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Pharmaceutical Development and Technology

ISSN: 1083-7450 (Print) 1097-9867 (Online) Journal homepage: http://www.tandfonline.com/loi/iphd20

Effect of formulation variables on design, in vitroevaluation of valsartan SNEDDS and estimation ofits antioxidant effect in adrenaline-induced acutemyocardial infarction in rats

Maha M. Amin, Omaima N. El Gazayerly, Nabaweya A. Abd El-Gawad, ShadyM. Abd El-Halim & Sally A. El-Awdan

To cite this article: Maha M. Amin, Omaima N. El Gazayerly, Nabaweya A. Abd El-Gawad, ShadyM. Abd El-Halim & Sally A. El-Awdan (2016) Effect of formulation variables on design, in vitroevaluation of valsartan SNEDDS and estimation of its antioxidant effect in adrenaline-induced acutemyocardial infarction in rats, Pharmaceutical Development and Technology, 21:8, 909-920, DOI:10.3109/10837450.2015.1078354

To link to this article: http://dx.doi.org/10.3109/10837450.2015.1078354

Published online: 31 Aug 2015. Submit your article to this journal

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ISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, 2016; 21(8): 909–920! 2015 Taylor & Francis. DOI: 10.3109/10837450.2015.1078354

RESEARCH ARTICLE

Effect of formulation variables on design, in vitro evaluation of valsartanSNEDDS and estimation of its antioxidant effect in adrenaline-inducedacute myocardial infarction in rats

Maha M. Amin1, Omaima N. El Gazayerly1, Nabaweya A. Abd El-Gawad2, Shady M. Abd El-Halim2, andSally A. El-Awdan3

1Pharmaceutics and Industrial Pharmacy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt, 2Pharmaceutics and Industrial Pharmacy

Department, Faculty of Pharmacy, October 6 University, Sixth of October City, Egypt, and 3Department of Pharmacology, National Research

Center, Cairo, Egypt

Abstract

Valsartan is a specific angiotensin II antagonist used for the treatment of hypertension. It suffersfrom low aqueous solubility and high variability in its absorption after oral administration. Theaim of this study was to improve the dissolution and thereby the bioavailability of Valsartanthrough the development of self nano-emulsifying drug delivery systems. Four ternary phasediagrams were constructed to identify the self-emulsification region of Capmul� MCM,Labrafil� M1944, Capryol� 90 and Labrafac� PG together with Cremophore� RH 40 andTranscutol� HP as oil, surfactant and co-surfactant, respectively. The effect of oil type, oil andsurfactant concentration on droplet size and in vitro Valsartan dissolution were studied. Theprotective effect of the optimum formula F5 in adrenaline-induced oxidative stress in ratsduring myocardial infarction was determined. Formula F5 exhibited globule size of (13.95 nm)with 76.07% ± 1.10 of Valsartan dissolved after five minutes compared to Disartan 80 mgcapsules (13.43%). Results revealed a significant reduction (p50.05) in serum aspartatetransaminase, creatine kinase myocardial band and malondialdehyde levels, while a significantincrease (p50.05) in serum glutathione in F5. Therefore, self nano-emulsifying drug deliverysystems could be considered as a promising approach to improve the dissolution and therebythe bioavailability of Valsartan.

Keywords

Angiotensin II antagonist, BCS class II,oxidative stress, ternary phase diagram

History

Received 3 June 2015Revised 21 July 2015Accepted 22 July 2015Published online 31 August 2015

Introduction

The administration of drugs via oral route is usually the mostpreferred route as about 80% of common pharmaceutical productsare being given orally1. The oral delivery of many drug moleculesis recurrently associated with complications of low watersolubility leading to poor and highly variable oral bioavailability.Different approaches have been attempted to increase watersolubility of poorly water soluble drugs, such as conversion of acrystalline molecule to its amorphous state, particle size reduc-tion, co-solvency, micellar solubilization, complexation, soliddispersion and formulation of lipid based systems2.

Lipid-based formulations, predominantly the self-nanoemulsi-fying drug delivery systems (SNEDDS), illustrate their potentialas alternative approaches for the delivery of hydrophobic drugs.SNEDDS formulations are isotropic mixtures of oil, surfactant,co-surfactant together with the drug. SNEDDS emulsify spontan-eously to produce fine o/w nanoemulsions with globule size (2–100 nm) when introduced into aqueous phase under gentleagitation provided by the peristaltic motility of gastrointestinaltract. The small size of the formed droplet provides a large

interfacial surface area for drug absorption and therefore increasesits bioavailability3.

The chosen surfactant in SNEDDS must be able to lower theinterfacial tension to a very small value with achieving a highsolubilization potency to aid the dispersion process during thepreparation of the nanoemulsion4. Co-surfactants also help indecreasing the interfacial tension between oil and water, adjust theflexibility of interfacial membrane and sometimes reduce theamount of the used surfactant5. Advantages of SNEDDS include:ease of preparation, thermodynamic stability, transparent andelegant appearance, enhanced penetration through the biologicalmembranes, increased bioavailability, less inter- and intra-indi-vidual variability in drug pharmacokinetics and bypassing the firstpass effect. Such advantages are the reason for which suchsystems are attractive for use in drug delivery6.

Several studies were carried out to formulate poorly solubledrugs into SNEDDS. Absorption and physical stability of anti-malarial compound Halofantrine had been improved when it wasformulated in the form of SNEDDS7. Cinnarizine SNEDDSformulations, the antihistaminic drug, showed higher dissolutionrate and therefore bioavailability compared to that of the marketedtablet, Stugeron�8. Optimized SNEDDS formulation of anti-hyperlipidimic Gemfibrozil showed a significant increase indissolution rate compared to conventional tablets9.

Valsartan is an antihypertensive agent which selectivelyinhibits type 1 angiotensin II receptor (AT1). It is a poorly

Address for correspondence: Dr Maha M. Amin, Pharmaceutics andIndustrial Pharmacy Department, Faculty of Pharmacy, Cairo University,Cairo, Egypt. E-mail: maha_amin2002@yahoo.com

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water soluble drug belonging to Class II, BiopharmaceuticalClassification System (BCS). Valsartan is rapidly absorbedfollowing oral administration with a bioavailability of about23% and mean half-life of 7.5 h. The usual daily dose ranges from80 to 160 mg. The drug is weakly acidic, pka¼ 3.9 and 4.7, andtherefore, it is poorly soluble in the acidic environment where itsabsorption window exists10.

Acute myocardial infarction (AMI) results from the prolongedmyocardial ischemia with necrosis of myocytes due to interrup-tion of blood supply to an area of heart. The metabolicdisarrangements that occur during ischemia predispose for theformation of free radicals and reactive oxygen species leading tothe formation of lipid peroxides, damage of cell membrane anddestruction of antioxidative defense system resulting in theincrease of serum malondialdehyde (MDA), AMI markers[aspartate transaminase (AST) and creatine kinase myocardialband (CK-MB)] and decrease of reduced glutathione (GSH)11.

The activity of the adrenergic system is increased in AMIwhich is reflected by raised plasma catecholamine concentration.A renin–angiotensin system (RAS) is present in the heart and itsactivation leads to increased in the formation of local AngiotensinII. Reactive oxygen species will be formed in excessive amountsresulting in excessive myocardial reperfusion injury. Blocking ofAngiotensin II receptors by Valsartan may therefore be effectivein reducing oxidative stress during AMI by reducing theformation of reactive oxygen species and thereby inhibiting celldamage which was further confirmed by a marked decrease inaspartate transaminase (AST) and creatine kinase myocardialband (CK-MB) levels in serum12.

The aim of this study was to develop Valsartan SNEDDS bystudying the effect of some formulation variables and to evaluatetheir effect on droplet size, in vitro Valsartan dissolution after fiveminutes (Q5min) and to elucidate the protective effect of Valsartanin the optimized SNEDDS formula (F5) in adrenaline-inducedoxidative stress in rats during myocardial infarction.

Materials and methods

Materials

Valsartan (Novartis Pharma, Egypt); Capmul� MCM EP andAcconon� MCM-2 EP/NF (Abitec Corp.); Capryol� 90,Labrafac� PG, Labrafil� M 1944 CS, Transcutol� HP,Lauroglycol� 90 and Labrasol� (Gattefosse, France);Cremophore� RH40 (BASF, Germany); Poly Ethylene Glycol400 (PEG400) (Fluka, Switzerland); hydrochloric acid (Merck,Darmstadt, Germany); methanol and methylene chloride (Analar,India); adrenaline 1 mg/ml S.C. ampoules (commercially avail-able, CID, Egypt); Rat AST ELISA Assay Kit (Cat. No. 201-11-0595, Sunred Biological Technology Co., Shanghai, China); RatCK-MB ELISA Assay Kit (Cat. No. KT-12247, KamiyaBiomedical Company, Seattle, WA); GSH Assay Kit (Cat. No.703002, Cayman Chemical, Ann Arbor, MI); MDA Assay Kit(Cat. No. ab118970, Abcam, Cambridge, UK).

Methods

Solubility studies in oils, surfactants and co-surfactants

The solubility of Valsartan in various oils (Capmul� MCM,Capryol� 90, Labrafil� M 1944 and Labrafac� PG), 10%surfactant solutions (Cremophore� RH40, Labrasol� andAcconon� MCM-2)13 and co-surfactants (CoS) (Transcutol�HP, Lauroglycol� 90 and Poly Ethylene Glycol 400) wasdetermined by dissolving an excess amount of Valsartan in 2 gof each of the four oils, surfactants and co-surfactants, respect-ively, in stoppered vials. The mixtures were continuously stirredusing vortex mixer (VM-300, Gemmy Industrial Corp., Taiwan)

for 10 min and kept at 37 ± 0.5 �C in water bath shaker (Model 25,Precision Scientific, Dallas, TX) for 72 h to attain equilibrium.The equilibrated samples were centrifuged (model 2041,Centurion Scientific, West Sussex, UK) at 3000 rpm for 15 minand the supernatant was filtered through 0.45 mm membrane filter.The supernatant was suitably diluted with methylene chloride incase of Labrafil� M 1944 and Labrafac� PG oils, or withmethanol in case of other excipients. Drug content was quantifiedspectrophotometrically (UV-1800, Schimadzu, Kyoto, Japan) at250 nm14 against a suitable blank15.

Screening of surfactants

The surfactant has a great effect on the emulsification process,nano-emulsifying region and droplet size. The surfactants werefurther screened based on their ability to emulsify the testedoils16. In our study, the aim to produce SNEDDS that immediatelyand rapidly spread in the aqueous medium to give o/wnanoemulsion requires the use of high hydrophilic–lipophilicbalance (HLB) surface active agents17. Three surfactants[Cremophore� RH40 (HLB¼ 15), Acconon� MC8-2(HLB¼ 14) and Labrasol� (HLB¼ 12)] were screened based ontheir ability to incorporate large amounts of water into oil:sur-factant mixtures. Each of the four different oils together with eachof the three tested surfactants were mixed at fixed ratio (1:1 w/w)to avoid the effect of oil or surfactant concentration on the amountof water incorporated, then water was titrated dropwise withcontinuous stirring using magnetic stirrer (MS-300HS, MisungScientific Co., Kyungkido, South Korea) until the mixturechanged from transparent to turbid. The maximum amount ofwater that could be incorporated into the oil/surfactant mixture (o/s mix.) served as one of the criteria in the final selection of thesurfactant for SNEDDS development being a second confirmatorytest. Percentage of water incorporated was calculated as follows18:

Percent of water incorporated

¼ wt: of added water

wt: of mixtureþ wt: of added waterð Þ

� �� 100

ð1Þ

Screening of co-surfactants

Co-surfactants were screened based on their efficacy to improvethe nano-emulsification ability of the selected surfactant3. Theselected surfactant (Cremophore� RH40) was combined witheach of the three different co-surfactants (Transcutol� HP,Lauroglycol� 90 and Poly Ethylene Glycol 400) at a S/Cos ratioof (1:1 w/w) to avoid the effect of surfactant or co-surfactantconcentration on the amount of water incorporated. Each S/Cosmixture together with each of the four oils was mixed at a ratio of(1:1 w/w). These mixtures were screened as discussed aboveunder screening of surfactants and the Cos which showed themaximum amount of water incorporated was further selected forthe construction of ternary phase diagram18.

Construction of ternary phase diagram

The selected components comprising the four different oilstogether with Cremophore� RH40 and Transcutol� HP assurfactant and co-surfactant, respectively, were mixed to constructthe four different phase diagrams. Phase diagrams were con-structed by weighing appropriate amounts of each component intosmall vials, vortex mixing for 10 min and gently stirring withheating at 37 �C to obtain a homogenous isotropic mixture. Foreach phase, 36 systems were prepared and stored under ambientconditions for 24 h before visual examination19. To determine the

910 M. M. Amin et al. Pharm Dev Technol, 2016; 21(8): 909–920

effect of drug addition on the nanoemulsion boundary, the phasediagrams were also constructed in the presence of Valsartan20

with constant drug loading of 4% w/w for all the preparedSNEDDS.

Characterization of the different selected Valsartan SNEDDS

Four formulae were selected from each phase diagram and thecomposition of the different selected SNEDDS (F1–F16) wasgiven in Table 1. The effect of oil type, oil concentration as wellas surfactant concentration on the droplet size and in vitroValsartan dissolution (Q5min) from the selected SNEDDS wasstudied.

Robustness to dilution. Robustness to dilution was confirmed bydiluting the different selected Valsartan SNEDDS 100 times with0.1 N HCl. The diluted SNEDDS were stored for 12 h andobserved for any sign of phase separation or drug precipitation21.

Assessment of self-emulsification efficiency. Turbidity measure-ment is required to identify the efficiency of the self-emulsifi-cation and whether the dispersion has reached equilibrium ornot22. The selected SNEDDS formulae were diluted 100 timeswith 0.1 N HCl. The percentage transmittance was measuredspectrophotometrically at 638 nm using 0.1 N HCl as a blank23.

Assessment of self-emulsification time. Self-emulsification timewas assessed using a standard USP dissolution apparatus II (TypePTW, Pharma Test, Hainburg, Germany). One hundred milli-grams of each of the selected SNEDDS formula containing 4% w/w Valsartan were loaded onto a watch glass and placed in thebottom of a dissolution vessel containing 500 mL 0.1 N HCl at37 ± 0.5 �C. Gentle agitation was provided by a standard dissol-ution paddle rotating at 50 rpm. Samples were withdrawn at thepredetermined time intervals (1, 3, 5, 7, 9, 11, 13, 15, 17, 19 min)for Valsartan analysis and replaced with equal volumes of fresh0.1 N HCl. The absorbance of the collected samples wasmeasured spectrophotometrically at wavelength of 250 nm14.The time required for emulsification was chosen as the time atwhich 90% Valsartan was measured24.

Viscosity determination. The viscosity of each of the selectedValsartan SNEDDS was determined using Anton Paar rheometer

(Model MCR 702, Graz, Austria) connected to spindle no PP25.A certain volume of each system was placed on the plate ofrheometer under ambient conditions. The viscosity of each systemwas determined by plotting the shear stress versus the shear ratevalues19.

Content uniformity. Content uniformity was done on the differ-ent selected Valsartan SNEDDS, where 500 mg of each systemwas added in volumetric flask containing suitable amount ofmethanol and mixed well with shaking, sonicated (Type USR3,Julabo Labortechnik, Seelbach, Germany) for 10–15 min and thenfiltered through a 0.2mm millipore filter. Aliquot of the filtratewas suitably diluted with methanol and drug content wasdetermined using UV-spectrophotometer at lmax 250 nm14 usingmethanol as a blank25.

Globule size, polydispersity index (PDI) and zeta potentialdetermination. The droplet size of the nanoemulsion is a crucialfactor in self-emulsification performance because it determinesthe rate of drug dissolution and thereby its absorption26. Thepolydispersity values give an indication about the uniformity ofdroplet size within each system27. Zeta potential is a veryimportant factor in characterizing the stability of colloidaldispersions. For the smaller droplet, a high zeta potential willconfer stability in the solution or dispersion by resistingaggregation28. The selected SNEDDS were diluted 100 timeswith double distilled water and sonicated, then each sample wasplaced directly in the module and analyzed by diffraction lightscattering at 25 �C and a scattering angle of 173� for either zetapotential or particle size determination and the average results(±SD) were recorded using Malvern Zetasizer (ver.6.20, Malvern,Worcestershire, UK)21.

In vitro dissolution studies. Each 500 mg SNEDDS system,containing 20 mg drug, was filled into capsules size 00. Dissolutionstudies of Valsartan from the prepared capsules were performedusing the USP Dissolution Tester, Apparatus II (Rotating paddle) ata rotation of 50 rpm over a period of 50 min. Studies were carriedout at 37 ± 0.5 �C in 1 L of 0.1 N HCl (pH 1.2) which was chosen asdissolution medium since it provided better discriminationbetween the different chosen systems. At appropriate time intervals(3, 5, 7, 9, 11, 13, 17, 21, 25, 29, 33, 41 and 49 min), 2 mL samplewas taken, filtered through a 0.2mm filter and analyzed forValsartan content by measuring the absorbance at the predeter-mined lmax 250 nm14 against 0.1 N HCl as blank.

All the dissolution data of Valsartan after 5 min (Q5min) fromthe selected SNEDDS filled into capsules were compared to thatof the commercially available Disartan 80 mg capsules. In vitrodissolution was done in triplicates and the average values (±SD)were taken.

A model independent parameter, the mean dissolution time(MDT), was employed for comparison of dissolution profiles ofthe different Valsartan SNEDDS with the commercially availableDisartan 80 mg capsule and it is calculated according to thefollowing equation:

MDTin vitro ¼Pn

i¼1 tmidDMPni¼1 DM

ð2Þ

where i is the sample number, n is the number of dissolutionsample times, tmid is the time at the midpoint between i and i� 1and DM is the additional amount of drug dissolved between i andi� 1. A low MDT value for a drug delivery system means that ithas a fast in vitro drug dissolution29.

The dissolution efficiency (DE) of a pharmaceutical dosageform, which is the area under the dissolution curve up to a certain

Table 1. Composition of the selected Valsartan SNEDDS systems.

Oil type

Formulano.

Capmul�

MCMLabrafil�

M1944Capryol�

90Labrafac�

PG **S **Cos

F1 10 – – – 10 80F2 10 – – – 80 10F3 10 – – – 40 50F4 30 – – – 40 30F5 – 10 – – 20 70F6 – 10 – – 80 10F7 – 10 – – 40 50F8 – 30 – – 40 30F9 – – 10 – 30 60F10 – – 10 – 80 10F11 – – 10 – 40 50F12 – – 30 – 40 30F13 – – – 10 20 70F14 – – – 10 80 10F15 – – – 10 40 50F16 – – – 30 40 30

**S indicates Surfactant (Cremophore� RH40); Cos indicates Co-surfactant (Transcutol� HP).

DOI: 10.3109/10837450.2015.1078354 Antioxidant effect of Valsartan SNEDDS 911

time t, is expressed as a percentage of the area of the rectangledescribed by 100% dissolution in the same time. DE for SNEDDScompared to the marketed product, Disartan 80 mg capsule, after5 min (Q5min) can be calculated from the following equation30:

DET ¼R T

0yt dt

y100 Tð3Þ

Another model independent parameter, the similarity factor‘‘f2’’ which is suggested in order to decide whether the differencebetween the dissolution profiles of the different systems comparedto the market product was being significant or not and wascalculated by the following equation:

f2 ¼ 50� log1þ 1

nPn

t¼1 Rt � Ttð Þ2

" #�0:5

�100

8<:

9=; ð4Þ

where n is number of dissolution sample times, Rt and Tt are thedissolution of reference and test products at time t. If ‘‘f2’’ value isbetween 50 and 100, this suggests that the data of two dissolutionprofiles are similar31. Statistical analysis of the in vitro dissolutionresults at Q5min of the different selected Valsartan SNEDDS wascomputed by one-way analysis of variance (ANOVA) followed bythe least significant difference (LSD) for multiple comparisons at(p50.05) and unpaired Student’s t-test. Statistical analysis wasperformed by the aid of statistical package for social sciences(SPSS) version 14.0 computer program.

Transmission electron microscopy (TEM). The morphology ofthe optimized SNEDDS formula (F5) was examined usingtransmission electron microscopy (TEM) (JEOL, JEM-1230,Japan). Briefly, an aliquot was prepared after its dilution 100times with distilled water, sufficient quantity of 1% phospho-tungstic acid was added and mixed gently. A drop of the mixturewas placed onto the carbon-coated grid and drained off the excess.The grid was allowed to dry. Photographs were taken at suitablemagnification32.

Determination of the antioxidant effect of the optimized ValsartanSNEDDS formula F5 in adrenaline-induced AMI in rats. Theantioxidant effect of Valsartan in adrenaline-induced AMI in ratswas determined for the optimized SNEDDS formula (F5) whichwas composed of Labrafil� M 1944 (10%), Cremophore� RH40(20%) and Transcutol� HP (70%) with drug loading (4% w/w)and was compared to Valsartan suspension. The study was carriedout according to the work published by Huda and Akhter12.

The research protocol was in accordance and approved byResearch Ethics Committee, Faculty of Pharmacy, CairoUniversity (REC-FOPCU). This study was performed on maleWistar Albino rats (n¼ 20, weight range 150–180 g). Rats werehoused in standard polypropylene cages five per cage understandard laboratory conditions of temperature, humidity and lightwith a free access to standard laboratory diet and water ad libitum.The rats were divided into four groups containing five rats each.

Group I: received distilled water (2 mL) orally throughintragastric tube daily for 14 consecutive days, then distilledwater was given subcutaneously in a single dose 24 h apart fortwo consecutive days from 15th day and served as normal.Group II: received distilled water (2 ml) orally throughintragastric tube daily for 14 consecutive days, then adrenaline(2 mg/kg) was given subcutaneously in a single dose 24 h apartfor two consecutive days from 15th day and served as control.Group III: received Valsartan SNEDDS formula F5 (equivalentto 30 mg Valsartan/kg)33 after its dispersion in 2 mL distilled

water orally through intragastric tube daily for 14 consecutivedays, then adrenaline (2 mg/kg) was given subcutaneously in asingle dose 24 h apart for two consecutive days from 15th dayand served as treatment.Group IV: received Valsartan (30 mg Valsartan/kg) suspendedin 2 mL distilled water orally through intragastric tube daily for14 consecutive days, then adrenaline (2 mg/kg) was givensubcutaneously in a single dose 24 h apart for two consecutivedays from 15th day and served as standard.All the rats were sacrificed 24 h after the last dose under light

anesthesia by ether. About 2 mL of blood from each rat wascollected in a clean and dry test tube by cervical decapitation. Theserum was separated by ultra-centrifugation (4000 rpm for 5 min)and collected by micropipette, transferred to labeled test tubes forbiochemical study as follows:

Estimation of AST level: AST level was estimated according tothe method of Sher and Hung34 using rat AST ELISA assaykit. The concentration of enzyme was measured spectrophoto-metrically at wavelength 340 nm.Estimation of CK-MB level: CK-MB level was estimated usingrat CK-MB ELISA assay kit. The concentration of enzyme wasmeasured spectrophotometrically at wavelength 450 nm.Estimation of GSH concentration: The concentration of GSHwas determined using GSH assay kit. The method depends onthe fact that GSH react with Ellman’s reagent to form a stableyellow color which can be measured spectrophotometrically atwavelength 412 nm12.Estimation of MDA level: MDA level was measured using thethiobarbituric acid reactive substances (TBARS) assay, asdescribed by Mihara and Uchiyama35. The principle of assaydepends on the colorimetric determination of a pink pigmentproduct resulted from the reaction and measured usingspectrophotometer at 532 nm36.All quantitative variables were expressed as mean ± SD.

ANOVA was done for statistical analysis. Post-hoc analysis ofdifferences was done by Least Significant Difference (LSD) test.

Results and discussion

Solubility studies in oils, surfactants and co-surfactants

The equilibrium solubility of Valsartan in different SNEDDScomponents was investigated and recorded in Table 2. Regarding thedifferent oils, Valsartan had the significantly highest solubility(p50.05) in Capmul� (952 ± 3.03) followed by Capryol� 90(363.43 ± 3.81), Labrafil� M 1944 (68.69 ± 2.91) and Labrafac�

PG (1.46 ± 2.14) mg/mL, respectively. Concerning Valsartan solu-bility in different surfactant solutions and co-surfactants, a signifi-cantly higher solubility (p50.05) in 10% Cremophore� RH40solution (29.08 ± 2.54) and Transcutol� HP (1732.85 ± 3.93) mg/mL, respectively. The four different oils were selected to constructthe ternary phase diagrams together with Cremophore� RH40 andTranscutol� HP as surfactant and co-surfactant, respectively.

Screening of surfactants

It is obvious from Figure 1 that Cremophore� RH40, thesurfactant with the highest HLB value, showed significantlyhighest (p50.05) percentage of water incorporated among theother screened surfactants (Labrasol� and Acconon� MCM-2)upon mixing in O:S ratio of (1:1 w/w). Cremophore� RH40 beingthe surfactant of choice is reported to be safe, nonionic with lowchronic toxicity16.

Screening of co-surfactants

Figure 2 shows the percentage of water incorporated in mixturesof (S:Cos mix) in a ratio (1:1w/w) together with each of the

912 M. M. Amin et al. Pharm Dev Technol, 2016; 21(8): 909–920

different used oils. It is clear that the mixture containingTranscutol� HP showed significantly (p50.05) highest percent-age water incorporated compared to the other screened co-surfactants (Lauroglycol� 90 and Poly Ethylene Glycol 400). It isobvious that the addition of co-surfactant led to an increase in thepercentage of water incorporated more than using surfactantalone. This might be due to the fact that the addition of co-surfactant facilitates the penetration of the oil phase into thehydrophobic region of the surfactant monomers, thereby furtherdecreasing the interfacial tension leading to an increase in thefluidity of the interface, enabling the take up of the differentcurvatures required to form nanoemulsions over a wide range ofcompositions37.

Characterization of the different selected ValsartanSNEDDS

Visual inspection

Figure 3(a) shows the different SNEDDS upon diluting 100 timeswith 0.1 N HCl. Only clear and translucent mixtures [grades (A)and (B)] were selected to determine the effect of drug loading (4%w/w) as illustrated in Figure 3(b). It is clear that grades (A) and(B) systems exhibit high percentage transmittance above 90%,which indicates good emulsification ability with globule size in

the nanometer range19. Visual observations revealed that no phaseseparation or drug precipitation was detected upon storage ofValsartan-loaded SNEDDS for 24 h at ambient temperature.

Assessment of self-emulsification efficiency

It has been reported that the drug incorporated into the SNEDDSmay have some effect on the self-emulsifying performance38. Inour study, a little reduction in the nanoemulsion region wasobserved after drug incorporation with SNEDDS composed ofLabrafac� PG or Capryol� 90 as oils with Cremophore� RH40and Transcutol� HP as surfactant and co-surfactant, respectively(Figure 3b). This might be due to the fact that the drug existing inthe surfactant may interfere with its capacity to decrease thesurface tension and hinder co-surfactant intercalation into themolecules of surfactant to form surface membrane, henceinfluencing the efficient self-nanoemulsion region39. On theother hand, no significant change in the nanoemulsion region wasobserved after Valsartan addition for Capmul� MCM andLabrafil� M, respectively. None of the prepared systems, loadedwith 4% w/w Valsartan, showed any drug precipitation within the12 h storage period after dilution for 100 times with 0.1 N HCl.

Generally, efficient emulsification was attained when the S/Cos concentration was 70% w/w or more and this is in accordancewith Oh et al.38 and could be explained by the stabilization of theo/w interface upon increasing the amount of surfactant or co-surfactant leading to the formation of a layer around the emulsiondroplets and reducing the interfacial energy as well as providing amechanical barrier to coalescence.

Assessment of self-emulsification time

Assessment of self-emulsification time of the selected Valsartan-loaded SNEDDS does not only indicate the time needed for theemulsification process under gentle agitation but also to ensurethe dispersion of Valsartan within the dispersed emulsion withoutfurther precipitation. For this purpose, the selected Valsartan-loaded SNEDDS previously mentioned in Table 1 were assessedfor their self-emulsification time.

Time needed for detecting 90% of Valsartan in 0.1 N HCl waschosen as the time required for emulsification as illustrated inFigure 4. Generally, systems with low surfactant concentration upto 30%w/w (F1, F5, F9 and F13) were emulsified in less than one

Figure 1. Histogram showing the percentage of water incorporated into mixtures of different surfactants with (a) Capmul� MCM, (b) Labrafil� M1944, (c) Capryol� 90 and (d) Labrafac� PG as oils at oil:surfactant ratio (O:S) of 1:1 w/w.

Table 2. Saturated solubility of Valsartan in different SNEDDScomponents.

Component Solubility (mg/mL) ± SD

OilsCapmul� MCM 952 ± 3.03Capryol� 90 363.43 ± 3.81Labrafil� M 1944 68.69 ± 2.91Labrafac� PG 1.46 ± 2.14

Surfactants (10% solution)Cremophore� RH40 29.08 ± 2.54Labrasol� 0.22 ± 2.10Acconon� MCM-2 0.15 ± 2.38

Co-surfactantsTranscutol� HP 1732.85 ± 3.93PEG 400 561.66 ± 4.25Lauroglycol� 90 324.44 ± 3.87

DOI: 10.3109/10837450.2015.1078354 Antioxidant effect of Valsartan SNEDDS 913

minute, in contrary to systems with high surfactant concentration(80% w/w) (F2, F6, F10 and F14) where a longer time (more thaneleven minutes) was taken for complete emulsification. Statisticalanalysis of the data revealed a significant difference in self-emulsification time (p50.05) between the above systems. Thismight be due to the fact that systems with high surfactantconcentration resulted in gel formation upon contact with water,such an effect was responsible for their slow dispersion, whilesystems with low surfactant concentration did not form gels and,therefore, dispersed immediately40.

In addition, systems with low oil concentration 10% w/w (F3,F7, F11 and F15) were emulsified in less than three minutes onthe other hand, systems with high oil concentration 30% w/w (F4,F8, F12 and F16) took more than three minutes to be emulsified.Statistical analysis of the data revealed a significant difference inself-emulsification time (p50.05) between these systems. Thismight be due to that increasing the oil concentration led to anincrease in system viscosity that required greater shear forces fordispersion and thus slowing down the system emulsificationprocess41 as shown in Table 3.

It was also reported that high concentration of oil (30% w/w)forms a poor emulsion with reduced ability to entrap water upondilution42–44. Also, systems with low oil concentration (10% w/w)contain higher co-surfactant concentration of 50% w/w more thansystems with high oil concentration that contain only 30% w/wco-surfactant concentration and as reported that increasing the co-surfactant concentration improved the drug dissolution whichmight be due to the penetration of the co-surfactant into thesurfactant monolayer interface, which further enhanced the self-emulsification performance of SNEDDS45.

Content uniformity

Concerning the content uniformity, the selected ValsartanSNEDDS filled into capsules were analyzed for their drugcontent. Results were within the official acceptable range of 85–115%. The actual Valsartan content was found in the average of98.01% and 102.03% of the labeled potency with standarddeviation of less than 2.28%, this means that all formulaecomplied with the parmacopoeial limits46.

Globule size, polydispersity index (PDI) and zeta potentialdetermination

The mean particle diameter, polydispersity index and zetapotential of the selected Valsartan SNEDDS are presented in

Table 4. All the selected systems produced nanoemulsion withglobule size ranged from 11.99 to 134.7 nm47.

Regarding the effect of oil concentration, it was observed thatsystems containing 30% w/w oil produced nanoemulsion withlarger particle size (17.09, 22.13, 21.81 and 134.70 nm) for F4,F8, F12 and F16, respectively, compared to systems containing10% w/w oil (13.90, 14.72, 18.75 and 118.10 nm) in F3, F7, F11and F15, respectively. This might be due to that the penetration ofoil molecules into surfactant chain affected the interfacial filminfluencing the surface curvature of the droplet leading to anincrease in particle size48.

Systems with high surfactant or co-surfactant concentrationproduced nanoemulsion with smaller particle size compared tosystems with low surfactant concentration as increasing theamount of surfactant or co-surfactant leading to the formationof a layer around the emulsion droplets and reducing theinterfacial energy as well as providing a mechanical barrier tocoalescence15. Tang et al. declared a linear relationship betweenthe emulsifier content and the average droplet size, where agradual decrease in droplet size was observed with increasingamount of emulsifier26.

The polydispersity index of all the selected SNEDDS wasfound to be below 0.3, which indicates a good uniformity in thedroplet size distribution49,50. The charge of the droplets was foundto be negative in value due to the presence of free fatty acids witha zeta potential range of �5.15 to �31 mV. Hence, the SNEDDSwould not exhibit any agglomeration as the nanoemulsion wasstabilized by a negative zeta potential and steric stabilizationeffect21.

In vitro dissolution studies

The in vitro dissolution studies of Valsartan from the selectedValsartan SNEDDS filled into capsules were performed in orderto ensure the rapid dissolution of Valsartan into the dissolutionmedium and to give an idea about the self-nanoemulsificationefficiency of the developed systems51. All the dissolution data ofValsartan from the selected SNEDDS were compared to that ofthe commercially available Disartan 80 mg capsules. All statis-tical analysis was done using one-way ANOVA and unpairedStudent’s t-test to study the effect of oil type, oil concentrationand surfactant concentration on the percentage of Valsartandissolved after 5 min (Q5min) from the different SNEDDS filledcapsules. The extent of Valsartan dissolved from the differentselected Valsartan SNEDDS is illustrated graphically in

Figure 2. Histogram showing the percentage water incorporated into (a) Capmul� MCM, (b) Labrafil� M 1944, (c) Capryol� 90 and (d) Labrafac�

PG as oils: [Cremophore� RH40/Cos (1:1 w/w)] mixtures at a ratio of 1:1 w/w.

914 M. M. Amin et al. Pharm Dev Technol, 2016; 21(8): 909–920

Figures 5–8. It is clear that most of the drug was dissolved withinthe first five minutes.

Regarding the effect of oil concentration, it is obvious thatincreasing oil concentration from 10% w/w (formulae F3, F7, F11and F15) to 30% w/w (formulae F4, F8, F12 and F16) significantly

retard the drug dissolution (p50.05), which might be due to thefact that increasing the oil concentration led to an increase in thesystem viscosity that slows down system emulsification41. This is inagreement with Qi et al. who reported that increasing the viscositywas the reason of retardation of drug dissolution52.

Figure 3. (a) Plain SNEDDS and (b) drug-loaded SNEDDS prepared with different oils using Cremophore� RH 40 as surfactant and Transcutol� HPas co-surfactant.

DOI: 10.3109/10837450.2015.1078354 Antioxidant effect of Valsartan SNEDDS 915

Also, systems with low oil concentration containing higher co-surfactant concentration (50% w/w) showed higher drug dissol-ution than systems with high oil concentration composed of 30%w/w co-surfactant. It had been reported that increasing the co-surfactant concentration improved the drug dissolution due to thepenetration of the co-surfactant into the surfactant monolayerinterface, which further enhanced the self-emulsification per-formance of SNEDDS45.

Regarding the effect of surfactant concentration, statisticalanalysis of the data revealed a significant difference in Q5min

(p50.05) between formulae (F1, F5, F9 and F13) containing lowsurfactant concentration (up to 30%) and formulae (F2, F6, F10and F14) containing high surfactant concentration (80%). Thismight be due to the fact that, systems with high surfactantconcentration were considered as the most viscous systems; asthey resulted in gel formation upon contact with water, such aneffect was responsible for the slow dispersion of these formulaeinto nanoemulsions. While systems with low surfactant concen-tration did not form gels and, therefore, dispersed immediately53.

Based on the in vitro dissolution results, formulae F1, F5, F9and F13 were chosen as the selected formulae since they containthe least surfactant concentration (up to 30%) and showed thehighest drug dissolution after five minutes. The dissolutionprofiles of Valsartan from the four selected formulae weresignificantly higher than the commercially available Disartan80 mg capsule at the selected time intervals (p50.05). After5 min, it was found that 78.99% ± 2.24, 76.07% ± 1.10,80.09% ± 1.81 and 69.43% ± 2.24 Valsartan were dissolved from

F1, F5, F9 and F13, respectively, while only 13.43% ± 0.57 wasdissolved from Disartan 80 mg capsule. The faster the dissolutionfrom SNEDDS might be attributed to the fact the drug waspresent in a solubilized form and upon exposure to the dissolutionmedium, small droplets were resulted leading to rapid drugdissolution5.

Regarding the effect of oil type, formula F13 prepared usingLabrafac� PG as oil showed significantly lower percentage69.43% ± 2.24 of drug dissolved after 5 min (p50.05), whencompared to the corresponding values of formulae F1, F5 and F9.This might be due to that formulae F1, F5 and F9 have muchlower globule size (18.30, 13.95 and 15.10 nm) than formula F13(121.60 nm), as previously mentioned in Table 4, and it is wellknown that the behavior of drug is due to the globule size which isinversely proportional to the surface area that means the lesser theglobule size the more is the surface area and thus the higher thedrug dissolution37.

A model independent parameter, the mean dissolution time(MDT), was used to characterize the drug dissolution rate from thedosage form and to predict DE of the prepared systems. LowerMDT for the selected SNEDDS formulae F1, F5, F9 and F13

Figure 4. Percentage of Valsartan detected in 0.1 N HCl from the selected Valsartan-loaded SNEDDS.

Table 4. Globule size, polydispersity index (PDI) and zeta potentialdetermination of the selected Valsartan SNEDDS.

Formula no.Particle

size (nm)Polydispersityindex (PDI)

Zetapotential (mv)

F1 18.30 0.23 �31.00F2 13.07 0.13 �15.50F3 13.90 0.12 �6.16F4 17.09 0.18 �9.65F5 13.95 0.11 �12.80F6 13.13 0.18 �12.30F7 14.72 0.13 �12.10F8 22.13 0.12 �7.61F9 15.10 0.16 �17.70F10 11.99 0.09 �11.70F11 18.75 0.21 �5.15F12 21.81 0.24 �15.00F13 121.60 0.14 �20.90F14 16.86 0.16 �9.47F15 118.10 0.15 �21.40F16 134.70 0.20 �20.90

Table 3. Viscosity of SNEDDS containing low and high oilconcentration.

SNEDDS Oil typeOil concentration

(% w/w)Viscosity(cp) ± SD

F3 Capmul� MCM 10 40.9 ± 0.35F4 Capmul� MCM 30 71.6 ± 0.28F7 Labrafil� M 1944 10 39.2 ± 0.24F8 Labrafil� M 1944 30 48.3 ± 0.26F11 Capryol� 90 10 35.4 ± 0.25F12 Capryol� 90 30 64.6 ± 0.31F15 Labrafac� PG 10 36.7 ± 0.21F16 Labrafac� PG 30 46.4 ± 0.21

916 M. M. Amin et al. Pharm Dev Technol, 2016; 21(8): 909–920

indicates higher DE and faster solubilization of the drug comparedto the marketed product Disartan, as shown in Table 525.

The DE was used to evaluate the dissolution performance ofthe different formulations54. Results of DE percent after fiveminutes (Table 5) revealed that DE of Valsartan from the selectedSNEDDS was increased fivefold compared to Disartan 80 mgcapsule (8.84%/min).

In order to decide whether the difference between thedissolution profiles for the selected SNEDDS comparedwith the marketed product is significant or not, anothermodel independent parameter, the similarity factor ‘‘f2’’ wasapplied. It is clear from Table 5 that all the selectedSNEDDS formulae showed significantly different dissolutionprofiles, ‘‘f2’’ value550, from that of marketed product, Disartan80 mg.

Based on the above results, formula F5 was selected as theoptimized SNEDDS formula taken for further investigations as it

exhibited the smallest globule size 13.95 nm with 76.07% ± 1.10of Valsartan dissolved after five minutes.

Transmission electron microscopy (TEM)

TEM image of the optimized SNEDDS formula (F5) post-dilutionin distilled water was performed. It could be seen from Figure 9that spherical globules were formed with mean globule size of13.8 nm. Furthermore, no signs of drug precipitation wereobserved inferring the stability of formed nanoemulsion37.

Determination of the antioxidant effect of Valsartan selectedSNEDDS formula F5 in adrenaline-induced AMI in rats

Table 6 shows the mean values of serum MDA, GSH, AST andCK-MB 24 h after last adrenaline dose injected. MDA level was0.62 ± 0.11, 1.13 ± 0.32, 0.62 ± 0.09 and 0.91 ± 0.19 mM/mg for

Figure 5. In vitro dissolution profiles of Valsartan from SNEDDS prepared using Capmul� MCM, Cremophore� RH40 and Transcutol� HP.

Figure 6. In vitro dissolution profiles of Valsartan from SNEDDS prepared using Labrafil� M 1944, Cremophore� RH40 and Transcutol� HP.

DOI: 10.3109/10837450.2015.1078354 Antioxidant effect of Valsartan SNEDDS 917

normal, control, treatment and standard groups, respectively.Statistical analysis revealed a significant difference (p50.05)between the group treated with Valsartan SNEDDS formula F5compared to the control and standard groups while a non-significant difference (p40.05) in mean MDA level between thetreatment and normal groups.

Figure 7. In vitro dissolution profiles of Valsartan from SNEDDS prepared using Capryol� 90, Cremophore� RH40 and Transcutol� HP.

Figure 8. In vitro dissolution profiles of Valsartan from SNEDDS prepared using Labrafac� PG, Cremophore� RH40 and Transcutol� HP.

Figure 9. Transmission electron microscopy (TEM) image of SNEDDSformula (F5) nanoemulsion post-dilution with distilled water.

Table 5. Mean dissolution time (MDT), dissolution efficiency (DE) andsimilarity factor (f2) of the selected SNEDDS formulae compared to themarket product (Disartan).

Formulae MDT (min) DE5min (%/min) f2

Disartan 194.61 8.84 –F1 3.77 45.67 9.07F5 3.34 43.47 9.43F9 2.99 48.17 7.70

918 M. M. Amin et al. Pharm Dev Technol, 2016; 21(8): 909–920

Twenty-four hours after the 2nd injection of adrenaline, GSHconcentration in the control, treatment and standard groups was1.24 ± 0.06, 2.77 ± 0.52 and 2.06 ± 0.43 mM/g, respectively,compared to 3.34 ± 0.42 mM/g for the normal group. The treat-ment and normal groups are not significantly different (p40.05)from each other while they are significantly different (p50.05)from the control and standard groups.

Regarding AST level, after the 2nd injection of adrenaline by24 h, it reached 110.50 ± 2.12, 33.00 ± 4.36 and 44.00 ± 7.00 U/Lfor the control, treatment and standard groups in contrary to20.40 ± 2.71 U/L for the normal group. It is obvious fromstatistical analysis that AST level in the treatment group issignificantly (p50.05) lower than its level in the control andstandard groups.

The normal group exhibited CK-MB level equal to3.30 ± 0.54 ng/mL. After the last dose of adrenaline by 24 h, thelevel of CK-MB was 22.00 ± 2.82, 6.47 ± 0.49 and 9.10 ± 1.03 forthe control, treatment and standard groups, respectively. Resultsrevealed that CK-MB level in the treatment group is significantlylower (p50.05) than its level in the control and standard groups.

These results proved that Valsartan SNEDDS formula F5 has ahighly protective effect in adrenaline-induced oxidative stress inrats during myocardial infarction.

Conclusion

The present study showed that formulation of Valsartan SNEDDScould be a promising strategy in improving its dissolution andhence its bioavailability. The optimized SNEDDS formula F5prepared using Labrafil� M 1944 (10% w/w) as oil together withCremophore� RH40 (20% w/w) and Transcutol� HP (70% w/w)as surfactant and co-surfactant, respectively, showed promisingresults in terms of globule size, zeta potential, DE and antioxidanteffect in adrenaline-induced AMI in rats.

Declaration of interest

The authors report no declarations of interest.

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*Means that the groups are significantly different (p50.05) than the treatment (Valsartan SNEDDS formula F5) group.

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