WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Patil et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 12, 948-956. Research Article ISSN 2278 – 4357

DEVELOPMENT AND VALIDATION OF UV SPECTROMETRIC

METHOD FOR SIMULTANEOUS ESTIMATION OF GALLIC ACID
AND PIPERINE IN HERBAL FORMULATION

Smita G. Patil*, Varsha M. Jadhav, Vilasrao J. Kadam

Mumbai University, Department of Quality Assurance, Bharati Vidyapeeth's College of Pharmacy,

Navi Mumbai, 400614, Maharashtra, India.

Article Received on ABSTRACT

26 September 2014,
Revised on 19 October 2014,
Accepted on 11 November
2014
*Correspondence for
Author
Smita G. Patil
Mumbai University,
Department Of Quality
Assurance, Bharati
Vidyapeeth's College Of
Pharmacy, Navi Mumbai,
400614, Maharashtra, India
A simple, rapid and accurate spectrometric method based on solving
simultaneous equation for simultaneous estimation of Gallic acid and
piperine was developed. Gallic acid and piperine showed maximum
absorbance at 273 and 343 nm respectively, absorbance was measured
at these wavelengths for estimation of gallic acid and piperine
respectively. Gallic acid and piperine both obeys beer Lambert's law in
the concentration range of 2-12µg/ml. method was developed and
validated as per ICH guidelines and can be applied for the routine
estimation of the gallic acid and piperine in herbal formulation.
KEYWORDS: Gallic acid, Piperine, Simultaneous equation, UV
spectrophotometer, Validation.

INTRODUCTION
Large number of herbal formulations are used for the treatment of various diseases.
Ayurvedic herbal formulations are generally available as single or mixture of more than one
plant constituents and it is necessary to quantify maximum number of markers present in
such formulation to achieve high quality for the product. Sanjeevani vati is official in
ayurvedic formulary of india. It is a polyherbal formulation containing ten ingredients i.e.
Amla (Emblica officinalis), bhallataka (Semicarpus anacardium) bibhitaka (Terminalia
bellerica), gudduci (Tinosporia coridifolia), krsna (Piper longum), nagara (Zingiber
officinalis), pathya (Terminalia chebula), vaca (Acrous calamus) Vidang (Embeliaribs), visa

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Patil et al. World Journal of Pharmacy and Pharmaceutical Sciences

(aconitum heterophylum). [1] This formulation is traditionally prescribed for the treatment of
fever, common cold, indigestion, stomach ache, worm infestation, etc. [2]

Gallic cid is chemically known as 3,4,5-trihydroxybenzoic acid (C6H2(OH)3COOH), mostly

found in gallnuts, sumac, witch hazel, grapes, oak bark tea leaves and hops. Gallic acid exist
in two forms as the free molecule and as part of tannins. Pure gallic acid is colourless
crystalline powder, salts and esters of gallic acid are termed as gallates. Gallic acid has
promising antioxidant activity due to which it is added to skin care products in the form of
natural herbal extracts. Gallic acid is commonly used in pharmaceutical industries. Gallic
acid shows cytotoxic activity against cancer cells, without damaging healthy cells and it can
be used to treat albuminuria and diabetes, it also has antifungal and antiviral properties,
which is used ad antioxidant and helps to protect the human cells against oxidative damage. It
is also used as standard substance in many antioxidant assays. It has also shown to possess
radical scavenging activity against several radicals. All these biological activities have
indicated the potential use of gallic acid. [3,4]

Piperine (Figure 2) is Chemically 1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]

[5,6]
piperidine and it is official in IP. It is major alkaloid component of Piper nigrum and
Piper longum which belongs to the family piperaceae. Pharmacological studies on piperine
have showed that this compound have many pharmacological activities such as analgesic,
[7]
antipyretic, hepatoprotective, bioavailability enhancer, anti-inflammatory. The piper
species have been also used in traditional medicine for intermittent fever and to promote
secretion of bile. They are also recommended for neurological bronco pulmonary and
gastrointestinal disorders. [8]

Figure 1: Structure of Gallic acid Figure 2: Structure of Piperine

[9]
Literature revels that several methods such as HPLC , HPTLC[10] for estimation of Gallic
[11,12]
acid are reported. Also UV , HPLC[13], HPTLC[7] methods are reported for

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Patil et al. World Journal of Pharmacy and Pharmaceutical Sciences

determination of piperine. No single UV or HPLC method is reported for simultaneous

estimation of gallic acid and piperine in herbal formulation.

MATERIAL AND METHODS

Apparatus: A UV visible spectrophotometer (Shimadzu), Digital balance, Ultra sonicator,
volumetric flask and pipettes.

Reagents and chemicals: Standard piperine was purchased from Sigma Aldrich And Gallic
acid was purchased from Research-Lab Fine Chem. industries. All other chemicals Used for
the study were of analytical grade.

Procedures
Preparation of stock solution and calibration curve: The standard stock solution of gallic
acid and piperine was prepared by dissolving 10mg of each drug in methanol, and the final
volume was adjusted with the same solvent in 10ml volumetric flask to get solution
containing 1000µg/ml of each drug. This stock solution was used to prepare working
standard solution of 10µg/ml of gallic acid and 10µg/ml of piperine. Both solutions were
scanned in entire UV range of 400-200 nm to determine λmax . Calibration curve as conc. vs.
absorbance were constructed to study Beer-Lambert's law and regression equations for Gallic
acid and piperine.

Simultaneous equation method: From the overlay spectra (Fig.3) of gallic acid (10µg/ml)
and piperine (10µg/ml), two wavelengths were selected as working wavelengths i.e. 273nm
as λmax for gallic acid and 343 as λ max for piperine, at which both the drugs showed
absorbance for each other. The absorbtivities of gallic acid and piperine was determined at
273 nm and 343 nm respectively. The concentration of two drugs in sample was calculated
using set of two simultaneous equations. [15]

..........(1)

...........(2)

Where Cx And Cy are concentrations of gallic acid and piperine in µg/ml respectively in
known sample solutions. A1 and A 2 Are absorbance's of sample solutions at 273 and 343 nm

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Patil et al. World Journal of Pharmacy and Pharmaceutical Sciences

respectively.ax1 and ax2 are absorptivity of gallic acid at 273 and 343 nm ,ay1 and ay2 are
absorptivity of piperine at 273 and 343 nm.

The concentration of Cx and Cy in herbal formulation can be obtained by solving equation

(1) and (2) respectively. Validity of above framed equation was checked by using mixture of
standards of pure drug sample of two drugs, measuring their absorbance at respective
wavelength and calculating concentration of two components using framed equation.

Analysis of herbal formulation: Accurately weighed quantity of Powdered herbal tablet

(100mg), was transferred in volumetric flask (10ml) and dissolved in methanol by sonication
for 10 mins. This sample solution was then filtered through syringe filter ( 0.45µ). This
solution was then diluted with appropriate quantity of methanol to get approximate
concentration of µg/ml of gallic acid and piperine each. The absorbance of the sample
solution were measured at 273 and 343nm against blank.

VALIDATION OF THE DEVELOPED METHOD[15]

Linearity and range: The standard stock solution containing 1000µg/ml each of Gallic acid
and Piperine was further diluted to get concentration of 2-12µg/ml for gallic acid and
piperine. Each concentration was analysed in triplicates. Calibration curve was plotted by
taking concentration on x-axis and absorbance on y-axis. The relation between drug and it's
absorbance is expressed by equation,
y= mx + c
where, m= slope and b= intercept.

Accuracy: To check the accuracy of the proposed method recovery studies were carried out
at 80, 100, 120% of the test concentration as per ICH guidelines. The recovery study was
performed three times at each level. The results of the recovery studies are reported in Table
2.

Precision
Interday and Intraday precision: The interday and intraday precision was determined by
assay of sample solutions on the same day and on different days at different time intervals
respectively (Six replicates). The results of the precision study are given in Table 3.

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Patil et al. World Journal of Pharmacy and Pharmaceutical Sciences

Ruggedness : It expresses precision within laboratory variations like altered analyst.

Ruggedness of the method was measured by spiking the standard three times by different
analysts using same equipment. The results are shown in Table 4.

Limit of detection: The detection limit is determined by the analysing the samples with
known concentration of analyte and by establishing the minimum level at which analyte can
be consistently detected.

where, σ = The standard deviation of the response

s = The slope of the calibration curve

Limit of Quantitation: The limit of quantitation can be determined by analysing samples

with known concentration of analyte and by establishing the minimum level at which the
analyte can be quantified with acceptable accuracy and precision.

RESULTS AND DISCUSSION

Figure 3 : Overlay Spectra Of Gallic acid and Piperine

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Figure 4: Calibration curve of Gallic Acid

Figure 5: Calibration Curve Of Piperine

Linearity range for gallic acid and piperine are 2-12µg/ml for each at respective selected
wavelength. The correlation coefficient for gallic acid at 273 nm and piperine at 343nm is
0.997 and 0.995 respectively. Both drugs shows good regression values at their respective
wavelength and the results of recovery study reveals that any small change in the drug
concentration in the solution can be accurately determined by the proposed method.
Percentage estimation of gallic acid and piperine in marketed herbal formulation was found
to be 0.0524 % ± 0.156338382 and 0.0479 % ± 0.256361021 respectively by this method.

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Table 1 : Results of Validation Parameters

Parameters Gallic acid Piperine
λmax 273nm 343nm
Linearity Equation y = 0.0432x + 0.1086 y = 0.1213x - 0.1181
Slope 0.0432 0.1213
R2 0.9965 0.9941
LOD 0.072603 0.433487
LOQ 0.220009 1.313597
Table 2 : Recovery studies
Gallic acid
Concentration of the drug Piperine Recovery
Recovery % RSD % RSD
added to formulation ±S.D.*
±S.D.*
99.91222222 99.60212857
80% 0.649407 1.156732
± 0.648837279 ± 1.15212959
99.75766667 99.4641385
100% 1.951438 0.355365
± 1.946709103 ± 0.353460994
98.69975758 99.61902621
120% 1.627579 1.009581
± 1.606416695 ± 1.005734314
*Average of three determinations

Table 3 : Intraday and Interday Precision

Intraday Precision Interday Precision
Drug % Amount % Amount
% RSD % RSD
Found ±S.D.* Found ±S.D.*
100.0728738 100.7008745
Gallic acid 0.91216 0.603063526
± 0.912665489 ± 0.62084355
99.29086135 98.85308846 ±
Piperine 0.899685225 0.692022851
± 0.892693859 0.684756468
*Average of three determinations at three different concentrations

Table 4 : Ruggedness study

ParameterDrug %Amount Found ±SD*
Gallic acid 99.09979424 ± 0.926193806
Analyst 1
Piperine 98.97865714 ± 1.161121325
Gallic acid 98.41049383 ± 1.190949164
Analyst 2
Piperine 98.36951543 ± 1.072192643
*Average of three determinations

Table 5 : Results of Analysis of Formulation

Formulation Drug Amount Found µg/ml ±S.D.*
5.243017418
Gallic acid
± 0.156338382
Sanjeevani Vati
4.790440098
Piperine
± 0.256361021
*Average of three determinations

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Patil et al. World Journal of Pharmacy and Pharmaceutical Sciences

CONCLUSION
The proposed spectrophotometric method is rapid, simple, accurate, precise and economic.
This method is validated as per ICH guidelines in terms of linearity, accuracy, precision,
specificity and reproducibility. This method can be successfully used for simultaneous
estimation of gallic acid and piperine.

AKNOLEDGEMENT
Authors are thankful to Bharati Vidyapeeth's College of Pharmacy for providing facilities to
complete this work successfully.

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