Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20

Self-nanoemulsifying drug-delivery system for

improved oral bioavailability of rosuvastatin using
natural oil antihyperlipdemic

Hadel A. Abo Enin

To cite this article: Hadel A. Abo Enin (2015) Self-nanoemulsifying drug-delivery system
for improved oral bioavailability of rosuvastatin using natural oil antihyperlipdemic, Drug
Development and Industrial Pharmacy, 41:7, 1047-1056

To link to this article: http://dx.doi.org/10.3109/03639045.2014.983113

Published online: 18 Nov 2014.

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 http://informahealthcare.com/ddi
ISSN: 0363-9045 (print), 1520-5762 (electronic)

Drug Dev Ind Pharm, 2015; 41(7): 1047–1056

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.983113

RESEARCH ARTICLE

Self-nanoemulsifying drug-delivery system for improved oral

bioavailability of rosuvastatin using natural oil antihyperlipdemic
Hadel A. Abo Enin1,2
1
Pharmaceutics Department, Taif University, Taif, Saudi Arabia and 2Pharmaceutics Department National Organization of Drug Control and
Downloaded by [University of Wisconsin - Madison] at 06:41 14 September 2015

Research (NODCAR), Giza, Egypt

Abstract Keywords
Aim: The aim is improving the antihyperlipidemic activity of Rosuvastatin Calcium (Rs) through Antihyperlipidemia, natural oil, rosuvastatin
improving its solubility using self-nanoemulsifying drug delivery system (SNEDDS) containing calcium, self-nanoemulsifying drug
natural oil full of unsaturated fatty acid and omega 3. delivery system
Methods: A 7  32 full factorial design was adopted for optimization of oil ratio, Surfactant:
Co-surfactant (S:CoS) ratio and oil:S/CoS ratio. Ternary phase diagrams were constructed for History
optimizing the system with drug loading (10 and 20%). The optimized SNEDD systems were
evaluated according to their physical evaluation and drug release. Furthermore, the anti- Received 30 April 2014
hyperlipidemia efficacy was compared with commercially marketed product on rates followed Revised 25 September 2014
by clinical study. Accepted 24 October 2014
Results: The system containing Tween 80:PEG 400 (3:1) and olive oil:garlic oil (1:1) as an oily Published online 18 November 2014
phase has droplet size less than 100 nm, ZP (+23.43 ± 2.58 mV), PDI (50.02) and cloud point
(490  C). In vitro drug release studies showed remarkable enhancement of the Rs release from
Rs-SNEDDS. The antihyperlipidemic effect of Rs-SNEDDS is greater than that of the commercial
tablets and the pure drug on rates and in hyperlipidemic patients.
Conclusion: Rs-SNEDDS is a promising drug delivery system for improving the drug solubility
and antihyperlipidemic effect using natural oils as (olive oil and garlic oil).

Introduction permeability of the gastrointestinal tract5,6. SNEDDS are able to

emulsify rapidly and spontaneously in the gastrointestinal fluids
Rosuvastatin (Rs) is the most effective statin at reducing LDL-C
and create fine oil/water emulsions under the gentle agitation
and triglycerides (TG) and at raising HDL-C levels thereby. It is a
provided by gastro-intestinal motion7,8. These fine droplets lead to
selective and a competitive inhibitor of HMG-CoA reductase; the
improve the drug solubility capacity in the stomach and its
rate limiting steps in cholesterol biosynthesis. It increases the
dissolution through providing a large interfacing surface area for
number of hepatic LDL receptor on the cell surface, enhances
partitioning the drug between the oil and GIT fluid as a lipid-
uptake and catabolism of LDL, in addition it inhibits the hepatic
based drug delivery system9. Small droplets of oil created by
synthesis of VLDL1.
SNEDDS also increase the drug diffusion into intestinal fluids
Rosuvastatin reveals that it has relatively low absolute oral
(because of large surface area). Moreover, the emulsion droplets
bioavailability (20%). It extensively metabolized by the liver via
lead to faster and more uniform distribution of drug in the GI
oxidation, lactonization and glucuronidation. Food reduces Rs
tract. They also minimize the mucosal irritation due to the contact
bioavailability by approximately 20%, but the extent of absorption
between the drug and the gut wall10.
is unchanged. It occurs at approximately three hours after multiple
SNEDDS are normally prepared as liquid dosage forms then
dosing. Rs is strongly and reversibly bound to plasma protein
incorporated in gelatin capsules. This combination offers the sum
(90%). It has a prolonged effect on hepatic cholesterol synthesis in
of the benefits of both SEDDS and solid dosage forms. As a rapid
animal models. Despite its crystalline nature, the low bioavail-
onset of action (there were no time for dispersion) and stability of
ability mainly attributed to its low solubility2–4.
drug molecules11,12.
Self-nanoemulsifying delivery systems (SNEDDS) are
In SNEDDS, isotropic mixtures of drug, lipid phase (oil),
employed to improving the oral exposure of poorly soluble and
surfactant and/or co-surfactant, forming very tiny emulsion/lipid
lipophilic drugs. It also improves the bioavailability of the drug
droplets, ranging in size between 20 and 300 nm13,14. The oily/
through improving its solubility and increasing the membrane
lipid component usually contains saturated, unsaturated hydro-
carbon or partially unsaturated fatty acid ester or a medium/long
chain, in liquid, semisolid or solid form at room temperature.
Address for correspondence: Hadel Abo El-Enin, Pharmaceutics They are natural or synthetic oils8,15.
Department, National Organization of Drug Control and Research
(NODCAR), 6 Abou Hazem St., Pyramids, Giza, Egypt. Tel: 002012
Natural oils prefer to synthetic oil to reduce the synthetic oils’
4294104. Fax: 00202 35855582. E-mail: hadelaboenin@yahoo.com side effects. For example, the saturated fatty acids and long-chain
 1048 H. A. A. Enin
alcohols showed detergent activity that can disrupt the cell
membranes. Also, oleic acid in high concentration can cause
excessive and continued inflammatory reaction by promoting the
secretion of pro-inflammatory16,17.
Thus, introducing of Rs in the SNEDDS using natural oil as
olive oil, fish oil, linoelic acid, nigella oil and garlic oil was a
promising. These oils are rich in unsaturated fatty acid and omega
3. They also have a significant reduction in total cholesterol (TC)
averaging 10%18–24. For example, an olive oil intake even with
high fat diet simultaneously decreases the cholesterol and has a
beneficial effect on the progression of hypercholesterolemia25.
Also, garlic oil significantly inhibited the hypercholesterolemia,
decreased tissue cholesterol and minimized the atheromatous
changes in the aorta26.
The aim of this study is to develop and characterize SNEDDS
formulations containing rosuvastatin calcium using natural oil
full of unsaturated fatty acid and omega 3. An efficient self-
nanoemulsifying system for Rs was selected and optimized using
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solubility tests and phase diagram construction. The formulations
were characterized by the evaluation of the self-emulsification
performance, droplet size analysis and in vitro drug release
characteristics. Finally, the antihyperlipidemic effect of Rs in
SNEDDS was evaluated on rats and human.
Materials and methods
Materials
Rs was purchased from Merck (Barcelona, Spain). Gelatin
capsules, No. 3, were kindly supplied by Arabia gelatinea,
Egypt. All other chemicals, Tween 80 (polyoxyethylene sorbitan
monooleate), Tween 20 (polyoxyethylene sorbitan monolaurate),
PEG 200 and PEG 400 (poly ethylene glycol) were obtained from
Merck, Mumbai. Cremophor El, propylene glycol and oils (Olive
oil, Fish oil, Linoelic acid, Nigella oil and Garlic oil) were
obtained from Sigma (St. Louis, MO), Fine-Chemicals Limited,
Mumbai. Other chemicals were in analytical grade.
Methods
Determination of rosuvastatin calcium (Rs)
Rs was determined by a modified validated high-pressure liquid
chromatography (HPLC) on an Agilent 1100 HPLC system with
an ultraviolet detector set at wavelength 242 nm. Separation was
achieved using a mobile phase consist of acetonitrile–water
(40:60, v/v) solution at a flow rate of 1.5 ml/min. Rs was
separated by C8 column (HypersilÕ BDS C8 analytical column).
The column was maintained at ambient temperature and injection
volume was 20 mL. The mobile phase was filtered through
0.45 mm filter before using. The linearity ranged from 0.01 to
10 mg/mL with a correlation coefficient 0.9996. Inter-day and
intra-day precisions were done as per ICH guidelines for 3 days.
All precision were below 2%27.
Solubility studies
The solubility of Rs in various buffer media, oils, surfactants (S)
and co-surfactants (CoS) was determined as in a previously
reported method28. An excess amount of Rs was added into each
vial containing 5 ml of the selected vehicle. Then, the mixture was
heated at 40  C in a water bath to facilitate the solubilization. The
systems were shaken at 25  C for 48 h. After equilibrium,
centrifugation of the mixtures was done at 1000 rpm for 10 min
and followed by filtration through a 0.45 -mm Millipore membrane
filter paper. The supernatant was diluted with methanol. The
dissolved Rs in various vehicles were quantified by a previously
validated HPLC method29.
Drug Dev Ind Pharm, 2015; 41(7): 1047–1056
Screening of oils and surfactants and co-surfactants
To evaluate the oil and surfactant capability to form an emulsion
spontaneously, oil was added to surfactant with vigorous vortex in
the 1:1 (w/w) ratio. The mixtures were heated at 40–50  C. Fifty
milligrams oil–surfactant mixtures were diluted with distilled
water in a Stoppard conical flask to 50 ml. The number of flask
inversions (time of emulsification) required to yield homogeneous
emulsion indicates the ease of emulsification. Emulsions were
kept standing for two hours and their percentage transmittance
was evaluated at 638.2 nm using distilled water as a blank.
Various co-surfactants were screened by mixing with surfactant
in 2:1 (w/w) ratio. Oily phase was added to this mixture in 1:1
(w/w). The mixture prepared and evaluated as previously
reported28,30.
Formulation optimization of a blank SNEDDS
The blank SNEDDS formulations were screened by the factorial
design using olive oil and garlic oil as an oil phase, T80 as a
surfactant and PEG 400 as a co-surfactant. Oil, surfactants and co-
surfactants were mixed at prefixed ratios in tubes with vigorous
vortex. A certain amount of the mixtures was added to distilled
water (v/v, 1:100). Four experiments were then performed for
each combination. The solubility test of the samples, emulsifica-
tion rate, turbidity and the mean particle size using a photon
correlation spectroscope were determined. All measurements
were done in triplicate.
Construction of ternary phase diagram
The self-emulsifying region of the selected system with different
drug loading was identified from ternary phase diagrams. The
system composed of olive:garlic ratio 1:1 and T80:PEG 400 3:1
was further studied. A series of self-emulsifying systems with
varying oil phase and S/CoS concentrations were prepared. Rs
was added in each system in different concentration (10–20% w/
w). The samples were then vortexed and sonicated until oily liquid
mixtures were obtained. Each sample was then added into
distilled water (v/v, 1:100) drop by drop. The self-emulsifying
performance was visually observed of the generated samples.
Samples that showed any cracking, drug precipitation or form
immediate coalescence of oil droplets when stirring were rejected.
Phase diagrams were plotted using the above criteria by Chemix
software version 3.5131 (Minnesota, MN).
Characterization and evaluation of SNEDDS
The mean droplet size and Zeta potential determination
The mean droplet size was measured by Laser Diffractometer
(LD) Mastersizer 2000 Version 2.00 (Malvern Instruments,
Malvern, UK). The data were presented in terms of median
(D50) volume and the span, which were calculated by Malvern
Software Version 3.0. For measurement, SNEDDS were analyzed
by dispersing it in 100 ml distilled water under slow agitation at
room temperature 25  C. All systems’ analysis was done in
triplicate.
The zeta potential z of the diluted SNEDDS formulations was
measured using a Malvern zetasizer (Malvern, UK). The
SNEDDS were diluted with distilled water (1:2500 v/v) and
mixed for one minute using a magnetic stirrer. Zeta potential of
each SNEDDS was determined in triplicate32.
Cloud-point measurement
The selected formulations were assessed for cloud-point value.
The formulations were diluted with distilled water (1:100) and
 DOI: 10.3109/03639045.2014.983113
placed in a water bath with gradual increase in temperature.
The drop in the sample percentage transmittance from zero point
(the cloud point) was measured spectrophotometrically at
638.2 nm33,34.
Physical robustness to dilution
Robustness of the selected formulations to dilution was assessed
in the absence and the presence of Rs (10 or 20%). The formulae
were exposed to different dilution folds (100, 500 and 1000) using
different media as distilled water, 0.1 N HCl and phosphate buffer
pH 6.8. Percentage transmittance was then measured spectro-
photometrically at 650 nm. The diluted nanoemulsions were
stored for six hours and monitored for any physical changes
(such as phase separation or precipitation)33,35.
Transmission electron microscopy
Transmission electron microscopy (TEM) was employed as a
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means for morphological examination and globular-size confirm-
ation. Before Rs-SNEDDS analysis, the sample was diluted in
distilled water (1:1000), and a sample drop was placed on a
copper grid. The excess was drawn off with filter paper.
Subsequently, samples were stained with saturated solution of
sodium phosphotungstate (0.2%w/w) for 30 s. The photograph of
drops was obtained using a transmission electron microscope
(JEM-100 CX II, Japan) under a high voltage electricity of
80 KV36.
In vitro drug release studies
Rs-SNEDDS was filled in hard gelatin capsules size ‘‘3’’. An
amount of Rs-SNEDDs equivalent to 10 mg drug was used for
drug release studies. In order to realize the results, the in vitro
drug release studies were also applied on plain Rs and commer-
cial tablets. The test was performed using USP dissolution
apparatus II (Dissolution tester, Pharma Test, PTZ, Germany).
The paddles were rotated at 50 RPM. Dissolution studies were run
using 100 ml of simulated intestinal fluid (pH 6.8 buffer) and
0.1 N HCl (simulated gastric fluid). No enzymes were added to
the media. The temperature was set at 37 ± 0.5  C. During the
release studies, a 1-mL sample of the medium was taken out and
estimated for drug content using the previous described HPLC
method. The samples were withdrawn at different time intervals.
Each study was conducted in triplicate36.
Antihyperlipidemic effects of SNEDDS
Animals and diets
Male rats were provided by the veterinary service NODCAR
(National Organization of Drug Control and Research, Egypt).
They were aged 8 weeks and weighed 245 ± 20 g. All animals
were handled in agreement with the ethical principles in animal
experimentation adopted by the Ethics Committees Accreditation
of laboratory Animal Experimentation Care (AAALAC) with
protocol no. 25/2002.
Eighty-four rats were randomly divided into seven groups
(GpP) of 12 each. Group A received only normal diets and
served as a vehicle. Group B received high fat diets and served
as the model group. Throughout the experimental periods, Group
B and other groups (group A, 1, 2, 3, 4 and 5) were fed high fat
diet. It contains 1% cholesterol, 10% lard, 0.2% methyl thiouracil
and 88.8% usual feed (moisture: 10%; protein: 20%; fat mix:
4%; calcium: 1.0–1.8%; phosphorus: 0.6–1.2; fiber: 5%;
essential amino acids: 2%) and were prepared by the authors.
The diet recipe was a classic formula for the hyperlipidemia
establishing, non-alcoholic fatty liver disease (NAFLD) and
Nanoemulsifying system of natural antihyperlipdemic oil 1049
related diseases recorded in pharmacological method (Third
edition)37.
Eighteen days after the start of the study (after insuring
of NAFLD case), two positive control groups were used [Group 1
(S/CoS) and oil (olive oil:garlic oil, 1:1) in Group 2]. Group 3
received Rs with the dose 10 mg/kg/day. The commercial market
tablets were tested in Group 4 (10 mg/kg/day). The selected
formula was tested in Groups 5. Each animal was received a drug
equivalence to 10 mg/kg/day. Animals received corresponding
treatments every day for another 24 days with continuous feeding
with high fat diet. All rats were fasted for two hours every day
before the therapeutic agent administration.
Assessment of total cholesterol, triglyceride, lipoprotein and liver
marker enzyme in blood
Samples of blood 1.5–2 mL in volume were collected from the
retro-orbital venous plexus once every 6 days throughout the
experiment. Animals were subjected to ether anesthetic, two hours
after administration of therapeutic agents. Animals were fasted for
12 hours in the morning. Serum was centrifuged at 16 000 RPM
for 15 min and analyzed instantly. Levels of AST, ALT, TG, TC,
LDL-C and HDL-C in serum were determined by an enzymatic
colorimetric method using commercial standard enzymatic assay
kits (Biosino Biotechnology & Science Inc., Human, Germany).
TG, TC, LDL-C and HDL-C were tested on days 0, 6, 12, 18, 24,
30, 36 and 42. AST and ALT were tested only on days 0, 18 and
42. All bioassays were carried out in triplicate38.
Clinical study
Thirty patients either male (55.74%) or female sex (44.26%),
normal weight, overweight and obesity according to their BMI
(29.37 ± 1.24), blood pressure: (140.8 ± 3.90/102.5 ± 1.86)
mmHg and heart rate: (63.6 ± 2.98 min1) were included in this
study.
The lipid profile parameters, HDL, LDL, TC and TG were
45.74 ± 4.87, 141.63 ± 21.21, 265.79 ± 45.28 and 232.46 ± 52.14,
respectively. They were interpreted as hyperlipidemic patients.
Also, the LDL/HDL ratio (3.11 ± 1.01), AST and ALT level were
evaluated before and after the experiment. The study protocol was
approved by the Human Ethics Committee of NODCAR. The
procedures followed in this study were with institutional guide-
lines. Written informed consent was obtained from all the enrolled
patients. The test was performed to insure the antihyperlipidemic
effect of the formulated RS-SNEDDS and to compare it with
10 mg commercial oral tablets.
The randomized, non-blind, two treatment study were fol-
lowed. Under this design, the patients were randomly divided into
two groups. Half of the subjects were given orally 10 mg
commercial oral tablets once daily at night and the other half
was given RS-SNEDDS capsule once daily at night (the
composition summarized in Table 4). The total duration of the
study was 12 weeks.
Blood samples were drawn from the patients by a specialized
laboratory technician after an overnight fast at baseline, 6 weeks
later and at the end of the study. A sample was sent for analysis of
triglycerides, total cholesterol (LDL, HDL) and creatine kinase
levels (ALT and AST).
Statistical analysis
All data in this study are expressed in the form of mean ± SD.
The data were evaluated by one-way analysis of variance
(ANOVA), and the differences between means assessed using
Duncan’s test with a significance level of p50.05, 50.01 and
50.001.
 1050 H. A. A. Enin Drug Dev Ind Pharm, 2015; 41(7): 1047–1056

Results and discussions
Solubility study (screening of oil)
The SNEDDs mainly consists of oil, surfactant, co-surfactant and
the drug. All components should produce a clear monophonic
solution when introduced in an aqueous phase at ambient
temperature to allow the presentation of the drug in solution39.
Solubility studies were aimed at identifying a suitable oily phase,
surfactant and co-surfactants for the Rs-SNEDDS develop. It also
used to determine the most suitable media for in vitro release
study. Identifying the suitable oil having the maximal solubilizing
potential for the drug under investigation is very important to
achieve optimum drug loading40. The solubility of Rs in various
oily phases and 10% (w/w) surfactant or co-surfactant solutions is
presented in Table 1.
From the various oily phases that were screened, linoleic
acid improves the maximum solubilizing potential. The solubil-
ity of Rs in it was about (25.026 ± 1.23 mg/mL). The solubility
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other hand, linoleic acid showed poor emulsification efficiency
with T80 as it requires a higher number of flask inversions
(Table 2).
It has been reported that the well-formulated SNEDDS is
dispersed within seconds under gentle stirring conditions which
finally depends on the emulsification ability of the used
surfactant42. The lower emulsification capacity of T20 than T80
was due to its HLB value (16.7). It has a more hydrophilic nature;
therefore, it has the minimum oil solubilization capacity. The
solubilization capacity of oils increases as the hydrophobic chain
length of surfactants increase (more hydrophobic)43. In addition,
T80 has a biological activity and, less affected by pH change in
gastrointestinal drug to facilitate the release profile of Rs44. The
aforementioned results suggested that, the use of garlic oil and
olive oil as an oily phase with T80 as a surfactant is a promised for
this study.
Preliminary screening of co-surfactants

of Rs in garlic oil and olive oil were 21.125 ± 1.16 and

The addition of a co-surfactant was reported to improve the
21.216 ± 0.65 mg/mL, respectively. Statistical analysis represents
dispersibility and the drug absorption45. As depicted in Table 2,
that no significant difference between the solubility of the drug in
co-surfactants, namely, PEG 400 and PG were compared (the
each oil (olive oil and garlic oil). The higher solubility of Rs in
highest drug solubility). Olive oil and garlic oil exhibited a good
surfactant was (16.74 ± 1.76 mg/mL and 17.87 ± 1.09 mg/mL) for
emulsification with them. PEG 400 showed the highest transmit-
T80 and T20, respectively. The maximum Rs solubility was in
tance (99.93%, 99.87%) followed by PG (98.68%, 98.64%).
PEG 400 (9.27 ± 0.34 mg/mL) and PG (7.45 ± 0.23 mg/mL) as a
Herein, the statistical analysis shows a significant difference
co-surfactant. The selection of surfactant and co-surfactant in the
between these co-surfactants. As the higher solubility co-surfac-
further study was governed by the emulsification efficiency. The
tant was the more efficient to penetrate the interface effectively
solubility of the drug was the highest in phosphate buffer pH 6.8.
and the hydrophilic co-surfactant mainly improves the emulsifi-
Therefore, the most suitable media used to study the dissolution of
cation capacity by penetrating the interfacial surface monolayer46.
the formulation was phosphate buffer pH 6.8 (as simulated
Therefore, PEG 400 was chosen as a co-surfactant.
enzyme-free intestinal fluid).
Development of Rs-SNEDDS
Preliminary screening of surfactants
The different systems of SNEDDS were prepared using mixtures
Non-ionic surfactants are considered less toxic than ionic
of garlic oil and olive oil as an oily phase in different ratios. The
surfactants. Therefore, they are accepted for oral administration41.
factorial 7  32 design was involved and represented in Table 3.
In this study, the non-ionic surfactants (T80 and T20) were
The S:CoS ratios were, 1:1, 2:1, 3:1, 1:2, 3:2, 1:3 and 2:3. While
selected out. Linoleic acid, olive oil and garlic oil were selected as
the oily phase represented different olive oil:garlic oil ratios as
oily phase. Results inferred that the oily phase; olive oil and garlic
1:1, 1:2 and 2:1. The oil%:S/Cos mix% was 10:90, 30:70 and
oil exhibited the highest emulsification efficiency with T80
50:50. Sixty-three different combinations were obtained. Each of
[%transparency: 100.02, 99.54 and 3 flask inversions (3 s),
the previous system was evaluated for their mean particle size of
respectively] to formulate a homogenous emulsion. On the
the samples, emulsification rate, turbidity and saturated solubility
of the drug (data not shown).
As the ratio of the surfactant T80 be more than the co-
surfactant (PEG 400) ratio as more stable self-nanoemulsifying
Table 1. The solubility of rosuvastatin calcium in
different media. system. In addition, the particle size became smaller. That agrees
with the previously reported; an excessive amount of co-surfactant
Media Solubility (mg/mL)
Oil
Fish oil 13.69 ± 0.54
Linoleic acid 25.026 ± 1.23 Table 2. Emulsification efficiency of various surfactant using different
Garlic oil 21.125 ± 1.16 oily phases.
Nigella oil 7.69 ± 0.07
Olive oil 21.216 ± 0.65 Oil Linoleic acid Garlic oil Olive oil
Surfactant Surfactant A* B** A* B** A* B**
Tween 80 16.74 ± 1.76
Tween 20 17.87 ± 1.09 Tween 80 40 5 99.54 3 100.02 3
Cremophor 15.29 ± 0.64 Tween 20 41.23 5 90.03 3 91.87 3
Co-surfactant Cremophor 48.34 6 78.54 5 77.45 6
PG 7.45 ± 0.23
PEG 200 6.41 ± 0.11 Co-surfactant:Tween 80 (1:1)
PEG 400 9.27 ± 0.34 Olive oil Garlic oil
Ethanol 1.47 ± 0.47 Co-surfactant A* B** A* B**
Dissolution media
0.1 N HCl 3.11 ± 0.27 PG 98.64 3 98.68 3
Phosphate buffer pH 6.8 5.29 ± 0.40 PEG 400 99.87 3 99.93 3
Phosphate buffer pH 7.4 4.04 ± 0.33
A*: % Transmittance. B**: Number of flask inversion.
 DOI: 10.3109/03639045.2014.983113 Nanoemulsifying system of natural antihyperlipdemic oil 1051

led to the system is less stable due to its intrinsic high aqueous
solubility which lead to an increase in droplet size47,48. Hence,
the selected surfactant to co-surfactant ratio would be 3:1. The
saturated solubility of the drug also increases with increase the
T80 ratio which related to the solubility test results. Also, increase
the olive oil ratio than garlic oil required increase of the T80 ratio.
This may be due to an olive oil which is composed of oleic acid
(monounsaturated fatty acid) has up to the 18-carbon (medium
chain carbon length)49. It was reported that the medium chain
carbon length with a high HLB value produces more stable
SNEDDS (small particle size and low turbidity)50. Therefore,
using S:CoS (3:1) ratio, the oil ratio 1:1 is the most promising.
Another reason for selecting this combination of S/CoS was
the presence of a lower amount of PEG 400. Mei et al.51 reported
that the possibility of instability of SNEDDS formulations which
are intended to be filled in hard gelatin capsule shells when they
contain PEG 400 at high concentrations. The selected system was
stable even with increasing oil ratio and produce high drug
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will lead to decrease the nanoemulsion area in the phase diagrams
(Figure 1a and b). The aim of optimization of the pharmaceutical
preparation was the determination of the best required criteria.
The highest drug solubility and the highest oil phase concentra-
tion are required, in addition, the most stable and the smallest
particle size. Based on the results shown in Figure 1, S1, S2, S3
and S4 were chosen for further studies (Table 4). They have a
large amount of oil ratio, the smallest particle size and the most
stable self-nanoemulsifying systems.
In vitro characterization of optimized SNEDDS
Droplet size and zeta potential determination
Droplet size distribution is a critical factor to evaluate a self-
nanoemulsion system. The particle size decrease gives the
meaning of enhancing the drug bioavailability. The mean
globule size of the selected Rs-SNEDDS formulations was
shown in Table 4. It is clear all formulae had an acceptable

solubility with smaller particle size. nanoparticle size ranged from 50 to 100 nm. This nanoparticle
size indicated the ability of the present formula to offers
Construction of ternary phase diagram large interfacing surface area needed for drug absorption52.
Smaller values of the polydispersity index (PI50.2; within
The pseudo ternary phase diagram of RS-SNEDDS in Figure 1
the acceptable limits) were used as a measure of homogeneity
indicates the nanoemulsion region. Increasing the drug loading
(Table 4). Formula S4 had the lowest mean particle size
(68 ± 4.11 nm; Figure 2a). The results showed a significant
difference between the different formulae.
Table 3. The factorial 7  32 design for formulation of the different The optimized Rs-SNEDDSs showed a significant different
SNEDDS systems were prepared using mixtures of garlic oil and olive oil zeta potential’s value as shown in Table 4. The zeta value and the
as an oily phase in different ratio.
positive charge of increases as the concentration of the drug
increase. This may be due to increasing the Ca ion concentration
Oil mixture (olive oil:garlic oil)
(rosuvastatin calcium). It was reported that the emulsion stability
Oil ratio:S/CoS mix is directly related to the magnitude of its’ surface charge53. In
in the system A (1:1) B (1:2) C (2:1) S*:CoS** general, an increase of electrostatic repulsive forces between
Low (10:90) 10A 10B 10C 1:1 nanoemulsion droplets prevents the droplets’ coalescence. On the
1:2 other hand, a decrease of electrostatic repulsive forces will cause
1:3 phase separation. This indicated that the formulation would
Medium (30:70) 30A 30B 30C 2:1 remain physically stable in storage. Also, the formulation’s zeta
2:3 potential positive charge undergoes an electrostatic interaction
High (50:50) 50A 50B 50C 3:1
3:2 with the mucosal surface of the intestine (mucus layer in the
lumen carries a negative charge) which would lead to enhance the
*S: Tween 80. **CoS: PEG 400 oral bioavailability of the drug54.

Figure 1. The pseudo ternary phase diagram of nanoemulsion region(Rs-SNEDDS). (a) 10% Rs drug loading. (b) 20% Rs drug loading.
 1052 H. A. A. Enin Drug Dev Ind Pharm, 2015; 41(7): 1047–1056

Table 4. The composition of the chosen formulae and their evaluation results.

Formulae S1 S2 S3 S4
Composition (W/W) 10% oil 10% oil 20% oil 20% oil
87.6% S/CoS 86.6% S/CoS 77.6% S/CoS 76.6% S/CoS
1.4% water 1.4% water 1.4% water 1.4% water
10 mg drug 20 mg drug 10 mg drug 20 mg drug
Emulsification rate 3 3 3 3
*Solubility% 50.21 ± 2.01 49.71 ± 3.12 53.65 ± 4.08 59.87 ± 3.54
*Mean particle size (nm) 73.68 ± 1.02 75.70 ± 2.01 69.1 ± 1.80 68 ± 4.11
*Polydispersity index (PI) 0.099 ± 0.014 0.1175 ± 0.024 0.1325 ± 0.03 0.2004 ± 0.02
Turbidity after dilution with water (1:100) (NTU) 4.65 11.465 3.58 1.23
*Zeta potential (mV) +15.65 ± 1.71 +16.43 ± 1.76 +20.65 ± 1.51 +23.43 ± 2.58
Mean Globule size (nm)
Physical robustness
*Water (100) 234 ± 23.81 103 ± 2.41 209 ± 6.98 87 ± 1.11
*Water (500) 571 ± 11.87 105 ± 1.44 611 ± 10.45 90 ± 0.99
*Water (1000) 843 ± 45.88 103 ± 1.45 798 ± 5.31 88 ± 1.02
*0.1 N HCl (1000) 914 ± 23.65 103 ± 3.81 960 ± 19.7 88 ± 2.43
Downloaded by [University of Wisconsin - Madison] at 06:41 14 September 2015

*Phosphate buffer pH 6.8 (1000) 886 ± 33.13 103 ± 8.36 988 ± 21.11 89 ± 1.23
Cloud-point ( C) 60.98 77.85 93 93.8
% Transmittance 93.14–28.14 91.34–23.64 90.67–44.23 –

*All results are mean ± SD and the standard deviation (n ¼ 3).

Physical robustness to dilution

Rs-SNEDDs formulations were exposed to different folds of
dilution in different media (Table 4). These media were
mimicking the in vivo condition after oral administration. S2
and S4 show no phase separation or cloudiness or even
precipitation in the different used media. S3 and S1 show slight
precipitation in different media. Thus, the results confirmed the
robustness of only formula S2 and S4 to dilution in different
media. Statistical analysis shows a significant difference between
the solubility of the drug in different media.

Cloud-point measurement
The cloud point is the temperature above which the formulation
clarity turns into cloudiness. At the cloud point, dehydration of
polyethylene oxide moiety of the non-ionic surfactant, phase
separation and a drop in sample percentage transmittance would
occur. Therefore, the drug solubility and the formulation stability
will decline. Therefore, the cloud point of the formulation should
be higher enough than 37  C (body temperature)35. Different
factors affect on the cloud point value such as drug hydropho-
bicity and mixing ratio of oils, surfactant and co-surfactant used55.
In our study, all formulations were stable as the formed clouds
were reversible after a few minutes. S2 and S4 exhibited the
highest cloudiness point at 93 and 93.8  C, respectively (Table 4).
There was no significant difference between both results. The
%transmittance dropped from 90.67% to 44.23% in S2. Hence, in
S4, the cloudiness did not persist enough time to be measured.
The results confirmed that the Rs-SNEDDS formula (S4) is stable
regarding the separation at gastrointestinal tract temperature.

Transmission electron microscopy

Figure 2. (a) Size distribution intensity of Rs-SNEDDS determined by
laser diffraction sizer after dilution to deionized water of S4. (b) Typical
Transmission electron microscopy of rosuvasatin in nanoemulsion
droplets of S4 at the dilution of 1:50 in deionized water. Bar, 100 nm,
original magnification 50 000.
As formula S4 exhibits the best character and the most
Rs-SNEDDs stability. Therefore, its morphology was observed
using TEM. As shown in the TEM image (Figure 2b), the
prepared Rs-SNEDDS after dilution with distilled water has
spherical particles, no sign of the drug precipitation and a mean
particle size of 50–100 nm. No sign of coalescence confirms the
efficiency of the prepared nanoemulsion. The nanoemulsion
droplets emerged dark and the surroundings were found to be
bright. The nanoemulsion particles closer analysis is surrounded
 DOI: 10.3109/03639045.2014.983113
Downloaded by [University of Wisconsin - Madison] at 06:41 14 September 2015
Figure 3. The in vitro release study of the prepared formula S4 (Rs-SNEDDs equivalent to 10 mg rosuvastatin calcium) was compared in this study
with respect to the plain drug and the commercial product (10 mg rosuvastatin calcium) in 0.1 N HCl media and in phosphate buffer pH 6.8.
by a thick monolayer. This result was explained by the previously
reported that reducing the interfacing energy, and barrier coales-
cence forming result from the formation of monolayer around the
emulsion droplets56.
In vitro dissolution studies
The in vitro release study of the prepared formula S4 was
compared in this study with respect to the plain drug and the
commercial product (Figure 3). The Rs-SENDDs have a higher
dissolution rate than the plain drug and the marketed product in
0.1 N HCl media and in phosphate buffer pH 6.8. After 10 min,
more than 85% of the drug was released from the prepared
formula while the plain drug released 530% in SGF and 40% in
SIF after 60 min. The statistical study of the dissolution result of
Rs-SENDDS (S4) did not show any significant difference between
SGF and SIF (p ¼ 0.324). By that comparison, the Rs-SNEDDS
completely dissolved in 10 min without pH influence. That can be
explained by the drug in both media was not only in free form, but
also entrapped in nanoemulsion particles (micelles form) which
helps in increasing the rate of the drug release through two
reasons. First, the very small particle size (nanoparticle) helps to
disperse rapidly in aqueous media. The other is the drug particle
dispersal in the interface formed by the surfactant and co-
surfactant.
Antihyperlipidemic effect of the selected SNEDDS
The antihyperlipidemic effect of the selected formula was
represented in Figure 4. The TG and TC contents in Group B
were higher than those of Group A on day 6. The TG and TC
contents of Group B were increased to the highest level on day 24,
359.9 ± 61.90 mg/dL and 300.258 ± 38.79 mg/dL, respectively,
almost three times than the control group (72.97 ± 3.12 and
64.63 ± 5.21) for TG and TC, respectively.
Also, LDL-C and HDL-C contents in Group B were higher
than in Group A from the day 6 and through the experiment. At
the end of the study, LDL-C and HDL-C were 260.97 ± 70.74 and
200.74 ± 67.28, respectively. Aspartate aminotransaminase (AST)
Nanoemulsifying system of natural antihyperlipdemic oil 1053
and alanine aminotransaminase (ALT) blood levels were
evaluated on days 0, 18 and 42 (data not shown). No change or
even increases in AST or ALT levels were observed, which
indicating no liver damage will occur during the research period.
The decrease in the raised levels of TG, TC, LDL-C and HDL-
C were illustrated in Figure 4. TC, TG, HDL-C and LDL-C levels
in all groups (G2–G5) were significantly lower than in group B
after treatment (except group 1). Group 1 shows no significant
difference than the GpB. The control and all other groups showed
similar AST and ALT levels, indicating the normal liver function
(data not shown).
At the end of the experiment, TG, TC, LDL-C and HDL-C
lowering effects were the greatest in Group 5 (Rs-SNEDDS
group), even after one week treatment. The TG, TC, LDL-C and
HDL-C levels in Group 5 were dropped to 188.0 ± 50.93,
87.94 ± 40.14, 89.70 ± 96.70 and 82.04 ± 10.14 mg/dL, respect-
ively, which was significantly lower than that of model group
(Group A; p50.001). The rats given market product, oil or even
the drug showed higher results than those gives Rs-SNEDDS at
the end of the study (Groups 2, 3 and 4; p50.05).
The results of the statistical analysis revealed that the
formulation had a significant effect on all the tested parameters
at p50.05 (F5, 17; n ¼ 11.424, 6.469, 2.002 and 43.890 for TC,
TG, LDL-C and HDL-C, respectively). On the other hand, for all
the tested parameters, there was no significant difference between
the subjects (rats).
Multiple comparisons using the Tukey’s HSD test revealed that
TG, TC, LDL-C and HDL-C results in all groups extremely
differed significantly from each other with the lowest value
observed for Rs-SNEDDS followed by the market product, drug
and finally the oil.
In our previous studies, Rs-SNEDDS showed more pro-
nounced effects on TG and TC regulation. The significant
decrease in the TG, TC, LDL-C and HDL-C in the Group 5 than
Group 3 or Group 4 (drug and market product, respectively) was
for many reasons. The first was poor oral absorption of the drug
and the market product (low solubility and large particle size)
compared to Rs-SNEDDs. The other is using an oil mixture (olive
 1054 H. A. A. Enin Drug Dev Ind Pharm, 2015; 41(7): 1047–1056

Figure 4. The antihyperlipidemic effect–time

plot of the selected formula S4 after a
multiple oral dose of 10 mg rosuvastatin
calcium. (a) The mean reduction of the total
cholesterol level. (b) The mean reduction of
the total triglycerides level. (c) The mean
reduction of the HDL-C level. (d) The mean
reduction of the LDL-C level. Group 1:
received (S/CoS) mix. Group 2: received
(O: G, 1:1) mix. Group 3: received Rs with
the dose 10 mg/kg/day. Group 4: received the
commercial market tablets (10 mg/kg/day).
Group 5: received the selected formula.
Group 1, 2, 3, 4 and 5 received high fat diets
throughout the experimental periods.
Downloaded by [University of Wisconsin - Madison] at 06:41 14 September 2015

oil and garlic) in Rs-SNEDDS. These oils were rich in unsaturated Clinical study
fatty acid and omega 3 which have a main effect on the TG and
In Table 5, the mean cholesterol levels, LDL levels and the TG
TC7,8. Also, allicin and diallyl sulfide in the garlic oil have a
levels decreased while the mean HDL levels also increased after
reducing effect on LDL and HDL-C levels than pure drug can be
treatment of the patients by Rs-SNEDDS and the market product.
attributed to their antioxidant effect24,57.
 DOI: 10.3109/03639045.2014.983113
Table 5. The antihyperlipidemic effect after oral administration of drug
in suspension and Rs-SNEDDS (S4) to hyperlipidemic patient
(Mean ± SD, n ¼ 30).
Mean difference
Variable RS-SNEDDS Commercial tabletsÕ p Value
TC 16.53 ± 1.56 5.31 ± 1.11 50.01
TG 39.98 ± 5.46 19.89 ± 2.87 50.01
LDL 2.64 ± 0.98 1.056 ± 0.57 50.01
HDL 9.08 ± 1.08 3.56 ± 1.98 50.01
LDL/HDL ratio 2.45 ± 2.01 2.81 ± 2.14 50.01
A statistical significant difference between these two groups was
also found for mean change p50.01.
In contrast to the above findings, the decrease in the same
parameters was highly significant (p50.01) when using Rs-
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SNEDDS in patients’ treatment. The absence of significant
differences in ALT and AST results, between the baseline and at
the end of the experiment, indicates that the absence of liver
damage after treatment with RS-SNEDDS formula.
These comparable results indicate that improving the anti-
hyperlipidemic effect of Rs by formulating it in self-nanoemulsi-
fying agent. The improvement of the drug solubility leads to
improve its bioavailability. The highest reduction in the lipid
content indicates using natural oils have a synergistic effect with
Rs in treatment of hyperlipidemic patients.
These findings could explain using natural oils full of omega 3
in introducing Rs in SNEDDS will improve the hyperlipidemic
patients’ therapy, but this needs further investigation in more
patient population with hyperlipidemia disease.
Conclusion
From the previous study, we conclude that the formulation of
rosuvastatin calcium in self-nanoemulsifying drug delivery
system using natural oils has antihyperlipidemic effect improved
its solubility and antihyperlipidemic effect. For optimizing the
system, S/Cos ratio (T80:PEG 400; 3:1) was used to decrease the
droplet size and the emulsification time. Using natural oil has
antihyperlipidemic effect been advisable. Hence, it improves the
antihyperlipidemic effect of rosuvastatin calcium. A mixture of
olive oil and garlic oil in the ratio 1:1 was optimized. It improves
the stability of the system and the solubility of the drug in the
system. SNEDDS of Rs showed a significant increase in the
release rate compared to the drug under the same condition,
about more than 85% released within the first 10 min. It is
concluded that Rs-SNEDDS containing olive oil and garlic oil
(1:1) as an oily phase (20% w/w), PEG 400 (19.15% w/w), T80
(57.45% w/w) and water (1.4% w/w) for oral administration would
be a promising dosage form. It has suitable in vitro pharmaceut-
ical results and enhances the antihyperlipidemic effect.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
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