Journal of Cereal Science 52 (2010) 282e287

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Journal of Cereal Science

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Physical properties of zein lms containing salicylic acid and acetyl salicylic acid
Narpinder Singh a, Dominique M.R. Georget b, *, Peter S. Belton c, Susan A. Barker b
a

Department of Food Science and Technology, Guru Nanak Dev University, Amritsar-143005, India School of Pharmacy, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK c School of Chemistry, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK
b

a r t i c l e i n f o
Article history: Received 5 March 2010 Received in revised form 7 May 2010 Accepted 7 June 2010 Keywords: Zein Films Salicylic Acetyl salicylic Drug release Protein conformation Tensile properties

a b s t r a c t
Zein lms containing salicylic acid (SA) and acetyl salicylic acid (ASA) between 2 and 10% (initial zein weight basis) with or without glycerol were evaluated for structure, mechanical and dissolution properties. The random coils, a helices and b sheets mainly governed the secondary structure of zein, depending on glycerol and level of model molecules. Adding ASA resulted in an increase in a helices whereas b sheets increased at the expense of a helices when SA was used. Including SA or ASA decreased the tensile strength and the stiffness of lms containing glycerol indicating the synergistic effect of SA and ASA. The strain at failure decreased with increasing content of SA but increased with increasing level of ASA. The dissolution properties were glycerol and drug dependent. ASA release in comparison to SA was quite low. The release was only observed above 10% ASA whereas it was detected in all lms containing SA. The possible interactions between active components and proteins are discussed together with their implications on the physical properties of zein lms. 2010 Elsevier Ltd. All rights reserved.

1. Introduction For the last few decades, studies on zein proteins and their applications in pharmacy have been steadily increasing (ODonnell et al., 1997). Being a food ingredient generally recognized as safe (www.fda.gov), these biopolymers would present no potential harm when included in a drug delivery system. Zein proteins are storage proteins of maize and comprise four sub-families: a zeins, b zeins, d zeins and g zeins. Despite the substantial annual tonnage of maize production (Lawton, 2002), the protein extraction is still expensive (Shukla and Cheryan, 2001). The requirement of maize starch is increasing because of expanding uses of maize starch in food (Tharanathan, 2005) and pharmaceutical (Hauschild and Picker, 2004) applications and especially as a source of biofuel (Farrell et al., 2006; Kim and Dale, 2004). Consequently there will be an increase in the production of zein proteins as co-products. Commercially available zein protein extracts are soluble in alcohol but insoluble in water, potentially making good candidates for a controlled oral delivery matrix of medicine. Our recent study (Georget et al., 2008) showed that zein proteins could be used as excipients in oral dosage forms for controlled drug release. Singh et al. (2009) also examined the use of zein lms together with

iodine for wound dressings and conrmed the real potential of this underutilized biopolymeric material. SA and ASA are molecules of therapeutic activity. Generally SA is used for its keratolytic properties in the treatment of skin conditions (Bashir et al., 2005) whereas ASA is very much known for its antiinammatory and analgesic functions. It has also been shown that it may be administered via the skin (Gerber et al., 2006). The topical route for drug administration might be a good choice as it by-passes the gastro-intestinal tract, and hence the associated side effects and thus may also be more accepted by non-compliant patients. The present study reports the potential use of zein in lms as a controlled release matrix in monolithic devices (that is a polymeric matrix in which an active pharmaceutical ingredient is fully dispersed or dissolved). Tests included measurements of the dissolution prole of ASA and SA from the lms. Additionally, we present here the results of the rst study of the effect of ASA and SA content on the conformation of zein proteins, observed by Fourier Transform Infrared (FTIR). Mechanical properties of zein-glycerol lms containing different levels of drug using a texture analyzer are also reported. 2. Materials and methods 2.1. Materials

* Corresponding author. Tel.: 1 603 593145; fax: 1 603 592003. E-mail address: d.georget@uea.ac.uk (D.M.R. Georget). 0733-5210/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2010.06.008

Zein puried material was from Acros Organics (Geel, Belgium) with a protein content of 97% determined by the Kjedhal method

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(N 6.5) and was used as is. SA, ASA and glycerol were purchased from SigmaeAldrich Company Ltd (Gillingham, UK) and BDH Laboratory Supplies (Pool, UK), respectively. 2.2. Film casting Film casting was done by dissolving zein (15%, w/v) in aqueous ethanol (90% v/v) with and without glycerol (30% on zein wt basis) as plasticizer. Based on the work from Gillgren et al. (2009), glycerol was chosen as the plasticizer. Aliquots (3 g) of this lm mix were weighed into 8.5 cm diameter plastic Petri dishes and gently swirled to coat the bottom of the dish. The Petri dishes were placed without lids on a level surface in an oven at 40  C for 24 h. Three replicate lms were cast for each lm mix. For casting zein lms containing active component, SA or ASA (3%) was dissolved in aqueous ethanol (90% v/v). The aqueous ethanol solution was replaced with SA or ASA solution to produce zein lms containing different concentrations of SA or ASA (2, 6, 8 and 10% on zein wt. basis). Prior to analysis, all lms were stored in a desiccator over P2O5 under vacuum for at least two weeks until constant weight was obtained; subsequent storage was always over P2O5. This ensured that sample moisture content was very close to zero as conrmed by thermogravimetric analysis. 2.3. Thermogravimetric analysis (TGA) The change in lm weight when heated was determined using a thermogravimetric analyzer TA Instruments HI-Res TGA 2950 at a heating rate of 10  C/min to 250  C. This allowed calculating the water loss when detected. Experiments were repeated three times to conrm reproducibility. 2.4. FTIR spectroscopy Spectra were recorded on a BioRad FTS 165 FTIR (Varian Limited, Oxford, UK) spectrometer with a mercury/cadmium/ telluride detector as described earlier (Georget and Belton, 2006). No evidence of any water signal was found in the IR spectra. Films were taken from the desiccator, placed on a single-reection diamond ATR (attenuated total reectance) accessory (SPECAC, Orpington, U.K.) and pressed down to ensure a good contact with the ATR crystal. The spectra (200 spectra at 4 cm1 resolution for each sample) were averaged. Three replicates of each sample were taken using the empty ATR crystal as a reference. Spectral analysis was performed using Omnic v6.1A software (Thermo Nicolet Cooperation, Madison, WI) as described earlier (Wellner et al., 1996). Each spectrum was baseline corrected following the method of Wellner et al. (1996) by ensuring that the spectrum was zeroed at 1800 cm1 where the baseline was relatively at. Fourier self-deconvolution (FSD) was also carried out with an enhancement factor of 1.3 and bandwidth of 30. Positions of the absorbance peaks located in the amide I region were determined using the second derivative (Herald and Smith, 1992). Values of the mean and the standard error of the mean were calculated. 2.5. Mechanical properties The mechanical properties of zein-glycerol lms containing different levels of ASA and SA were measured in tensile mode using a texture analyzer (TAXT2, Stable Microsystems, Godalming, UK) with a 5 Kg load cell as described earlier (Georget et al., 2001). The speed was kept constant at 1 mm/min. In order to measure the tensile properties, lms of a well-dened geometry were used. Film strips 5 mm wide and 50 mm long were cut. The average thickness

of lms was 0.095 0.005 mm, which was measured using a micrometer (outside, range 0.01e0.25 mm). Film strips were single edge notched to a depth of 2.5 mm midway along their length. Ends were glued to two metal plates using cyanoacrylate glue. The mechanical tests were performed to obtain force/ displacement data and tensile strength, strain at failure and estimated modulus. Tensile strength was measured using the following equation:

sfail

Fmax w at

where Fmax is the maximum force associated with failure, w is the width of the strip, a is the notch length and t is the thickness of the strip. Strain at failure is dened as follows:

3fail

Dlmax
l

where Dlmax is the change in length at Fmax and l is the initial length. Estimated modulus is dened as follows:

Eest

dF l w at Dl

where Dl/l is the strain obtained from a t of the initial linear portion of the force/displacement curve and dF is the force at this particular strain. Ten replicates were tested and values of the mean and the standard error of the mean were reported. 2.6. Dissolution properties The lm dissolution studies were based on a modication of the dissolution method described by British Pharmacopoeia (2010). They were carried out in distilled water (200 mL) at 37.0 0.5  C using a mesh stainless steel dissolution basket which was cylindrical in shape (3.6 cm long and 2.2 cm diameter). One lm specimen weighing approximately 100 mg was placed inside one basket. The stirring was done at a constant speed of 480 rpm using a magnetic stirrer (Velp Scientica, Italy). At appropriate time intervals, 2 mL aliquots of dissolution medium were withdrawn and ltered using a 0.22 mm lter (Millipore, Cork, Ireland) and the UV absorbance measured at 296 and 265 nm corresponding to the lmax of SA and ASA, respectively using a spectrophotometer (S-22 Boeco UV/Vis, Boeckel and Co, Hamburg, Germany). The experiments were carried out in triplicate. The dissolution proles of ASA and SA were corrected using the absorbance values obtained from the drug free lms. The values of the mean and the standard error of the mean are reported. 2.7. Statistical analysis The data were analyzed statistically using ANOVA with the software package SPSS. Using a Bonferroni test, signicant difference was considered at the level of p < 0.05. 3. Results and discussion 3.1. Cast lms Transparent pale yellow zein lms containing ASA or SA were produced. The visual examination revealed that the appearance of the lms with or without glycerol was not affected by increasing

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the drug content. The water content of the dried lms irrespective of glycerol content was very close to 0% (wet weight basis).

3.2. Secondary structure of zein Zein protein showed three major bands in the amide I region at 1685, 1650 and 1615 cm1. Based on the assignment of the infrared absorbance peaks previously reported (Forato et al., 2003; Georget and Belton, 2006; Wellner et al., 1996), the secondary structure of zein is mainly governed by a helices (1650 cm1) with some b sheets (1615 cm1). This is consistent with the results of Singh et al. (2009) who used the amide I band of zein lms to determine the secondary structure. The shoulder observed at 1685 cm1 can also be attributed to b sheets (Georget and Belton, 2006). A band at 1745 cm1 corresponding to lipids was also detected. Two discreet bands in the amide II region of similar intensity were observed at 1545 and 1515 cm1. The FTIR spectra for ASA and SA (spectra not shown) were performed. Between 1700 and 1500 cm1, they showed three typical peaks of medium intensity at 1678, 1609 and 1580 cm1 due to the COOH for ASA and 1653, 1605 and 1576 cm1 for SA also due to COOH. These did not affect the analysis of the zein conformation for ASA containing lms. It could be more problematic with SA containing lms, for which an IR absorbance peak at 1653 cm1 is detected which is in the amide I region of zein. However as will be further shown, there was no statistical change in the relative intensity ratio 1650/1615, indicating that the contribution from SA is negligible. The zein lms containing glycerol and 8% ASA showed a prominent trough in the second-derivative curve at 1650 cm1 (associated with random coils and a helices) followed by a valley at around 1615 cm1 (assigned to b sheets) (Fig. 1). Similar spectra were observed for zein lms containing glycerol and SA. A shift from 1645 cm1 to 1650 cm1 and from 1610 cm1 to around 1620 cm1 in the amide I region was observed when the zein powder was cast into lms indicative of a change in chemical environment due to the addition of glycerol, SA and ASA. The trough observed on the second derivative at circa 1650 cm1 was detected and did not signicantly shift irrespective of the inclusion of glycerol, ASA or SA in the lms. However the 1615 cm1 valley detected in zein lm alone shifted towards high frequency values (1620 cm1) upon the addition of glycerol, glycerol with drug or drugs on their own, indicating that the chemical environment of the b sheets had changed. This would

0.004 0.002 0
Arbitrary units

suggest that weak interactions such as hydrogen bonding are more predominant in the absence of plasticizer, resulting possibly in tighter intermolecular b sheets whereas in the presence of glycerol, ASA and SA, there is a solvation effect. The ratio values of the intensities of the absorbance peaks at 1650 cm1 and 1615 cm1 (1650/1615) and the ratio values of the intensities of the absorbance peaks at 1540 cm1 and 1515 cm1 (1540/1515) are reported in Table 1. In lms containing only increasing levels of ASA there is a consistent increase in 1650 cm1intensity relative to the intensity at 1615 cm1. This might suggest that formation of a helices was enhanced; however, an increase in random coil content might also be the cause of such a change in relative intensity. Upon the addition of glycerol, the 1650/1615 ratio is not signicantly affected. With regard to the 1540/1515 ratio, increasing the level of ASA had no effect. When glycerol is included as a plasticizer, the ratio increases as reported by Gao et al. (2006) and Gillgren et al. (2009). The change in intensities of the amide II bands (Table 1) is not what would be expected if the protein is being solvated and protein/ protein hydrogen bonding is being replaced by protein/plasticizer hydrogen bonding (Almutawah et al., 2007; Gillgren et al., 2009). A possible explanation of the two sets of data might therefore be that ASA causes a change in protein structure towards random coil and enhances protein/protein interactions. When glycerol is added, there is some solvation and the ratio 1540/1515 increases. This reduction of protein/protein interaction then allows the relaxation of the system and the formation of more random coils and or a helices. With increasing SA level, the ratio 1650/1615 remains unaltered. Additionally, glycerol has little effect. However, when compared to ASA containing lms, the ratio is signicantly lower, indicating that more b sheets are formed when SA is added, irrespective of glycerol level. The increase in b sheets has been associated with protein aggregation (Gao et al., 2005). The ratio 1540/1515 is comparable to that calculated for ASA containing lms. In the case of SA, the changes in both the amide I and amide II bands (Table 1) are consistent: SA enhances protein/protein interactions with an increase of interprotein hydrogen bonding at the expense of random coil/a helical structure. Once again the effects are mitigated by the addition of glycerol. The work from Hsu et al. (2005) demonstrated, using Raman spectroscopy, that benzoic acid incorporated in zein lms may interact with tyrosine residues on the zein proteins. SA being very similar in structure to benzoic acid, might favor such interactions with the exposed tyrosine on zein proteins, whereas ASA might bind to the proteins to a lesser extent due to steric hindrance of the methyl ester. Casting resulted in SA zein lms with more b sheets, indicative of aggregation of the zein proteins whereas in the ASA containing lms, a helices/random coils predominate.

-0.002 -0.004 -0.006 -0.008 -0.01 -0.012 1750 1700

1685 cm-1 1650 cm-1 1615 cm-1

Table 1 FTIR intensity ratios (1650/1615 and 1540/1515) for zein lms containing ASA, SA and glycerol. For a given intensity ratio and active ingredient, means with the same letter are not signicantly (p < 0.05) different. Drug 1650/1615 No glycerol Control ASA 0% 2% 6% 8% 10% 2% 6% 8% 10% 1.34 1.36 1.42 1.44 1.50 1.20 1.20 1.25 1.30 0.04a 0.03a 0.01a,b 0.04a,b 0.05b 0.01b 0.08b 0.08a,b 0.10a,b With glycerol 1.40 1.44 1.57 1.56 1.54 1.27 1.24 1.22 1.18 0.03a 0.04a 0.01b 0.06b 0.04b 0.08b 0.13b 0.03b 0.08b 1540/1515 No glycerol 1.18 1.11 1.08 1.07 1.04 1.01 1.03 1.11 1.09 0.04a 0.03a,b 0.02a,b 0.03a,b 0.04b 0.04b 0.03b 0.02a,b 0.03a,b With glycerol 1.33 1.31 1.29 1.28 1.25 1.27 1.25 1.23 1.19 0.04a 0.01a 0.03a 0.01a 0.03a 0.08a,b 0.09a,b 0.03a,b 0.08b

1650

1600
-1

1550

Wavenumbers (cm )
Fig. 1. Typical FTIR second-derivative spectra in the amide I region of zein lm (thick black line), zein-glycerol lm (thick grey line), zein containing 8% SA (medium-thick black line), zein-glycerol containing 8% SA (medium-thick grey line), zein containing 8% ASA (thin black line) and zein-glycerol containing 8% ASA (thin grey line). SA

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3.3. Film mechanical properties Due to the brittle nature of the zein lms free of glycerol, the mechanical properties could not be measured. The tensile strength, strain at failure and modulus of zein-glycerol lms containing different levels of ASA and SA are illustrated in Fig. 2. Zein-glycerol lm without ASA/SA showed a tensile strength value of 23.9 MPa which decreased to 12.0 MPa and 16.0 MPa with the incorporation of 10% SA and ASA respectively (Fig. 2a). This demonstrates that inclusion of a model drug in the zein lms might have a synergistic effect with glycerol. The results for strength of drug free lms are in line with values reported by Ghanbarzadeh and Oromiehi (2008) who found values of 12 MPa (tensile strength) for zein lms with 30% glycerol at 50% relative humidity. It also compares to a published value of 20 MPa for dry zein lms containing a similar level of glycerol (Lawton, 2004) and with a similar order of magnitude with published values from Gillgren and Stading (2008) for strength for

30 25
a a b b b b b b b

fail (MPa)

20 15 10 5 0 0 2

10

% ASA or SA (initial zein weight basis)

0.05 0.04

fail

0.03 0.02 0.01 0

a a,c b b b a,b b

a,c b

10

defatted zein lms containing lactic acid, PEG and glycerol at 50% relative humidity, suggesting that the experimental conditions crucially inuence the mechanical properties of zein lms. With regard to the present study, statistically, the effect is similar when both model drugs are included. The strain at failure for drug free zein lms was just below 0.03 (Fig. 2b) which is very low when compared to results from Gillgren and Stading (2008). In their study, the water content was 20% which denitely affected the strain at failure. Indeed the strain at failure will increase with increasing water content. In the present study, the water content determined by TGA was close to 0, hence this would explain this low value of strain at failure as also reported by Gao et al. (2006) who investigated the strain at breakage of karin lms and found a value of 0.026 for dry karin lms. Lawton (2004) and Parris and Cofn (1997) reported comparable values for dry zein lms and lms conditioned at 50% relative humidity, respectively. With the incorporation of SA, strain at failure for zein lms consistently decreases whereas, upon addition of ASA, the strain at failure decreased but further increased with increasing ASA content to reach a value of strain at failure very similar to that for drug free zein lms (Fig. 2b). These results on strain at failure indicate that again model molecules with similar structure resulted in less deformable zein lms. However the trend was reversed when ASA content increased. Moduli of lms (Fig. 2c) containing different levels of SA were signicantly lower than lm without SA. Incorporation of ASA also showed a similar effect on estimated modulus at levels greater than 2%. Based on the results presented in this study, the mechanical properties are affected by the extent of polymer chain associations in the lm matrix. It can only be speculated that SA and ASA might have a plasticizing effect on the zein lms. Further work such as thermal analysis would be required to conrm this effect. This might be compared to the work from Moss et al. (2006). In their study, lms comprising polymethylvinylether, maleic anhydride and hydroxyethylcellulose with tetracaine ranging from 1.5 to 10% (w/w) from initial casting gel were produced. They found that increasing levels of tetracaine resulted in a decrease in tensile strength, elastic modulus and strain at failure. Given that the conformation of the zein proteins in the lms are different; that is, more random coils/a helices in zein lms containing ASA and more b sheets (interproteins bonding) in zein lms with SA, the high deformation properties of the lms remain comparable except for the strain at failure. The mechanical properties represent an overall macroscopic behaviour whereas the FTIR results indicate changes in the secondary structure of zein at a microscopic level. 3.4. Release of ASA and SA from lms Release of both ASA and SA was examined over a period of 75 min at 37  C in distilled water. The release of ASA was only detected at 10% ASA level amongst the lms containing different levels of drug (data not shown). Release of ASA was the lowest in lms containing glycerol. In the absence of glycerol, there was a steady increase in release to 1% at 75 min whereas with glycerol, ASA dissolution only started after 40 min to reach a value of 0.5% at 75 min. SA release continuously increased with increasing dissolution time and was signicantly greater than that of ASA. Release of 75% SA in lms with 8% SA without glycerol (Fig. 3) was observed in comparison to 61% with glycerol. In the absence of glycerol at 2, 6 and 10% SA the dissolution proles were very similar (results not shown for clarity) whereas in the presence of glycerol, the fastest release was observed for the lowest SA loading. The difference in release for the two model molecules could be due to the effect of molecular weight. As reported by Petersson et al. (2007), the release of sugars from karin lms was lowest with the greatest

% ASA or SA (initial zein weight basis)

2000 1500
a a b a b b b b b

Eest (MPa)

1000 500 0 0

10 2 6 8 % ASA or SA (initial zein weight basis)

Fig. 2. Tensile strength (a), strain at failure (b) and estimated modulus (c) of zeinglycerol lms as a function of SA (grey) and ASA (white) concentration. For a given level of glycerol, means with the same letter are not signicantly (p < 0.05) different.

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100 90 80
% drug release

4. Conclusion The conformation, mechanical and dissolution properties of zein lms are affected by glycerol as well as ASA and SA levels. The inclusion of the model drugs signicantly affects the secondary structure of zein, with a relative increasing content in a helices when ASA was added, whereas b sheets predominated when SA was used, indicative of protein aggregation. The lms were weaker with the drugs, with SA containing lms being the strongest. From the dissolution proles observed in this study, the drug release was the greatest in the presence of SA. It is postulated that both drugs differently interact with zein. ASA might be physically trapped in the zein proteins whereas SA might interact with tyrosine via weak bonding. This study demonstrates the importance of the chemical structure of active molecules and warrants in further investigation. From a biomedical perspective, zein lms could act as drug release devices providing that a thorough understanding of the interactions between zein proteins and the active ingredient is established.

70 60 50 40 30 20 10 0

20

40 Duration (mins)

60

80

Fig. 3. SA release from zein lms without (open grey triangle: 8% SA) and with glycerol (open square: 2%; solid square: 6%; solid triangle: 8%; solid diamond: 10%).

molecular weight. This is also supported by the work from Rujiravanit et al. (2003) who found that the release of drugs using chitosan/silk broin lms depended on the molecular weight of the therapeutic molecules, with SA having the lowest molecular weight and amoxicilline trihydrate the highest. In this report, glycerol was used as a plasticizer and it is possible that glycerol can be released from the zein lm at the same time as the drug being released (Petersson et al., 2007). Based on the FTIR data, inclusion of ASA led to an increase in random coils/a helices at the expense of b sheets. Gao et al. (2005) reported that karin with a signicant level of a helices would pack more effectively. If this is the case, it follows that zein lms containing ASA with a relatively high content of a helices would show a certain degree of packing. It is plausible that ASA might become trapped within the packs and hence its release during dissolution slightly hindered. FTIR results suggested that in SA lms, zein proteins tend to aggregate more probably due to the interactions between exposed tyrosine (Hsu et al., 2005) and SA during casting, resulting in protein aggregation. It is also interesting to note that for zein lms containing either ASA or SA, the use of plasticizer led to a signicant decrease in drug release. This effect was unexpected. Visual inspection of the test specimen after dissolution showed that lms though highly exible, remain intact irrespective of the glycerol and drug content. Due to glycerol, a possible increase in viscosity of the diffusion layer might be responsible for the limited diffusion of the drugs into the dissolution medium and this could be perfectly explained by StokeseEinstein equation (Hrter and Dressman (1997) which describes the inverse proportionality between the viscosity and the diffusion coefcient. In parallel, glycerol will also diffuse into the dissolution medium, increasing the viscosity of the dissolution medium and hence negatively affecting ASA and SA release. It is noteworthy that the dissolution volume of this study is four times lower than that described by British Pharmacopoeia (2010). Consequently the release of glycerol in this case will have a greater effect such as an increase in viscosity. Given that the mechanical properties of the lms are comparable irrespective of SA and ASA, the difference in the dissolution properties of lms whether SA or ASA is included can only be explained by the difference in conformation of the zein proteins. The dissolution results put together with the change in conformation and the high deformation analysis would suggest that the physical properties of zein lms signicantly depend on the chemical nature of the active molecules. Interactions might occur either by physical means such as entrapment or by chemical bonds such as weak interactions with the zein proteins.

Acknowledgements Financial assistance from The Royal Society, UK and INSA, New Delhi, India to NS is acknowledged.

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