Vijaya Saradhi Settaluri et al. / Journal of Pharmacy Research 2012,5(3),1380-1382 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Novel spectroscopic methods for estimation of Using HRP

Vijaya Saradhi Settaluri1*, Douglas Daniel kumar1 and Naga Koumudi Vankamamidi1 Department of Biotechnology, K L University, Greeenfields, Vaddeswaram-522502,Andhra Pradesh, India Received on:10-12-2011; Revised on: 15-01-2012; Accepted on:12-02-2012

ABSTRACT Tannic acid is chemically 3, 5-dihydroxy-2-(3, 4, 5-trihydroxybenzoyl). Tannic acid has numerous food and pharmacological applications. It is an additive in medicinal products, is used as a flavoring agent and as an anti-oxidant in various foods and beverages. A thorough literature survey revealed only very few analytical methods like high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) for estimation of tannic acid. However designing a reaction with the application of an enzyme was rare to find. In the present study, two simple, sensitive and cost effective bioanalytical methods are proposed for routine determination of tannic acid. These are based on the formation of colored species on reaction of tannic acid with n- Methyl benzo thiazolone hydrazone (MBTH )and ferric chloride to produce green colored chromogen (lmax at 500 nm) in the presence of enzyme horseradish peroxidase (HRP) for method A and 1,10 phenanthroline , ferric chloride and ortho phosphoric acid coupled with the enzyme horseradish peroxidase to produce an orange colored chromogen (l max at 510 nm) for method B. Results of analysis were validated statistically and the relative standard deviation (RSD) of the proposed methods were 1.89 for method A and 1.162 for method B. Based on the RSD values obtained the method is highly reproducible and could be effectively employed for the determination of tannic acid and extended for recovery studies of tannic acid from various biological samples.

Key words: Tannic acid, HRP, Spectrophotometer, Molar absorptivity, Beer’s law.

INTRODUCTION Tannic acid Tannic acid is chemically called [3, 5-dihydroxy2-[3, 4, 5-tri hydroxyl ben- Biomedical applications of Tannic acid zoyl]. Tannic acid is a specific commercial form of , a type of polyphe- are considered as one of the basic ingredients in the chemical stain nol. It’s weak acidity is due to numerous phenol groups in the structure ing of wood [3]. The presence of tannins in the bark of red wood (sequoia) is .The chemical formula for commercial tannic acid is often given as a strong natural defense against wild life, decomposition and infestation by [1] certain insects such as termites[4] .Tannic acid is a common mordant used in C76H52O46 , which corresponds with decaalloyl , but in fact it is a mixture of polygalloyl glucose or polyalloyl quinic acid esters with the the dyeing process for cellulosic fibers such as cotton ,often combine with number of galloyl moieties per molecule ranging from 2 up to 12 depending alum or iron[5] It is also an alternative for Fluoro carbon after treatments to on the plant source used to extract the tannic acid , commercial tannic acid impart anti-staining properties to polyamide yarn or carpets .A particular is usually extracted from any of the mentioned plant parts like Tara and unique niche textile application of tannic acid is the activation of flock pods(cesalpinia spinosa), gallnuts from rhus semialata or quercus infectoria ,which is basically an anti static after treatment[6]. Tannic acid is used in the or Sicilian sumac leaves, etc.., it’s a yellow to light brown color amorphous conservation of ferrous based metal objects to passivate and inhibit corro- powder which is highly soluble in water sion[7] . Tannic acid is used in commercially iron or steel corrosion treat ments such as Hammerite Kurust.

Tanic acid as food source Use of tannic acid in food applications is far more widespread and signifi- cant amounts are use as process aids in beer clarification, aroma compounds in soft drinks and juices.

Tannic acid in medication Tannic acid was once used as a treatment for many toxic substances [8] such as strychnine, mushroom and ptomaine poisonings in the late 19th and early 20th centuries. Today tannic acid is still used in pharmaceutical applications to produce albumin which is used as anti-diarrhea agent. Tannic acid is also used to produce a tannate salts of lextain anti-histamins and anti- tunives to impart increased stability (or) slow release properties to the Fig 1: Structure of Tannic Acid active pharmaceutical ingredient (API) soaking feet in tannic acid can treat Distribution [9] Amla, Chest nut, Pomegranate, Kusburnu, Neetle, Wall nuts, Wood berries, (or) prevent blisters, foot odor an rough (or) dry feet . It has anti-bacterial, anti-enzymatic and astringent properties. It has constringing action on Chinese galls, Red tea. Oak wood is very rich in tannic acid. High levels of mucous tissues such as tongue and inside of mouth. The ingestion of tannic tannic acids are found in some plant galls .These is formed by plants when [2] acid causes constipation and can be used to treat diarrhea. Externally tannic they are infected by certain insects . acid is used to treat ulcers, toothache and wounds.

*Corresponding author. HORSE RADISH PEROXIDASE Dr. S. Vijaya Settaluri The enzyme Horseradish peroxidase (HRP) found in edible vegetable horse Department of Biotechnology, radish is used extensively in biochemistry applications primarily for its K L University, Greeenfields, ability to amplify a weak signal and increase detects ability of a target Vaddeswaram-522502, molecule. Horse radish peroxidase is a 44,173.9 Dalton glycoprotein with 4 Andhra Pradesh, India lysine residues [10]

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1380-1382 Vijaya Saradhi Settaluri et al. / Journal of Pharmacy Research 2012,5(3),1380-1382 The sliced pieces were homogenized in 200 ml of buffer solution in a blender at high speed for 15 minutes .the extract is clarified by centrifuga- tion (10-15000 rpm/ 10min) and filtered through whattman no.1 filter pa- per [12]. The extract for stability was stored in toluene for at least a week at 40C . The extract was suitably diluted for further experimental analysis.

PREPARATION OF REAGENTS All the chemicals used were of analytical grade .All solutions were freshly prepared with millipore double distilled water and always used for analy- sis. Following aqueous solutions were used.

METHOD A: MBTH (0.2% W/V), Fecl3 (0.7% W/V), HCl (0.1N)

METHOD B: 1, 10 Phenanthroline (0.01M), Fecl3 (0.003M), Ortho phos- phoric acid (0.2M)

Assay procedure

Fig 2 Three dimensional structure of HRP METHOD A: Into a series of 25 ml calibrated tubes; enzyme solution

(0.5ml W/V), FeCl3, MBTH, HCL (1ml) were taken. These tubes were APPLICATIONS incubated for 10 minutes for color development. Aliquots of 0.4-2.0 ml of It produces a colored, flourometric or luminescent derivative of a labeled standard tannic acid solution were transferred to the incubate solution and molecule, allowing it to be detected and quantified [11]. HRP is often used as the absorbance of green colored chromogen were measured at 500 nm against conjugates to determine the presence of a molecular target. Horse radish the reagent blank. peroxidase is also commonly used in techniques such as ELISA and immune histo chemistry .In recent years the technique of marking neurons with the METHOD B: Into a series of 25 ml calibrated tubes; 0.5 ml of HRP en- enzyme HRP has become a major tool. zyme, FeCl3, 1, 10 phenanthroline (1ml) each were taken. These tubes were kept in a hot water bath for 15 minutes at 1000C and then cooled to EXPERIMENTAL room temperature .to develop initial color orthophosphoric acid solution After due calibration of the instruments, spectral and absorbance measure- (2ml) was added to each tube .aliquots of 0.4-2.0 ml of standard tannic acid ments are made using ELICO UV-Visible spectrophotometer model SL- solution were transferred to the resulting solution and the absorbance of 159, Mumbai, India. All the chemicals used were of analytical grade. All the orange colored complex were measured at 510nm against reagent blank. solutions were freshly prepared using double distilled water. Freshly pre- pared solutions were used for analysis. In the proposed methods aqueous RESULTS AND DISCUSSIONS solutions of MBTH, ferric chloride(0.7% w/v)and 0.1 N HCl were used for The results of analysis for method A and B were validated through system- method A and 1,10 phenanthroline ,ferric chloride ,ortho phosphoric acid atic statistical analysis and results are tabulated .The statistical analysis were used for method B. values are reported in Table -1 and assay and recovery results for this methods are tabulated in Table-2. STANDARD AND SAMPLE SOLUTION OF TANNIC ACID: About 100mg of tannic acid was accurately weighed on a single pan balance METHOD A: The proposed method is based on the mechanism of oxida- and dissolved in 100 ml of double distilled water in a volumetric flask to tion followed by complex formation, where in the initial reaction the anti- prepare a standard solution with concentration equal to1mg/ml, and further oxidant undergoes oxidation in the presence of ferric chloride and then the dilutions were made to obtain a solution of 0.1 mg/ml for method A and oxidized anti-oxidant reacts with MBTH to form a green colored complex method B. which exhibits maximum absorption at wavelength of 500 nm. The mecha- nism of the reaction is represented in scheme-1 EXTRACTION OF CRUDE ENZYME: One Radish weighing 40g was peeled, washed and cut into 1 inch cubes.

Fig 3: Mechanism of reaction between Tannic acid and MBTH

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1380-1382 Vijaya Saradhi Settaluri et al. / Journal of Pharmacy Research 2012,5(3),1380-1382

METHOD B: The proposed method is based on the mechanism of oxida- CONCLUSION tion in the presence of ferric chloride where the anti-oxidant gets oxidized Performance recovery experiments and percent recovery values obtained and the oxidized Anti- oxidant reacts with 1, 10 phenanthroline to form an indicated the absence of interference from the commonly encountered addi- orange colored complex which exhibits maximum absorption at wavelength tives and excipients, thus the proposed methods A and B for determination 510nm. of tannic acid are reproducible, simple and sensitive with reasonable preci- sions and accuracy and can be suitably adapted for the determination of tannic acid in quality control analysis.

ACKNOWLEDGEMENTS: The authors are grateful to the management of Koneru Lakshmaiah Univer- sity, Vaddeswaram, Guntur district, for their continuous support and en- couragement and for providing the necessary facilities (infrastructural and chemicals) for carrying out this work.

REFERENCES: 1. Veronika Kozlovskaya, Eugenia Kharlampieva, Irina Drachuk, Derek Cheng and Vladimir V. Tsukruk* Responsive microcapsule reactors based on hydrogen-bonded tannic acid layer-by-layer as- semblies, Int. J. Electrochem. Sci., 5 (2010) 1236 – 1245 2. Banerjee D, Mona KC, and Pati R, production and characterization of extracellular and intracellular tannase from newly isolated As- pergillus aculeatus ,DBF 9.J.Basic Microbial, 41 (2001) 313-318 Fig 4: Mechanism of reaction between Tannic acid and 1, 10 Phenanthroline 3. Banerjee R, Mukherjee G, Patra K, Microbial transformation of tannin-rich substrate to through co-culture method, For these methods optical characteristics such as absorption maxima, Beer’s BioresourTechnol, 96 (2005), : 949-953. law limits, molar absorptivities, regression analysis using the method of 4. Haïdopoulos M, Turgeon S, Sarra-Bournet C, Laroche G, Mantovani least squares, slopes (a), intercept (b) and correlation coefficients (r) ob- , Development of an optimized electrochemical process for subse- tained from different concentrations are summarized in Table-1.The preci- quent coating of 316 stainless steel for stent applications, J. Mater. sion and accuracy were found by analyzing five replicate samples contain- Sci. – Mater. Med, 17, (2006), 647-57. ing known amounts of the anti-oxidant and the results summarized in Table- 5. F Lewis, P Horny , P Hale , S Turgeon , M Tatoulian and D 1. The accuracy of these methods in the case of formulations were thor- Mantovani Study of the adhesion of thin plasma fluorocarbon coat- oughly studied by recovery experiments and the results were presented in ings resisting plastic deformation for stent applications. Journal of Table-2.Additional checks on the accuracy of these methods were analyzed Phys, 41 (2008), PP 4. by adding known amounts of pure anti –oxidant to pre analyzed formula- 6. Hale P, Turgeon S, Horny P, Lewis F, Brack N, Van Riessen G, tions. Pigram P, Mantovani D. X-ray photoelectron emission micros- copy and time-of-flight secondary ion mass spectroscopy analysis Table-1: Optical characteristics, precision and accuracy of tannic acid. of ultrathin fluoropolymer coatings for stent applications. Langmuir, Parameter M1 M2 24(15), (2008), 7897-905. 7. M. Touzin, P. Chevallier, F. Lewis, S. Turgeon, S. Holvoet, Name of the method MBTH, ferric chloride 1,10, Phenanthroline Ferric chloride G. Laroche, D. MantovanI. Study on the stability of plasma-poly- merized fluorocarbon ultrathin coatings on stainless steel in water, l max 500nm 510nm Beers law limits (UG/ml) 0.4-2.0 0.4-2.0 Science direct, 202, (2008), 4884. Molars absorptivity (1mol1cm-1) 4.02×104 7.09×104 8. Bigat, T.K., and Saifer, A., some methodological modifications of Sendels sensitivity 0.042 0.023 (UG/cm2/0.001 absorbance Unit) the technicon “SMA 12-60 Auto Analyzer” system. Clinical Chem- Regression equation (Y=a+bc) istry18 (1972), 630. Slope (b) 0.00219 0.0213 9. Cox, L., & Cox, J. Ecobeauty: Scrubs, rubs, masks, and bath bombs Intercept (a) -0.0062 -2.027 for you and your friends. Keley Ber [Calif.]: Ten Speed Press, (2009). Correlation coefficient(r) 0.998 0.9998 Standard deviation 5.3×103 1.5×103 219. Relative standard deviation 1.89 1.16 10. Veitch, Nigel C., Horseradish peroxidase: a modern view of a classic % range of error(confidence enzyme, Photochemistry, 65, (2010), 249-259. Limits)0.05 level ±1.5803 ±0.9699 11. Condeelis, J. How is actin polymerization nucleated in vivo, Trends 0.01 level ±2.3380 ±1.4350 Cell Biol, 11, (2001), 288 - 93. Table-2. Assay and recovery of tannic acid. 12. P. Pant, V. Saradhi. S, S.Felice, B. Gurung, V. Divya G, N.Rao M Formulations Labeled amount Amount found *(%) Recovery by Developed ,Spectrophotometric Determination of Acyclovir through Oxida- ( mg/vial) (mg/vial) method tive Coupling of with 2,2 Bipyridine by Horseradish Peroxidase Method-A Method-B Method –A Method-B (HRP) ,journal of applied chemical research, 10 ,(2009) , 7-12. sample -1 50 µg 48 45 92.00 90.01 sample-2 70 µg 68 65 97.14 92.85 sample -3 90 µg 88 85 97.71 94.40 Source of support: Nil, Conflict of interest: None Declared *Recovery amount is the average of five determinations

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1380-1382