SENSITIVE AND SELECTIVE METHODS FOR DETERMINATION OF PROTEOLYTIC ACTIVITY OF FORMULATION CONTAINING BROMELAIN AND TRYPSIN AS PROTEOLYTIC ENZYMES

SENSITIVE AND SELECTIVE METHODS FOR DETERMINATION OF PROTEOLYTIC ACTIVITY OF FORMULATION CONTAINING BROMELAIN AND TRYPSIN AS PROTEOLYTIC ENZYMES

S. S. Wani and R. C. Mashru*

Pharmacy Department, Faculty of Technology and Engineering, The M.S. University of Baroda, Near Dandia Bazar, Kalabhavan, Vadodara-390001, Gujarat state, India.

ABSTRACT: Accurate and sensitive assay methods for quantitative determination of Rutoside trihydrate (rutin), Trypsin and Bromelain in tablet formulation based on UV-spectrophometric analysis were developed and validated. This three drug combination has anti-inflammatory properties and hence is used for treatment of edema and inflammations. Rutin was analyzed directly by UV-Vis spectrophotometer at 257nm because Trypsin and Bromelain are not UV-active therefore they don’t interfere in the analysis of rutin. Trypsin and Bromelain being proteolytic enzymes, Casein was used as substrates for their analysis. Casein was standardized according to Bradford assay using Bovine serum albumin as a standard. Casein was hydrolyzed to amino acid tyrosine by both the proteolytic enzymes. Tyrosine formed was further reacted with Folin-Ciolcalteu (F-C) reagent and absorbance was recorded at 785nm. Trypsin was inhibited by specific trypsin inhibitor Nα-p-tosyl-L-lysine chloromethyl Ketone (TLCK) and specific proteolytic activity of Bromelain was analysed using casein. Bromelain was inactive towards N-a-benzoyl-DL-arginine p-nitroaniline (BApNA) therefore; BApNA was used as a specific substrate for analysis of Trypsin. BApNA was hydrolyzed to pNA by Trypsin which was further diazotized with N (1 Naphthyl) Ethylene Diamine Dihydochloride (NEDD) and absorbance was recorded at 545nm. Intra- and inter-day precision of all the methods developed were within acceptable limits. All methods were validated as per ICH guidelines and can be adopted for the routine analysis of Trypsin, Rutoside trihydrate and Bromelain in tablet formulations

Trypsin, Bromelain, Rutoside trihydrate, Casein, N-a-benzoyl-DL-arginine p-nitroaniline (BApNA), Nα-p-tosyl-L-lysine chloromethyl Ketone (TLCK)

INTRODUCTION: This tablet formulation is a combination of two proteolytic enzymes Typsin and Bromelain together with plant flavonoid Rutoside which has anti-inflammatory properties. It is indicated for treatment of edema and inflammation of traumatic origin such as contusions, lacerations and cuts. It is also indicated for treatment of edema and inflammation following surgery, tooth extraction, cellulitis, abscess, sport injuries and sprains.

The combination of Trypsin, Bromelain, and Rutoside trihydrate can also be used in the treatment of Osteoarthritis, Rheumatoid Arthritis, Spondylopathies, Tendonitis and Bursitis.

Rutin:

Rutoside trihydrate also known as Rutin (3-[[6-O-(6-Deoxy-α-L-mannopyranosyl)βDglucopyranosyl] oxy]-2-(3, 4-dihydroxyphenyl)-5 7-dihydroxy-4H-1 benzopyran-4-one) is a flavonoid of the flavonol type¹. It is found in many typical nutrimental plants such as buckwheat, apple and black tea. Rutin helps preventing hemorrhages and ruptures in the capillaries and connective tissues, and is therefore often used to treat chronic venous insufficiency, hemorrhages and epitaxis ^{2, 3}. Similar to many flavonoid derivatives it also displays a remarkable array of pharmacological and biological activities, such as antioxidant, antiinflammatory, anticarcinogenic, antithrombic and vasoprotective activities Figure 1.

FIGURE 1: STRUCTURE OF RUTOSIDE TRIHYDRATE

 Trypsin:

Trypsin is a proteolytic enzyme obtained by the activation of trypsinogen extracted from the pancreas of healthy mammals. It is a member of mammalian “serine” protease family ⁴.

It hydrolyses protein hence it is generally used to digest proteins into peptides⁵. It improves humoral response & and prevents growth of pathogens by removing dead and necrotic tissue. It inhibits the C-reactive protein titres and prevents increase in serum acute phase proteins⁶. Thus, it has anti-inflammatory activity.

Bromelain:

Bromelain belongs to a group of protein digesting enzymes obtained commercially from the fruit or stem of pineapple⁷.  “Bromelain” refers usually to the “stem bromelain” and is a member of cysteine proteinase family⁸. Bromelain exhibits various fibrinolytic, antiedematous, antithrombotic, and anti-inflammatory activities.

Bromelain is considerably absorbable in the body without losing its proteolytic activity and without producing any major side effects. Preferential cleavage site is the carbonyl end of lysine, alanine, tyrosine and glycine. So far, a number of analytical techniques have been described for determination of rutin, trypsin and bromelain. However, no method is described for estimation of rutin with both proteolytic enzymes in any dosage form.

This study describes accurate and precise methods for determination of trypsin, bromelain and rutoside trihydrate in oral dosage forms. The method has been validated with respect to linearity range, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy¹⁰. The proposed methods have been applied to the analysis of all the three drugs in commercially available pharmaceutical preparations Enzomac (Trypsin 48mg, Bromelain 90 mg, Rutoside Trihydate 100mg.)

MATERIAL AND METHODS:

Apparatus:

A Shimadzu model 1700 double beam UV-Visible spectrophotometer connected with computer and also with spectral width of 1 nm, wavelength accuracy of ± 0.1 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software (Ver.2.34). The samples were weighed on electronic analytical balance (A×120, shimadzu). pH meter  (Lab india), Sonicator (SelecXT543), Centrifuge (REMI).

Reagents and chemicals:

Rutin, Trypsin, Bromelain, Folin-Ciocalteu reagent, Coomassie brilliant blue G-250, Trichloroacetic acid, Casein, Tris base Buffer, Bovine serum albumin, p-nitroaniline, DMSO, Phosphate buffer standards were purchased from Loba Chemie. BApNA and TLCK were purchased from Sigma Aldrich. Methanol (AR grade) was purchased from Rankem. Bromelain was obtained as a gift sample from Meteoric Life Sciences, Ahmedabad. Tablet formulation (Enzomac) was purchased from local market. All the chemicals used were of analytical grade.

Preparation of Reagents:

Bradford reagent- Bradford reagent was made by dissolving 10 mg Coomassie Blue G-250 in 5 ml 95% methanol, 10 ml 85% (w/v) phosphoric acid was added to this solution and the mixture was diluted to 100 ml with water. CuSO₄ mixture- Equivalent volume of 0.1M NaOH + 2% w/w Na₂CO₃ + 0.5% sodium potassium tartarate + 0.5% sodium dodecyl sulphate was added to 1% w/w CuSO₄.5H₂O as per 10:1 ratio. Folin-ciocalteu reagent- 1N F-C reagent was prepared by diluting 2N solution with distill water as per 1:1 ratio. BApNA solution- 5mg/ml BApNA solution was prepared by first dissolving BApNA in DMSO and making up the volume with tris buffer.

General Procedures:

Procedure for analysis of Rutin:

The stock standard solution of rutin was prepared by dissolving 10 mg of rutin in 100ml Methanol to get a solution containing 100 μg/ml of rutin¹⁰. Series of dilutions were prepared by aliquoting 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0ml of the standard stock solution and diluted with methanol to yield 10mL of standard solutions containing 5 to 50 μg/ml of rutin, respectively. The absorbance of resulting solution was measured at its λmax. A calibration curve as concentration vs. absorbance was constructed to study the Beer-Lambert’s Law and regression equation.

Method for analysis of bromelain and trypsin (Total proteolytic activity):

Casein was used as a substrate for analysis of total proteolytic activity of Bromelain and Trypsin and was standardized according to Bradford assay ¹¹.

Bradford Assay:

Procedure:

A 100ppm standard solution of BSA was prepared by dissolving 10 mg BSA in 100ml 0.2M phosphate buffer pH 7.5. Series of dilutions were prepared in the same buffer to get concentrations ranging from 10 to 50μg/ml respectively.

FIGURE2: CALIBRATION CURVE OF STANDARD BSA SOLUTIONS

2 ml of each diluted solution was taken and 8 ml Bradford reagent was added. Samples were incubated at room temperature for 5 min. Absorbance was measured at 585nm using UV-Vis spectrophotometer. The concentration of protein was determined using y=mx+b equation. Casein solution, 50μg/ml was prepared in Phosphate buffer and procedure was carried out in a similar manner Figure 2.

 Procedure for analysis of Trypsin and Bromelain (Determination of total proteolytic activity):

5ml of casein solution (6.5mg/ml prepared in 0.2M Phosphate buffer) was taken and incubated at 37ᵒC for 5 minutes. 1ml of freshly prepared enzyme solution in 10mM Na acetate solution was added & kept at 37ᵒC for 10min. Reaction was inhibited by addition of 4ml of 1M trichloroacetic acid and kept for 30min at room temperature. The solution was centrifuged at 2000 rpm for 7 minutes¹².

The supernatant was collected and was neutralized by 0.1N NaOH. Tyrosine which was formed due to hydrolysis was analyzed further in following manner. 1 ml of each dilution containing tyrosine was taken. To this 5 ml of CuSO₄ mixture was added and shaken well. This solution was kept at room temperature for 10 min. 0.5 ml of diluted F.C reagent (1:1) was added and solutions were shaken. All the samples were kept at room temperature for 30 minutes and absorbance was measured within 45 minutes at 785nm. A calibration curve as concentration vs. absorbance was constructed using standard tyrosine solutions to study the Beer-Lambert’s Law and regression equation.

Determination of specific proteolytic activity of Bromelain:

Specific proteolytic activity was determined by inhibiting Trypsin by TLCK¹³. 5mg/ml of TLCK solution was prepared in distill water. 5ml of this solution was added to 500μg/ml of total proteolytic enzyme concentration and incubated at 37ᵒC for 10 min¹⁴. Casein was added after 10 min. and further procedure was carried out in a similar manner for hydrolysis of casein.

METHOD FOR ANALYSIS OF SPECIFIC PROTEOLYTIC ACTIVITY OF TRYPSIN:

Analysis of trysin by hydrolysis of substrate BApNA:

0.6ml of enzyme solution prepared in distill water was taken and 3ml of 50mM tris buffer with 0.6 ml of distill water was added¹⁵. Solution was shaken well. 0.6 ml of freshly prepared BApNA solution was added and incubated at 35-40ᵒc for 30 min. Reaction was stopped by adding 2ml of 3N HCL. BApNA was hydrolyzed to pNA which was analyzed in following manner. 1ml of each diluted solution containing pNA was taken and 4ml of 0.1% sodium nitrite solution was added. This was kept in icebath for 10 min.

Then 4ml of 0.5% ammonium sulfamate was added, shaken well and again kept in icebath for 5 min. Finally, 1ml of 1% NEDD dye was added and the solution was kept at room temperature for 15 min. The absorbance of resulting solution was measured at its analytical wavelength 545nm. A calibration curve as concentration vs. absorbance was constructed using standard pNA solutions to study the Beer-Lambert’s Law and regression equation.

Kinetic studies of Trypsin:

Kinetic studies were carried out for trypsin enzyme to determine Km and Vmax value^{17, 18}. 6 appropriate concentrations 0.001, 0.003, 0.01, 0.03, 0.05, 0.06 mM were used. The Michaelis-Menten constant Km and Vmax (moles product/mg protein/minute) were evaluated by non-linear regression analysis using the graphpad prism5 software package.  Kcal was calculated from equation¹⁹ Figure 3, 4 and Table1.

TABLE 1: SUMMARY OF KINETIC PARAMETERS

FIGURE 3: MICHAELIS MENTEN PLOT OF TRYPSIN

FIGURE 4: LINEWEAVER-BURK PLOT OF TRYPSIN

Optimization of Parameters:

Contour graphs were plotted for optimization of parameters with the help of Design Expert software version 7.0.0 by considering 3 variables temperature, time and concentration for enzyme trypsin. Optimization was carried out by calculating average velocity (abs/min) Figure 5 and Table 2.

TABLE 2: SUMMARY OF OPTIMIZATION OF PARAMETERS

FIGURE 5: CONTOUR GRAPHS FOR OPTIMIZED PARAMETERS OF TRYPSIN

Analysis of Marketed formulation:

Validity of the proposed methods was tested for pharmaceutical preparation by assaying Enzomac tablets (labeled to contaion 100mg of rutoside trihydrate and proteolytic activity of Bromelain and Trypsin not less than 1095 FIP units).

VALIDATION OF DEVELOPED METHODS:

Validation of all the methods was carried out according to ICH guidelines Q2B

Accuracy:

Forstudyingtheaccuracyoftheproposedmethods,andforcheckingtheinterferencefrom excipientsusedinthedosageforms,recoveryexperimentswerecarriedoutbythestandard addition method. This study was evaluated through the percentage of recovery of known amounts of rutin, bromelain and trypsin added to solutions of the commercial product. The analyzed samples were spiked with extra 80, 100 and 120 % of standard rutin, Bromelain and trypsin solution. Accuracy was calculated from the following equation: [(spiked concentration−mean concentration)/ spiked concentration] ×100 Table3.

TABLE 3: RESULT OF RECOVERY STUDY OF RUTIN, BROMELAIN AND TRYPSIN BY DEVELOPED METHODS

*mean value ofthree determination

Precision:

Intra-dayprecisionandinter-dayprecisionforthedevelopedmethodsweremeasuredinterms of%R.S.D.Theexperimentswererepeatedthreetimesadayforintra-dayprecisionand on3 differentdaysforinter-dayprecision.Theconcentrationvaluesforbothintra-day precisionandinter-day precision werecalculatedthree times separately and% R.S.D. were calculated Table 4.

Linearity:

For Estimation of Rutin:

For rutin, appropriate dilutions of standard solutions were assayed as per the developed method. The Beer- Lambert’s concentration range was found to be 5-50 μg/ml for rutin Table 4.

For estimation of Bromelain:

Casein was hydrolysed to tyrosine, the BEER-Lambert’s concentration range for tyrosine was found to be 20-70μg/ml Table 4.

For estimation of Trypsin:

BApNA was hydrolysed to pNA, the Beer-Lambert’s concentration range for pNA was found to be 5-30 μg/ml Table 4.

LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ):

Limitofdetection(LOD)andlimitofquantitation(LOQ)werecalculatedaccordingtothe3s/m and10s/mcriterions,respectively,wheresisthestandarddeviationofintercept(n=6)ofthe sample andmis the slope ofthe corresponding calibration curve Table 4.

TABLE 4: SUMMARY OF VALIDATION PARAMETERS BY DEVELOPED METHODS

  RESULTS AND DISCUSSIONS:

Analysis of Bromelain and Trypsin:

Bromelain and Trypsin hydrolyzes casein into small peptides at alkaline pH so pH was kept above 7. This tyrosine formed cannot be precipitated with a specific reagent. Therefore, after incubation (10 min at 35 °C) undigested casein which can be precipitated is removed by acidifying the solution with trichloroacetic acid and the amount of peptides remaining in solution was than reacted with F-C reagent in presence of copper sulphate which is then  determined by UV-Vis spectrophotometer at 785nm.

Analysis of Bromelain:

Trysin was inhibited by specific trypsin inhibitor TLCK as it inhibiths serine which is end amino acid of trypsin. Bromelain is unaffected by TLCK so bromelain remaining in the solution is determined by hydrolysis of casein.

Analysis of Trypsin:

Trypsin hydrolyses BApNA to pNA at alkaline pH. Arginine is not a preferential cleavage site for Bromelain so it doesn’t hydrolyze BApNA. The reaction is inhibited by acididfying the solution by addition of 3N HCL. pNA formed is the diazotized by NEDD to give a colored product which is then determined by UV-Vis spectrophotometer at 545nm Figure 6.

FIGURE 6: REACTION MECHANISM FOR HYDROLYSIS OF BApNA BY TRYPSIN

Optimization parameters of Trypsin:

Trypsin reaction with BapNA was optimized by determining appropriate temperature, time and concentration with the help of contour plots. They indicate that with increasing temperature and substrate concentration average velocity increases as showed by the darkened area. The optimized temperature, time and substrate concentration was found to be 0.03mM, 30min and 35ᵒc respectively.

Determination of Proteolytic activity:

Proteolytic activity was determined in terms of FIP units. 1 FIP unit of enzyme is the amount of enzyme that hydrolyzes protein under the standard conditions into not acid-precipitable peptides at an initial rate such that there is liberated per minute an amount of peptides which gives the same absorbance as 1 °mole of that peptide.

1Ph.Eur. Unit = 1 BP Unit = 1 FIP Unit ∼ 62.5 USP Units

1FIP unit = 1 U/g

For Bromelain:

5.0 FIP units/mg ≈ 2500 GDU/g ≈ 1560 CDU/mg,

On the basis of these conversions FIP units in tablet was calculated.

Validationwas carried out according to ICHguideline. Table3 and Table 4 exhibits resultsofmarketed formulation,summaryofvariousvalidationparameters andresults ofaccuracy studies for all methodsrespectively.

Results of Marketed formulations:

The content of Rutin based on mean value of three determinations was found out to be 100.61± 0.485% w/w. Total proteolytic activity was found to be 1124 FIP units. Specific proteolytic activity of Bromelain was 1028 FIP units while that of Trypsin was found out to be 96 FIP units.

CONCLUSIONS: The proposed methods are simple, accurate, rapid and selective for routine analysis of Rutin, Bromelain and Trypsin in tablet formulations. The methods are more selective than reported spectrophotometric methods and are free from interferences from the common excipients. The statistical parameters and the recovery data reveal good accuracy and precision of the methods. The developed methods can be used as general methods for determination of proteolytic activity of proteolytic enzyme in any dosage form or formulation.

 REFERENCES:

1. Kuntic V, Pejic N, Ivkovic B, Vujic Z, Ilic K, Micic S, Vukojevic V: Isocratic RP-HPLC method for rutin determination in solid oral dosage forms.Journal of Pharmaceutical and Biomedical Analysis 2007; 43: 718–21.
2. Wang CH, Wang YX, Liu HJ: Validation and application by HPLC for simultaneous determination of vitexin-o-glucoside, vitexin-o-rhamnoside, rutin, vitexin and hyperoside. Journal of Phrmaceutical Analysis 2011; 1(4): 291-96.

3. Araujo KC, Costa EM, Valadares MC: Bioconversion of quercetin and rutin and the cytotoxicity activities of the transformed products. Food and Chemical Toxicology 2013; 51: 93-6
4. Fernandez M, Fragoso A, Cao R, Banos M, Schumacherc M, Hartmeierc W, Villalonga R: Functional properties and application in peptide synthesis of trypsin modified with cyclodextrin-containing dicarboxylic acids. Journal of Molecular Catalysis B: Enzymatic 2004; 31: 47–52.
5. Andrew MJ, Stewart EJ, Take ZS, Doucette AA: Critical assessment of the spectroscopic activity assay for monitoring trypsin activity in organic-aqueous solvent. Analytical Biochemistry 2013; 435: 131–36.
6. Avanzo P, Sabotic J, Anzlovar S, Popovic T, Leonardi A, Pain RH, Kos J, Brzin J: Trypsin-specific inhibitors from the basidiomycete clitocybe nebularis with regulatory and defensive functions. Microbiology 2009; 15: 3971–8.
7. Gautam SS, Mishra SK, Dash V, Goyal AK, Rath G: Comparative study of extraction, purification and estimation of bromelain from stem and fruit of pineapple plant. Thai J. Pharm. Sci 2010; 34: 67-76.
8. Kalaiselvi M, Gomathi D, Uma C: Occurrence of Bioactive compoyunds in Ananus comosus(L) Standardization by HPTLC. Asian Pacific Journal of Tropical biomedicine 2012; S: 1341-6.
9. International Conference on Harmonization, Validation of analytical procedures: Text and methodology. ICH Harmonized Tripartite Guidelines Q2 (R1), November 2005.
10. Hassan HN, Barsoum BN, Habib IH: Simultaneous spectrophotometric determination of rutin, quercetin and ascorbic acid in drugs using a Kalman Filter approach. Journal of Pharmaceutical and Biomedical Analysis 1999; 20: 315-20.
11. Bradford D, MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemestry 1976; 722: 248-54.
12. Corzo CA, Waliszewski KN, Welti-Chanes J: Pineapple fruit bromelain affinity to different protein substrates. Food Chemistry 2012; 133: 631–35.
13. Hosseininaveh V, Bandani A, Hosseininaveh F: Digestive proteolytic activity in Sunn Pest Eurygaster Integriceps. Journal of Insect Science 2009; 9(70):
    1-11.
14. Perera E, Casariego JR, Guez-Viera LR, Calero J, Morales RP, Mancera JM: Lobster (Panulirus argus) Hepatopancreatic trypsin isoforms and their digestion efficiency. Biol. Bull 2012; 222: 158-70.
15. Lin ZY, Yu ZH, Chen C, Dong QX, Wang MZ, Yu YQ: An HPLC method for the assay of trypsin inhibitors and its applications to the study of Momordica cochinchinensis extract. Journal of chinese Pharmaceutical Sciences 2011; 20: 397–403.
16. Jeffery GH, Bassett J, Mendham J, Denney RC: Textbook of Quantitative inorganic analysis. Vogel’s, Edition 7: 693-98.
17. Hau PV, Benjakul S: Purification and characterization of trypsin from pyloric caeca of Bigeye Snapper (Pricanthus Macracanthus). Journal of Food Biochemistry 2006; 30: 478-95.
18. Bosnic OM, Gopcevic KR, Vrvic MM, Karadzic IM: Inhibition of trypsin by heparin and dalteparin, a low molecular weight heparin. Journal of the Serbian Chemical Society 2009; 74(4): 379–88.
19. Sharifi M, Chitgar MG, Ghadamyari M, Ajamhasani M: Identification and characterization of midgut digestive proteases from the rosaceous branch borer, osphranteria coerulescens redtenbacher (coleoptera: cerambycidae). Rom. J. BIOCHEM 2012; 49(1): 33–47.
    

    ---

    How to cite this article:

    Wani SS and Mashru RC:Sensitive and Selective Methods for Determination of Proteolytic Activity of Formulation Containing Bromelain and Trypsin as Proteolytic Enzymes. Int J Pharm Sci Res2014; 5(11): 4838-45.doi: 10.13040/IJPSR.0975-8232.5 (11).4838-45.

    ---

     

    All © 2014 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---