Development and Validation of RP-HPLC Method for Simultaneous Determination of Resveratrol and Curcumin in Pure Form

 

Manish Patidar, Gopkumar P*., Sridevi G., C.C. Behera and Sujit Pillai

G.R.Y. Institute of Pharmacy, Borawan, Khargone 451228

*Corresponding Author E-mail: gopnitk@gmail.com

Cancer chemopreventive agents are designed to reduce the incidence of tumorigenesis by intervening at one or more stages of carcinogenesis. Recently, resveratrol, a natural product found in the diet of humans, has been shown to function as a cancer chemopreventive agent. Resveratrol was first shown to act as an antioxidant and antimutagenic agent, thus acting as an anti-initiation agent. Resveratrol belongs to a class of polyphenolic compounds called stilbenes.(3,5,4'-trihydroxy-trans-stilbene) is a natural compound found in red grape skin, Japanese knotweed (polygonum cuspidatum), peanuts and blueberries. A naturally occurring polyphenolic phytoalexin Curcumin, [1,7-bis (4-hydroxy-3-methoxyphenyl)-1, 6- heptadiene-3, 5-dione] is the major yellow pigment extracted from turmeric, a commonly used spice, derived from the rhizome of Curcuma longa Linn. In animal studies, curcumin has been shown to increase the survival of tumor-bearing rodents by inhibiting tumor growth and impeding metastasis.

 

 At present study a simple, precise, rapid and accurate reverse phase HPLC method developed for the estimation  of Resveratrol and Curcumin in combination as well as individually were developed and validated. The retention time of Resveratrol and Curcumin were found to be 2.90 and 4.11 minutes. Detection was carried out at 424 and 306nm. The regression coefficient value of Resveratrol and Curcumin is 0.9904 and 0.9937 which was found to be linear in the detection range. Limit of detection and limit of quantification of Resveratrol was found to be 0.08µg/ml and 0.32 µg/ml and Curcumin  is 0.05 µg/ml and 0.17 µg/ml. Analysis was performed using a C18 column (250 X 4.6 mm) at room temperature in isocratic mode. The mobile phase used was Citric acid (pH adjusted to 3.5): Acetonitrile (40:60) at flow rate of 1.0 ml/min.

 

 

 

INTRODUCTION:

Resveratrol belongs to a class of polyphenolic compounds called stilbenes. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural compound. It is a powerful antioxidant produced by some plants to protect them against environmental stresses. Resveratrol, a naturally occurring polyphenolic phytoalexin, is present in many plants and fruits, including red grapes, eucalyptus, spruce, blueberries, mulberries, peanuts, and giant knotweed. It is an effective antioxidant with strong anti-inflammatory and antiproliferative properties. It is a fat soluble compound that occurs in a Trans and a cis configuration¹.

 

Curcumin is the principal curcuminoid of the popular Indian spice turmeric, which is a member of the ginger family (Zingiberaceae).

 

Turmeric's other two curcuminoids are desmethoxy- curcumin and bis-desmethoxycurcumin. The curcuminoids are natural phenols that are responsible for the yellow color of turmeric. Curcumin can exist in several tautomeric forms, including a 1,3-diketo form and two equivalent enol forms. The enol form is more energetically stable in the solid phase and in solution².

 

Increasing interest in the use of phytochemicals to reduce prostate cancer led to investigate two potential agents, curcumin and resveratrol as cancer chemo preventive agents. . Several In vitro assays studies using PTEN-CaP8 cancer cells were performed to investigate the combined effects curcumin with resveratrol on (i) cell growth, apoptosis and cell cycle (ii) impact on activated p-Akt, cyclin D1, m-TOR and androgen receptor (AR) proteins involved in tumor progression. And results of these studies indicated positive anticancer effects of these drugs^3-4. Thus in the present investigation simultaneous estimation method for these two drugs in combination was developed and validated. And value was given to develop a simple, sensitive and repeatable estimation method^.

 

EXPERIMENTAL:

Preparation of standard stock solution

The stock solution containing 1mg/ml of Curcumin and Resveratrol were prepared in mobile phase. The stock solution store in light resistant containers. Aliquots of Curcumin (5-50µg/ml) and Resveratrol (10-100µg/ml) were prepared in the mobile phase.

 

Preparation of combine standard stock solution

An accurately weight quantity equivalent to 100mg of Resveratrol and 50mg of Curcumin were transferred to 100 ml volumetric flask the mixture were dissolved with small quantity of mobile phase and diluted upto the mark and sonicated for 10 minutes at room temperature. The final concentration of Stock solution is 1mg/ml. From this stock solution different dilution of Resveratrol-Curcumin between the range 10-100µg/ml and 5-50 µg/ml respectively, concentration solutions were prepared. These prepared mixture of dilutions were used to determine the linearity using peak area calibration. A plot of peak area versus the respective concentration gives the calibration graph.

 

Figure: 1structure of Resveratrol

 

Figure: 2 structure of Curcumin

 

Chromatographic condition

The HPLC system included a Yung lin instrument 900’ UV730D solvent delivery system (pump), injector with a 20μL loop volume. The class VP 6.01 data station software was utilized for integration. Separation was achieved using a Princeton C₁₈ column (250 X 4.6 mm, 5μ ID), (Merck, India). The solvent system consists of Citric acid solution (pH 3.5): acetonitrile (40:60 v/v) was pumped isocratically at a flow rate of 1.0 mL/ Min^5-8.

 

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Resveratrol and Curcumin. Parameters that were studied to evaluate the suitability of the system are given in Table 1

 

Table 1: Combination of Resveratrol and Curcumin

Parameter	Resveratrol	Curcumin
Theoretical plates	587.60	1778.5
Tailing factor	1.1818	1.5000
Retention time(min)	2.90	4.11
LODµg/ml	0.08	0.05
LODµg/ml	0.32	0.17

 

Figure: 3 Representative Chromatogram for Resveratrol and Curcumin combination by HPLC

 

Figure: 4 Calibration curve indicating linearity range of Resveratrol by HPLC analysis

 

Figure 5: Calibration curve indicating linearity range of Curcumin in HPLC analysis

Chromatograms eluted from the developed method indicated the retention time of Resveratrol and Curcumin was 2.90 and 4.11 min respectively. A mixture of Acetonitrile and Citric acid solution (pH adjust to 3.5) in the ratio of 60:40 was found to be most suitable. The selected mobile phase was efficient in resolving two drugs to obtain  well defined  peaks free from tailing. In the present developed HPLC method, the sample preparation required less time. A good linear relationship for both the drugs in combination i.e  for Resveratrol (r²=0.9904) was observed  between the concentration in the linear range of 20-100µg/ml and that of  Curcumin (r²=0.9937) was between linear range  of  10-50µg/ml. 

 

Analytical Method Validation Parameters

The method was validated as per ICH guidelines (Q2 R1).

 

Linearity and Range

Linearity of method is its ability, within a given range, to obtain test results that are directly, or through a mathematical transformation, proportional to concentration of analyte . Good linear correlations were obtained between absorbance and concentration in the selected range of Resveratrol and Curcumin is 10 –100 and 5-50μg/ml respectively.

 

Precision

The precision of method expresses the degree of scatter between a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. Intraday precision refers to the use of analytical procedure within a laboratory over a short period of time using the same operator with the same equipment whereas Interday precision involves estimation of variations in analysis when a method is used within a laboratory on different days, by different analysts . Repeatability (intraday) was assessed by analyzing these three different Concentrations of Resveratrol (20, 40, 60 μg/ml) Curcumin(10,20,30µg/ml) three times a day. Intermediate precision (Interday) was established by analyzing these three different concentrations of Resveratrol (20, 40, 60 μg/ml) Curcumin(10,20,30µg/ml), three times a day for at least three different days. The Standard Deviation, % RSD  for the intra-day Resveratrol and Curcumin 0.96 and0.61 Respectively.

 

Accuracy

Accuracy of method is the closeness of test results to true value. It was determined by the application of procedure to recovery studies, where known amount of standard is spiked in preanalyzed samples solutions .The % recovery of the Resveratrol and Curcumin is 98.37% and 99.15% Respectively .

 

LOD and LOQ

LOD and LOQ were calculated according to the formulae:

For Resveratrol

LOD=3.3 σ / S =0.08µg/ml

LOQ=10 σ / S = 0.32µg/ml

 

For Curcumin

LOD=3.3 σ / S =0.05µg/ml

LOQ=10 σ / S = 0.17µg/ml

 

CONCLUSION:

The development method of estimation indicates that it is simple sensitive and repetitive and major advantage being use of inexpensive solvents. The mixture of solvents used indicated well separation of two peaks without any tailing effects. The developed method can be easily applied for routine analysis of these two drugs Resveratrol and Curcumin in free or formulation dosage forms. 

 

ACKNOWLEDGMENT:

The authors are thankful to JNCET’S trust and Principal, GRY Institute of Pharmacy, Borawan for providing all facilities and kind support to successful completion of Research project.

 

REFERENCES:

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3.        Pezzuto JM. Plant-derived anticancer agents. Biochem Pharmacol 1997;53:121–33.

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Accepted on 07.07.2013         © RJPT All right reserved