCl2, 0.2; FeSO4, 0.1; Glucose, 20; K2HPO4, 3.0; KH2PO4, 1.0; MgSO4, 0.2; pH adjusted to 7.0 was used for bioconversion of hydrocortisone succinate. Medium (20 ml) was dispensed in a series of 100 ml conical flasks and sterilized at 121 ºC for 15 min and allowed to cool to room temperature. The flasks were inoculated with actively growing culture of the strains of microorganism in the same medium, allowed to grow overnight at 30 ºC on a shaking platform at 100 rpm and hydrocortisone succinate dissolved in 0.2 ml acetone was added. Samples were drawn at specified intervals and extracted twice with equal volumes of ethyl acetate. The organic layer was decanted, dried over sodium sulphate and solvent evaporated by keeping the tubes in a boiling water bath. The residues were dissolved in 3 ml sulphuric acid, chromogen developed, absorbance determined at selected wavelength and mole % conversion was calculated.  Results and Discussion  The sulphuric acid chromogens showed stable absorbance in 98% sulphuric acid after 30 min reaction in boiling water bath. The overlay spectra (fig 1) of the two compounds showed minimum interference of Kirti Pawar et al./Rec Res Sci Tech 3 (2011) 01-04  hydrocortisone succinate after 400 nm. To determine the best wavelength for estimation, absorbance of chromogens was determined at 400, 460 and 545 nm (fig 2 & 3) and difference in absorbance at each concentration determined. Maximum difference was observed at 545 nm at highest concentration of the chosen range and hence selected for determination of the appearance of product in the bioconversion medium.  Fig 1: Overlay spectra of Hydrocortisone succinate and Prednisolone succinate                   Fig 2: Absorbance of different amounts of hydrocortisone succinate at selected wavelengths                               Fig 3: Absorbance of different amounts of prednisolone succinate at selected wavelengths          Kirti Pawar et al./Rec Res Sci Tech 3 (2011) 01-04   The concentration of the precursor was not allowed to increase above 100 μg ml -1 in the extracts of bioconversion media and compensated by appropriate dilution of extract before producing sulphuric acid chromogens whenever higher substrate concentrations had to be added.  Similar simultaneous estimation method for Androstenedione and its dehydrogenated product has been developed by Patil et al (1991) and for solasonine and solasodine estimation by Patil et al (1992) and successfully applied for simultaneous estimation of precursor and product steroids during bioconversion. The mole % conversion hydrocortisone succinate bioconversion by Comamonas acidovorans MTCC 3364 using different substrate concentrations have been shown in table 1. The maximum accumulation of product was observed at 0.5 mg l -1 substrate concentration after 72 hr incubation. Thin layer chromatography revealed rapid disappearance of the accumulated product after achieving maximum bioconversion. The production of prednisolone succinate decreased with increasing substrate concentration. Nadia Naima et al. (2003) used Pseudomonas fluorescens cells entrapped in poly-hydroxyethylmethacrylate (Poly-HEMA), which converted hydrocortisone to prednisolone with maximum bioconversion efficiency at a substrate concentration of 2 mg l-1 medium. The result also complies with observation of Jiradej Manosroi et al. (1998) who reported reduction in prednisolone production with increasing hydrocortisone concentration during bioconversion by immobilized cells of a three bacterial strains (Bacillus sphaericus ATCC 13805, Bacillus sphaericus SRP III and Arthrobacter simplex 6946) in an aqueous medium. There was decrease in amount of accumulated product on further incubation with the organism indicating degradation to non steroidal compounds. Similar reduction of accumulated steroidal products has been reported by Patil et al (1991).  Table 1: Mole % conversion of hydrocortisone succinate by growing cells of Comamonas acidovorans MTCC 3364  Substrate concentration ( mg l-1) Mole % conversion after incubation (hr)  24 hrs  48 hrs 72 hrs               0.5  31.68 ± 3.0 47.85 ± 2.3 69.55 ± 1.8               1.0  63.00 ± 1.7 41.65 ± 1.4 56.80 ± 1.1               1.5  50.88 ± 5.5 17.39 ± 5.7 35.46 ± 5.7               2.0  09.81 ± 1.5 37.37 ± 0.9 37.40 ± 1.1               5.0  09.81 ± 1.1 10.99 ± 0.5 05.75 ± 2.7 Data are mean of three replicates ± SD Comamonas acidovorans MTCC 3364 was found to 1,2-dehydrogenate hydrocortisone succinate effectively as an actively growing culture The results do not coincide with the findings of Srivastava and Patil (1998) who indicated the dependence of steroid 1(2) dehydrogenation on the C-17 side chain during cholesterol metabolism by immobilized Mycobactrium fortuitum. It can be indicated that 1(2) dehydrogenation of hydrocortisone succinate by Comamonas acidovorans MTCC 3364 is not affected by presence of aliphatic side chain at C-17 position.  Acknowledgement The authors acknowledge Charutar Vidyamandal, Vallabh Vidyanagar, Anand for providing the infrastructure, chemical, computational and other necessary facilities for the successful completion of this work. Authors are also thankful to Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India for providing the microbial culture used for the study. Thanks are due to Wyeth Limited, Verna Goa, India for gifting some of the samples of steroids required for this study.  References Jiradej Manosroi, Pattana Sripalakit & Aranya Manosroi (1998) Bioconversion of hydrocortisone to prednisolone by immobilized bacterial cells in a two-liquid-phase system. Journal of Chemical Technology & Biotechnology; 73, ( 3); (203–210). Kaul Rajni and Mattiasson Bo (1986) Extractive bioconversion in aqueous two phase system. Applied Microbiology and Biotechnology, 24; (259-265). Nadia Naima, Nehad Z. Adhama, Hassan Abd El-Rehimb and AbeerAbd El-Hadya (2003) Prednisolone production using Pseudomonas Kirti Pawar et al./Rec Res Sci Tech 3 (2011) 01-04  fluorescens cells immobilized with polymer carrier produced by radiation polymerization. Process Biochemistry, Vol. 38, (7), 28; (1083-1089) Patil S., Srivastava A., Shukla A, and Phase N. (1991) Spectrometric estimation of 4-androstene, 3,17-dione and 1,4-androsta3,17-dione during C1,2-dehyrogenation by Mycobacterium fortuitum NRRL B 8153 ; World Journal of Microbiology and Biotechnology, 7, (626-627). Patil S., Shukla A, Srivastava A., and Phase N. (1992) Spectrophotmetric determination of solasonine and its bioconversion product  solasodine ; Indian Jounal of Chemistry, 32B, (1083-1085) Pinheiro H. M. and Cabral J. M. S. (1990) Effect of solvent molecular toxicity and microenvironment composition on the Δ1 dehydrogenation activity of Arthrobacter simplex cells. Biotechnology and Bioengineering, vol. 37; (94-102). Srivastava A and  Patil S. (1998) Dependence of steroid 1(2)-dehydrogenation on the C-17 side chain during cholesterol metabolism by immobilized Mycobacterium fortuitum; World Journal of Microbiology and Biotechnology; 11(3), (284-286),DOI:10.1007/BF00367099.   

Simultaneous Spectrophotometric Estimation of 11Î²,17Î±-dihydroxy -4-pregnene-3,20-dione- 21-succinate and its 1(2)-Dehydrogenated Product During Bioconversion by Comamonas acidovorans MTCC 3364

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Simultaneous Spectrophotometric Estimation of 11Î²,17Î±-dihydroxy -4-pregnene-3,20-dione- 21-succinate and its 1(2)-Dehydrogenated Product During Bioconversion by Comamonas acidovorans MTCC 3364

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