enzymatic spectrophotometric determination of diacerein using ...

G. Vijaya Raja et al., IJSID 2011, 1 (2), 278-282

ISSN:2249-5347

IJSID International Journal of Science Innovations and Discoveries Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info

ENZYMATIC SPECTROPHOTOMETRIC DETERMINATION OF DIACEREIN USING PEROXIDASE/MBTH SYSTEM G.Vijaya Raja1, Sk.Jaffar2*, Viswanath Reddy Pyreddy3, MD.Zaheeruddin2, A.Ramu4, MD.Ibrahim5, M.Guruprasad6 and M.Kondala Rao7. 1Centre

for Biotechnology, Acharya Nagarjuna University, Nagarjunanagar–522 510, 2Dept. of Biochemistry, Acharya Nagarjuna University, Nagarjunanagar–522 510, 3Dept. of Biochemistry, Sri Krishnadevaraya University, Anantapur, AP, India, 4Ultra College of Pharmacy, Madurai, TN, India, 5Shadan Institute of Medical Science, Hyderabad, AP,India, 6Regional Agriculture Research Station, Tirupathi, A.P, India and 7Dept of Zoology Acharya Nagarjuna University, Guntur, AP, India.

Received: 06.08.2011

ABSTRACT

Modified: 11.10.2011 Published: 27.10.2011

Simple, sensitive and inexpensive spectrophotometric method for the determination of diacerein was proposed. Method was based on

*Corresponding Author

enzyme catalyzed oxidative coupling reaction between the drug and MBTH in presence of hydrogen peroxide and horse radish peroxidase enzyme to produce yellow colored complex with absorbance maxima at 340 nm. The chromogen intensity was linear with the concentration of diacerein over the range 2-10µg/ml .The relative standard deviation was 1.3. The proposed method has been successfully applied for the determination of Address: Name: Sk. Jaffar Place: Guntur, AP, India E-mail: [email protected]

diacerein in a pharmaceutical formulation without interference from its potential impurities. Keywords: Diacerein, Relative standard deviation, MBTH.

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INTRODUCTION Diacerein (1,8-diacetoxy-3-carboxyanthraquinone) (Fig. 1), is a novel anti-inflammatory drug with pharmacological properties different from those of classical non- steroidal anti-inflammatory drugs.1 Clinical studies have suggested that diacerein exerts a beneficial effect on the symptomatic treatment of osteoarthritis.2,3 It is also known to inhibit the production of interleukin-1 and to prevent cartilage breakdown in a mouse granuloma model,4 and to slow the progression of cartilage lesions in a canine model of osteoarthritis5. Diacerein is known to be completely metabolized by animals and humans into rhein, an active metabolite of diacerein found in plasma and synovial fluid6. Moreover, Cruz and Pastrak have related the use of diacerein or rhein to treat and prevent vascular diseases7.For its potential pharmaceutical effects on health, the development of a simple, rapid and sensitive method for the determination of diacerein in pharmaceutical preparations would therefore be highly desirable. Giannellini etal8 reported a validated HPLC stability-indicating method for the determination of diacerein in bulk drug substances.

Fig: 1 Structure of Diacerein EXPERIMENT: Apparatus: Elico UV – Visible Double beam spectrophotometer model SL-159. Materials and Reagents: All the chemicals used were of analytical grade. All the solutions were freshly prepared in distilled water. Standard drug solution: Accurately weighed 100mg of Diacerein was dissolved in 100mL distilled water to give a concentration of 1mg/mL. The final concentration was brought to 100 µg/mL. Assay of pharmaceutical formulations: Tablet powder equivalent to 100 mg was accurately weighed and dissolved in water and filtered. The filtrate was made up to 100 mL and appropriate aliquots of the drug solution were treated as described above and the results were tabulated. Reagents: •

0.2% MBTH

•

Phosphate buffer pH 7 International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011

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•

0.01M Hydrogen peroxide

•

Horse radish peroxidase Enzyme

Enzyme extraction: 40g of peeled turnip pieces were homogenized in 200ml of buffer in a high speed blender for 15min. the extract is centrifuged and filtered through Whatman no . 1 filter paper. For stability the was stored in toluene at 4oC and the extract was suitably diluted for further use. Assay procedure for the determination of Diacerein: Method : In to a series of volumetric flasks transfer 2ml buffer, 1.5 ml MBTH, 1ml hydrogen peroxide and 1.5ml enzyme extract shake well and kept for 10 min then add aliquots (0.5 – 2.5 ml) of standard drug solution .The contents were made up to 10ml with distilled water and the absorbance was measured after complete color development at 340m against reagent blank and the calibration curve was constructed. RESULTS AND DISCUSSION: The proposed method is based on peroxidase enzyme catalyzed oxidative coupling reaction in which MBTH acts as electron donor for the conversion of hydrogen peroxide to H2O by the enzyme and is converted to highly reactive oxidized form which couples to the drug forming a stable, water soluble, yellow colored product having λmax at 340nm.The concentration of the colored product is proportional to the drug. The schematic reaction mechanism for the proposed method was shown in fig: 2. The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity and Sandell’s sensitivity for the proposed method is presented in Table-1.The regression analysis using the method of least squares was made for the slope (a) and intercept (b) obtained from different concentrations are summarized in Table-1. The precision and accuracy were found by analyzing six replicate samples containing known amounts of the drug and the results are summarized in Table-1 The accuracy of these methods was ascertained by comparing the results obtained with the proposed and reference methods in the case of formulation are presented in Table-2. Recovery experiments indicated the absence of interferences from the commonly encountered pharmaceutical additives and excipients.


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TABLE – 1: Optical Characteristics, Precision and Accuracy of Proposed Method Parameters Values λ max (nm) 340 Beer’s law limit (μg/ mL) 2-10 Sandell’s Sensitivity (μg/cm2/0.001 abs. unit) 0.0246 Molar absorptivity(Litre.mole-1.cm-1) 1.491 x 104 Stability of Color (hours) 24 Regression equation (Y)* Intercept (a) 0.0066 Slope(b) 0.0039 $ % RSD 1.352 % Range of errors ( 95% confidence limits): 0.05 significance level 1.132 0.01 significance level 1.672 * Y = a + bx, where Y is the absorbance and x is the concentration of Diacerein in μg/ mL $ For six replicates International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011

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TABLE – 2: Assay and Recovery of Diacerein in Pharmaceutical Formulations Formulations

Labeled Recovery by reference Recovery by proposed amount(mg) method*(%) method (%) ** Diasol 50 99.95 99.90 Diacer 50 99.90 99.89 * Reference method was UV method developed in the laboratory, ** Recovery amount was the average of six determinants. CONCLUSION The proposed methods are useful due to negligible interference for the common excipients found in drug formulations. The proposed methods do not require any elaborate treatment of the drug for the formation of colored chromospheres of the drug with the interacting reagents. In the proposed method the enzyme extraction is only the tedious prosses.This was the only demerit of the proposed method. ACKNOWLEDGEMENTS The authors are grateful to Centre for Biotechnology, Acharya Nagarjuna University, Guntur, and Sri Durga Malleswara Siddhartha Mahila Kalasala, Vijayawada for providing their continuous support. REFERENCES 1. 1.Tamura,T., Shirai,N., Kosaka,N., Ohmori,K.,Takafumi,N.Eur.J.Pharma col.,2002; 448 (81). 2. Nguyen, M., Dougados, M., Berdah, L., Amor, B. Arthritis Rheum. 1994; 37: 526. 3. Plletier,J.P.,Yaron,M., Haraoui,B., Cohen,P., Nahir,M.A.,Choquette,D.,Wigler,I., Rosner, I.A., Beauleu, A.D. Arthritis Rheum. 2000; 43: 2339. 4. Moore, A.R., Greenslade, K.J., Alam, C.A., Willoughby, D.A., osteoarthr.Cartilage. 1998; 6: 19. 5. Brandt, K.D., Smith, G., Kang, S.Y., Myers, S., O Connor, B., Albrecht, M. osteoarthr.Cartilage. 1997; 5: 438. 6. Debord, P., Louchahi, K., Tod, M., Cournot, A., Perret, G., Petitjean, O., Eur. J. Drug Metab.Ph. 1994; 19: 13. 7. Cruz, T., Pastrak, A.PCT int.Appl. 2003; P26. 8. Giannellini, V., Salvatore, F., Bartolucci.G. Coran, S. A., Bambagiotti-Alberti, M. J. Pharm.Biomed., 2005; 39: 776.


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