Self-nanoemulsifying drug delivery system of sinapic acid: In vitro and in vivo evaluation

Journal of Molecular Liquids 224 (2016) 351–358

Contents lists available at ScienceDirect

Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Self-nanoemulsifying drug delivery system of sinapic acid: In vitro and in vivo evaluation Faiyaz Shakeel a,b,⁎, Mohammad Raish b, Md. Khalid Anwer c, Ramadan Al-Shdefat c a b c

Deanship of Scientific Research, King Saud University, Riyadh 11451, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 23 August 2016 Received in revised form 3 October 2016 Accepted 4 October 2016 Available online 06 October 2016 Keywords: Antioxidant activity Anti-inflammatory effects Self-nanoemulsifying drug delivery system Sinapic acid

a b s t r a c t Sinapic acid (SA) has been reported as a poorly soluble bioactive compound due to its dissolution rate and oral bioavailability, which are very poor. Therefore, in the current research work, different self-nanoemulsifying drug delivery systems (SNEDDS) of SA were developed for enhancement of its in vitro dissolution (drug release) and bioactivity. Different SA-loaded SNEDDS were prepared by low-energy emulsification methods, characterized and evaluated for various physicochemical and in vitro parameters such as thermodynamic stability, selfnanoemulsification efficiency, droplet size, polydispersity index (PDI), zeta potential (ZP), refractive index (RI), the % of transmittance (% T) and drug release profile. In vitro dissolution rate of SA was significantly enhanced from SNEDDS in comparison with SA suspension. The optimized SNEDDS of SA with droplet size of 12.4 nm, PDI value of 0.158, ZP value of − 32.8 mV, RI value of 1.335, % T value of 98.7% and drug release profile of 96.8% was selected for in vitro antioxidant and in vivo anti-inflammatory studies. The 2,2-diphenyl-1picrylhydrazyl (DPPH) scavenging assay indicated significant antioxidant activity of optimized SA SNEDDS (IC50 = 4.52 μg/ml) in comparison with free SA (IC50 = 7.62 μg/ml) and standard ascorbic acid (IC50 = 40.78 μg/ml). In vivo anti-inflammatory studies in rats also indicated that SA in optimized SNEDDS was significantly efficacious than SA suspension. Free SA as well as SA in SNEDDS was proved to be an excellent antioxidant in comparison with standard ascorbic acid. The results obtained in the current research work indicated the great potential of SNEDDS in the enhancement of in vitro dissolution rate and therapeutic efficacy of poorly soluble bioactive compounds such as SA. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Sinapic acid (SA) is a potent nutraceutical or natural bioactive compound which is commonly present in the human diet [1,2]. It is a derivative of hydroxycinnamic acid (HCA) which has been reported as the potent antioxidant by many researchers [3–7]. Its antioxidant efficacy has been considered superior to that of ferulic acid, a HCA derivative already reported as a natural antioxidant [1,3,8] and comparable to that of caffeic acid (another HCA derivative) [4,9,10]. It is obtained from various plant sources such as rye, fruits and vegetables [11–13]. SA showed the variety of therapeutic activities including anti-inflammatory [13], antioxidant [14,15], antibacterial [16–18], antimicrobial [19,20], antianxiety [21], cardioprotective [2,22,23] and antitumor activity [24]. The antioxidant and anti-inflammatory activities of SA have been well reported in the literature [13,25]. In the literature, the mechanistic anti-inflammatory effects of SA were investigated by Yun et al. They indicated that anti-inflammatory effects of SA in rats and mice were possible due to ⁎ Corresponding author at: Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (F. Shakeel).

http://dx.doi.org/10.1016/j.molliq.2016.10.017 0167-7322/© 2016 Elsevier B.V. All rights reserved.

inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and proinflammatory cytokines inductions [13]. SA is a poorly soluble bioactive compound in water (mole fraction solubility has been reported as 6.22 × 10−5 at 25 °C) due to its in vitro dissolution and oral bioavailability, which are expected to be poor [26]. The maximum plasma concentration of SA has been reported as 40 nM with bioavailability of 3% of total phenolics [27,28]. Recently, self-nanoemulsifying drug delivery systems (SNEDDS) have been reported as potential colloidal drug carriers for enhancing solubility/in vitro dissolution, bioavailability and bioactivity of various poorly soluble drugs or natural bioactive compounds [29–38]. SNEDDS/nanomulsions are prepared using low-energy emulsification (aqueous phase titration and phase inversion temperature methods) and high-energy emulsification techniques (high-pressure homogenization, microfluidization and ultrasonication) [30,39]. Aqueous phase titration methods offer several advantages over high-energy methods such as ease of preparation, low cost and high stability, and hence this method was applied in this study [31]. SNEDDS were selected in this study because they are more stable than other colloidal dispersions such as emulsions and suspensions [30,38]. Moreover, they are known to enhance the solubility of poorly soluble compounds due to their

352

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358

lower droplet size, higher surface area and the presence of solubilizers such as surfactants and cosurfactants [36–38]. In spite of several bioactivity of SA, its formulations approach had rarely been reported in the literature for solubilization and bioactivity/ bioavailability enhancement. Moreover, the antioxidant and anti-inflammatory effects of SA have not been investigated in the literature via NEDDS of SA. Therefore, in the current research work, various SNEDDS formulations of SA were developed by low-energy emulsification/aqueous phase titration technique in order to enhance its in vitro antioxidant and in vivo anti-inflammatory effects. Different SNEDDS of SA were developed using safe and non-toxic components such as Triacetin® (oil phase), Triton-X100® (Surfactant), Carbitol® (cosurfactant) and ultra-pure water (aqueous phase). 2. Experimental 2.1. Materials SA, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 1-butanol, 2-butanol, isopropyl alcohol (IPA), Cremophor-EL® (polyoxyl-35 castor oil), Tween20® [polyoxyethylene (20) sorbitan monolaurate] and Tween-80® [polyoxyethylene (20) sorbitan monooleate] were obtained from Sigma Aldrich (St. Louis, MO). Triacetin® (glycerol triacetate) was procured from Alpha Chemica (Mumbai, India). Labrafil-M1944CS® (oleoyl macrogol-6-glyceride), Capryol-90® (propylene glycol monocaprylatetype II), Capryol-PGMC® (propylene glycol monocaprylate-type I), Lauroglycol-90® (propylene glycol monolaurate-type II), LauroglycolFCC® (propylene glycol monolaurate-type I), Labrafac-PG® (propylene glycol dicaprylocaprate), Labrasol® (caprylocaproyl macrogol-8-glyceride) and Carbitol® (diethylene glycol monoethyl ether) were obtained from Gattefossé (Lyon, France). Triton-X100® (isooctylphenoxypolyethoxyethanol), castor oil and olive oil were obtained from BDH Laboratories (Liverpool, UK). Propylene glycol (PG), ethylene glycol (EG) and polyethylene glycol-400 (PEG-400) were obtained from E-Merck (Berlin, Germany). Ready-to-use dialysis bags (molecular weight cutoff 12,000 g/ mol) were obtained from Spectrum Medical Industries (Mumbai, India). Ultra-pure water was obtained from Milli-Q water purification unit. 2.2. Screening of components for SA SNEDDS development The most important criterion for the screening of components is the solubility of drug molecule in different components [37,38]. Therefore, various components were selected on the basis of solubility profile of SA. Therefore, the equilibrium solubility of SA in different oils (Triacetin®, olive oil, castor oil, Capryol-90®, Capryol-PGMC®, Labrafac-PG®, Lauroglycol-90®, Lauroglycol-FCC® and Labrafil-M1944CS®), different surfactants (Tween-80®, Tween-20®, Triton-X100®, Labrasol® and Cremophor-EL®), different cosurfactants (ethanol, PG, PEG-400, EG, IPA, 1-butanol, 2-butanol and Carbitol®) and water was determined by an isothermal method reported in the literature [40]. For the solubility determination, the excess quantity of SA was added in 1.0 g of each component in 4.0 ml capacity glass vials in triplicates. The glass vials were transferred to a biological shaker (Julabo, MA) for continuous shaking at the shaking speed of 100 rpm at 25 ± 0.5 °C for 3 days to reach equilibrium [26]. After 3 days, all the samples were taken out from the shaker and allowed to settle SA particles overnight. The supernatants were carefully taken from each sample and subjected for the quantification of SA content spectrophotometrically at 322 nm [26].

use of water as the aqueous phase in previous literature [36–38]. In order to identify infinitely diluted SNEDDS zones for SA, pseudo-ternary phase diagrams were constructed by low-energy emulsification/aqueous phase titration method [38]. Surfactant (Triton-X100) and cosurfactant (Carbitol) were mixed in different mass ratios such as 1:2, 1:1, 2:1 and 3:1. Triacetin (oil phase) and a specific mass ratio of Triton-X100 to Carbitol (Smix) were further mixed in the mass ratios of 1:9 to 9:1. The mixture of Triacetin and a specific Smix were then titrated by slow addition of ultra-pure water. Pseudo-ternary phase diagrams were then constructed for a specific Smix ratio. The physical state of infinitely diluted SNEDDS was marked on a phase diagram of each Smix ratio by representing the different components in three different axis of phase diagram. The highest infinitely diluted SNEDDS zones were recorded for 1:1 Smix ratio; therefore, different SNEDDS of SA were prepared using 1:1 Smix ratio. Hence, different SNEDDS of SA with formulation codes of SA1–SA5 were selected precisely from phase diagram. With respect to oil phase concentration, the entire range of infinitely diluted SNEDDS zones in the phase diagram were considered and varied concentrations of Triacetin (8.0%, 12.0%, 16.0%, 20.0% and 24.0% w/w) with minimum Triton-X100 (18.0% w/w) and Carbitol (18.0% w/w) concentration were selected. A total of 40 mg of SA was dissolved in Carbitol due to highest solubility and other components such as Triacetin, Triton-X100 and ultra-pure water were added by vortexing at 1000 rpm at 25 ± 1 °C till clear and transparent SNEDDS of SA obtained [37,38]. The compositions of different SA SNEDDS coded as SA1–SA5 are presented in Table 1.

2.4. Thermodynamic stability and self-nanoemulsification efficiency of SA SNEDDS Developed SA SNEDDS (SA1-SA5) were subjected to different thermodynamic stability tests in order to remove unstable or metastable formulations. Three different thermodynamic tests, namely, centrifugation (at 500 rpm for 30 min), heating and cooling cycles (3 cycles between 45 °C and 25 °C) and freeze–pump–thaw cycles (3 cycles between −25 °C and 25 °C) were performed on SA SNEDDS by adopting the procedure given in our previously published articles [30–32]. Upon thermodynamic stability tests, formulations were observed visually for any phase separation, drug precipitation, coalescence and conversion into emulsion formations. Formulations which were recorded stable at centrifugation, heating and cooling cycles and freeze–pump–thaw cycles were further subjected for self-nanoemulsification test. The objective of this test was to investigate any phase separation or drug precipitation upon dilution with different diluents (aqueous media) such as water (simple diluent), 0.1 N HCl (to mimic stomach conditions) and phosphate buffer [pH 6.8] (to mimic intestinal conditions) [33,35]. In order to perform self-nanoemulsification efficiency test on SA SNEDDS, 1 ml of each formulation (SA1–SA5) was diluted sufficiently (1:500) with each diluent. Because 500 ml of dissolution media was used in drug release studies, 1:500 dilution ratios was used for this test in order to maintain similar conditions. The efficiency of each

Table 1 Formulation composition of SA SNEDDS (SA1–SA5) developed using Triacetin, TritonX100, Carbitol and water. SNEDDS composition (% w/w)⁎

2.3. Construction of pseudo-ternary phase diagrams and preparation of SA SNEDDS Based on the highest solubility of SA in different components, Triacetin, Triton-X100 and Carbitol were selected as oil phase, surfactant and cosurfactant, respectively, for the preparation of SA SNEDDS. Ultra-pure deionized water was used as aqueous phase due to frequent

Smix ratio

Code

Triacetin

Triton-X100

Carbitol

Water

SA1 SA2 SA3 SA4 SA5

8.0 12.0 16.0 20.0 24.0

18.0 18.0 18.0 18.0 18.0

18.0 18.0 18.0 18.0 18.0

56.0 52.0 48.0 44.0 40.0

⁎ Forty milligrams of SA was incorporated into each formulation.

1:1 1:1 1:1 1:1 1:1

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358

formulation was then investigated with the help of A–E grading systems as described below [30,31,38]: A: Rapidly forming clear nanoemulsions (which emulsified within 1 min) B: Rapidly forming slightly less clear nanoemulsions (which emulsified within 2 min) C: Milky emulsions (which takes N2 min for emulsification) D: Slowly forming dull/grayish emulsions (which takes N3 min for emulsification) E: Formulations with poor emulsification having large oil droplets at the surface (which takes N4 min for emulsification) Formulations which passed this test at grades A and B were selected for further evaluation. 2.5. Physicochemical investigation of SA SNEDDS Prepared SA SNEDDS were characterized in terms of various physicochemical parameters such as droplet size distribution, polydispersity index (PDI), zeta potential (ZP), refractive index (RI) and the percent of transmittance (% T). The mean droplet size and PDI of SA SNEDDS (SA1–SA5) were measured using Malvern Particle Size Analyzer (Malvern Instruments Ltd., Holtsville, NY) at room temperature (25 ± 1 °C) and scattering angle of 90° by adopting the procedure presented in our previous article [30]. The ZP value of SA SNEDDS (SA1–SA5) was determined using Malvern Zetazizer (Malvern Instruments Ltd., Holtsville, NY) by using the procedure presented in the literature [31]. The RI value of SA SNEDDS (SA1–SA5) was determined with the help of Abbes type Refractometer (Precision Testing Instruments Laboratory, Germany) at room temperature (25 ± 1 °C) by following the procedure reported in the literature [30,38]. The % T value of SA SNEDDS (SA1–SA5) was measured spectrophotometrically at 550 nm by following the procedure reported in the literature [30,31]. 2.6. In vitro release (dissolution) of SA In vitro drug release or dissolution studies on SA SNEDDS (SA1–SA5) were performed through ready-to-use dialysis bag in order to make comparison between the release profile of SA from different SNEDDS and its suspension formulation, all having 40 mg of SA [29]. In vitro drug release studies were performed in 500 ml of phosphate buffer (pH 6.8) [dissolution media] using United States Pharmacopoeia (USP) XXIV method [38]. These studies were carried out at 100 rpm and the temperature of the dissolution media was maintained at 37 ± 0.5 °C. During these studies, 1 ml of each formulation was transferred to a dialysis bag and immersed into dissolution media. Three milliliters of sample from each formulation were withdrawn at regular intervals of time and the same amount of SA-free fresh phosphate buffer (pH 6.8) was replaced at each time interval. The amount of SA in each sample was quantified spectrophotometrically at 322 nm [26]. The calibration curve was plotted between the concentration of SA and spectrophotometric absorbance. From the calibration curve, the amount of SA in dissolution media (released media) was determined at different time intervals. 2.7. Drug release kinetics for SA In the current research work, the analysis of kinetics of SA from different SNEDDS and SA suspension was carried out using different mathematical models (zero order, first order, Higuchi model, Hixon–Crowell model and Korsemeyer–Peppas model) [41–43]. These mathematical models are expressed using Eqs. (1)–(5) as described below: Zero order

Q t ¼ Q 0 þ K0t

ð1Þ

First order Higuchi

353

Log C ¼ Log C 0 −

K1t 2:303

Q t ¼ kt 0:5

Hixon−Crowell

ð3Þ

 1  1 W 30 −W 3t ¼ kh t

Korsemeyer−Peppas

ð2Þ

Qt −kp t n Q∞

ð4Þ ð5Þ

In which, Q0, Qt and Qω are the amounts of SA released initially, at time t and at time ω, respectively. However, C0 and C are the amounts of drug initially and at time t, respectively. W0 and Wt represent the amounts of the SA in SNEDDS formulations initially and at time t, respectively. K0, k1, k, kh and kp represent the rate constants for zero order, first order, Higuchi model, Hixon–Crowell model and Korsemeyer–Peppas model, respectively. The exponent n is the diffusion coefficient which is used in the characterization of the mechanism of drug release. 2.8. In vitro antioxidant assay On the basis of lowest droplet size (12.4 ± 1.04) nm, least PDI value (0.158), optimal ZP value (−32.8 mV), optimal RI value (1.335 ± 0.03), highest % T value (98.7 ± 0.2%) and highest drug release profile (96.8 ± 7.30% after 24 h), the SNEDDS SA1 was selected for in vitro antioxidant assay against DPPH scavenging activity. DPPH scavenging assay of SA suspension (free SA), optimized SNEDDS SA1 and standard ascorbic acid was determined by adopting the method reported by Ye et al. in the literature [44]. In order to conduct this assay, 100 μl of different concentrations of free SA (1–15 μg/ml), optimized SNEDDS SA1 (1–8 μg/ml) and ascorbic acid (1–80 μg/ml) were added to 3 ml of DPPH solution (0.3 mM). The respective mixtures were shaken at room temperature (25 ± 1 °C) for about 40 min. The spectrophotometric absorbance of each solution was measured at 517 nm [44]. The DPPH scavenging activity was determined by adopting the standard formula reported in the literature [44,45]. 2.9. In vivo anti-inflammatory effects In vivo anti-inflammatory studies were also performed on optimized SNEDDS SA1 in order to compare its anti-inflammatory effects with SA suspension and standard indomethacin. Twenty-four male Wistar rats (weighing from 200 to 250 g) were collected from the Animal Care and Use Center of College of Pharmacy at King Saud University, Riyadh, Saudi Arabia and institutional guidelines were strictly followed for these studies. All the rats were provided standard laboratory conditions of temperature and relative humidity. The animals were kept in plastic cages with free access to standard laboratory pellet diet and water ad libitum. The anti-inflammatory effects of SA suspension, optimized SNEDDS SA1 and standard indomethacin were evaluated by the carrageenan-induced hind paw edema method reported by Winter et al. 1965 [46]. The animals were randomly divided into 4 groups each containing 6 rats. Group I animals received carrageenan only and it was served as control. Group II, III and IV animals received SA suspension, optimized SNEDDS SA1 and standard indomethacin, respectively. Group II and III animals served as test groups while the group IV animals served as standard. SA suspension (30 mg/kg), optimized SNEDDS SA1 (30 mg/kg) and standard indomethacin (10 mg/kg) were administered orally half an hour before subplantar injection of carrageenan in the right paw. The control group received carrageenan only. Paw edema was induced by injecting 0.1 ml of a 1% w/v suspension of carrageenan which was prepared in ultra-pure water. The paw volume was measured at regular intervals of time (1, 2, 3, 4 and 6 h) after injection with the help of a digital plethysmometer (Ugo Basile, Italy). Percent inhibition of edema produced by each formulation-treated

354

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358 Table 2 Equilibrium solubility of crystalline SA in different components at 25 °C (n = 3). Components

Solubility ± SD (mg/g)

Triacetin® Castor oil Capryol-90® Olive oil Labrafil-M1944CS® Capryol-PGMC® Lauroglycol-90® Lauroglycol-FCC® Labrafac-PG® Tween-80® Tween-20® Labrasol® Cremophor-EL® Triton-X100® Carbitol® PEG-400 PG EG IPA 1-Butanol 2-Butanol Water

46.41 ± 3.74 4.13 ± 0.14 24.27 ± 2.56 6.02 ± 0.85 28.12 ± 3.04 27.12 ± 2.81 32.30 ± 2.91 30.08 ± 3.04 18.40 ± 1.95 39.31 ± 3.16 43.24 ± 4.84 24.15 ± 2.02 29.21 ± 2.69 63.42 ± 5.98 51.30 ± 3.80 33.17 ± 1.97 13.40 ± 0.83 21.50 ± 1.08 7.97 ± 0.52 10.80 ± 0.64 12.40 ± 0.76 0.77 ± 0.01

group was then calculated with respect to control group by adopting the standard formula reported in the literature [46,47].

2.10. Statistical analysis The results of this work were analyzed statistically by adopting the one-way analysis of variance (ANOVA) analysis followed by Dennett's test using GraphPad Instat software.

3. Results and discussion 3.1. Screening of components for SA SNEDDS development In the current research work, the solubility of SA in different components was the main criterion for the components screening [30–32]. The equilibrium solubility profile of solid SA in different components including ultra-pure water at 25 °C is listed in Table 2. It was observed that the highest solubility of solid SA was recorded in Triacetin (46.11 ± 3.74 mg/g) followed by Lauroglycol-90, Lauroglycol-FCC, Labrafil-M1944CS, Capryol-PGMC, Capryol-90, Labrafac-PG, olive oil and castor oil among different oil phases evaluated. However, the highest solubility of solid SA was recorded in Triton-X100 (63.42 ± 5.98 mg/g) followed by Tween-20, Tween80 and Labrasol among different surfactant molecules evaluated. While the highest solubility of solid SA was recorded in Carbitol (51.30 ± 3.80 mg/g) followed by PEG-400, EG, PG, 2-butanol, 1-butanol and IPA among different cosurfactants evaluated. The equilibrium solubility of solid SA in ultra-pure water was recorded as 0.77 ± 0.01 mg/g. Based on the results presented in Table 2, Triacetin, Triton-X100 and Carbitol were selected as oil phase, surfactant and cosurfactant, respectively. However, ultra-pure water was selected as the aqueous phase.

3.2. Construction of pseudo-ternary phase diagrams and preparation of SA SNEDDS For the preparation of SA SNEDDS, pseudo-ternary phase diagrams were constructed separately for each Smix ratio and results are presented in Fig. 1. The Smix ratio of 1:0 was not investigated in this work because most of the formulations prepared by this Smix ratio have been reported unstable or metastable in the literature [31–38]. From the

Fig. 1. Pseudo-ternary phase diagrams developed by low-energy emulsification/aqueous phase titration method showing infinitely diluted SNEDDS zones for oil phase (Triacetin)], aqueous phase (water), surfactant (Triton-X100) and cosurfactant (Carbitol) at Smix ratios of (A) 1:2; (B) 1:1; (C) 2:1 and (D) 3:1.

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358 Table 3 Physicochemical investigation of SA SNEDDS (SA1–SA5). Characterization parameters Code

Droplet size ± SD (nm)

PDI

ZP (mV)

RI ± SD

% T ± SD

SA1 SA2 SA3 SA4 SA5

12.4 ± 1.04 20.8 ± 1.9 32.3 ± 2.8 54.8 ± 3.9 94.3 ± 8.7

0.158 0.195 0.211 0.242 0.258

−32.8 −33.7 −34.6 −37.3 −39.4

1.335 ± 0.03 1.339 ± 0.05 1.340 ± 0.06 1.342 ± 0.07 1.344 ± 0.08

98.7 ± 0.2 98.2 ± 0.3 97.0 ± 0.4 94.4 ± 0.3 91.4 ± 0.1

355

nanoemulsification tests were conducted to evaluate any phase separation or drug precipitation upon dilution with three different diluents. It was observed that SNEDDS formulations SA1–SA4 passed self-nanoemulsification test with grade A in the presence of ultrapure water, 0.1 N HCl and phosphate buffer (pH 6.8). However, SA SNEDDS SA5 passed self-nanoemulsification efficiency test with grade B in the presence of all three diluents investigated.

3.4. Physicochemical investigation of SA SNEDDS results presented in Fig. 1, it can be seen that the Smix of 1:2 (Fig. 1A) showed relatively low SNEDDS zones. However, in case of Smix ratio of 1:1 (Fig. 1B), SNEDDS zones were increased rapidly in comparison with 1:2 ratio. In case of Smix ratio of 2:1 (Fig. 1C), SNEDDS zones were found to be decreased in comparison with Smix ratio of 1:1 but increased in comparison with 1:2 ratio. Finally, when the Smix ratio of 3:1 was investigated (Fig. 1D), the SNEDDS zones were found to be decreased again in comparison with Smix ratios of 1:1 and 2:1. From these results, it was observed that the maximum SNEDDS zones were exposed by Smix ratio of 1:1 (Fig. 1B). Therefore, different SNEDDS formulations were selected from Fig. 1C. With respect to the oil phase, the entire SNEDDS zones in Fig. 1B were considered and different oil compositions (8.0%, 12.0%, 16.0%, 20.0% and 24.0% w/w) with fixed Smix concentration (36.0% w/w) were precisely selected from phase diagram. Around 40 mg of solid SA was dissolved in each formulation. Different SA SNEDDS were coded as SA1–SA5 and their compositions are presented in Table 1.

3.3. Thermodynamic stability and self-nanoemulsification tests Thermodynamic tests were conducted to remove any metastable or unstable SNEDDS of SA. All of the prepared SNEDDS (SA1–SA5) of SA were found to be thermodynamically stable at centrifugation, heating and cooling cycles and freeze–pump–thaw cycles. Self-

The resulting data of physicochemical investigation of SA SNEDDS is presented in Table 3. The droplet size of developed SA SNEDDS (SA1– SA5) recorded by Malvern Particle Size Analyzer was observed in the range of 12.4–94.3 nm. The impact of the concentration of surfactants on droplet size was not investigated because it was kept constant in all formulations. However, the impact of the concentration of oil phase (Triacetin) on droplet size of SA SNEDDS was investigated in this work. It was observed that the droplet size of SA SNEDDS reduced significantly with decrease in the concentration of Triacetin (P b 0.05). The value of the largest droplet size was recorded in formulation SA5 (94.3 ± 8.7 nm). This observation was possible due to the presence of highest concentration of oil phase, i.e. Triacetin in formulation SA5 (24.0% w/w). However, the lowest value of droplet size was recorded for formulation SA1 (12.4 ± 1.04 nm). The lowest droplet size of SA1 was possible due to the presence of the lowest concentration of oil phase in formulation SA1 (8.0% w/w). The PDI values of SA SNEDDS (SA1–SA5) were recorded in the range of 0.158–0.258. The values PDIs for all SNEDDS (SA1–SA5) were low, which indicated good uniformity of oil droplets within the developed formulations. The SNEDDS SA1 had the least PDI value (0.158), indicating the highest uniformity of oil droplets in SA1. However, the highest value of PDI was observed in formulation SA5 (0.258). The ZP values of SA SNEDDS (SA1–SA5) were observed in the range of −39.4 to −32.8 mV (Table 3). The lowest value of ZP was observed in formulation SA5 (− 39.4 mV). However, the highest value of ZP was

Fig. 2. In vitro release profile of SA from developed SNEDDS (SA1–SA5) and SA suspension through dialysis bag (n = 3; mean ± SD).

356

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358

Table 4 Drug release kinetic parameters in terms of correlation coefficients (R2), respective rate constants and exponential value (n) for drug release of SA from SNEDDS (SA1–SA5) and SA suspension. Formulation

SA1 SA2 SA3 SA4 SA5 Suspension

Zero order

Higuchi

Hixon–Crowell

Peppas

K0

R2

k1

First order R2

R2

R2

R2

n

13.10 11.61 10.94 8.34 7.43 4.52

0.903 0.904 0.908 0.943 0.958 0.966

2.17 1.73 1.61 1.33 1.27 1.13

0.983 0.978 0.976 0.976 0.970 0.959

0.950 0.951 0.955 0.969 0.960 0.905

0.973 0.961 0.959 0.967 0.968 0.962

0.987 0.986 0.985 0.987 0.988 0.995

0.842 0.902 0.921 0.991 0.886 0.912

Zero order rate constant (K0) and first order rate constant (k1).

observed in formulation SA1 (−32.8 mV). The negative ZP values of all formulations were possibly due to the presence of Triacetin in all formulations [30,31]. The RIs values of SA SNEDDS (SA1–SA5) were recorded in the range of 1.335–1.344 (Table 3). The highest RI value was recorded for formulation SA5 (1.344 ± 0.08). However, the lowest RI value was recorded for formulation SA1 (1.335 ± 0.03). The RIs of all formulations were very close with RI of water (RI = 1.33), indicating transparent nature and oil-water type behavior of SA SNEDDS [31]. The % T values of SA SNEDDS (SA1–SA5) were observed in the range of 91.4%–98.7% (Table 3). The highest % T value was recorded for formulation SA1 (98.7 ± 0.2%). However, the lowest % T value was recorded for formulation SA5 (91.4 ± 0.1%). The results of % T indicated transparent nature of all formulations investigated [30]. 3.5. In vitro drug release/dissolution studies In vitro drug release/dissolution studies were conducted through ready-to-use dialysis bag on SA SNEDDS (SA1–SA5) and SA suspension. The results of SA release from SNEDDS (SA1–SA5) and SA suspension are shown in Fig. 2. It was observed that the initial release of SA from all SNEDDS and SA suspension was rapid. However, the statistical difference in the release profile of SA between SNEDDS (SA1–SA5) and SA suspension was highly significant (P b 0.05). More than 75% of SA was found to be released from SA SNEDDS SA1–SA3 as compared to only 31.2% from SA suspension after 8 h (Fig. 2). However, SA SNEDDS SA4 and SA5 showed 58.3% and 52% of SA release, respectively, after 8 h of study. After 8 h, all SA SNEDDS and SA suspension showed slower release of SA (i.e. sustained release profile of SA). Formulation SA1 presented the highest release profile of SA in comparison with other SNEDDS and SA suspension. The cumulative % release of SA that was recorded from formulation SA1 after 24 h of study was 96.8% as compared to only 36.2% from SA suspension. 91.1% of SA was released from formulation SA1 after 8 h of drug release studies. The highest release of SA from formulation SA1 was possible due to its lowest droplet size (12.4 nm), least PDI value (0.158) and the presence of the lowest Triacetin concentration (8% w/w). The release profile of SA in two steps from developed SNEDDS (SA1–SA5) and SA suspension showed the diffusion-controlled dissolution rate of SA from all formulations investigated [30]. The two-step release profile of SA from different SNEDDS was similar to those reported for ibrutinib, 5-fluorouracil conjugate, isoniazid analogue, tadalafil and indomethacin from SNEDDS [30,33,37,48,49]. 3.6. Drug release kinetics for SA The release data of SA from different SNEDDS (SA1–SA5) and SA suspension was fitted using different mathematical models and the resulting data of this analysis are presented in Table 4. It has been reported that if the value of n = 0.5, this indicates a Fickian diffusion

mechanism. However, if the value of n N 0.5 but b1.0, it indicates nonFickian diffusion mechanism. On the other hand, if the value of n N 1.0, it indicates supercase II transport mechanism [41]. In the current research work, the drug release analysis showed that all SNEDDS as well as SA suspension followed Korsemeyer–Peppas model with non-Fickian diffusion mechanism because the value of n was obtained in the range of 0.842–0.991. Moreover, the R2 values for SNEDDS (SA1–SA5) and SA suspension were also higher for Peppas model in comparison with zero order, first order, Higuchi model and Hixon–Crowell model. The results obtained in this work were in good agreement with those reported for drug release kinetics of tadalafil and ibrutinib from SNEDDS [33,36]. 3.7. In vitro antioxidant assay In vitro DPPH method was applied for the evaluation of antioxidant activity of optimized SA SNEDDS (SA1), SA suspension and standard ascorbic acid because this method has been reported as an appropriate method [50,51]. The results of antioxidant assay of SA suspension, optimized SA SNEDDS SA1 and standard ascorbic acid in terms of respective IC50 values are presented in Table 5. The IC50 value of SA SNEDDS SA1 (IC50 = 4.52 ± 0.67 μg/ml) was highly significant in comparison with SA suspension (IC50 = 7.62 ± 0.89 μg/ml) and standard ascorbic acid (IC50 = 40.78 ± 3.92 μg/ml) (P b 0.05). The antioxidant effects of optimized SA SNEDDS SA1 were significant in comparison with SA suspension and standard ascorbic acid over the entire concentration range investigated (P b 0.05). The IC50 values of SA and standard ascorbic acid have been reported as 7.21 and 39 μg/ml, respectively, against DPPH assay [25,52]. In the current research work, the IC50 values of SA and standard ascorbic acid were recorded as 7.62 and 40.78 μg/ml, respectively, against DPPH assay. These values were very close with literature values [25,52]. The antioxidant activity of SA was possible due to the presence of phenolic groups as reported in the literature [25]. The enhanced antioxidant activity of SA from optimized SNEDDS SA1 was possible due to lower droplet size and rapid absorption of SA from SNEDDS SA1. Overall, SA and SA SNEDDS SA1 were found to be better antioxidants than standard ascorbic acid. 3.8. In vivo anti-inflammatory effects In vivo anti-inflammatory effects of optimized SA SNEDDS SA1 upon oral administration were compared with those of SA suspension and

Table 5 In vitro antioxidant activity in terms of IC50 value of SA, optimized SNEDDS SA1 and standard ascorbic acid against DPPH assay (n = 3). Sample matrices

IC50 ± SD (μg/ml)

SA SA1 Ascorbic acid

7.62 ± 0.89 4.52 ± 0.67 40.78 ± 3.92

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358

357

Fig. 3. In vivo anti-inflammatory effects of SA suspension, optimized SNEDDS SA1 and indomethacin (standard) in rats (n = 6; mean ± SD).

standard indomethacin. The results of in vivo anti-inflammatory effects are presented in Fig. 3. The anti-inflammatory effects of SA, SA1 and indomethacin were found to be increased with respect to time for up to 3 h. However, after 3 h of oral administration, these effects were started to decrease slightly as shown in Fig. 3. The % inhibition value after 3 h of oral administration was found to be 92.4% for optimized SNEDDS SA1 as compared to only 41.7% for SA suspension. This value of SNEDDS SA1 was highly significant in comparison with SA suspension (P b 0.05). The % inhibition value for standard indomethacin after 3 h of oral administration was found to be 96.8% (Fig. 3). Overall, the anti-inflammatory effects of optimized SNEDDS SA1 were highly comparable with standard indomethacin. The enhanced anti-inflammatory effects of optimized SNEDDS SA1 were possible due to the rapid absorption of SA from SNEDDS due to the presence of solubilizers such as Triton-X100 and Carbitol in comparison with SA suspension. The anti-inflammatory effects of SA and optimized SNEDDS SA1 were possible due to inhibition of iNOS, COX-2 and proinflammatory cytokines inductions as reported previously in the literature [13]. 4. Conclusions In the current research work, different SNEDDS formulations of SA were developed and investigated in order to enhance its in vitro antioxidant and in vivo anti-inflammatory effects. Different SNEDDS of SA were prepared by low-energy emulsification method. Based on the lowest droplet size (12.4 ± 1.04 nm), least PDI value (0.158), optimal ZP value (− 32.8 mV), optimal RI value (1.335 ± 0.03), the highest % T (98.7 ± 0.2), the highest release profile of SA (96.8 ± 7.30%) and the presence of the lowest Triacetin concentration (8% w/w), SNEDDS formulation SA1 was selected for in vitro antioxidant and in vivo anti-inflammatory studies. DPPH scavenging assay indicated significant antioxidant activity of optimized SA SNEDDS in comparison with free SA and standard ascorbic acid based on IC50 values. In vivo anti-inflammatory studies in rats also indicated that SA in optimized SNEDDS was highly efficacious than SA suspension. However, the anti-inflammatory effects of optimized SNEDDS were comparable to standard indomethacin. Free SA as well as SA in optimized SNEDDS was proved to be an excellent antioxidant in comparison with standard ascorbic acid. Overall, the results obtained in the current research work showed that the SNEDDS could be successfully used in the enhancement of in vitro

dissolution rate and therapeutic efficacy of poorly soluble bioactive compounds such as SA. Declaration of interest The authors report no declaration of interest associated with this manuscript. Acknowledgement The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding the research through research group project no. RG-1435-005. References [1] N. Niciforovic, H. Abramovic, Sinapic acid and its derivatives: natural sources and bioactivity, Compr. Rev. Food Sci. Food Saf. 13 (2014) 34–51. [2] T. Silambarasan, J. Manivannan, M.K. Priya, N. Suganya, S. Chatterjee, B. Raja, Sinapic acid protects heart against ischemia/reperfusion injury and H9c2 cardiomyoblast cells against oxidative stress, Biochem. Biophys. Res. Comm. 456 (2015) 853–859. [3] M.E. Cuvelier, H. Richard, C. Berset, Comparison of the antioxidative activity of some acid-phenols: structure–activity relationship, Biosci. Biotechnol. Biochem. 56 (1992) 324–325. [4] F. Natella, M. Nardini, M. Di Felice, C. Scaccini, Benzoic and cinnamic acid derivatives as antioxidants: structure–activity relation, J. Agr. Food Chem. 47 (1999) 1453–1459. [5] S.S. Pekkarinen, H. Stockmann, K. Schwarz, I.M. Heinonen, A.I. Hopia, Antioxidant activity and partitioning of phenolic acids in bulk and emulsified methyl linoleate, J. Agr. Food Chem. 47 (1999) 3036–3043. [6] H. Kikuzaki, M. Hisamoto, K. Hirose, K. Akiyama, H. Taniguchi, Antioxidant properties of ferulic acid and its related compounds, J. Agr. Food Chem. 50 (2002) 2161–2168. [7] Y. Zou, A.R. Kim, J.E. Kim, J.S. Choi, H.Y. Chung, Peroxynitrite scavenging activity of sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) isolated from Brassica juncea, J. Agr. Food Chem. 50 (2002) 5884–5890. [8] R.J. Robbins, Phenolic acids in foods: an overview of analytical methodology, J. Agr. Food Chem. 51 (2003) 2866–2887. [9] O. Firuzi, L. Giansanti, R. Vento, C. Seibert, R. Petrucci, G. Marrosu, R. Agostino, L. Saso, Hypochlorite scavenging activity of hydroxycinnamic acids evaluated by a rapid microplate method based on the measurement of chloramines, J. Pharm. Pharmacol. 55 (2003) 1021–1027. [10] N. Nenadis, O. Lazaridou, M.Z. Tsimidou, Use of reference compounds in antioxidant activity assessment, J. Agr. Food Chem. 55 (2007) 5452–5460. [11] M.F. Andreasen, A.K. Landbo, L.P. Christensen, A. Hansen, A.S. Meyer, Antioxidant effects of phenolic rye (Secale cerealeL.) extracts, monomeric hydroxycinnamates,

358

[12]

[13]

[14]

[15] [16]

[17] [18]

[19] [20]

[21] [22]

[23]

[24]

[25]

[26]

[27] [28] [29]

[30]

[31]

[32]

F. Shakeel et al. / Journal of Molecular Liquids 224 (2016) 351–358 and ferulic acid dehydrodimers on human low-density lipoproteins, J. Agric. Food. Chem. 49 (2001) 4090–4096. C. Lu, S. Yao, N. Lin, Studies on reactions of oxidizing sulfulsulful three-electronbond complexes and reducing alphaamino radicals derived from OH reaction with methionine in aqueous solution, Biochim. Biophys. Acta 16 (2001) 89–96. K.J. Yun, D.J. Koh, S.H. Kim, S.J. Park, J.H. Ryu, D.G. Kim, J.Y. Lee, K.T. Lee, Anti-inflammatory effects of sinapic acid through suppression of inducible nitric oxide synthase, cyclooxygenase-2 and proinflammatory cytokines expression via nuclear factor-kB inactivation, J. Agr. Food Chem. 56 (2008) 10265–10272. H. Nowak, R. Kujava, R. Zadernowski, B. Roczniak, H. Kozlowska, Antioxidative and bactericidal properties of phenolic compounds in rapeseeds, Eur. J. Lipid Sci. Technol. 94 (1992) 149–152. J. Teixeira, A. Gaspar, E.M. Garrido, J. Garrido, F. Borges, Hydroxycinnamic acid antioxidants: an electrochemical overview, Biomed. Res. Int. 2013 (2013), E251754. S. Tesaki, S. Tanabe, H. Ono, E. Fukushi, J. Kawabata, M. Watanabe, 4-hydroxy-3nitrophenyllactic and sinapic acids as antibacterial compounds from mustard seeds, Biosci. Biotechnol. Biochem. 62 (1998) 998–1000. C.E. Maddox, L.M. Laur, L. Tian, Antibacterial activity of phenolic compounds against the phytopathogen Xylella fastidiosa, Curr. Microbiol. 60 (2010) 53–58. C. Engels, A. Schieber, M.G. G¨anzle, Sinapic acid derivatives in defatted oriental mustard (Brassica juncea L.) seed meal extracts using UHPLC-DADESI-MSn and identification of compounds with antibacterial activity, Eur. Food Res. Technol. 234 (2012) 535–542. M.S. Barber, V.S. McConnell, B.S. DeCaux, Antimicrobial intermediates of the general phenylpropanoid and lignin specific pathways, Phytochem. 54 (2000) 53–56. M.L. Johnson, J.P. Dahiya, A.A. Olkowski, H.L. Classen, The effect of dietary sinapic acid (4-hydroxy-3, 5-dimethoxy-cinnamic acid) on gastrointestinal tract microbial fermentation, nutrient utilization, and egg quality in laying hens, Poul. Sci. 87 (2008) 958–963. B.H. Yoon, J.W. Jung, J.J. Lee, Y.W. Cho, C.G. Jang, C. Jin, T.H. Oh, J.H. Ryu, Anxiolyticlike effects of sinapic acid in mice, Life Sci. 81 (2007) 234–240. S.J. Roy, P.S.M. Prince, Protective effects of sinapic acid on cardiac hypertrophy, dyslipidaemia and altered electrocardiogram in isoproterenol-induced myocardial infarcted rats, Eur. J. Pharmacol. 699 (2013) 213–218. T. Silambarasan, J. Manivannan, M.K. Priya, N. Suganya, S. Chatterjee, B. Raja, Sinapic acid prevents hypertension and cardiovascular remodeling in pharmacological model of nitric oxide inhibited rats, PLoS One 9 (2014), E115682. E.A. Hudson, P.A. Dinh, T. Kokubun, M.S.J. Simmonds, A. Gescher, Characterization of potentially chemopreventive phenols in extracts of brown rice that inhibit the growth of human breast and colon cancer cells, Cancer Epidem. Biomar. Preven. 9 (2000) 1163–1170. A. Gaspar, M. Martins, P. Silva, E.M. Garrido, J. Garrido, O. Firuzi, R. Miri, L. Saso, F. Borges, Dietary phenolic acids and derivatives: evaluation of the antioxidant activity of sinapic acid abd its alkyl esters, J. Agr. Food Chem. 58 (2010) 12273–12280. F. Shakeel, N. Haq, M. Raish, M.K. Anwer, R. Al-Shdefat, Solubility and thermodynamic analysis of sinapic acid in various neat solvents at different temperatures, J. Mol. Liq. 222 (2016) 167–171. C. Chen, Sinapic acid and its derivatives as medicine in oxidative stress-induced diseases and aging, Oxidat. Med. Cell. Long. 2016 (2016), E3571614. L. Trnkova, I. Bousova, V. Kubicek, J. Drsata, Binding of naturally occurring hydroxycinnamic acids to bovine serum albumin, Natural Sci. 2 (2010) 563–570. F. Shakeel, N. Haq, A. Al-Dhfyan, F.K. Alanazi, I.A. Alsarra, Double w/o/w nanoemulsion of 5-fluorouracil for self-nanoemulsifying drug delivery system, J. Mol. Liq. 200 (2014) 183–190. F. Shakeel, N. Haq, M. El-Badry, F.K. Alanazi, I.A. Alsarra, Ultra fine super selfnanoemulsifying drug delivery system (SNEDDS) enhanced solubility and dissolution of indomethacin, J. Mol. Liq. 180 (2013) 89–94. F. Shakeel, N. Haq, F.K. Alanazi, I.A. Alsarra, Impact of various nonionic surfactants on self-nanoemulsification efficiency of two grades of Capryol (Capryol-90 and Capryol-PGMC), J. Mol. Liq. 182 (2013) 57–63. F. Shakeel, N. Haq, F.K. Alanazi, I.A. Alsarra, Polymeric solid self-nanoemulsifying drug delivery system of glibenclamide using coffee husk as a low cost biosorbent, Powder Technol. 256 (2014) 352–360.

[33] F. Shakeel, M. Iqbal, E. Ezzeldin, Bioavailability enhancement and pharmacokinetic profile of an anticancer drug ibrutinib by self-nanoemulsifying drug delivery system, J. Pharm. Pharmacol. 68 (2016) 772–780. [34] F. Shakeel, G.A. Shazly, M. Riash, A. Ahmad, M.A. Kalam, N. Ali, M.A. Ansari, G.M. Elosaily, Biological investigation of a supersaturated self-nanoemulsifying drug delivery system of Piper cubeba essential oil, RSC Adv. 5 (2015) 105206–105217. [35] F. Shakeel, N. Haq, F.K. Alanazi, I.A. Alsarra, Surface-adsorbed reverse micelle-loaded solid self-nanoemulsifying drug delivery system of talinolol, Pharm. Develop. Technol. 21 (2016) 131–139. [36] M. El-Badry, N. Haq, G. Fetih, F. Shakeel, Solubility and dissolution enhancement of tadalafil using self-nanoemulsifying drug delivery system, J. Oleo Sci. 63 (2014) 567–576. [37] F. Shakeel, N. Haq, M. Raish, N.A. Siddiqui, F.K. Alanazi, I.A. Alsarra, Antioxidant and cytotoxic effects of vanillin via eucalyptus oil containing self-nanoemulsifying drug delivery system, J. Mol. Liq. 218 (2016) 233–239. [38] S. Shafiq, F. Shakeel, S. Talegaonkar, F.J. Ahmad, R.K. Khar, M. Ali, Development and bioavailability assessment of ramipril nanoemulsion formulation, Eur. J. Pharm. Biopharm. 66 (2007) 227–243. [39] F. Shakeel, S. Shafiq, N. Haq, F.K. Alanazi, I.A. Alsarra, Nanoemulsions as potential vehicles for transdermal and dermal delivery of hydrophobic compounds: an overview, Expert Opin. Drug Deliv. 9 (2012) 953–974. [40] T. Higuchi, K.A. Connors, Phase-solubility techniques, Adv. Anal. Chem. Instr. 4 (1965) 117–122. [41] P. Costa, J.M.S. Lobo, Modeling and comparison of dissolution profiles, Eur. J. Pharm. Sci. 15 (2001) 123–133. [42] S. Dash, P.N. Murthy, L. Nath, P. Chowdhury, Kinetic modeling on drug release from controlled drug delivery systems, Acta Pol. Pharm. 67 (2010) 217–223. [43] O. Nuchuchua, U. Sakulku, N. Uawongyart, S. Puttipipatkhachorn, A. Soottitantawat, U. Ruktanonchai, In vitro characterization and mosquito (Aedes aegypti) repellent activity of essential-oils-loaded nanoemulsions, AAPS Pharm. Sci. Tech. 10 (2009) 1234–1242. [44] H. Ye, K. Wang, C. Zhou, J. Liu, X. Zeng, Purification, antitumor and antioxidant activities in vitro of polysaccharides from the brown seaweed Sargassum pallidum, Food Chem. 111 (2008) 428–432. [45] C.B.S. Oliveira, Y.S.R. Meurer, M.G. Oliveira, W.M.T.Q. Medeiros, F.O.N. Silva, A.C.F. Brito, D.D.L. Pontes, V.F. Andrade-Neto, Comparative study on the antioxidant and anti-toxoplasma activities of vanillin and its resorcinarene derivative, Mol. 19 (2014) 5898–5912. [46] C.A. Winter, Anti-inflammatory testing methods: comparative evaluation of indomethacin and other agents, Nonsteroid. Anti-inflammat. Drugs 82 (1965) 190–202. [47] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, S. Shafiq, Enhanced anti-inflammatory effects of celecoxib from a transdermally applied nanoemulsion, Pharmazie 64 (2009) 258–259. [48] M.A. Bhat, M. Iqbal, A. Al-Dhfyan, F. Shakeel, Carvone Schiff base of isoniazid as a novel antitumor agents: nanoemulson development and pharmacokinetic evaluation, J. Mol. Liq. 203 (2015) 111–119. [49] F. Shakeel, F.K. Alanazi, M. Raish, N. Haq, A.A. Radwan, I.A. Alsarra, Pharmacokinetic and in vitro cytotoxic evaluation of cholesterol-rich nanoemulsion of cholesterylsuccinyl-5-fluorouracil, J. Mol. Liq. 211 (2015) 164–168. [50] K. Mishra, H. Ojha, N.K. Chaudhury, Estimation of antiradical properties of antioxidants using DPPH assay: a critical review and results, Food Chem. 130 (2012) 1036–1043. [51] G. Clarke, K.N. Ting, C. Wiart, J. Fry, High correlation of 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging, ferric reducing activity potential and total phenolics content indicates redundancy in use of all three assays to screen for antioxidant activity of extracts of plants from the Malaysian rainforest, Antioxidants 2 (2013) 1–10. [52] S. Mukherjee, N. Pawar, O. Kulkarni, B. Nagarkar, S. Thopte, A. Bhujbal, P. Pawar, Evaluation of free radical quenching properties standard Ayurvedic formulation Vayasthapana Rasayana, BMC Compl. Alt. Med. 11 (2011), E38.