INTRODUCTION:

Flavonoids consist of a large group of polyphenolic compounds having a benzo-𝛾-pyrone structure present in plants. It has presently recognized to be a unique class of therapeutic molecules due to their diverse therapeutic properties. Flavonoids are important antioxidants which promote several health effects. These are used as antiviral, anticancer, anti inflammatory, anti allergic agents.

It reduces the risk of atherosclerosis by inhibiting the oxidation of low density lipoproteins. Flavonoids are also essential for plants for nitrogen fixation, cell cycle inhibition and producing the colors needed to attract pollinating insects.^{1, 2, 3}

Rutin has been explored for a number of pharmacological effects. Rutin is also known as vitamin P or rutoside (Fig-1). Rutin is one of the active constituents of tea leaves, apples etc. Rutin shows anticancer, antioxidant, anti-inflammatory, anticateract, diuretic, antidiabetic and many more therapeutic effects. Rutin is hydrophobic in nature. In the human body, rutin can remove excess iron and copper. It inhibits the endothelial growth factor in subtoxic concentrations and helps to prevent the formation of cancer cell.^{3, 4, 5}

Fig 1: Rutin

Resveratrol is a naturally occurring polyphenolic compound (Fig-2). It has been reported as a cardio protective, neuroprotective, chemopreventive agent along with antiageing properties. Studies have been carried out using in vitro and animal models for studying the action of resveratrol on cancer cells and cancer related pathways. Innumerable in vitro studies exist in the literature on the action of resveratrol in various types of cancers like breast cancer, gastric and colorectal cancer, lung cancer, skin cancer, prostate cancer, pancreatic cancer, hepatoma, fibrosarcoma, neuroblastoma and leukemia.^{4,
6, 7}

Fig 2: Resveratrol

Rutin and resveratrol both are poorly water soluble drug. Liposome permeability plays a significant role in the encapsulation efficiency of hydrophobic drugs in liposome because the lipid bilayer in the liquid crystalline phase is more permeable to the encapsulated material than the other. And if the permeability is more then the bioavailability of the drugs is also increased. Rutin and resveratrol share similar biosynthesis pathways in spite of different biological origin. Both compounds exhibit similarity in features considering their molecular topography which supports the hypothesis that the targets for absorption or efflux may be shared between them. With reference to molecular structures, rutin and resveratrol show close similarities with presence of several phenolic groups as well as unsaturated carbon chains attached to the hydroxyl groups.^{4, 8, 9}Moreover both drugs have anticancer properties.^{3, 6} Therefore co-administration of these two compounds by liposomal encapsulation is thought to have synergistic effect.

Chaudhari et al., (2014) reported validation of UV spectrophotometric method for simultaneous estimation of rutin and quercetin in niosome formulation based on solving simultaneous equation in niosome formulation. As Rutin and Quercetin show absorbance maximum at 257 and 372 nm respectively, absorbance was measured at these wavelengths for estimation of Rutin and Quercetin respectively. They found that both Rutin and Quercetin obey the Beer- Lambert's law in the concentration ranges of 2-20 μg/ml.¹⁰A new absorption subtraction method for simultaneous estimation of resveratrol and benzoyl peroxide using UV spectrophotometry was formulated by Manju et al., (2017). The method involved absorption subtraction method using two wavelengths, with one being of benzoyl peroxide (234 nm, ) and the other being the isoabsorptive point of both drugs (246 nm). The results of analysis have been validated statistically and by recovery study. The method was found precise, reproducible with accuracy range for resveratrol between 103.16 and 104.49% and for benzoyl peroxide 104.05 to 110.49. ¹¹Salunke et al., (2013) proposed two simple, precise and economical UV methods for the simultaneous estimation of Lopinavir and Ritonavir in bulk and pharmaceutical dosage form. Method A was based on absorbance maxima and method B rely on area under the curve (AUC). The results were validated statistically as per ICH Q2 R1 guideline and were found to be satisfactory. The proposed methods were successfully applied for the determination of for Ritonavir and Lopinavir in commercial pharmaceutical dosage form.¹²Chitlange et al., (2011) developed three simple UV spectrophotometric methods for simultaneous estimation of nimesulide and drotaverine from tablet dosage form. Method-I involves, formation of Q-absorbance equation at 349 nm (isoabsorptive point) and 298.5 nm (ƛ max of nimesulide); Method-II simultaneous equation method involves the measurement of absorbances at two wavelengths 298.5 nm (ƛ max of nimesulide) and 245 nm (ƛ max of drotaverine) in ethanol (95%) and Method-III multicomponent mode of analysis involves the measurement of absorbances at two wavelengths 298.5 nm (_max of nimesulide) and 362.5 nm (ƛ max of drotaverine). All methods were found to be rapid, specific, precise and accurate and successfully applied for the routine analysis of nimesulide and drotaverine in bulk and combined dosage form.¹³

MATERIALS AND METHOD:

MATERIALS:

Rutin was purchached from Central Drug House, New Delhi. Resveratrol was supplied by Yarrow Chem Products, Mumbai. All other chemicals and solvent used were of analytical grade.

Instrument:

A double beam UV-Visible spectrophotometer (Shimadzu-1800), Japan with 1 cm quartz cell was used for all analysis.

Experimental:

1. Preparation of standard stock solutions:

Standard stock solution (1000𝜇g/ml) of Rutin and Resveratrol was prepared separately by dissolving carefully weighed 10mg of drug in 10ml volumetric flask and diluting up to the mark with methanol. 5 ml of this solution was diluted up to 50ml with phosphate buffer 6.8. And from that again take 10 ml of solution and diluted upto 100 ml to get working stock solution (10𝜇g/mL).

2. Determination of isoabsorptive point and wavelength of maximum absorbance (𝜆max).

Solutions of 10𝜇g/ml of both drugs were prepared from working stock solution and scanned in the range of 200 nm to 400 nm against phosphate buffer 6.8 as blank. The overlaying spectrum was also obtained to determine isoabsorptive point.

3. Standard calibration curve (linearity):

A calibration curve was plotted over a concentration range of 1–20 𝜇g/ml for rutin, 1-10 𝜇g/ml for resveratrol and 1-10 𝜇g/ml for mixture rutin and resveratrol (1:1). Accurately measured stock solution of rutin (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.6, 2.0 ml), stock solution of resveratrol (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0 ml) and stock solution of rutin and resveratrol (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0ml) were transferred to separate series of 10ml volumetric flask and diluted up to the mark with phosphate buffer 6.8. The absorbance of all solutions was taken at their respective 𝜆max and at isoabsorptive point. The calibration curves were constructed by plotting concentration against absorbance where each reading was an average of three determinations.

Iso absorptive point method (Absorption Ratio Method):

This method is the modification of simultaneous equation method. According to this method, the ratio of absorbance at any two wavelengths of the substance, which obeys Beer’s law is constant and the value is independent of concentration and path length. This constant is known as Q value. This method involves the measurement of absorbance at two wavelengths, one is the 𝜆max of one component (𝜆₂) and other being a wavelength of equal absorptivity of both the components, called Iso absorptive point.^{11, 12, 13, 14, 15}

The concentration of each component can be calculated by mathematical equation:

C_x= (Q_m- Q_y/ Q_x- Q_y)*A/ a₁

C_y= (Q_m- Q_x/ Q_y- Q_x )*A/a₂

C_xand C_y: Concentration of X and Y respectively.

A: Absorbance of the sample at iso absorptive point.

a₁ and a₂ : Absorptivity of X and Y.

Absorbance of sample solution at 𝜆max of one component (𝜆₂)

Q_{m =- ----------------------------------------------------------------------------------------------------} Absorbance of sample solution at isoabsorptive point

Absorptivity of X at 𝜆max of one component (𝜆₂)

Q_x=_{--------------------------------------------------------------------------------------------------}

Absorptivity of X at isoabsorptive point

Absorptivity of Y at 𝜆max of one component (𝜆₂)

Q_y=_{--------------------------------------------------------------------------------------------------}

Absorptivity of Y at isoabsorptive point

Analytical validation by uv spectroscopic method: ^{10, 11, 12, 13, 14}

1. Linearity and Range:

Linearity, consisting of the basic elements input → converter → output, is the assumption that there is a straight line relationship between the input and output variables that can be written mathematically by the expression if the straight line crosses through the origin or by the expression if the straight line does not cross through the origin. The linear range corresponds to the valid interval of functional dependence of the signal on concentration or mass which assumes homoscedasticity of the measurements over the linear range. The linear response of rutin and resveratrol was determined by analyzing the calibration curve in the range of 1-20 μg/mL and1-10μg/ml.

2. Precision:

The term precision is defined by the ISO International Vocabulary of Basic and General Terms in Metrology (ISO-VIM) and ICH as the closeness of agreement between quantity values obtained by replicate measurements of a quantity under specified conditions [10]. Assessing the precision implies expressing numerically the random error or the degree of dispersion of a set of individual measurements by means of the standard deviation, the variance, or the coefficient of variation.

3. Repeatability (Within-Run Precision):

It is the concordance of a series of measurements of the same quantity when the experiments are conducted under same conditions (analyst, apparatus, instrument, and day) in a rapid succession. For this experiment, standard solution of Rutin and Resveratrol (5 + 5𝜇g/mL) was prepared and analyzed six times as per the proposed method.

4. Intermediate Precision (Between-Run Precision):

It is the concordance of a series of measurements of the same quantity when the experiments are conducted within the same laboratory under different conditions (analyst, apparatus, instrument, and day). Standard solution of Rutin and Resveratrol (5 + 5 𝜇g/mL) was prepared and analyzed as per the proposed method.

5. Accuracy (%recovery):

The accuracy was tested by recovery experiments. Recovery studies were carried out at 100 % level by adding a known quantity of pure drug to the preanalyzed formulation and the proposed method was followed. From the amount of drug found, percentage recovery was calculated.

Process:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The recovery experiments were carried out in triplicate by spiking previously analyzed samples with three different concentrations of standards.

6. Ruggedness:

Ruggedness was determined by carrying out analysis by two different analyst and the respective percentage recovery was noted and the results was indicated as % RSD.

7. Limit of Detection (LOD) and Limit of Quantification (LOQ):

The detection limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be detected but not necessarily quantitated as an exact value. The quantification limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ of the proposed method were determined by using calibration curve:

3.3σ 10σ

LOD =--------- , LOQ =---------

S S

Where, ơ is the standard deviation of the response (Y intercept) and S is the slope of the calibration curve.^11,16

8. Specificity:

Specificity of the methods was achieved by the analysis of different laboratory prepared mixtures of rutin and resveratrol within the linearity range.

RESULTS AND DISCUSSION:

The solutions of 10𝜇g/mL of rutin and resveratrol were analyzed and the 𝜆max was found to be 257 nm and 306 nm, respectively. The isoabsorptive point 267 nm was found in overlaying spectra (Fig-3). The calibration curve of rutin and resveratrol individually and the mixture of both drugs at 257 nm and 306 nm were plotted. The relationship between the absorbance and the concentration of rutin and Resveratrol was found to be linear in the range of 1-20 µg/ml at wavelengths 257 nm (table-1, fig-4)and in the range of 1-10 µg/ml at wavelength 306 nm (table-2,fig-5)respectively. The representative linear equations and correlation coefficients have indicated very good linearity. Evaluation of repeatability, intermediate precision, accuracy, ruggedness, specificity done and percent relative standard deviation (%RSD) values were calculated. These values were found to be less than two (%RSD< 2), indicating good precision.

The limit of detection of rutin and resveratrol at isoabsorptive point (267 nm) was found to be 20.30μg/ml and 10.43μg/ml. The LOD of rutin at 257 nm was found to be 19.82μg/ml and of resveratrol at 306 was 10.19μg/ml. The limit of quantification of rutin and resveratrol at isoabsorptive point (267 nm) was found to be 61.51μg/ml and 31.63μg/ml respectively. The LOQ of rutin at 257 nm was found to be 60.06μg/mL and of resveratrol at 306 nm was 30.88μg/mL.

Fig 3: Determination of iso apsorptive point.

Fig 4: Calibration curve of rutin at 257 nm

Table -1: Standard calibration curve of rutin at 257nm

Serial no	Concentration(µg/ml)	Absorbance	%RSD
1	2	0.0755	0.0105
2	4	0.1432	0.0129
3	6	0.2093	0.0156
4	8	0.2747	0.0208
5	10	0.3345	0.0205
6	12	0.402	0.0153
7	16	0.5346	0.0761
8	20	0.6635	0.0338

Table -2: Standard calibration curve of rutin at 267nm.

Serial no	Concentration(µg/ml)	Absorbance	%RSD
0.1118	2	0.0701	0.011
0.1435	4	0.1235	0.0154
0.188	6	0.1990	0.0189
0.2461	8	0.2567	0.0112
0.2984	10	0.3188	0.0073
0.3854	12	0.3804	0.0136
0.494	16	0.494	0.0318
0.6217	20	0.6217	0.0275

Fig 5: Calibration curve of rutin at 267 nm

Table -3: Standard calibration curve of resveratrol at 306 nm.

Serial no	Concentration(µg/ml)	Absorbance	%RSD
1	2	0.1999	0.0149
2	4	0.3612	0.0231
3	6	0.5433	0.0064
4	8	0.7122	0.0370
5	10	0.9017	0.0064

Fig 6: Calibration curve of resveratrol at 306 nm

Table -4: Standard calibration curve of resveratrol at 267 nm.

Serial no	Concentration(µg/ml)	Absorbance	%RSD
1	2	0.0869	0.0147
2	4	0.1682	0.0263
3	6	0.2456	0.0113
4	8	0.324	0.0254
5	10	0.4012	0.0156

Fig 7: calibration curve of resveratrol at 267 nm

Precision

Table-5: Repeatability and Intermediate Precision study.

Serial no	Precision	% recovery of rutin	% RSD	% recovery of resveratrol	% RSD
1	Repetability	104.269	0.565	101.106	1.070
2	Intermediate precision (day1-day6)	104.289	0.535	103.236	0.0850

Accuracy

Table -6: Results of recovery studies of rutin and resveratrol.

Serial no	Amount of mixture taken	Amount of rutin added	% recovery	%RSD
1	5 µg/ml	1 µg/ml	111.263	0.991
2	5 µg/ml	2 µg/ml	108.436	1.010
3	5 µg/ml	3 µg/ml	107.202	0.984

Serial no	Amount of mixture taken	Amount of resveratrol added	% recovery	% RSD
1	5 µg/ml	1 µg/ml	102.002	0.865
2	5 µg/ml	2 µg/ml	104.241	0.984
3	5 µg/ml	3 µg/ml	102.292	1.006

Ruggedness:

Ruggedness was determined by carrying out analysis by two different analyst and the respective percentage recovery was noted and the results were indicated as % RSD.

Table-7: Results of Ruggedness of rutin and resveratrol.

Serial no		% recovery of rutin	% RSD	% recovery of resveratrol	% RSD
1	Analyst 1	101.106	1.070	104.269	0.565
2	Analyst 2	100.589	0.943	105.345	0.684

Table -8: Limit of Detection (LOD) and Limit of Quantification (LOQ).

Serial no	Name of the drug	257 nm	257 nm	306 nm	306 nm	267 nm	267 nm
Serial no	Name of the drug	LOD	LOQ	LOD	LOQ	LOD	LOQ
1	Rutin	0.08	0.242	-	-	0.042	0.129
2	Resveratrol	-	-	0.025	0.090	0.057	0.175

Specificity:

Specificity of the methods was achieved by the analysis of different laboratory prepared mixtures of rutin and resveratrol within the linearity range.

Table-9: Specificity of different laboratory prepared mixture.

Serial no

Ratio

Rutin: Resveratrol

% recovery of rutin

%RSD

% recovery of resveratrol

%RSD

2:1

109.864

1.064

102.639

1.003

1:2

98.443

0.843

100.210

0.728

Application of the proposed method for the determination of rutin and resveratrol in combined liposomes:

The drug content determination of the liposome formulation containing rutin and resveratrol was performed by using this UV spectrophotometric iso absorptive point method. The formulation contains 100 mg drug (rutin + resveratrol) in 30 ml, therefore 1 ml should contain 3.33 mg or 3300 µg/ml. From the 30 ml we had taken 1 ml of the formulation and diluted upto 100 ml and observed the absorbance.. The absorbance was used to calculate % drug content applying the equation of rutin and resveratrol at iso absorptive pointlation.

The absorbance was found to be 0.5404. So for rutin and resveratrol it was found to be 1781 µg/ml and 1375 µg/ml. Therefore total amount of drug present in the formulation was 3156 µg/ml. Therefore, percent drug content = (3156*100/3300) = 95.63%

CONCLUSION:

The UV spectrophotometric absorption ratio or iso absorptive point method was developed and validated for the simultaneous estimation of Rutin and Resveratrol. The method was validated as per ICH guidelines. The results together established that the method is simple, accurate, precise, reproducible, rapid, and sensitive. The standard deviation and % RSD calculated by this method is <2, indicating high degree of precision of the method. The method could be applied successfully and economically for the simultaneous estimation of Rutin and Resveratrol for the combination formulations of these two drugs in the future.

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 07.06.2018 Accepted on 22.08.2018

Asian J. Pharm. Ana. 2018; 8(3): 147-152.

DOI: 10.5958/2231-5675.2018.00027.3