Asian J. Research Chem. 3(3): July- Sept.

2010

ISSN 0974-4169 www.ajrconline.org

RESEARCH ARTICLE

New Visible Spectrophotometric Method for Estimation of

Serratiopeptidase from Tablet Formulations
Parula Patel* and Bhavisha Rabadiya
Atmiya Institute of Pharmacy, Kalawad Road, Rajkot, Gujarat, India.
*Corresponding Author E-mail: pbpatel@aip.edu.in

ABSTRACT:
A simple, economical, precise and rapid visible spectrophotometric method has been developed for the determination
of Serratiopeptidase in tablet dosage forms. Developed method is based on the formation of stable colored complex of
drug with Biuret reagent. A wavelength maximum was found to be 531 nm. Linearity was observed in the
concentration range of 0.5-5 mg /ml. Correlation coefficient was found to be 0.9993. The result of analysis has
been validated statistically and also by recovery study.

KEYWORDS: Serratiopeptidase, Visible spectrophotometric method, Biuret reagent

INTRODUCTION: Preparation of calibration curve:

Serratiopeptidase, a proteolytic enzyme, offer a powerful Preparation of Working Biuret Reagent solution (WBR):
treatment for pain and inflammation with widespread use in Working Biuret Reagent solution was prepared as per
arthritis, fibrocystic breast disease, chronic bronchitis, standard method8.
sinusitis, atherosclerosis, wound debridement and carpal
tunnel syndrome1-3. Literature survey revealed report of UV Preparation of Standard stock solution (SSS):
spectrophotometric4, UV microplate formation5, HPLC6 and Standard drug solution (10 mg/ml) was prepared in 7.2 pH
SEC7 methods for the estimation of Serratiopeptidase from phosphate buffer (SSS). For the calibration curve nine 10
pharmaceutical formulation. ml volumetric flasks were taken and labeled as 1 to 8 and
Blank. Quantity of SSS, 7.2 pH phosphate buffer and WBR
Enzymes are protein by nature. Proteins react with cupric were added to the volumetric flasks as specified in the
ions in alkaline medium to form a violet colored complex. Table I:
The intensity of the color produced is directly proportional
to the concentration of protein present in the specimen and Content of the flasks were mixed well and allowed to stand
can be measured using visible spectrophotometer or by for 15 mins. Wavelength maxima of all solutions were
using green filter of colorimeter8. This method was applied determined against reagent blank using Shimadzu 1700 UV
for the determination of proteins in the body fluids by many Visible spectrophotometer and it was found to be 531 nm
researchers. In present work an attempt has been made to (Fig 1). The absorbance was measured at 531 nm by
develop a simple colorimetric method using this principle spectrophotometers against reagent blank. Calibration curve
for estimation of Serratiopeptidase in it's tablet formulations. was prepared by plotting concentration of drug vs.
measured absorbance.
MATERIALS AND METHODS:
Shimadzu 1700 UV-Visible spectrophotometer, Systronic Analysis of tablet formulations:
visible spectrophotometer and Systronic photoelectric Randomly sampled twenty tablets were accurately weighed
colorimeter were used for the present work. The chemicals and average weight of tablets was determined. Tablets were
used were of analytical grade. Commercially available crushed to fine powder and tablet powder equivalent to 100
tablets of Serratiopeptidase were procured from local mg of Serratiopeptidase was accurately weighed, extracted
market. Gift sample of standard Serratiopeptidase drug was with 15ml 7.2 pH phosphate buffer and filtered through
procured from Wintech Pharmaceuticals. 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
Received on 10.02.2010 Modified on 19.02.2010 solution, 5 ml was transferred in another 10 ml volumetric
Accepted on 12.03.2010 © AJRC All right reserved flask, to it 5 ml WBR was added, mixed well and was
Asian J. Research Chem. 3(3): July- Sept. 2010; Page 631-633 allowed to stand for 15 mins. The absorbance was measured

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 Asian J. Research Chem. 3(3): July- Sept. 2010

at 531 nm by spectrophotometers against reagent blank. Table II: Validation parameters.

Concentration of samples was calculated from the Parameter Values
calibration curve. The analysis procedure was repeated five max (nm) 531
Beer’s law limit(mg/ml) 0.5 to 5
times for both marketed formulations.
Regression equation y = 0.097x + 0.0026
The analysis was repeated by using colorimeter (green Slope 0.097
filter). Intercept 0.0026
Correlation coefficient 0.9993
Limit of detection (µg/ml) 100
Table I: Addition of reagents for preparation of calibration curve. Limit of quantification (µg/ml) 400
Flask no. SSS, ml Phosphate Buffer 7.2 WBR, ml Precision
pH, ml Interday (%CV) 0.5612
1 1.5 3.5 5 Intraday (%CV) 0.2605
2 2 3 5
3 2.5 2.5 5 RESULTS AND DISCUSSION:
4 3 2 5 In present research work a colorimetric method has been
5 3.5 1.5 5
6 4 1 5 developed for determination of Serratiopeptidase from its
7 4.5 0.5 5 tablet formulations. The developed method was based on
8 5 0 5 formation of stable colored complex of drug with Biuret
Blank 0 5 5 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).

CALIBRATION CURVE

0.6

0.5

0.4
Absorbance

0.3

0.2
y = 0.097x + 0.0026
0.1 R2 = 0.9993
0
0 1 2 3 4 5 6
Serratiopeptidase (m g/ml)
Fig 2: Calibration curve of Serratiopeptidase
Fig 1: Specrta of Serrassiopeptidase
Percentage label claim estimated for two tablet formulations
were found to be in the range of 98.14 -101.14% of
Method validation: Serratiopeptidase (Table III).
The developed method was validated for its accuracy and
precision. Accuracy of the method was determined by This method can be satisfactorily applicable with UV
performing recovery studies for the tablet formulation. Visible spectrophotometer, Visible Spectrophotometer or
Also, the experiment was repeated three times in a day to Photoelectric Colorimeter. The result suggests that present
determine intra-day precision and on three different days to method can be satisfactorily applicable for the estimation of
determine inter-day precision. The percent coefficient of Serratiopeptidase in its tablet dosage forms. It is simple
variance (%CV) was calculated at each concentration level. precise, rapid and economical.
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
ACKNOWLEDGMENT:
The authors are thankful to M/S Wintech Pharmaceuticals,
Table II.
Mumbai for providing the gift sample of Serratiopeptidase.

Table III: Results of analysis of commercial formulations:

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
* Average of six determinations

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 Asian J. Research Chem. 3(3): July- Sept. 2010

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