INTRODUCTION:

Flavonoids are the polyphenolic compounds, present abundantly in various plants, and majorly exhibit activities like anti-oxidant, anti-inflammatory and anti-tumour. Fisetin (3,3’, 4’7-tetrahydroxyflavone) is one of the flavonoids belongs to the category flavanol. It is a pale-yellow powder having molecular weight 286.26 g/mol and melting point 330°C (fig. 1). It is soluble in methanol, acetone and acetic acid but very slightly soluble in water.

It is present in various fruits and vegetables and the highest concentration is reported in strawberries (160 µg/g) followed by apple (26.9µg/g) and persimmons (10.5µg/g)^1. It possesses pharmacological activities like antioxidant, anti-inflammatory, hypolipidemic, anti-adipocyte differentiation, inhibition of allergic airway inflammation^2,3,4,5, neurotropic activity⁶ and anti-depressant⁷. Despite having various pharmacological properties, there are some limitations like low aqueous solubility and oral bioavailability. As per literature, liquid self nano emulsifying drug delivery system (L-SNEDDS) are helpful to improve dissolution rate and bioavailability of poorly water-soluble drugs. SNEDDS are the homogenous mixture of oil, surfactant and co-surfactant in which drug is dissolved, which upon ingestion in the stomach produces oil in water micro or nano-emulsion. The nano size of droplets provide greater surface area for the absorption of the entrapped drug^8,9.

In past various analytical methods have been developed and reported for estimation of fisetin. Moolakkadath developed HPTLC method for the evaluation of fisetin in active pharmaceutical ingredients and marketed capsule formulation. Toluene, ethyl acetate, formic acid and methanol in the ration of (3:5.5:1:0.5 v/v/v/v) were used as mobile phase¹⁰. Jo developed and validated a method for the evaluation of fisetin in mouse plasma using LC-MS/MS using 0.1%v/v acetonitrile as solvent A and 01 % v/v formic acid in water as solvent B¹¹. In another study by Bouzid, HPLC method was developed to estimate the amount of fisetin present in the extract of Arbutus using methanol and water in the ratio of 50:50 (pH 2.8)¹². Similarly, another HPLC method was developed by Touil for evaluation of pharmacokinetic parameters of fisetin in mice plasma and tumour cells using 52% v/v methanol and 2% v/v acetic acid as mobile phase at 1ml/min flow rate and uses 360 nm wavelength for detection¹³. In another study LC/MS method was developed for estimation of fisetin in extract of Glycyrrhiza glabra¹⁴. The methods reported above are very sensitive methods. However, they are expensive and require skilled person to operate the instruments. So, there is a need to develop simple, cost-effective and sensitive UV method for estimation of fisetin in pharmaceutical samples. Hence, a simple, cost effective and validated analytical method for the estimation of fisetin has been developed by UV spectrophotometer.

Figure 1: Structure of fisetin

MATERIALS AND METHODS:

Materials:

Fisetin was purchased from the Tokyo Chemical Industries, Japan. Methanol was purchased from the LOBA Chemie Mumbai, India. Castor oil, Tween 80 were purchased from CDH, Mumbai, Lauroglycol and Transcutol were provided as gift samples from Gattefosse Company. The analytical method has been developed using double beam UV–Visible spectrophotometer (Shimadzu 1800, Japan).

Method Development:

A stock solution of fisetin was prepared by dissolving 10 mg of drug in 10mL of methanol which was further diluted to give a solution of concentration 100µg/mL. For the development of calibration curve, working solutions of fisetin having different concentration (4, 6, 8, 10, 12µg/mL) was prepared by withdrawing suitable aliquots from the standard stock solution. The spectrum was scanned and overlay was recorded. The study was carried out in triplicate and mean data was recorded.

Method Validation:

For the validation of the method, recovery of the compound was determined in triplicate at three concentration levels i.e. 80%, 100 % and 120% of the median concentration (8µg/mL). For this first stock solution of 100µg/mL was prepared, from this further dilution of 20µg/mL was prepared. From the diluent 20 µg/mL solution suitable volumes were withdrawn in order to prepare the working solution of final concentration that is 6.4, 8 and 9.6µg/mL which eventually represents the 80%, 100% and 120% of the mid value of the method concentration. The precision study was performed at intermediate days and by inter analyst.

Estimation of LOD and LOQ:

LOD and LOQ were determined by standard deviation of response (sigma) and slope of calibration curve (S). Standard deviation of Y intercepts of regression line was used as standard deviation. Eq. 1 and 2 for LOD and LOQ, respectively, are as follow:

LOD=3.3σ/S (1)

LOQ=10 σ/S (2)

Development of formulation:

To develop the formulation, in a clean glass vial 120µL of castor oil, 80µL of lauroglycol FCC, 450µL of tween 80 and 350µL of Transcutol P were added and mixed well. To this homogenous mixture 5mg of fisetin were added and vortexed to mix well. Now, the prepared formulation was stored for further evaluation^8,9.

Calculation of drug loading:

L-SNEDDS were prepared by adding 5mg of fisetin selected batch containing 1 mL mixture of castor oil, lauroglycol, Tween-80, and Transcutol® P. These were vortexed using vortex mixer for 15 min, and then, added to 500mL of double distilled water being stirred at 500g at a temperature of 37°C. Sample (5mL) was withdrawn and centrifuged at 10000g for 15 min for removal of the undissolved fisetin. The absorbance of supernatants were accurately measured and fisetin concentration was estimated by using calibration curve^8,9.

The percentage drug loading was calculated as per the following formula

RESULTS:

All the prepared dilutions were scanned in the range of 200–400nm. The absorbance maxima of all the dilutions was found at 362nm. The overlay of their spectra is shown in fig. 2. The developed method was validated as per ICH Q2 (R1) guidelines^15,16.

Figure 2: Overlay of different concentration of fisetin in methanol

Accuracy:

The determination of accuracy was done measuring the drug recovery in triplicate at three levels 80%, 100% and 120% of the median concentration (8µg/mL). The mean recoveries of fisetin expressed in term of the percentage recovery (table 1). The percentage relative standard deviation was found to be less than 2% which indicate that the data was precised. Moreover, the % recovery was found more than 95% in all cases.

Linearity:

Figure 3: Calibration curve of fisetin

The calibration curve was plotted by analysing the samples of concentration 4, 6, 8, 10, 12 µg/mL at 362 nm. The study was carried out in triplicate and the data was found linear with correlation coefficient of 0.999 fig. 3. The regression equation obtained was Y= 0.0753x+0.0067.

Precision:

Precision was evaluated with respect to repeatability (inter day) and intermediate precision. Repeatability was tested by six determination at three levels 80%, 100% and 120% of the median concentration that is 8 µg/mL. For intermediate repeatability the study was performed by two other analysts at three different days at three levels of 80%, 100% and 120% respectively. The percentage of relative standard deviation of responses were calculated and found to be less than 2%. The results are shown in (table 1).

Table 1: Method Validation Data

Interday Repeatibility	Day	Conc. (µg/ml)	Absorbance						Mean	STD	SEM	% Recovery	%RSD
	Day 1	Conc. (µg/ml)	1	2	3	4	5	6	Mean	STD	SEM	% Recovery	%RSD
		6.4	0.483	0.49	0.491	0.486	0.486	0.504	0.490	0.007	0.003	100.286	1.522
		8	0.606	0.615	0.616	0.612	0.6	0.63	0.613	0.010	0.004	100.675	1.665
		9.6	0.713	0.73	0.738	0.732	0.709	0.745	0.728	0.014	0.006	99.758	1.938
	Day 2	6.4	0.521	0.511	0.513	0.517	0.519	0.512	0.516	0.004	0.002	105.578	0.793
		8	0.632	0.636	0.634	0.635	0.642	0.639	0.636	0.004	0.001	104.521	0.568
		9.6	0.705	0.729	0.741	0.711	0.713	0.709	0.718	0.014	0.006	98.398	1.944
	Day 3	6.4	0.511	0.509	0.499	0.489	0.497	0.501	0.501	0.008	0.003	102.569	1.617
		8	0.626	0.635	0.629	0.63	0.622	0.625	0.628	0.005	0.002	103.110	0.722
		9.6	0.711	0.721	0.719	0.73	0.707	0.716	0.717	0.008	0.003	98.306	1.124
Inter analyst repeatibility, Analyst 1	Day 1	6.4	0.466	0.47	0.469	0.467	0.471	0.46	0.467	0.004	0.002	95.548	0.850
		8	0.592	0.589	0.591	0.589	0.601	0.608	0.595	0.008	0.003	97.659	1.306
		9.6	0.689	0.687	0.692	0.697	0.711	0.721	0.700	0.014	0.006	95.839	1.943
	Day 2	6.4	0.459	0.466	0.468	0.46	0.468	0.472	0.466	0.005	0.002	95.203	1.085
		8	0.574	0.579	0.581	0.592	0.577	0.585	0.581	0.006	0.003	95.391	1.102
		9.6	0.692	0.694	0.709	0.701	0.721	0.719	0.706	0.012	0.005	96.738	1.755
	Day 3	6.4	0.478	0.483	0.487	0.493	0.487	0.495	0.487	0.006	0.003	99.698	1.288
		8	0.61	0.615	0.623	0.63	0.624	0.632	0.622	0.009	0.003	102.197	1.366
		9.6	0.705	0.716	0.711	0.721	0.719	0.722	0.716	0.007	0.003	98.075	0.917
Inter analyst repeatibility, Analyst 2	Day 1	6.4	0.459	0.452	0.468	0.472	0.466	0.473	0.465	0.008	0.003	95.099	1.742
		8	0.587	0.581	0.602	0.577	0.586	0.589	0.587	0.009	0.003	96.331	1.458
		9.6	0.711	0.725	0.719	0.732	0.731	0.73	0.725	0.008	0.003	99.320	1.140
	Day 2	6.4	0.461	0.468	0.473	0.476	0.477	0.481	0.473	0.007	0.003	96.690	1.518
		8	0.573	0.582	0.59	0.591	0.594	0.597	0.588	0.009	0.004	96.470	1.504
		9.6	0.735	0.714	0.711	0.726	0.717	0.71	0.719	0.010	0.004	98.513	1.361
	Day 3	6.4	0.496	0.487	0.502	0.499	0.508	0.495	0.498	0.007	0.003	101.912	1.423
		8	0.616	0.598	0.622	0.631	0.629	0.628	0.621	0.012	0.005	101.920	1.996
		9.6	0.717	0.703	0.725	0.735	0.728	0.729	0.723	0.011	0.005	99.067	1.571

STD: Standard deviation, RSD: Relative standard deviation, SEM: Standard error mean

LOD, LOQ and Drug loading:

The LOD was found to be 0.28 and LOQ was found to be 0.86 µg/mL. The above developed and validated method was used to calculate the concentration of fisetin in the sample volume, which further utilized for the estimation of the drug loading. The drug loading in the formed L-SNEDDS was found to be 93.66%.

DISCUSSION:

A simple. Sensitive and cost effective method for the estimation of fisetin was developed and validated as per the ICH guidelines¹⁵. The validation data represents that the method was accurate, precise and robust. The method further used for the estimation of fisetin in the formulated L-SNEDDS, which reflects that method was specific for the fisetin and there is no interference of the excipients of the formulation. The L-SNEDDS shows good drug loading, which will help to reduce the dose of fisetin as it will also improve the bioavailability. Various other methods were also reported for the estimation of fisetin, but the developed method was based on the use of economical instrument and to reduce the time of analysis.

CONCLUSION:

The present study described successfully development and validation of simple, cost effective and sensitive UV method for estimation of fisetin in pharmaceutical dosage form, with good linearity, accuracy and precision and its application in the estimation of fisetin in the L-SNEDDS.

CONFLICT OF INTEREST:

Declared none.

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Received on 16.07.2019 Modified on 10.09.2019

Research J. Pharm. and Tech 2020; 13(3):1179-1182.

DOI: 10.5958/0974-360X.2020.00217.6