INTRODUCTION:

Serratiopeptidase, a proteolytic enzyme, offer a powerful treatment for pain and inflammation with widespread use in arthritis, fibrocystic breast disease, chronic bronchitis, sinusitis, atherosclerosis, wound debridement and carpal tunnel syndrome^1-3. Literature survey revealed report of UV spectrophotometric⁴, UV microplate formation⁵, HPLC⁶ and SEC⁷ methods for the estimation of Serratiopeptidase from pharmaceutical formulation.

Enzymes are protein by nature. Proteins react with cupric ions in alkaline medium to form a violet colored complex. The intensity of the color produced is directly proportional to the concentration of protein present in the specimen and can be measured using visible spectrophotometer or by using green filter of colorimeter⁸. This method was applied for the determination of proteins in the body fluids by many researchers. In present work an attempt has been made to develop a simple colorimetric method using this principle for estimation of Serratiopeptidase in it's tablet formulations.

MATERIALS AND METHODS:

Shimadzu 1700 UV-Visible spectrophotometer, Systronic visible spectrophotometer and Systronic photoelectric colorimeter were used for the present work. The chemicals used were of analytical grade. Commercially available tablets of Serratiopeptidase were procured from local market. Gift sample of standard Serratiopeptidase drug was procured from Wintech Pharmaceuticals.

Preparation of calibration curve:

Preparation of Working Biuret Reagent solution (WBR): Working Biuret Reagent solution was prepared as per standard method⁸.

Preparation of Standard stock solution (SSS):

Standard drug solution (10 mg/ml) was prepared in 7.2 pH phosphate buffer (SSS). For the calibration curve nine 10 ml volumetric flasks were taken and labeled as 1 to 8 and Blank. Quantity of SSS, 7.2 pH phosphate buffer and WBR were added to the volumetric flasks as specified in the Table I:

Content of the flasks were mixed well and allowed to stand for 15 mins. Wavelength maxima of all solutions were determined against reagent blank using Shimadzu 1700 UV Visible spectrophotometer and it was found to be 531 nm (Fig 1). The absorbance was measured at 531 nm by spectrophotometers against reagent blank. Calibration curve was prepared by plotting concentration of drug vs. measured absorbance.

Analysis of tablet formulations:

Randomly sampled twenty tablets were accurately weighed and average weight of tablets was determined. Tablets were crushed to fine powder and tablet powder equivalent to 100 mg of Serratiopeptidase was accurately weighed, extracted with 15ml 7.2 pH phosphate buffer and filtered through Whatman filter paper no. 41. Filter paper was washed twice with 4 ml of the buffer. Washings were added to the filtrate and volume was made to 25 ml with the same. From this solution, 5 ml was transferred in another 10 ml volumetric flask, to it 5 ml WBR was added, mixed well and was allowed to stand for 15 mins. The absorbance was measured at 531 nm by spectrophotometers against reagent blank. Concentration of samples was calculated from the calibration curve. The analysis procedure was repeated five times for both marketed formulations.

The analysis was repeated by using colorimeter (green filter).

Table I: Addition of reagents for preparation of calibration curve.

Flask no.	SSS, ml	Phosphate Buffer 7.2 pH, ml	WBR, ml
1	1.5	3.5	5
2	2	3	5
3	2.5	2.5	5
4	3	2	5
5	3.5	1.5	5
6	4	1	5
7	4.5	0.5	5
8	5	0	5
Blank	0	5	5

Fig 1: Specrta of Serrassiopeptidase

Method validation:

The developed method was validated for its accuracy and precision. Accuracy of the method was determined by performing recovery studies for the tablet formulation. Also, the experiment was repeated three times in a day to determine intra-day precision and on three different days to determine inter-day precision. The percent coefficient of variance (%CV) was calculated at each concentration level. Limit of detection (LOD) and limit of quantification (LOQ) were calculated by repeating the blank measurements twelve times. The data of method validation are given in Table II.

Table II: Validation parameters.

Parameter	Values
λ max (nm)	531
Beer’s law limit(mg/ml)	0.5 to 5
Regression equation	y = 0.097x + 0.0026
Slope	0.097
Intercept	0.0026
Correlation coefficient	0.9993
Limit of detection (µg/ml)	100
Limit of quantification (µg/ml)	400
Precision
Interday (%CV)	0.5612
Intraday (%CV)	0.2605

RESULTS AND DISCUSSION:

In present research work a colorimetric method has been developed for determination of Serratiopeptidase from its tablet formulations. The developed method was based on formation of stable colored complex of drug with Biuret reagent. Wavelength maxima of Serratiopeptidase was found to be at 531 nm. The developed color is stable after 10 mins. and up to 5 hrs. Linearity was observed in concentration range of 0.5-5 mg/ml (Fig 2).

Fig 2: Calibration curve of Serratiopeptidase

Percentage label claim estimated for two tablet formulations were found to be in the range of 98.14 -101.14% of Serratiopeptidase (Table III).

This method can be satisfactorily applicable with UV Visible spectrophotometer, Visible Spectrophotometer or Photoelectric Colorimeter. The result suggests that present method can be satisfactorily applicable for the estimation of Serratiopeptidase in its tablet dosage forms. It is simple precise, rapid and economical.

ACKNOWLEDGMENT:

The authors are thankful to M/S Wintech Pharmaceuticals, Mumbai for providing the gift sample of Serratiopeptidase.

REFERENCES:

1. Kee W.H., Tan S.L., Lee V., Salmon Y.M.: The treatment of breast engorgement with serrapeptase: a randomized double blind controlled trial, Singapore Med J, 1989, 30, 48-54. (Through PubMed)

2. Majima Y., Hirata K., Takeuchi M.: Effects of orally administered drugs on dynamic viscoelasticity of human nasal mucus, Am Rev Respit Dis, 1990, 141: 79-83.

3. Sherry S., Fletcher A.P.: Proteolytic enzymes: a therapeutic evaluation, Clin Pharmacol Ther, 1960, 1: 202-206.

4. Saudagar R.B., Rawat M., Singh D., Saraf S., Saraf S.: Development and validation of derivative spectrophotometric method for determination of serratiopeptidase in pharmaceutical formulation, Oriental J Chem, 2007, 23(3): 1057-1060.

5. Sandhya K.V., Gayathridevi S., Mathew S.T, Quantitation of Serrapeptase in Formulations by UV Method in the Microplate Format, Current Drug Delivery, 2008, 5(4): 303-305.

6. Dallas P., Rekkas D., Choulis N.H., HPLC determination of serratiopeptidase in biological fluids, Pharmazie, 1989, 44(4): 297.

7. Ijitsu T., Yonezawa K., Ueno M., Improved analysis of serrapeptase by high performance steric exclusion chromatography (SEC), Yakugaku Zasshi, 1986, 106(1): 95-98.

8. Godkar P.B., Godkar D.P., Textbook of Medical Laboratory Technology, 2^nd edition by Bhalani Publishing House, 2004, 265-267.

Received on 10.02.2010 Modified on 19.02.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 631-633

Formulation	Label claim (mg)	*Label claim estimated (mg)**	%Recovery	% Assay	SD	RSD	%CV
A	10	10.14	99.08	101.40	0.0006	0.0029	0.2896
B	10	9.81	99.1	98.14	0.0010	0.0052	0.5181