Water Solubility and Dissolution Enhancement of Fisetin by Spherical Amorphous Solid Dispersion in Polymer of Cyclodextrin

Research Article

Austin J Biotechnol Bioeng. 2021; 8(1): 1106.

Water Solubility and Dissolution Enhancement of Fisetin by Spherical Amorphous Solid Dispersion in Polymer of Cyclodextrin

Skiba M*, Gasmi H, Milon N, Bounoure F and Lahiani-Skiba M

Normandy University, UNIROUEN, France

*Corresponding author: Skiba M, Normandy University, UNIROUEN, DC2N INSERM U1239-Galenic Pharmaceutical Team, UFR of Health, 22 boulevard Gambetta 76000 Rouen, France

Received: March 08, 2021; Accepted: March 29, 2021; Published: April 05, 2021


Fisetin (3, 7, 3', 4'-tetrahydroxyflavone) is an active ingredient characterized by a large spectrum of biological activities with a wide range of nutraceutical properties, including neuroprotecting, antidiabetic and suppression or prevention of tumors. The only drawback limiting its use is its low water solubility. The present work was focused on the solubility improvement of fisetin using spray-drying solid dispersion method, using also cyclodextrin polymers and which is extensively employed as technique to enhance poorly-water soluble compounds. Fisetin solubility was evaluated in the presence of poly-aβ-CD, poly-aγ-CD, poly-aβγ-CD and poly-methyl-β-CD by Higuchi solubility study. Spherical Amorphous solid dispersion formulations were prepared using polymethyl- β-CD (choice of the polymer was based on the better results of solubility study) at different ratios drug: polymer (1:1) and (1:3). In addition, different formulations were investigated using several techniques such as: Fourier Transformed Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), NMR and Scanning Electron Microscopy (SEM). The in vitro dissolution was performed in water at 37°C. The results of dissolution studies showed a complete dissolution observed with the formulation ratio 1:3 while the formulation ratio 1:1 showed a maximum dissolution rate about 59%. This difference is due to the formation of watersoluble inclusion complex and on the other hand to the high hydrophilicity of the cyclodextrin polymer.

Keywords: Fisetin; Cyclodextrin-polymer; Spherical amorphous solid dispersion; Spray-drying


Fisetin (3, 7, 3', 4'-tetrahydroxyflavone) is a flavonol that belongs to the flavonoid group of polyphenols which is present in some fruits and vegetables [1] including strawberries [2], apples [3] and grapes [4]. It is characterized by a large spectrum of biological activities, which include: anti-inflammatory [5], antioxidant [6], apoptotic and anti-proliferative [7,8] action. Recently, fisetin has shown anti-cancer activity, in studies performed in vitro and in vivo, against several cancer types such as: lung cancer [9], prostate cancer [10], colon cancer [11] and breast cancer [12]. Fisetin is Class II drug according to BCS classification, its low solubility (<1 mg/mL) [13] and its low bioavailability are the limiting factors of its administration in vivo. To improve the dissolution rate of poorly water soluble drugs, several techniques were used such as particle size reduction (formation of micro- and nano-systems), formation of water soluble complexes [14] and amorphous solid dispersion preparations [15].

The solid dispersion technique is one of the most strategies used to improve oral bioavailability of poorly-water soluble drugs. It offers several advantages for enhancing drug solubility such as: (i) reduced particle size and thus the surface area is improved and increased dissolution rate ; (ii) improved wettability and this parameter dependent on the characteristic of drug carrier; (iii) transferring the drug from crystalline to amorphous state [16]. Various solid dispersion preparations of hydrophilic carrier/poorly water soluble drugs demonstrating the improvement of its solubility have been reported in the literature. Borba et al. showed that sodium alginate was successfully used as carrier in solid dispersion and demonstrated to be able to improve the apparent solubility of telmisartan (up to 16.7 fold) [17]. Another study was performed by Yuvaraja et al. which consists of using native cyclodextrins in solid dispersion formulation to enhance the solubility of carvedilol [18].

In the case of fisetin, several studies have been made to improve its water solubility and therefore its bioavailability. Among the latter, the nanoemulsion formation [19], liposome formulation [20,21], complexation with dimer of cyclosophoroase [22], cocrystallization with another substance [23] and formation of inclusion complex with natural cyclodextrins [24,25].

Native cyclodextrins a, β and γ are cyclic oligosaccharides derived primarily from starch by enzymatic degradation of this later and constituted by six, seven and eight glucopyranoside units; respectively. Cyclodextrins have a truncated conical shape and characterized by a hydrophilic outer surface and a hydrophobic internal cavity thus able to host other hydrophobic molecules forming inclusion complexes. The formation of the inclusion compounds greatly modifies the physical and chemical properties of the guest molecule, mostly in terms of water solubility. This is the reason why cyclodextrins have attracted much interest in many fields, especially pharmaceutical applications [26]. In the literature, some published papers have reported the interaction between fisetin and native cyclodextrins (mainly β and γ cyclodextrins) [24,27].

The aim of this work was to improve the solubility and the dissolution rate of fisetin by preparing amorphous solid dispersion using new polymers based on cyclodextrins (poly-aβ-CD, poly- aγ-CD, poly-aβγ-CD and poly-methyl-β-CD) as a hydrophilic carrier, which is characterized by higher water solubility and greater complexing property than nature cyclodextrins. The physicochemical characterizations were performed using different techniques such as DSC, TGA, FT-IR spectroscopy, NMR and SEM. The in vitro dissolution was performed in water at 37°C to evaluate the efficacy of the polymers on fisetin release. The Sink conditions were provided throughout the experiments.

Experimental Methods


Native cyclodextrins a, β and γ were ordered from Wacker (France). Citric acid and sodium phosphate dibasic were supplied by Sigma Aldrich (France). Fisetin was received from BiosynTec (China). Ethanol was obtained from VWR (France).

Preparation of co- and terpolymer Poly-aβ-CD, Poly-aγ- CD, poly-aβγ-CD and poly-methyl-β-CD

The copolymer Poly-aγ-CD was synthesized according to M.Skiba [28]. Briefly, a mixture of known amount (w : w) of cyclodextrin a, γ, citric acid and sodium phosphate dibasic was transferred into a reactor which was maintained at temperature ranging between 140- 150 °C for 15 to 30 min. the obtained solid form was dissolved in water and dialyzed using polyether sulfate membrane filter with molecular weight cut of 10000 Da. The dialysis was controlled by measuring the conductivity of the purified water at T0 and after 4 hours of dialysis, the resulted solution was spray-dried using Büchi Mini Sprayer Dryer B-290. Spray-dryer parameters were validated by preliminary works and were as follow: inlet temperature: 130°C; outlet temperature 80- 85 °C; aspiration 70%; pump: 10% and pressure: (-45 mbar). The same procedure was used to synthesis the poly-aβ-CD, poly-aβγ-CD and poly-permethyl-β-CD.

Phase solubility study

Phase solubility studies of fisetin were performed according to the method reported by Higuchi and Connors. Excess amounts of fisetin were added to amber glass flasks containing 5 mL of an aqueous solution of increased concentration of cyclodextrin copolymer: poly-aβ-CD, poly-aγ-CD, poly-aβγ-CD and poly-permethyl-β-CD, agitated in a thermostatically water bath maintained at 37±0.2 °C during 7 days. Samples were centrifuged, diluted and analyzed by UV-spectrophotometry (λ=360 nm; UV-1600 PC).

Solid dispersion preparation

A Büchi 290 nozzle type mini spray dryer (Flawil, Switzerland) was used for the preparation of fisetin-loaded solid dispersion using cosolvent method. Two solid dispersions were prepared with 1 g of fisetin and 1-3 g of poly-methyl-β-CD polymer in the weight ratio 1:1 and 1:3. Fisetin was dissolved in 230 mL of ethanol and the polymer was dissolved in 460 mL of water. The two solutions were mixed during 1h and delivered to the nozzle with 1.4 mm diameter, flow rate of pump at 15 % and spray-dried at 95±1 °C inlet temperature and 55±1 °C outlet temperature. The flow rate of the drying air was maintained at the aspirator setting of 80 % which indicated the pressure of the aspirator filter vessel as -45 mbar. The direction of air flow was the same as that of sprayed solution.

Preparation of physical mixture

The Physical Mixture (PM) was prepared through simple homogenization of fisetin and polymer powders with a mortar for 10 min in the ratio of 1:1 and 1:3 (w/w) until obtaining an apparent homogeneous powder and preserved in closed glass flasks.

Characterization of solid dispersions

Thermogravimetry analysis: The thermal stability of complexe fisetin/poly-methyl-β-CD, polymers (poly-methyl-β-CD) and also the physical mixtures were investigated using TGA 4000 thermogravimetric analyzer (TA instrument, PerkinElmer, USA). 1-3 mg of sample was heated at a rate of 10°C/min from 30°C to 400°C under dynamic nitrogen atmosphere. The upper limit of thermal stability of samples was taken as the initial sample weight loss.

Differential scanning calorimetry: Thermal analysis was conducted on PerkinElmer 6 DSC (USA). Samples (5-6 mg) were hermetically sealed in a flat-bottomed aluminum pan and heated from 30 to 400°C at a scanning rate of 10°C/min under a nitrogen gas flow. An empty aluminum pan was used as reference. The DSC cell was calibrated with indium (Tpeak 156°C) and zinc (Tpeak 419°C).

Fourier transformed infrared spectroscopy: The FT-IR spectra of fisetin, polymers, physical mixture and solid dispersion were acquired on a PerkinElmer SpectrumTM One Fourier-Transform Infrared (FT-IR) Spectrophotometer. Samples were scanned at room temperature from 4000 to 450 cm-1 and at a spectral resolution of 2 cm-1 and 10 scans were performed for each sample.

Scanning electron microscopy: The external morphology of solid dispersion formulations was studied using a FEI QUANTA 200 scanning electron microscope. Samples were fixed with a ribbon carbon double-sided adhesive and covered with a fine layer of carbon and analyzed.

NMR spectroscopy: 1H NMR spectra was used to determine the chemical shift changes of drug protons resulting of its interaction and insertion into the hydrophobic cavity of cyclodextrin. It was recorded at 22°C on a Bruker AVANCE III 400 NMR spectrometer using a presaturation of the water resonance, 32 K data points, a recycling delay of 2 s and a spectral width of 10 ppm. Samples were prepared by dissolving 1 to 2 mg of solid samples in Deuterated Water (D2O). Setting the water resonance at 4.75 ppm referenced the chemical shifts. 1H-NMR peak assignment was established from published data and by using basic correlation experiments.

In vitro dissolution

The dissolution study was performed in USP type II dissolution apparatus II (Vankel, VK7000). Briefly 22 mg of free fisetin or its equivalent of fisetin-loaded spray-dried dispersion formulations and physical mixtures were added to vessel containing 1 L of water at 37±0.2 °C with paddle speed of 100 rpm for 90 min. Two milliliters sample was withdrawn, replaced with fresh medium and then analyzed using UV-spectrophotometry (λ=360 nm; UV-1600 PC). Each experiment was conducted in triplicate and Sink conditions were provided throughout the experiments.


Phase solubility studies

The phase solubility diagram at 37°C was obtained by plotting the equilibrium concentrations of fisetin against polymer cyclodextrins concentrations. Its solubility profile in various concentrations of cyclodextrin co- and ter-polymers was significantly increased as it was shown in Figure 1. A better result was observed with polymethyl- β-CD and poly-aγ-CD. According to Higuchi an Connors phase-solubility diagram classification [29], the solubility diagram of the complex fisetin/poly-methyl-β-CD and fisetin/ poly-aγ-CD can be classified as type An which is characterized by the formation of soluble inclusion complex between the guest and the host molecules and it was present in the aqueous solution [30]. The slope value was calculated from the linear plot of the phase solubility diagrams of poly-methyl-β-CD and poly-aγ-CD; it was equal to 0.015 and 0.013; respectively. Moreover, the slope values were lower than one which indicate that the inclusion complex was about 1:1 molar ratio between the fisetin and the cyclodextrin polymers, as it was reported by Doile et al. [31]. The fisetin solubility in water was increased to 3.71 mg/ mL and 2.96 mg/mL so 42-fold and 33-fold when poly-methyl-β-CD and poly-aγ-CD; respectively; were used in concentration of 800 mg/ mL compared to its initial solubility in water without CDs which is around 89 μg/mL. According to these results, poly-methyl-β-CD was chosen to be used for the preparation of solid dispersion formulations at different ratios.