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Jaldip et al.
Research Article
Article Received on
10 March 2014,
Jaldip Jasoliya
Department of Pharmaceutical
Sciences, Saurashtra
India-360005
respectively. The method was found to be linear over the range of 1050 g/ml and 1-5 g/mlwith correlation co-efficient (r2) 0.999 & 0.999
for Resveratrol and Piperine respectively. Percentage recoveries obtained for both the drugs
were 99.49-100.25% and 99.52-100.53% for Resveratrol and Piperine respectively. The
%RSD for precision and accuracy of the method was found to be less than 2%.The method
was validated according to the ICH guidelines with respect to specificity, linearity, accuracy,
precision and robustness. The method developed can be used for the routine analysis of
Resveratrol and Piperine from their combined dosage form.
Key Words: Resveratrol, Piperine, ICH guideline, simultaneous determination, RP-HPLC
method.
INTRODUCTION
The international conference on harmonization (ICH) guide-lines emphasizes that the purity
and assay of drug susceptible to change during storage, must be determined by using
validated
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Jaldip et al.
prevention of disease.In context of antioxidant, especially derived from natural sources such
as Indian medicinal plant and herbal drugs derived from them require special attention.
Antioxidant have many potential application especially in relation to human health, both in
term of prevention and treatment of disease.
Resveratrol is chemically 3,5,4-trihydroxystilbenepossesses antioxidant activities in
vitro. It is obtained from Roots of polygonum cuspidatum and Leaves of veratrum
grandiflorum. It dose-dependently inhibited the generation of peroxyl, hydroxyl, peroxides,
and lipid peroxidation products in cell free systems. Oxidative burst of whole human blood
stimulated with PMA, fMLP, OpZ, and A23187 was inhibited in a concentration-dependent
way, indicating suppression of both receptor and nonreceptor activated chemiluminescence
by resveratrol. Results from isolated human neutrophils revealed that resveratrol was active
extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen
species. Resveratrol is useful as Anti-cancer, Anti-diabetic, Anti-inflammatory, Anti-viral,
Cardioprotective effect, Neuroprotective effect etc.., Molecular weight of Resveratrol is
228.4 gm/mol and formula is C14H12O3.
.
Resveratrol, It increaseas its bioavailability and intracellular residency timefound that the
degree of exposure (i.e. AUC) to resveratrol was enhanced to 229% and the maximum serum
concentration (C(max) ) was increased to 1544% with the addition of piperine. it is official in
Indian pharmacopeia 2010. Molecular weight of Piperine is 285.34 gm/mol and formula is
C17H19NO3. Literature Review revels that there was no reported Stability Indicating RPHPLC method for Simultaneous Estimation of Resveratrol and Piperine in combined dosage
form
[9-13]
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Accuracy
The accuracy of the methods was determined by calculating recoveries of Resveratrol and
Piperine by the standard addition method.
LOD&LOQ
The limit of detection (LOD) and the limit of quantification (LOQ) of the Resveratrol and
Piperine were calculated using the standard deviation of responses (N) and slopes (S) of
respective calibration curves using signal-to-noise ratio.
LOD = 3.3 N/SLOQ = 10 N/S
Robustness
The evaluation of robustness should be considered during the development phase and
depends on the type of procedure under study. It should show the reliability of an analysis
with respect to deliberate variations in method parameters. Robustness of method is
determine by change in Saturation Time, Analytical Wavelength & mobile phase composition
there is no change in peak area & shape data show in table 4.
Assay of the tablet dosage form
The proposed validated method was successfully applied to determine Resveratrol and
Piperine in capsule dosage form. Three replicates of sample solution (10:1g/ml of each)
were injected. From the peak area of the Resveratrol and Piperine, amounts of drugs in
samples were computed. %Assay is given in to table 5.
RESULTS AND DISCUSSION
Table 1 :Data of Repeatability of Resveratrol and Piperine by HPLC(n=6)
Sr.No
1
2
3
4
5
6
Mean
SD
%
RSD
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Concentration(g/Ml)
Resveratrol
Piperine
30
30
30
30
30
30
3
3
3
3
3
3
Area
Resveratro
Piperine
l
4892096
609945
4893126
612689
4892259
598402
4900135
608931
4889580
616734
489627
599876
4893912
607762.8
3734.64
7220.84
0.076
1.18
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Jaldip et al.
Conc.
(g/Ml)
Piperine
1
3
5
Mean Area
SD (N=3)%
Resveratrol
164089018533
489508747423
819087973955
Mean Area
SD (N=3)%
Piperine
1991063612
5972146543
9755274447
%RSD
Resveratrol
1.134
0.970
0.902
%RSD
Piperine
1.81
1.09
0.45
Conc.
(g/ml)
Mean Area
SD (n=3)%
%RSD
Mean Area
SD (n=3)%
%RSD
Resveratrol
Piperine
Resveratrol
Resveratrol
Piperine
Piperine
10
30
50
1
3
5
163745016537.6
490508754823.2
815717963955.0
1.09
1.11
0.78
2011062072
6022676822.4
9411277127.1
1.03
1.13
0.75
Mean of % Recovery
Resveratrol Piperine
99.79
100.073
99.54
99.79
99.32
99.69
Amount of
drug found (mg)%
Amount found
(n=3) SD
Criteria
1
2
3
Theoretical plates
Resolution
Tailing factor
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Results
RESVERATROL PIPERINE
3380.7
4001
4.2
1.701
1.11
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Jaldip et al.
RP-HPLC method
RESVERATROL
PIPERINE
10-50 g/ml
1-5 g/ml
Y=31170X+3786 y=34446X+44477
0.999
0.999
0.076 %
1.18 %
0.90-1.13%
0.45-1.81%
0.78-1.11%
0.75-0.1.03%
99.49-100.25%
99.52-100.53%
0.43-0.97%
0.40-1.09%
0.70-0.984%
0.80-1.068%
0.26 g/ml
0.18g/ml
0.78 g/ml
0.55g/ml
99.48%
100.02%
2/
mAU
3.5 254nm,4nm (1.00)
3.0
2.5
2.0
3/
1.5
7/
5/
0.5
6/
1/
4/
1.0
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
min
4.5
min
1/RT2.779
275
250
225
200
175
150
125
100
75
2/RT3.710
50
25
-25
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
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1/
mAU
306nm,4nm (1.00)
500
450
400
350
300
250
200
150
100
50
-50
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
min
mAU
340nm4nm (1.00)
37.5
35.0
32.5
30.0
27.5
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
-7.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
mi n
mAU
281nm
60 286nm
291nm
296nm
50 301nm
306nm
311nm
40 316nm
321nm
326nm
30 331nm
20
10
0
3.50
3.75
4.00
min
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Jaldip et al.
mAU
281nm
286nm
291nm
400 296nm
301nm
306nm
311nm
300 316nm
321nm
326nm
331nm
200
100
0
2.50
2.75
3.00
min
uV
1100000
1000000
900000
800000
700000
600000
500000
400000
300000
200000
100000
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
min
Fig 10: Calibration graph for the Resveratrol (Peak Area Vs Conc.)
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10. Rupali L.Choudekar, Mahajan, M. P, Sawant Validated RP-HPLC Method for the
Estimation of Piperine fumarate in Bulk and Tablet Dosage FormDer Pharmachemica
2012;4(3):1047-1053.
11. Vaiddya ashok D. B,, Devasagam Thomas P.,A,current status of
Siddharth and K.
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