Simultaneous Spectrophotometric Estimation of Curcumin and Quercetin in mixture

 

Shaveta Sharma¹, Vimal Arora²*

¹Chandigarh College of Pharmacy, CGC Campus, Landran, Mohali, Punjab (India) -140307.

²University Institute of Pharma Sciences, Chandigarh University, NH-95,

Chandigarh Ludhiana Highway, Mohali, Punjab (India) -140413.

 

ABSTRACT:

Plants bioactive have been found to be useful in therapeutic management of various diseases such as Cancer, Inflammation and diabetes. Analysis part becomes difficult when use their combination Analysis forms essential exercise before any formulation development. Various analytical techniques such as Chromatographic, Electrochemical and Spectrophotometric have been applied in the analysis of pharmaceuticals. However chromatographic methods are time consuming processes for separation of compounds and costly to these as require high quantity of solvent. Moreover, electrochemical methods are sensitive to temperature. Herein simple, rapid, economical efficient method has been reported for simultaneous estimation of curcumin and quercetin. This simultaneous equation method or Vierordt’s method involves use of methanol as solvent and further dilutions were made using phosphate buffer pH6.8 and were measured at two wavelengths 324nm and 426nm. The linearity was noted in concentration range of 2-12µg/ml for both the bioactives. Calibration curves of both were found to be linear with regression coefficient (r2) values as 0.991 for Quercetin and 0.999 for Curcumin. Linearity, accuracy and precision was validated for this method as per ICH guidelines. The noted values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. UV-Spectrophotometric method was proposed and validated for simultaneous quantification of both Curcumin and Quercetin throughout formulation progress presently initiated in our laboratories. The proposed analytical method is easy, economical and experimentally prevents loss of time.

 

 

 

INTRODUCTION:

Polyphenols plays an essential role in treatment of various diseases. Curcumin (Figure 1 (I)) is a poorly soluble, bright yellowish compound, chemically known as diferuloylmethane derived from the rhizomes of Curcuma longa (Family-Zingiberaceae), shows anti-inflammatory, antioxidant and antiangiogenic activities ¹. Various research studies demonstrated that curcumin is poorly absorbed from the gut, suppresses Cytochrome P450 isoenzyme and metabolized by glucuronidation². Major metabolites are glucuronides of tetra hydrocurcumin and hexahydrocurcumin, with dihydroferulic acid and ferulic acid present as minor metabolites³.

 

Quercetin (Figure 1 (II)), also known as Meletin, Xanthaurine, Sophoretin with IUPAC name 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one, is another polyphenolic flavonoid, yellow colored powder and belonging to Biopharmaceutics Classification System (BCS) Class II drugs. It has been explored in cancer, diabetes management and act as an anti-inflammatory, analgesic, antibacterial, anti-oxidant.

 

Figure 1. Chemical Structure of Curcumin and Quercetin

 

Various Curcumin and Quercetin formulations include nanoparticles, liposomes phytosomes, improves bioavailability solubility, dissolution rate.The binary mixture must be more effective in controlling diseases. Analytical techniques tend to provide information of the constituents of particular formulation mentioned in the label. For separation and estimation of multi-components of a formulation, various techniques, like chromatographic and Spectrophotometric, have been reported^5,6 Chromatographic techniques like HPLC being quite more complicated, time-consuming, and require high consumption of organic solvents⁷ while in HPTLC, plate length is limited so separation can take place up to certain length. Earlier methods reported for simultaneous estimation of Curcumin and Quercetin in nanoformulations, Traditional Chinese medicine and Polyherbal Churna by HPLC was reported but not a single UV – Visible Spectrophotometer method was so far reported. UV Visible Spectrophotometer has advantages than other analytical techniques as it is one of simple, rapid, method for quantitative determination. UV Spectrophotometric method are reported in numerous mixture of drugs such .as Cefixime and Ofloxacin⁸, Cefixime and Lisinopril¹⁰, Rosuvastatin Calcium and Glimepride⁹, Nimesulide and Diclofenac Sodium¹⁰, Metformine Hydrochloride and Pioglitazine ¹¹, Tizanidine and Aceclofenac¹², Levosulpride and Esomeprazole¹³, Diazepam and Propranolol¹⁴ in bulk and Tablet dosage form and alone, Triamcealone¹⁵, Ziprasidone ¹⁶ in tablet dosage form. Present work emphasizes on validating of UV Visible Spectrophotometric analytical method for curcumin and quercetin, in combination.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Curcumin was procured from Loba chemie, Mumbai while Quercetin from Himedia Pvt. Ltd. Mumbai India and analytical grade of Methanol was used from M/s Merck Ltd., Mumbai, India

 

Instrumentation:

The proposed work was carried on Shimadzu UV-1800; UV spectrophotometer which possess double beam double detector configuration with a1 cm quartz matched cell. All weighing was done on calibrated analytical balance.

 

Selection of Solvents:

On the basis of solubility study methanol was selected as the solvent for dissolving Curcumin and Quercetin.

 

Preparation of binary mixture:

The binary mixtures Curcumin and Quercetin in a 1:1 proportion by weight. were prepared and mechanically homogenized with a mortar and pestle

 

Preparation of standard stock solution of Curcumin and Quercetin:

Curcumin Stock Solution An accurately weighed quantity of Curcumin (50 mg) was taken in 50 mL volumetric flask and dissolved in methanol (20 mL) with the help of ultrasonication for about 10 min. Then the volume was made up to the mark using methanol to get Curcumin standard stock solution (1 mg / mL).

 

Curcumin Working Standard Solution Curcumin standard stock solution 5 mL was diluted to 50 mL using Phosphate buffer pH 6.8to get working standard solution 100 µg / mL

 

Quercetin Stock Solution An accurately weighed quantity of Quercetin (50 mg) was taken in 50 mL volumetric flask and dissolved in methanol (20 mL) with the help of ultrasonication for about 10 min. Then the volume was made up to the mark using methanol to get Quercetin standard stock solution (1 mg / mL).

 

Quercetin Working Standard Solution Quercetin standard stock solution 5 mL was diluted to 50 mL using phosphate buffer pH 6.8 to get working standard solution 100 µg / Ml

 

Determination of λ Max of Individual Component: An appropriate aliquot portion of Quercetin and Curcumin (0.2 mL) were transferred to two separate 10 mL volumetric flasks, the volume was made up to the mark using Phosphate buffer pH 6.8 to obtain Curcumin and Quercetin 2 µg/ml dilution Drug solutions were scanned separately between 200 nm to 800 nm. Quercetin shows λ max at 324nm while Curcumin at 426 nm, respectively (Figure2(a)). Solutions were filtered using wahtmann filter paper before analysis.

 

Figure 2. Spectra of Curcumin and Quercetin

 

Overlay Spectra of Quercetin and Curcumin: The overlain spectrum of both drugs (2 µg/ml) was recorded (Fig.4) and two wavelengths 426.0 nm (λ max of Curcumin) and 324 nm (λ max of Quercetin) were selected for further study.

 

Figure 3. Overlay Spectra of Curcumin and Quercetin

 

Linearity Study for Curcumin:

An accurately measured aliquot portion of working standard solution of Curcumin was transferred to six separate 10mL volumetric flasks. The volume was made up to the mark using Phosphate buffer pH 6.8 to obtain concentrations (2-12µg/mL). Absorbance of these solutions was measured at 426 nm, (Table1) Calibration curve was plotted, absorbance Vs concentration as shown in (Fig. 3).

 

Linearity Study for Quercetin:

Accurately measured aliquot portions of working standard solution of Quercetin were transferred to six separate 10mL volumetric flasks. The volume was made up to the mark using Phosphate buffer pH 6.8 to obtain concentrations (2-12µg/mL). Absorbance of these solutions was measured at 324nm, (Table 1). Calibration curve was plotted, absorbance Vs concentration as shown in (Fig. 4).

 

Figure 4. Calibration curve of Curcumin and Quercetin

 

Linear regression analysis of the data attained 2-12 µg/ml which depicts highly linear relationship as the correlation coefficient (R²) value were around 0.999 for Curcumin and 0.991 for Quercetin. Calibration Curve equation, Y= .005x+.001 for quercetin and Y= .005x+.002 for curcumin at λ_max 324nm for quercetin and 428 for curcumin

 

Simultaneous Equation method:

25mg each of mixture Curcumin and Quercetin was weighed correctly into a volumetric flask of 50 ml and dissolved in small quantity of methanol (20mL) with the help of ultrasonication for about 10min. Then the volume was made up to the mark using methanol to get mixture standard stock solution (1mg/mL). From standard stock solution 5 mL was diluted to 50mL using Phosphate buffer pH 6.8to get working standard solution 100µg/mL An appropriate aliquot portion of Quercetin and Curcumin (0.2mL) were transferred to 10mL volumetric flasks, the volume was made up to the mark using phosphate buffer pH6.8 to obtain 2µg/ml.

 

The concentration of each component was determined by using simultaneous equation method (Vierodt’s method).

 

A1 = ax₁bc_x + ay₁ bc_y   --------- (at λ1–324nm)

A2 =ax₂bc_y + ay₂bc_y      --------- (at λ2–426 nm)

 

Amount of each drug was estimated using following equations,

C_x = A₂ × ay₁ - A₁ × ay₂ / ax₂ ay_{1 –} ax₁ ay₂

Cy = A₁ × ax₂- A2× ax₁ / ax₂ ay_{1 –} ax₁ ay₂

A1= absorbance value (324nm) A2= absorbance value (426nm)

ax₁= absorptivity of Quercetin at 324nm ay₁= absorptivity of Curcumin at 324 nm

ax₂= absorptivity of Quercetin at 254nm ay₂= absorptivity of Curcumin at 275nm

C1= concentration of Quercetin in μg/ml C2= concentration of Curcuminin μg/ml

The absorptivity values for simultaneous equation are enclosed in Table 2

 

Table 1. Absorptivity Values for Simultaneous Estimation of Quercetin and Curcumin

S. No	Drug	Parameters		Absorptivity
1	Quercetin	ax1	ax2	0.015	0.013
2	Curcumin	ay1	ay2	0.018	0.029
3	Mixture	A1	A2	0.066	0.084

Each value is an average of three determinants.

C1 and C2 were found to be 2 μg/ml in mixture

 

Validation of Proposed Method:

The Proposed method was validated as per the ICH guidelines.

 

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value and the value found. It is an agreement between value found and an excepted reference value Accuracy of the method was investigated by preparing stock solution of 1 µg/ml then from that make 100 µg/ml then 25 %,50 % and 75 % dilutions were prepared and analysed at these different levels. It exhibits excellent reproducibility with the percent relative standard deviation (%RSD) of less than 2.00%. The accuracy established within the range as shown in table no. 3

 

Table 2. Accuracy Study

Accuracy Parameters	Quercetin			Curcumin
Conc. taken	3 µg/ml (25%)	6 µg/ml (50%)	9 µg/ml (75%)	3 µg/ml (25%)	6 µg/ml (50%)	9 µg/ml (75%)
Conc. Observed± SD	3.08±0.012	5.9±0.017	9.03±0.023	3.1±0.025	6.07±0.019	9.0±0.017
RSD	0.38%	0.28%	0.25%	0.80%	0.31%	0.18%
Each value is an average of three determinants.

 

Precision: It is defined as the degree of repeatability of the method under standard operating conditions Three different concentration equivalent to 25%, 50% and 75 % diluted solution were analyzed at diverse time-points on the same day and by repeating the analysis on the next subsequent day. Values of SD and %RSD were computed for validating the precision of the developed method¹⁷ It was estimated for 3 concentration levels (2,4,6μg/ml) covering the entire linearity range by intermediate (inter-day) as well as repeatability (intra-day) studies and was reported as %RSD. The results indicate that the method is highly precise. The precission value are tabularized in Table No. 4,5

 

Table 3. Curcumin and Quercetin Precision Study

Curcumin Precision Study
Conc. taken	Conc. Observed± SD	RSD
3 µg/ml	3.18±0.017	0.53%
6 µg/ml	6.13±0.029	0.47 %
9 µg/ml	9.04±0.012	0.132 %
Intermediate Precision Evening (Day1)
Conc. taken	Conc. Observed± SD	RSD
3 µg/ml	3.06±0.026	0.84%
6 µg/ml	6.08± 0. 015	0.24%
9 µg/ml	9.19±0.019	0.20%
Intermediate Precision Morning Inter day (Day 11)
Conc. taken	Conc. Observed± SD	RSD
3 µg/ml	3.13± 0.027	0.86%
6 µg/ml	6.26±0.012	0.19%
9 µg/ml	9.28±0.016	0.17%
Quercetin Precision Study
Conc. taken	Conc. Observed± SD	RSD
3 µg/ml	3.07± 0.014	0.45%
6 µg/ml	6.29± 0.019	0.30 %
9 µg/ml	9.17± 0.011	0.11%
Intermediate Precision Evening (Day1)
Conc. taken	Conc. Observed± SD	RSD
3 µg/ml	3.24± 0.015	0.46%
6 µg/ml	6.19± 0.012	0.19
9 µg/ml	9.37±0.017	0.18
Intermediate Precision Morning Inter day (Day 11)
Conc. taken	Conc. Observed± SD	RSD
3 µg/ml	3.30± 0.028	0.84%
6 µg/ml	6.24±0.019	0.30
9 µg/ml	9.24±0.014	0.15%

 

Robustness:

It is used as a parameter characterizing the stability of the method with respect to variations of the internal factors of the method. It was examined by analyzing a drug concentration of 100µg/ml with minute changes like pH of phosphate buffer. It has ability to remain unaffected by minute changes in ± 0.2 phosphare buffer pH in analytical parameters. Conct. taken is 3µg/ml which was observed 3.01 in quercetin and 3.09 in curcumin by changing buffer pH

 

Ruggedness:

Spectrophotometric analysis of different concentrations equivalent to 25%, 50% and 75% diluted solution was performed by two analysts assess the ruggedness of the developed method. All analytical parameters are remain unaffected even by performed by another analyst. As Conct. taken is 3µg/ml by both analyst and observed is 3.04 and 3.08

 

Sensitivity:

LOD: Minimum concentration of analyte in a test sample that can be identified but not quantified. LOQ is lowest concentration of the standard curve that can be calculated with satisfactory accuracy and precision. The noise to signal ratio for LOQ¹⁸ should be 1:10 The values of LOD and LOQ, therefore, were computed from

 

Equation 1.

LOD=3.3σ/S; LOQ=10σ/S σ is standard deviation S is slope

 

The LOD and LOQ for Quercetin in phosphate buffer were found to be 0.024µg/ml and 0.045µg/ml, respectively and values for Curcumin were 0.024µg/ml and 0.037 µg/ml, respectively which indicates adequate sensitivity of method

 

ACHKNOWLEDGEMENT:

The Authors are grateful to the authorities of Chandigarh College of Pharmacy, Landran and Chandigarh University for the facilities.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 18.05.2021           Modified on 11.08.2021

Accepted on 21.09.2021         © RJPT All right reserved