The Preparation Of (E)-Resveratrol Nanoemulsion And In Vitro Release

Yue Zhang1,2,a, Jungang Gao*1,b, Danpeng Ma2,c

1College of Chemistry and Enviromental Science, Hebei University, Baoding 071002, China

2School of Chemical and Pharmaceutical Engineering, Hebei University of Science and

Technology, Shijiazhuang 050018, China

ayuezhang02@163.com, bgaojg@hbu.edu.cn, c19870224dandan@163.com

Key words: nanoemulsion, (E)-resveratrol, pseudo ternary phase diagram, in vitro release

Abstract. Here a novel procedure to prepare stable (E)-resveratrol nanoemulsion is reported. The

optimum (E)-resveratrol nanoemulsion formulation was sifted based on the pseudo ternary phase

diagrams and particle size distribution. An optimized prescription was given as (E)-resveratrol

0.35%, EL-40 22.6%, 1,2-propylene glycol 5.03%, IPM 9.21% and water 62.81% (mass ratio), with

the mean particle size 47.3 nm. The morphology of the (E)-resveratrol nanoemulsion was

characterized by TEM. The test results demonstrate that the nanoemulsion could dramatically

improve the stability and release of (E)-resveratrol.

Introduction

(E)-Resveratrol was firstly isolated from the roots of white hellebore [1,2] and later many

researches have demonstrated that resveratrol is a chemopreventive and therapeutic agent for

cancers, cardiovascular disease, and ischemia [3]. While its poor stability and solubility in water

lead to poor release in vivo and bioavailability. Therefore, it is necessary to seek for a new vehicle

to elevate its stability and solubility in water and promote its absorption rate from the

gastrointestinal lumen. Nanoemulsion is a frequently used drug delivery system carrier possessing

high solubility and thermodynamic stability [4,5]. It can not only protect medicine from oxidation

and hydrolysis, but also improve the bioavailability of the poorly water-soluble drugs [6-8]. Some

present studies on resveratrol nanoemulsion have been reported only for enhancing its stability [9].

The aim of the present work is to select an optimal binding of surfactant and cosurfactant to form a

nanoemulsion with a better particle size distribution at low concentrations of emulsifier. Formula

optimization is based on the ternary phase diagrams and the particle size distributions studies. The

particles formed by optimized formulation are spherical in shape verified by transmission electron

microscopic (TEM) analysis. The tests of stability and in vitro release of the (E)-resveratrol

nanoemulsion have been also investigated.

Experimental details

(E)-Resveratrol was purchased from Xi’an Sino-Herb Bio-Technology Co.LTD (purity 99%),

whose molecular structure was shown in Fig.1. Isopropyl myristate (IPM) was bought from

Shanghai LEASUN chemical Co.LTD. Polyoxyethylene sorbitan fatty acid esters (Tween80),

Polyoxyethylenated castor oil (EL-40) and 1, 2-propylene glycol were purchased from Yongda

Chemical Reagent Development Center and Kangkede Com., Tianjin, China. Other chemical

reagents were all of analytically pure grade and purchased from Modern Reagent Co., Shijiazhuang,

China. Olive oil was made in COFCO, Tianjin, China. Here the twice distilled water was used.

Advanced Materials Research Vols. 236-238 (2011) pp 2357-2361Online available since 2011/May/12 at www.scientific.net© (2011) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMR.236-238.2357

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Fig. 1 Structure of (E)-resveratrol Fig. 2 Solubility of (E)-resveratrol in various components

Pure (E)-resveratrol was dissolved in cosurfactant, by adding surfactant and oil to this mixture,

the combination was mixed in a magnetic mixer for 15 min at 30 oC. Finally the distilled water was

added dropwise to the oily phase with constant stirring to obtain the nanoemulsion.

Results and Discussions

The solubility studies of (E)-resveratrol in various kinds of components are shown in Fig. 2. These

data show that (E)-resveratrol has good solubility both in castor oil and IPM, while later study

shows that the nanoemulsion system containing IPM has an obvious transitional phase inversion

and a considerable stability compared to the one formed by castor oil, which may due to that the

length of its carbon chain and viscosity can promote (E)-resveratrol’s dissolution and distribution.

EL-40 and Tween-80, whose HLBs range is from 8 to 16 and have considerable solubility to

(E)-resveratrol, can be used as surfactants. Surfactant and cosurfactant are mixed at different ratios

as 9:1 and 8:2 respectively. Obviously, nanoemulsions formed by EL-40 along with three kinds of

cosurfactants possessed a better clarity compared to that by Tween-80, which could only arrange

two kinds of cosurfactants in pairs to generate visual transitional phase inversions. In addition, the

maximum amount of oil emulsifies by EL-40 is higher at the two specific ratios (Fig. 3). In the final

analysis, a better stability and a larger nanoemulsion region can be formed by EL-40 along with 1,

2-propylene glycol, and it is favorable to oral administration due to their gentle properties and

non-toxicity to human body.

As seen from Fig. 4, when surfactant and cosurfactant are combined at ratio of 3.5:1 (a), a very

low amount of oil (8.9% w/w) can be emulsified. When surfactant concentration in Smix is

increased to 4:1 (b), the maximum amount of oil emulsified is 13.0% w/w with 31.3% w/w of Smix,

whereas the total area of nanoemulsion somewhat decreases compared to that observed at the ratio

of 3.5:1. An appreciable increase in the nanoemulsion region is observed for further increasing the

proportion of surfactant in the Smix to 4.5:1 (c), and the maximum emulsified amount of oil can be

increased to 13.3% w/w. In contrast, on further increasing the proportion of surfactant in the Smix

from 4.5:1 to 5:1 (d), it is observed that the nanoemulsion region drastically reduce, the amount of

emulsified oil increases to 14.4% w/w with a significantly higher Smix concentration of 34.5% w/w.

No appreciable increase in the nanoemulsion region is observed for further increasing the

proportion of surfactant in the Smix to 5.5:1 (e) or 6:1 (f) and the maximum amount of emulsified

oil can be almost remained the same (Table 1).

2358 Application of Chemical Engineering

Fig. 3 Regions of nanoemulsions formed by EL-40 and Tween-80 along with different cosurfactants

Table 1 Composition of nanoemulsion formulation with different Kms

Km Percentage of different components in formulation with the maximum oil emulsified/w/w

Oil Smix Aqueous

3.5:1 8.9 35.3 55.8

4.0:1 13.0 31.3 55.7

4.5:1 13.3 31.0 55.7

5.0:1 14.4 34.5 51.9

5.5:1 14.3 34.0 51.7

6.0:1 14.0 33.0 53.0

Note: L1: W/O nanoemulsion area; L2: O/W nanoemulsion area; G1: viscous but clarified area; G2: viscous

but opacified area; E: ordinary emulsion area; W: the turbid area.

Fig. 4 Pseudo ternary phase diagrams indicating O/W nanoemulsion region at different Smix ratios

With the favorable Km (4.5), nanoemulsion with different ratios (2.5:1, 2.7:1, 3:1, 5.5:1) of

Smix to oil were prepared, and the droplet size distributions observed by light-scattering studies

are shown in Fig. 5. At the ratio of 2.5:1, the mean droplet size of emulsion is 191.5 nm with

considerable amount of droplets in the range of 3.5~ 5.5µm, this can be explained by the fact that

the amount of Smix is highly insufficient to decrease interfacial tension so as to obtain smaller

droplets. When changing the ratio of Smix to oil in a large scale (5.5:1), two branches of

nanoemulsion with significantly different droplet sizes are obtained, one branch ranging from 14 to

Advanced Materials Research Vols. 236-238 2359

24 nm and the other ranging from 60 to 110 nm, indicating a sharp decrease in interfacial tension

which couldn’t allow forming an ideal nanoemulsion. When the amount of Smix is 3 times to oil,

the nanoemulsion possesses a narrow droplet size distribution ranged from 10 to 100 nm (Fig. 6).

While at the lower ratio of Smix to oil (2.7:1), the mean droplet size is 151.5 nm, similar with the

first one.

Fig. 5 Light-Scattering Studies of nanoemulsion formed at different ratios of Smix to oil

Fig. 6 Light-Scattering Studies of optimized (E)-resveratrol nanoemulsion

Fig. 7 TEM photograph of (E)-resveratrol nanoemulsion Fig. 8 Release test of (E)-resveratrol nanoemulsion

2360 Application of Chemical Engineering

Table 2 Mean particle size, clarity and concentration of (E)-resveratrol in optimized formulation

Time (day) Mean droplet size (nm) Clarity Concentration of (E)-resveratrol (µg.ml-1)

0 47.3±0.07 Yes 37.49±0.05

30 47.3±0.13 Yes 37.49±0.12

60 47.8±0.05 Yes 37.13±0.17

90 49.3±0.17 Yes 36.77±0.13

180 49.5±0.11 Yes 36.73±0.13

Finally we screened the optimal prescription of nanoemulsion formulation with (E)-resveratrol

0.35%, EL-40 22.6%, 1,2-propylene glycol 5.03%, IPM 9.21% and water 62.81% (mass ratio).

TEM shows that particles have spherical shape; the core of the particle namely the darker region

reveals that (E)-resveratrol has been enfolded by this system (Fig. 7). The stability tests show that

the nanoemulsion has the excellent physical stability with no physical phase separation (Table 2).

The release tests show that nanoemulsion in phosphate buffer (pH 7.4) provides the highest

release of 66.5% after 12 h; more than 60% of the (E)-resveratrol is released in the initial 6 h in

comparison to only 3.8% of (E)-resveratrol suspension (Fig.8). Obviously (E)-resveratrol

suspensions show unsatisfied releasing results neither in acetate buffer (pH 3.6) nor in phosphate

buffer (pH7.4), with 11.4% and 2.5% respectively after 12 h, and that should not be ignored is a

decreasing trend after 8 h (pH 7.4), which indicates that pure (E)-resveratrol is poorly stable and

may be degrades under this circumstance, while as a delivery system, nanoemulsion enhances the

stability of (E)-resveratrol and shows an optimal releasing result.

Conclusion

The low energy emulsifying method can prepare (E)-resveratrol nanoemulsion. The studies of

pseudo ternary phase diagrams and droplet size distribution can help us to select the optimal

prescription with a low concentration of emulsifier and more over, a favorable Km and Smix/oil

ratio. TEM analysis and stability studies are performed in characterization section. In vitro release

test, the (E)-resveratrol nanoemulsion shows a characteristic sustained release and accumulative

release ratio can reaches to 66.5%.

Acknowledgements

The authors are thankful to He Bei Normal University for supporting in TEM analysis.

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