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W emulsion by NMR and its influence to emulsion stability
Accepted Manuscript Title: The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability Author: Yiqiao Xie Jisheng Chen Shu Zhang Kaiyan Fan Gang Chen Zerong Zhuang Mingying Zeng De Chen Longgui Lu Linlin Yang Fan Yang PII: DOI: Reference:
S0378-5173(16)30031-X http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.032 IJP 15498
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
17-9-2015 2-1-2016 14-1-2016
Please cite this article as: Xie, Yiqiao, Chen, Jisheng, Zhang, Shu, Fan, Kaiyan, Chen, Gang, Zhuang, Zerong, Zeng, Mingying, Chen, De, Lu, Longgui, Yang, Linlin, Yang, Fan, The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability Yiqiao Xie1 [email protected], Jisheng Chen2 [email protected], Shu Zhang1 1
1
[email protected], Kaiyan Fan [email protected], Gang Chen [email protected], Zerong
Zhuang1 [email protected], Mingying Zeng1 [email protected], De Chen1 1
[email protected], Longgui Lu [email protected], Linlin Yang [email protected], Fan Yang 1
1*
3*
[email protected]
Department of Pharmaceutics, Guangdong Pharmaceutical University, Guangzhou,
Guangdong 510006, China. 2
Department of Pharmacy, First Affiliated Hospital of Guangdong Pharmaceutical
University, Guangzhou, Guangdong 510006, China. 3
Guangdong province maternity and child care hospital, Guangzhou, Guangdong
510006, China. Yiqiao Xie and Jisheng Chen can contribute equally as first authors. Both Fan Yang and Linlin Yang are the corresponding authors. *Corresponding author: Linlin Yang: Tel.: +86 137-2535-2700 Fan Yang: Tel.: +86 139-2213-5439
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weak
Stability of emulsion
Graphical Abstract
“
”—egg yolk lecithin
“
”—HS15
Monolayer bilayer
strong
Dosage of co-emulsifier increase
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Abstract Purpose: This paper discussed the influence of microstructure of emulsion membrane on O/W emulsion stability. Methods: O/W emulsions were emulsified with equal dosage of egg yolk lecithin and increasing dosage of co-emulsifier (oleic acid or HS15). The average particle size and centrifugal stability constant of emulsion, as well as interfacial tension between oil and water phase were determined. The microstructure of emulsion membrane had been studied by 1H/13C NMR, meanwhile the emulsion droplets were visually presented with TEM and IFM. Results: With increasing dosage of co-emulsifier, emulsions showed two stable states, under which the signal intensity of characteristic group (orient to lipophilic core) of egg yolk lecithin disappeared in NMR of emulsions, but that (orient to aqueous phase) of co-emulsifiers only had some reduction at the second stable state. At the two stable states, the emulsion membranes were neater in TEM and emulsion droplets were rounder in IFM. Furthermore, the average particle size of emulsions at the second stable state was bigger than that at the first stable state. Conclusions: Egg yolk lecithin and co-emulsifier respectively arranged into monolayer and bilayer emulsion membrane at the two stable states. The microstructure of emulsion membrane was related to the stability of emulsion. Abbreviations O/W emulsion, oil-in-water emulsion; 1H NMR, proton nuclear magnetic resonance;
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C NMR,
carbon nuclear magnetic resonance; TEM, transmission electron microscopy; IFM, inverted fluorescence microscopy; DPPC, dipalmitoyl phosphatidyl choline
Keywords: emulsion membrane; microscopic structure; O/W emulsion; stability; 1H NMR/13C NMR spectroscopy
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1 Introduction O/W emulsion was a heterogeneous liquid dispersion system, consisting of two immiscible fluids through emulsification process. As a carrier of lipophilic ingredients, O/W emulsion had many advantages: (1) enhancing bioavailability of drugs; (2) relieving irritation of components to skin and mucous membrane; (3) covering bad smell of components. Due to these virtues, O/W emulsions had been widely used in various fields, including food[1-3], cosmetic[4-8] and pharmaceutical[9-11] industries. However, the emulsion itself was thermodynamically unstable, possessing a higher interfacial free energy, which resulted in the tendency of separating back into component oil and aqueous phases[12]. In order to maintain the state that oil droplets dispersed homogeneously in aqueous phase, emulsifier and co-emulsifier would form emulsion membrane between oil and aqueous phase. Therefore, the microscopic structure of the emulsion membrane was of great importance to the stability of emulsion. Recently, some studies had proved that, the elements composition and surface topography of emulsion membrane would influence the stability of emulsion with the use of X-ray photoelectron spectroscopy[13-15] and atomic force microscopy[16-19]. But current studies hadn’t exposed the true reasons why the two factors would cause the change of emulsion’s stability. In fact, elements composition and surface topography of emulsion membrane were based on the arrangement of emulsifier and co-emulsifier at oil-water interface. So, to study the arrangement of emulsifier and co-emulsifier at oil-water interface would be helpful to grasp the relationship between microscopic structure of emulsion membrane and the stability of emulsion. In this paper, egg yolk lecithin was chosen as the emulsifier and oleic acid or HS15
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as co-emulsifier to prepare O/W emulsions. Average particle size[20-22], centrifugal stability constant[23] and interfacial tension[24] as recognized indicators were used to evaluate stability of emulsion. Then NMR, TEM and IFM were adopted to study the arrangement of emulsifier and co-emulsifier at oil-water interface. In all of these methods, NMR played the most important role in this study. NMR, as a strong qualitative tool, its application had gradually extended to the field of microscopic structure of emulsion[25-28]. NMR would present the functional groups of the substances in the form of signal peaks at different chemical shift values. For emulsifier and co-emulsifier, the signal intensity was related to the degree of exposure of characteristic functional groups in emulsion[29]. Moreover, the degree of exposure of characteristic functional groups was depended on the arrangement of emulsifier and co-emulsifier at oil-water interface. Therefore, the arrangement of emulsifier and co-emulsifier would be demonstrated according to variation of signal intensity of characteristic functional groups in 1H NMR and 13C NMR. TEM and IFM were both used to observe the microscopic structure of emulsion droplets from different perspectives. TEM had a resolution between 0.1 to 0.3 nm and was used to observe the thickness and shape of emulsion membrane[30-32] , which would be used to infer the arrangement of emulsifier and co-emulsifier at oil-water interface. The size and shape of emulsion droplets could be observed using IFM when emulsifier molecules had been labeled with fluorescence. DPPC was the main composition of egg yolk lecithin, and in this paper, it had been labeled with fluorescent reagent, becoming fluorescent lipid analogue BODIPY-PC[33] (as shown in Figure 1), which could be used to label egg yolk lecithin. This was the first time that IFM was used to study the size and shape of emulsion droplets.
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Analyzing three stability indicators and results of three novel techniques mentioned above comprehensively, the microscopic structure of emulsion membrane could be shown more clearly, and also, the relationship between microscopic structure of emulsion membrane and stability of emulsion could be grasped better. Therefore, the results would have a profound guiding significance for preparing emulsions of high quality. 2 Materials and Methods 2.1 Materials Materials included egg yolk lecithin (Lipoid E80, Lot 510300-2133385/935; German Lipoid Company), HS15 (Lot 96146968E0; German Basf Company), oleic acid (Lot 520200-2120664; German Lipoid Company), soybean oil (Tieling North Oil Co. Medicinal), glycerol (Lot 20100918; Tianjin Kermel Chemical Reagent Co., Ltd.), distilled water (laboratory made), NaOH solution (0.1 mol/L; laboratory made), D2O (Sigma, USA), CDCl3 (Tenglong Weibo Technology, Qingdao), tungstophosphoric acid hydrate (Meilan Industrial Co., Ltd., Shanghai), BODIPY-PC (Lot 1480483; Life technologies, New York) 2.2 Methods 2.2.1 Preparation of emulsions The O/W emulsions were prepared with a two-step emulsification method[34]. Table Ⅰ presented the prescriptions which adopted in the emulsion preparation. Briefly, all surfactants, egg yolk lecithin, oleic acid and HS15, were dissolved in soybean oil to form oil phase. Glycerol was dissolved in distilled water to form aqueous phase. The oil phase and aqueous phase were separately heated up to 70 °C and stirred for a certain time (RET basic, Germany IKA company). Subsequently, the oil phase was
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added drop wise into the water phase under moderate stirring (10000 rpm) (FJ 200-S digital high-speed dispersion homogenizer, Shanghai Specimen Model Plant). The coarse emulsion was passed six times through a 1000 Pa pressure homogenizer (AH-2010, Canada ATS Industrial Systems Co., Ltd.). After homogenization, the pH value of emulsion was adjusted to 7.0 using 0.1 mol/L NaOH solution (Leici PHS-2F pH meter, Shanghai Electronics Scientific Instrument Co., Ltd.).
2.2.2 Average particle size measurement Laser diffraction was used to determine average particle size of emulsions. The measurements were performed with an Average Nanoparticle Analyzer (Delsa Nano C, BECKMAN COULTER). Prior to the measurements, 33 μL of emulsion was diluted by a factor of 300 with distilled water in a 10 mL brown volumetric flask to avoid multi-scattering events. Each measurement was performed in triplicate. Then three measurement results were averaged. 2.2.3 Centrifugal stability constant measurement A certain amount of emulsion was diluted by a factor of 500 with distilled water in a 10 mL brown volumetric flask, and its absorbance value (A) was measured at the wavelength of 500 nm after mixing, using an ultraviolet light–visible spectrophotometer (UV-2550, Shimadzu Corporation). A 10-mL aliquot of the same emulsion was transferred into a centrifuge tube and centrifuged at 4000 rpm for 15 min in a high-speed centrifuge (3-30K, Germany Sigma Corporation). A certain amount of subnatant was diluted by a factor of 500 with distilled water in a 10 mL brown volumetric flask, and its absorbance value (A0) was measured at the wavelength of 500 nm after mixing. The centrifugal stability constant (Ke) was calculated using the Eq. 1. Ke A A0 A0 100%
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(1)
2.2.4 Interfacial tension measurement So far, existing instruments and methods could only measure the interfacial tension between oil phase and water phase before forming emulsion. Therefore, a series of oil phase solutions and water phase solutions were prepared according to the prescriptions showed in Table I. The oil-water interfacial tension was determined at 25 °C by the pendant drop method, which was performed using an automatic interfacial tensiometer (OCA40 Micro, German DataPhysics Company) in conjunction with the Drop Shape Analysis software of SCA20. A drop of oil phase solution was formed in water phase solution at the tip of syringe by pressing the oil phase solution out by means of a setscrew. The drop shape analysis was performed as follows: a drop profile was extracted from the drop image; a curve-fitting program was then used to compare the experimental drop profile with a theoretical profile to derive the corresponding interfacial tension value. 2.2.5 Nuclear magnetic resonance (NMR) High resolution proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) of samples were obtained on AVANCE III spectrometer (Swiss Bruker Company) operating at 500 MHz and 25 °C. All the test solutions prepared as Table Ⅱ were sonicated using a CNC ultrasonic instrument (KQ5200DB; Kunshan Ultrasonic Instrument Co., Ltd.) for 30 cycles of 30 s on and 30 s off each, and then transferred to NMR tubes for measurement. Nuclear magnetism reagent filled in glass capillaries was added for field login.
2.2.6 Transmission electron microscopy (TEM) To confirm the presence of O/W emulsion droplets and characterized the thickness and shape of their emulsion membranes, TEM was applied. A total of 20 µL of
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emulsion was diluted in distilled water to a final volume of 1 mL. One drop of the diluted solution was placed on a carbon-coated copper grid (200 meshes) for 1 min and then stained with the tungstophosphoric acid hydrate solution for 1 min. The analysis was carried out with a JEM-100CXII instrument (JEOL, Tokyo, Japan) at magnifications of 200,000×. 2.2.7 Inverted fluorescence microscopy (IFM) Before preparing fluorescent labeled emulsions, BODIPY-PC was dissolved in methanol (10 μg/mL stock solutions). A volume of stock solutions containing the equivalent of 10-4 mol % unlabelled egg yolk lecithin BODIPY-PC was doped in oil phase. In the process of stirring oil phase, the solvent of methanol was volatilized and BODIPY-PC was left to label egg yolk lecithin. The subsequent steps were the same as those described in 2.2.1. 10 µL fluorescent labeled emulsion was diluted with distilled water to 1 mL. One drop of the diluted solution was placed on glass slide. The analysis was carried out with an AX10 instrument (ZEISS, Germany) at magnifications of 400×. 3 Results 3.1 Average particle size As was seen in Figure 2-I, the average particle size of emulsions decreased after adding co-emulsifier. However, the average particle size of emulsions was not sustained decrease with the dosage of co-emulsifier increased. The average particle size measurement revealed that 0.2 % or 1.8 % HS15 made average particle size further decreased. When the dosage of oleic acid increased, the variation trend of average particle size was the same as that of HS15. Emulsions containing 0.2 % or 0.6 % oleic acid had smaller average particle size.
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3.2 Centrifugal stability constant As was shown in Figure 2-II, the emulsions containing oleic acid or HS15 as co-emulsifier had smaller centrifugal stability constants. However, the emulsions were not more and more stable when the dosage of co-emulsifier increased. Obviously, the centrifugal stability constant of emulsion emulsified with 0.2 % or 1.8 % HS15 was smaller than other combinations’. When the dosage of oleic acid was 0.2 % or 0.6 %, the centrifugal stability constant of emulsion also appeared a further decline. 3.3 Interfacial tension In general, with increasing dosage of co-emulsifier, the interfacial tension between oil and water phase was on the decline. However, the oil-water interfacial tension wouldn’t constantly decrease, which had been verified in this study. As was displayed in Figure 2-III, the variation trend of oil-water interfacial tension was in wavy shape. The smaller interfacial tension achieved when 0.2 % or 1.8 % HS15 was added. Similarly, the interfacial tension became smaller at 0.2 % or 0.6 % oleic acid.
3.4 Nuclear magnetic resonance (NMR) 3.4.1 Proton nuclear magnetic resonance (1H NMR) The 1H NMR of emulsifier and co-emulsifiers were given in Figure 3-IV. The assignment of NMR signals to egg yolk lecithin and HS15 was performed in agreement with the literature[29] (as shown in Figure 3-I/II). And the assignment of NMR signals to oleic acid was performed in agreement with Spectral Database for Organic Compounds (as shown in Figure 3-III). The 1H NMR (as shown in Figure 3-IV) of egg yolk lecithin and oleic acid in CDCl3 showed the most prominent signals correspond to methyl (signal a/A; -CH3: 0.9 ppm) and methylene (signal b/B; -CH2-: 1.3 ppm) protons which were characteristic for
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alkyl chain of fatty acids. The ppm value of the methyl protons of the trimethyl ammonium group (signal e; -N+-(CH3)3: 3.3 ppm) in egg yolk lecithin was shifted to higher ppm due to the vicinity of nitrogen. The 1H NMR (as shown in Figure 3-IV) of HS15 in D2O displayed three distinct signals. Signal α at 0.9 ppm and signal β at 1.3 ppm could be attributed to the methyl and methylene protons of the alkyl chain of 12-hydroxystearic acid. The ppm value of the methylene protons of polyoxyethylene chain (signal γ; -O-CH2-CH2-O-: 3.7 ppm) in HS15 was shifted to higher value due to the vicinity of oxygen. Therefore, the characteristic functional group of egg yolk lecithin was trimethyl ammonium group (signal e; -N+-(CH3)3: 3.3 ppm) and the characteristic functional group of HS15 was polyoxyethylene chain (signal γ; -O-CH2-CH2-O-: 3.7 ppm). However, as the characteristic functional group of oleic acid, carboxyl group didn’t appear in 1H NMR because active hydrogen of carboxyl group would exchange with nuclear magnetism reagent. 1
H NMR of emulsions was shown in Figure 4. For the emulsions stabilized by egg
yolk lecithin and HS15, the signal intensity of polyoxyethylene chain of HS15 had some reduction at 1.8 % HS15 rather than continuously increased with increasing dosage of HS15. While the signal intensity of trimethyl ammonium group of egg yolk lecithin showed a wavy shape variation trend, which first disappeared (point A) then appeared and disappeared (point B) again. For the emulsions stabilized by egg yolk lecithin and oleic acid, the signal intensity of trimethyl ammonium group of egg yolk lecithin had the same variation trend as mentioned above. As was shown in Figure 3-I/II, most functional groups of oleic acid were the same as those of egg yolk lecithin except carboxyl group, however, the signal
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of carboxyl group disappeared in 1H NMR. In order to study how the signal intensity of carboxyl group of oleic acid would change with increasing dosage of oleic acid,
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C
NMR was carried out. 3.4.2 Carbon nuclear magnetic resonance (13C NMR) The 13C NMR of egg yolk lecithin and oleic acid in CDCl3 were given in Figure 5-I. Carboxyl group (signal a; -COOH: 179 ppm) and ester group (signal b; -COOR: 171 ppm) were the characteristic functional groups for oleic acid and egg yolk lecithin respectively. The
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C NMR of emulsions stabilized with egg yolk lecithin and oleic acid were
shown in Figure 5-II. After forming emulsion, the signal of carboxyl group of oleic acid was disappeared. While the signal intensity of ester group of egg yolk lecithin was on the rise except that 0.6% oleic acid was added with the dosage of oleic acid increased. In theory, the signal intensity of ester group should remain unchanged because the dosage of egg yolk lecithin was fixed. But in fact, the signal intensity of ester group changed. In order to confirm whether the carboxyl group of oleic acid would reacted with NaOH as pH regulator to produce ester group (-COONa), the
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C NMR of emulsions
prepared without adjusting pH value was conducted. In Figure 5-III, the signal of carboxyl group of oleic acid appeared at 179 ppm, which indicating that NaOH was the cause of carboxyl group transforming into ester group. The signal intensity of carboxyl group also had some reduction at 0.6 % oleic acid rather than continuously increased with increasing dosage of oleic acid. This time the signal intensity of ester group didn’t change.
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3.5 Emulsion droplet morphology on TEM TEM examination of emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 was shown in Figure 6. Emulsion droplets were presented in core-shell structure, but the thickness and shape of shells were different in TEM. At 0.2 % or 1.8 % HS15 as co-emulsifier, the shells of emulsion droplets were more continuous and neater. However, at other dosages of HS15, the shells of emulsion droplets were displayed in various shapes and oil droplets were pressed into different shapes.
3.6 Emulsion droplet morphology on IFM IFM examination of emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 was shown in Figure 7. Under blue fluorescence excitation, the emulsion droplets presented green fluorescence. With the dosage of HS15 increased, the shape and size of emulsion droplets were different in IFM. At 0.2 % or 1.8 % HS15 as co-emulsifier, the emulsion droplets were rounder than those of other dosages. The emulsion droplets of emulsion containing 1.8 % HS15 were bigger than those containing 0.2 % HS15.
4 Discussions So far, O/W emulsions had been so ubiquitous that it was hard to imagine life without them. And the stability of O/W emulsion was worthy of close attention. Because the stability of emulsion was closely related to the microscopic structure of emulsion membrane. The arrangement of emulsifier and co-emulsifier at oil-water interface might directly influence the stability of emulsion. In order to study this viewpoint, we firstly used average particle size, centrifugal stability constant and oil-water interfacial tension measure methods to study the stability
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of emulsions. In Figure 2, three measurement results showed that they had the same variation trend. Keeping the dosage of egg yolk lecithin constant, average particle size and centrifugal stability constant of emulsion, as well as interfacial tension between oil and water phase showed a wavy shape variation, which firstly decreased then increased and decreased again with increasing dosage of oleic acid or HS15. In this wavy variation trend, two low points appeared, at which the emulsions had better stability. As was shown in Figure 2, the average particle size of point A was smaller than that of point B, but the centrifugal stability constant and oil-water interfacial tension of point A were bigger than that of point B, which illustrated that the stability of emulsions was better at point B. So we surmised that emulsifier and co-emulsifier might arrange into stable monolayer and bilayer emulsion membrane at point A and point B respectively. What’s more, the emulsion with bilayer emulsion membrane had larger particle size, but smaller oil-water interfacial tension and centrifugal stability constant, indicating a better stability. In order to study whether the arrangement of emulsifier and co-emulsifier at oil-water interface would change with increasing dosage of co-emulsifier, 1H NMR and 13
C NMR were used. For emulsions stabilized with egg yolk lecithin and HS15, the
characteristic signal of trimethyl ammonium group of egg yolk lecithin disappeared at 0.2 % and 1.8 % HS15, but the characteristic signal of polyoxyethylene chain of Solutol HS15 never disappeared (as shown in Figure 4-I) in 1H NMR. So, it could be speculated that the trimethyl ammonium group of egg yolk lecithin should tend toward lipophilic core, while the polyoxyethylene chain of Solutol HS15 should tend to aqueous phase (as
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shown in Figure 8). For emulsions stabilized by egg yolk lecithin and oleic acid, the signal of carboxyl group of oleic acid would be observed in 13C NMR. But the signal of carboxyl group of oleic acid appeared only when NaOH wasn’t used to adjust the pH value of emulsion (as shown in Figure 5-II/III). This meant that carboxyl group of oleic acid would react with NaOH to produce ester group (-COONa). As NaOH just exist in aqueous phase, it suggested that the carboxyl group should also tend toward aqueous phase (as shown in Figure 8). Combining the analysis results above with the amphiphilic of surfactants, the directional diagram of emulsifier and co-emulsifier molecules at oil-water interface was deduced as Figure 8. And then, the arrangement of emulsifier and co-emulsifiers at oil-water interface would also be inferred through 1H NMR and 13C NMR.
As was shown in Figure 4, similar with the results of average particle size, centrifugal stability constant and oil-water interfacial tension, the signal intensity of trimethyl ammonium group of egg yolk lecithin had the same wavy variation trend in 1
H NMR of emulsions containing 1.2 % egg yolk lecithin and increasing dosage of
different co-emulsifier (HS15 or oleic acid). And the signal of trimethyl ammonium group disappeared at two low points (point A: 0.2 % HS15/oleic acid; point B: 1.8 % HS15 /0.6 % oleic acid). As was proved above, the trimethyl ammonium group of egg yolk lecithin should tend to lipophilic core. So at the two low points, the dosage of co-emulsifier should be appropriate, which could guarantee the egg yolk lecithin arranging around oil droplet in a closer and more ordered array. As a result, the signal of trimethyl ammonium group tending to lipophilic core disappeared. Furthermore, as the average particle size of
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emulsions at point A was smaller than that at point B, it could be speculated that monolayer and bilayer emulsion membrane respectively formed at point A and B. In other cases, the signal of trimethyl ammonium group could be detected, which demonstrated that the dosage of co-emulsifier was inappropriate, leading to the increase of free egg yolk lecithin. Two reasons might account for why the dosage of free egg yolk lecithin increased. When co-emulsifier was at low concentration, such as 0.1 % HS15 or oleic acid, the emulsion membrane was loose and egg yolk lecithin could escape from emulsion membrane. When co-emulsifier was at high concentration, such as at 0.8/2.0 % HS15 or 0.4/0.8 % oleic acid, co-emulsifier competed against egg yolk lecithin at oil-water interface and some of egg yolk lecithin squeezed out from emulsion membrane. After egg yolk lecithin escaping or squeezing out from emulsion membrane, the trimethyl ammonium group would be exposed and its signal could be detected. The signal intensity of polyoxyethylene chain of HS15 had some reduction at 1.8 % HS15 rather than continuously increased with increasing dosage of HS15 in 1H NMR of emulsion. According to the above explanation, 1.8 % HS15 formed bilayer emulsion membrane with egg yolk lecithin. Furthermore, the polyoxyethylene chain has been proved to tend to aqueous phase, so polyoxyethylene chains which located in inner layer of emulsion membrane were hard to avoid being covered by outer molecules. When monolayer emulsion membrane formed by 0.2 % HS15 and egg yolk lecithin, polyoxyethylene chains were still exposed. So the signal intensity of polyoxyethylene chain became stronger from 0.1 % to 0.2 % HS15. This result supported the viewpoint about monolayer and bilayer emulsion membrane. The signal intensity of carboxyl group of oleic acid decreased at 0.6 % oleic acid rather than continuously increased with increasing dosage of oleic acid in 13C NMR of
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emulsion. According to the above explanation, 0.6 % oleic acid formed bilayer emulsion membrane with egg yolk lecithin. Similarly, the carboxyl group was tended to aqueous phase, so carboxyl groups locating in inner layer of emulsion membrane were hard to avoid being covered by outer molecules. When monolayer emulsion membrane formed by 0.2 % oleic acid and egg yolk lecithin, carboxyl groups were exposed. So the signal intensity of carboxyl group didn’t decrease at 0.2% oleic acid. The viewpoint of monolayer and bilayer emulsion membrane had been verified again. On the base of the analysis above, it could be concluded that both the stability of emulsion and arrangement of emulsifier and co-emulsifier molecules at oil-water interface would change with the dosage of co-emulsifier increased. And these changes could be visually presented with the help of TEM and IFM. In TEM, the shell of emulsion droplet observed was the emulsion membrane which was formed by emulsifier and co-emulsifier. As for 0.2 % and 1.8 % HS15, emulsion membranes were more continuous and neater, and the oil droplets were rounder in TEM photomicrographs. In addition, the emulsion droplets were also rounder in IFM photomicrographs. So in the two cases, the arrangements of emulsifier and co-emulsifier were in order. What’s more, the emulsion droplets of emulsion containing 1.8 % HS15 were bigger than those containing 0.2 % HS15 both in TEM and IFM photomicrographs, which was consistent with the result of average particle size. Thereout, 0.2 %/1.8 % HS15 and egg yolk lecithin respectively arranged into monolayer and bilayer emulsion membrane. While in other dosages of HS15, the thickness of emulsion membranes were uneven and oil droplets had edges and corners in shape in TEM photomicrographs. In addition, the shapes of emulsion droplets weren’t round in IFM photomicrographs. So in
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these cases, the arrangements of emulsifier and co-emulsifier were in disorder. These phenomena in TEM and IFM all reflected arrangement of emulsifier and co-emulsifier molecules at oil-water interface and approved the view about monolayer and bilayer emulsion membrane. 5 Conclusions The microscopic structure of emulsion membrane was inherently connected to the stability of emulsion. When emulsifier and co-emulsifier arranged orderly into monolayer or bilayer emulsion membrane, the stability of emulsions would enhance. When emulsifier and co-emulsifier arranged in disorder at oil-water interface, the stability of emulsions would decrease. This discovery had an important significance for increasing efficiency and minimizing cost during the development of stable emulsions.
Acknowledgements The project were supported by Major Scientific and Technological Project of Guangzhou technology bureau (Item number: 201300000046) and Natural Sciences Fund of The First Affiliated Hospital of Guangdong Pharmaceutical University (Item number: GYFYLH201310).
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lipid oxidation in oil-in-water emulsions. Food Hydrocolloid. 2013; 33: 99-105. [20] Fratter A, Semenzato A. New association of surfactants for the production of food and cosmetic nanoemulsions: preliminary development and characterization. Int J Cosmetic Sci. 2011; 33: 443–449. [21] Mahrhauser D, Nagelreiter C, Baierl A, et al. Influence of a multiple emulsion, liposomes and a microemulsion gel on sebum, skin hydration and TEWL. Int J Cosmetic Sci. 2015; 37: 181–186. [22] Kowalska M, Ziomek M, Żbikowska A. Stability of cosmetic emulsion containing different amount of hemp oil. Int J Cosmetic Sci. 2015; 37: 408–416. [23] Mahmood T, Akhtar N, Manickam S. Interfacial film stabilized W/O/W nano multiple emulsions loaded with green tea and lotus extracts: systematic characterization of physicochemical properties and shelf-storage stability. J Nanobiotecg. 2014; 12: 1-8. [24] Tsai C, Lin LH, Kwan CC. Surface properties and morphologies of pheohydrane/liquid crystal moisturizer product. Int J Cosmetic Sci. 2010; 32: 258-265. [25] Bernewitz R, Schmidt US, Schuchmann HP, et al. Structure of and diffusion in O/W/O double emulsions by CLSM and NMR–comparison with W/O/W. Colloid Surface A. 2014; 458: 10–18. [26] Di Bari V, Norton JE, Norton IT. Effect of processing on the microstructural properties of water-in-cocoa butter emulsions. J Food Eng. 2014; 122: 8–14. [27] Verbi Pereira FM, Rebellato AP, Pallone JAL, et al. Through-package fat determination in commercial samples of mayonnaise and salad dressing using time-domain nuclear magnetic resonance spectroscopy and chemometrics. Food. Control. 2014; 48: 62-66. [28] Giovanna FC, Renzo CS, Lúcio LB, et al. Characterisation and selection of
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demulsifiers for water-in-crude oil emulsions using low-field 1H NMR and ESI–FT-ICR MS. Fuel. 2015; 140: 762-769. [29] Schubert MA, Harms M, Müller-Goymann CC. Structural investigations on lipid nanoparticles containing high amounts of lecithin. Eur J Pharm Sci. 2006; 27: 226-236. [30] Victoria K, Claudia V, Nadejda B. Analytical electron microscopy for characterization of fluid or semi-solid multiphase systems containing nanoparticulate material. Matsko Pharm. 2013; 5: 115-126. [31] Imura T, Nakayama M, Taira T, et al. Interfacial and emulsifying properties of soybean peptides with different degrees of hydrolusis. J Oleo Sci. 2014; 12: 1-7. [32] Arujo FA, Kelmann RG, Araujo BV, et al. Development and characterization of parenteral nanoemulsions containing thalidomide. Eur J Pharm Sci. 2011; 42: 238–245. [33] Sach R, Boldyrev I, Johansson LBA. Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers. Phys Chem Chem Phys. 2010; 12: 6027–6034. [34] Piao JK, Adachi SJ. Stability of O/W emulsions prepared using various monoacyl sugar alcohols as an emulsifier. Innov Food Sci Emerg. 2006; 7: 211-216.
22
Figure Captions
Figure 1 The structure of fluorescent lipid analogue BODIPY-PC.
23
(I)
(II)
(III) Figure 2
(I) Average particle size of O/W emulsions, (II) Centrifugal stability constant of
O/W emulsions, (III) Interfacial tension between oil and water phase that were prepared
24
according to the prescriptions showed in Table I: with egg yolk lecithin (1.2 %, w/v) and increasing dosage of (left) HS15; (right) oleic acid.
25
(I)
(II)
(III)
(IV)
26
Figure 3
(I) The structure of Egg yolk lecithin; (II) The structure of Oleic acid; (III) The
structure of HS15; (IV) 1H NMR of emulsifier and co-emulsifiers.
27
(I)
(II)
Figure 4 1H NMR of emulsions: (I) emulsified with egg yolk lecithin and HS15; (II) emulsified with egg yolk lecithin and oleic acid.
28
(I)
(II)
(III) Figure 5
13
C NMR of (I) egg yolk lecithin and oleic acid; (II) emulsions emulsified with egg
29
yolk lecithin and oleic acid and its amplified spectra (right); (III) emulsions emulsified with egg yolk lecithin and oleic acid and its amplified spectra (right), but hadn’t its pH value adjusted.
30
Figure 6 TEM photomicrographs of the emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 at magnifications of 200,000×. The dosage of HS15 was: (A) 0.1 %; (B) 0.2 %; (C) 0.8 %; (D) 1.8 %; (E) 2.0 % (w/v).
31
Figure 7 IFM photomicrographs of the emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 at magnifications of 400×. The dosage of HS15 was: (A) 0.1 %; (B) 0.2 %; (C) 0.8 %; (D) 1.8 %; (E) 2.0 % (w/v).
32
Figure 8 Directional diagram of emulsifier and co-emulsifiers at oil-water interface of O/W emulsion.
33
Tables Table Ⅰ Composition of O/W emulsions Excipients (%, w/v)
Formulation 1
Formulation 2
Soybean oil
10
10
Egg yolk lecithin
1.2
1.2
Oleic acid
—
0.1/0.2/0.4/0.6/0.8
HS15
0.1/0.2/0.8/1.8/2.0
—
Glycerol
2.25
2.25
Distilled water
86.45/86.35/85.75/84.75/84.55
86.45/86.35/86.15/85.95/85.75
34
Table Ⅱ Preparation of test solutions for NMR Nuclear sequence Test objects
Test project
magnetism
number reagent 1
Egg yolk lecithin
1
H NMR/13C NMR
CDCl3
2
Oleic acid
1
H NMR/13C NMR
CDCl3
3
HS15
1
H NMR
D2O
H NMR/13C NMR
D2O
Emulsion stabilized by egg yolk lecithin 4
1
(1.2 %, w/v) and oleic acid (0.1/0.2/0.4/0.6/0.8 %, w/v) Emulsion stabilized by egg yolk lecithin
5
(1.2 %, w/v) and Solutol HS15 (0.1/0.2/0.8/1.8/2.0 %, w/v)
35
1
H NMR
D2O
S0378-5173(16)30031-X http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.032 IJP 15498
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
17-9-2015 2-1-2016 14-1-2016
Please cite this article as: Xie, Yiqiao, Chen, Jisheng, Zhang, Shu, Fan, Kaiyan, Chen, Gang, Zhuang, Zerong, Zeng, Mingying, Chen, De, Lu, Longgui, Yang, Linlin, Yang, Fan, The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability Yiqiao Xie1 [email protected], Jisheng Chen2 [email protected], Shu Zhang1 1
1
[email protected], Kaiyan Fan [email protected], Gang Chen [email protected], Zerong
Zhuang1 [email protected], Mingying Zeng1 [email protected], De Chen1 1
[email protected], Longgui Lu [email protected], Linlin Yang [email protected], Fan Yang 1
1*
3*
[email protected]
Department of Pharmaceutics, Guangdong Pharmaceutical University, Guangzhou,
Guangdong 510006, China. 2
Department of Pharmacy, First Affiliated Hospital of Guangdong Pharmaceutical
University, Guangzhou, Guangdong 510006, China. 3
Guangdong province maternity and child care hospital, Guangzhou, Guangdong
510006, China. Yiqiao Xie and Jisheng Chen can contribute equally as first authors. Both Fan Yang and Linlin Yang are the corresponding authors. *Corresponding author: Linlin Yang: Tel.: +86 137-2535-2700 Fan Yang: Tel.: +86 139-2213-5439
1
weak
Stability of emulsion
Graphical Abstract
“
”—egg yolk lecithin
“
”—HS15
Monolayer bilayer
strong
Dosage of co-emulsifier increase
2
Abstract Purpose: This paper discussed the influence of microstructure of emulsion membrane on O/W emulsion stability. Methods: O/W emulsions were emulsified with equal dosage of egg yolk lecithin and increasing dosage of co-emulsifier (oleic acid or HS15). The average particle size and centrifugal stability constant of emulsion, as well as interfacial tension between oil and water phase were determined. The microstructure of emulsion membrane had been studied by 1H/13C NMR, meanwhile the emulsion droplets were visually presented with TEM and IFM. Results: With increasing dosage of co-emulsifier, emulsions showed two stable states, under which the signal intensity of characteristic group (orient to lipophilic core) of egg yolk lecithin disappeared in NMR of emulsions, but that (orient to aqueous phase) of co-emulsifiers only had some reduction at the second stable state. At the two stable states, the emulsion membranes were neater in TEM and emulsion droplets were rounder in IFM. Furthermore, the average particle size of emulsions at the second stable state was bigger than that at the first stable state. Conclusions: Egg yolk lecithin and co-emulsifier respectively arranged into monolayer and bilayer emulsion membrane at the two stable states. The microstructure of emulsion membrane was related to the stability of emulsion. Abbreviations O/W emulsion, oil-in-water emulsion; 1H NMR, proton nuclear magnetic resonance;
13
C NMR,
carbon nuclear magnetic resonance; TEM, transmission electron microscopy; IFM, inverted fluorescence microscopy; DPPC, dipalmitoyl phosphatidyl choline
Keywords: emulsion membrane; microscopic structure; O/W emulsion; stability; 1H NMR/13C NMR spectroscopy
3
1 Introduction O/W emulsion was a heterogeneous liquid dispersion system, consisting of two immiscible fluids through emulsification process. As a carrier of lipophilic ingredients, O/W emulsion had many advantages: (1) enhancing bioavailability of drugs; (2) relieving irritation of components to skin and mucous membrane; (3) covering bad smell of components. Due to these virtues, O/W emulsions had been widely used in various fields, including food[1-3], cosmetic[4-8] and pharmaceutical[9-11] industries. However, the emulsion itself was thermodynamically unstable, possessing a higher interfacial free energy, which resulted in the tendency of separating back into component oil and aqueous phases[12]. In order to maintain the state that oil droplets dispersed homogeneously in aqueous phase, emulsifier and co-emulsifier would form emulsion membrane between oil and aqueous phase. Therefore, the microscopic structure of the emulsion membrane was of great importance to the stability of emulsion. Recently, some studies had proved that, the elements composition and surface topography of emulsion membrane would influence the stability of emulsion with the use of X-ray photoelectron spectroscopy[13-15] and atomic force microscopy[16-19]. But current studies hadn’t exposed the true reasons why the two factors would cause the change of emulsion’s stability. In fact, elements composition and surface topography of emulsion membrane were based on the arrangement of emulsifier and co-emulsifier at oil-water interface. So, to study the arrangement of emulsifier and co-emulsifier at oil-water interface would be helpful to grasp the relationship between microscopic structure of emulsion membrane and the stability of emulsion. In this paper, egg yolk lecithin was chosen as the emulsifier and oleic acid or HS15
4
as co-emulsifier to prepare O/W emulsions. Average particle size[20-22], centrifugal stability constant[23] and interfacial tension[24] as recognized indicators were used to evaluate stability of emulsion. Then NMR, TEM and IFM were adopted to study the arrangement of emulsifier and co-emulsifier at oil-water interface. In all of these methods, NMR played the most important role in this study. NMR, as a strong qualitative tool, its application had gradually extended to the field of microscopic structure of emulsion[25-28]. NMR would present the functional groups of the substances in the form of signal peaks at different chemical shift values. For emulsifier and co-emulsifier, the signal intensity was related to the degree of exposure of characteristic functional groups in emulsion[29]. Moreover, the degree of exposure of characteristic functional groups was depended on the arrangement of emulsifier and co-emulsifier at oil-water interface. Therefore, the arrangement of emulsifier and co-emulsifier would be demonstrated according to variation of signal intensity of characteristic functional groups in 1H NMR and 13C NMR. TEM and IFM were both used to observe the microscopic structure of emulsion droplets from different perspectives. TEM had a resolution between 0.1 to 0.3 nm and was used to observe the thickness and shape of emulsion membrane[30-32] , which would be used to infer the arrangement of emulsifier and co-emulsifier at oil-water interface. The size and shape of emulsion droplets could be observed using IFM when emulsifier molecules had been labeled with fluorescence. DPPC was the main composition of egg yolk lecithin, and in this paper, it had been labeled with fluorescent reagent, becoming fluorescent lipid analogue BODIPY-PC[33] (as shown in Figure 1), which could be used to label egg yolk lecithin. This was the first time that IFM was used to study the size and shape of emulsion droplets.
5
Analyzing three stability indicators and results of three novel techniques mentioned above comprehensively, the microscopic structure of emulsion membrane could be shown more clearly, and also, the relationship between microscopic structure of emulsion membrane and stability of emulsion could be grasped better. Therefore, the results would have a profound guiding significance for preparing emulsions of high quality. 2 Materials and Methods 2.1 Materials Materials included egg yolk lecithin (Lipoid E80, Lot 510300-2133385/935; German Lipoid Company), HS15 (Lot 96146968E0; German Basf Company), oleic acid (Lot 520200-2120664; German Lipoid Company), soybean oil (Tieling North Oil Co. Medicinal), glycerol (Lot 20100918; Tianjin Kermel Chemical Reagent Co., Ltd.), distilled water (laboratory made), NaOH solution (0.1 mol/L; laboratory made), D2O (Sigma, USA), CDCl3 (Tenglong Weibo Technology, Qingdao), tungstophosphoric acid hydrate (Meilan Industrial Co., Ltd., Shanghai), BODIPY-PC (Lot 1480483; Life technologies, New York) 2.2 Methods 2.2.1 Preparation of emulsions The O/W emulsions were prepared with a two-step emulsification method[34]. Table Ⅰ presented the prescriptions which adopted in the emulsion preparation. Briefly, all surfactants, egg yolk lecithin, oleic acid and HS15, were dissolved in soybean oil to form oil phase. Glycerol was dissolved in distilled water to form aqueous phase. The oil phase and aqueous phase were separately heated up to 70 °C and stirred for a certain time (RET basic, Germany IKA company). Subsequently, the oil phase was
6
added drop wise into the water phase under moderate stirring (10000 rpm) (FJ 200-S digital high-speed dispersion homogenizer, Shanghai Specimen Model Plant). The coarse emulsion was passed six times through a 1000 Pa pressure homogenizer (AH-2010, Canada ATS Industrial Systems Co., Ltd.). After homogenization, the pH value of emulsion was adjusted to 7.0 using 0.1 mol/L NaOH solution (Leici PHS-2F pH meter, Shanghai Electronics Scientific Instrument Co., Ltd.).
2.2.2 Average particle size measurement Laser diffraction was used to determine average particle size of emulsions. The measurements were performed with an Average Nanoparticle Analyzer (Delsa Nano C, BECKMAN COULTER). Prior to the measurements, 33 μL of emulsion was diluted by a factor of 300 with distilled water in a 10 mL brown volumetric flask to avoid multi-scattering events. Each measurement was performed in triplicate. Then three measurement results were averaged. 2.2.3 Centrifugal stability constant measurement A certain amount of emulsion was diluted by a factor of 500 with distilled water in a 10 mL brown volumetric flask, and its absorbance value (A) was measured at the wavelength of 500 nm after mixing, using an ultraviolet light–visible spectrophotometer (UV-2550, Shimadzu Corporation). A 10-mL aliquot of the same emulsion was transferred into a centrifuge tube and centrifuged at 4000 rpm for 15 min in a high-speed centrifuge (3-30K, Germany Sigma Corporation). A certain amount of subnatant was diluted by a factor of 500 with distilled water in a 10 mL brown volumetric flask, and its absorbance value (A0) was measured at the wavelength of 500 nm after mixing. The centrifugal stability constant (Ke) was calculated using the Eq. 1. Ke A A0 A0 100%
7
(1)
2.2.4 Interfacial tension measurement So far, existing instruments and methods could only measure the interfacial tension between oil phase and water phase before forming emulsion. Therefore, a series of oil phase solutions and water phase solutions were prepared according to the prescriptions showed in Table I. The oil-water interfacial tension was determined at 25 °C by the pendant drop method, which was performed using an automatic interfacial tensiometer (OCA40 Micro, German DataPhysics Company) in conjunction with the Drop Shape Analysis software of SCA20. A drop of oil phase solution was formed in water phase solution at the tip of syringe by pressing the oil phase solution out by means of a setscrew. The drop shape analysis was performed as follows: a drop profile was extracted from the drop image; a curve-fitting program was then used to compare the experimental drop profile with a theoretical profile to derive the corresponding interfacial tension value. 2.2.5 Nuclear magnetic resonance (NMR) High resolution proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) of samples were obtained on AVANCE III spectrometer (Swiss Bruker Company) operating at 500 MHz and 25 °C. All the test solutions prepared as Table Ⅱ were sonicated using a CNC ultrasonic instrument (KQ5200DB; Kunshan Ultrasonic Instrument Co., Ltd.) for 30 cycles of 30 s on and 30 s off each, and then transferred to NMR tubes for measurement. Nuclear magnetism reagent filled in glass capillaries was added for field login.
2.2.6 Transmission electron microscopy (TEM) To confirm the presence of O/W emulsion droplets and characterized the thickness and shape of their emulsion membranes, TEM was applied. A total of 20 µL of
8
emulsion was diluted in distilled water to a final volume of 1 mL. One drop of the diluted solution was placed on a carbon-coated copper grid (200 meshes) for 1 min and then stained with the tungstophosphoric acid hydrate solution for 1 min. The analysis was carried out with a JEM-100CXII instrument (JEOL, Tokyo, Japan) at magnifications of 200,000×. 2.2.7 Inverted fluorescence microscopy (IFM) Before preparing fluorescent labeled emulsions, BODIPY-PC was dissolved in methanol (10 μg/mL stock solutions). A volume of stock solutions containing the equivalent of 10-4 mol % unlabelled egg yolk lecithin BODIPY-PC was doped in oil phase. In the process of stirring oil phase, the solvent of methanol was volatilized and BODIPY-PC was left to label egg yolk lecithin. The subsequent steps were the same as those described in 2.2.1. 10 µL fluorescent labeled emulsion was diluted with distilled water to 1 mL. One drop of the diluted solution was placed on glass slide. The analysis was carried out with an AX10 instrument (ZEISS, Germany) at magnifications of 400×. 3 Results 3.1 Average particle size As was seen in Figure 2-I, the average particle size of emulsions decreased after adding co-emulsifier. However, the average particle size of emulsions was not sustained decrease with the dosage of co-emulsifier increased. The average particle size measurement revealed that 0.2 % or 1.8 % HS15 made average particle size further decreased. When the dosage of oleic acid increased, the variation trend of average particle size was the same as that of HS15. Emulsions containing 0.2 % or 0.6 % oleic acid had smaller average particle size.
9
3.2 Centrifugal stability constant As was shown in Figure 2-II, the emulsions containing oleic acid or HS15 as co-emulsifier had smaller centrifugal stability constants. However, the emulsions were not more and more stable when the dosage of co-emulsifier increased. Obviously, the centrifugal stability constant of emulsion emulsified with 0.2 % or 1.8 % HS15 was smaller than other combinations’. When the dosage of oleic acid was 0.2 % or 0.6 %, the centrifugal stability constant of emulsion also appeared a further decline. 3.3 Interfacial tension In general, with increasing dosage of co-emulsifier, the interfacial tension between oil and water phase was on the decline. However, the oil-water interfacial tension wouldn’t constantly decrease, which had been verified in this study. As was displayed in Figure 2-III, the variation trend of oil-water interfacial tension was in wavy shape. The smaller interfacial tension achieved when 0.2 % or 1.8 % HS15 was added. Similarly, the interfacial tension became smaller at 0.2 % or 0.6 % oleic acid.
3.4 Nuclear magnetic resonance (NMR) 3.4.1 Proton nuclear magnetic resonance (1H NMR) The 1H NMR of emulsifier and co-emulsifiers were given in Figure 3-IV. The assignment of NMR signals to egg yolk lecithin and HS15 was performed in agreement with the literature[29] (as shown in Figure 3-I/II). And the assignment of NMR signals to oleic acid was performed in agreement with Spectral Database for Organic Compounds (as shown in Figure 3-III). The 1H NMR (as shown in Figure 3-IV) of egg yolk lecithin and oleic acid in CDCl3 showed the most prominent signals correspond to methyl (signal a/A; -CH3: 0.9 ppm) and methylene (signal b/B; -CH2-: 1.3 ppm) protons which were characteristic for
10
alkyl chain of fatty acids. The ppm value of the methyl protons of the trimethyl ammonium group (signal e; -N+-(CH3)3: 3.3 ppm) in egg yolk lecithin was shifted to higher ppm due to the vicinity of nitrogen. The 1H NMR (as shown in Figure 3-IV) of HS15 in D2O displayed three distinct signals. Signal α at 0.9 ppm and signal β at 1.3 ppm could be attributed to the methyl and methylene protons of the alkyl chain of 12-hydroxystearic acid. The ppm value of the methylene protons of polyoxyethylene chain (signal γ; -O-CH2-CH2-O-: 3.7 ppm) in HS15 was shifted to higher value due to the vicinity of oxygen. Therefore, the characteristic functional group of egg yolk lecithin was trimethyl ammonium group (signal e; -N+-(CH3)3: 3.3 ppm) and the characteristic functional group of HS15 was polyoxyethylene chain (signal γ; -O-CH2-CH2-O-: 3.7 ppm). However, as the characteristic functional group of oleic acid, carboxyl group didn’t appear in 1H NMR because active hydrogen of carboxyl group would exchange with nuclear magnetism reagent. 1
H NMR of emulsions was shown in Figure 4. For the emulsions stabilized by egg
yolk lecithin and HS15, the signal intensity of polyoxyethylene chain of HS15 had some reduction at 1.8 % HS15 rather than continuously increased with increasing dosage of HS15. While the signal intensity of trimethyl ammonium group of egg yolk lecithin showed a wavy shape variation trend, which first disappeared (point A) then appeared and disappeared (point B) again. For the emulsions stabilized by egg yolk lecithin and oleic acid, the signal intensity of trimethyl ammonium group of egg yolk lecithin had the same variation trend as mentioned above. As was shown in Figure 3-I/II, most functional groups of oleic acid were the same as those of egg yolk lecithin except carboxyl group, however, the signal
11
of carboxyl group disappeared in 1H NMR. In order to study how the signal intensity of carboxyl group of oleic acid would change with increasing dosage of oleic acid,
13
C
NMR was carried out. 3.4.2 Carbon nuclear magnetic resonance (13C NMR) The 13C NMR of egg yolk lecithin and oleic acid in CDCl3 were given in Figure 5-I. Carboxyl group (signal a; -COOH: 179 ppm) and ester group (signal b; -COOR: 171 ppm) were the characteristic functional groups for oleic acid and egg yolk lecithin respectively. The
13
C NMR of emulsions stabilized with egg yolk lecithin and oleic acid were
shown in Figure 5-II. After forming emulsion, the signal of carboxyl group of oleic acid was disappeared. While the signal intensity of ester group of egg yolk lecithin was on the rise except that 0.6% oleic acid was added with the dosage of oleic acid increased. In theory, the signal intensity of ester group should remain unchanged because the dosage of egg yolk lecithin was fixed. But in fact, the signal intensity of ester group changed. In order to confirm whether the carboxyl group of oleic acid would reacted with NaOH as pH regulator to produce ester group (-COONa), the
13
C NMR of emulsions
prepared without adjusting pH value was conducted. In Figure 5-III, the signal of carboxyl group of oleic acid appeared at 179 ppm, which indicating that NaOH was the cause of carboxyl group transforming into ester group. The signal intensity of carboxyl group also had some reduction at 0.6 % oleic acid rather than continuously increased with increasing dosage of oleic acid. This time the signal intensity of ester group didn’t change.
12
3.5 Emulsion droplet morphology on TEM TEM examination of emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 was shown in Figure 6. Emulsion droplets were presented in core-shell structure, but the thickness and shape of shells were different in TEM. At 0.2 % or 1.8 % HS15 as co-emulsifier, the shells of emulsion droplets were more continuous and neater. However, at other dosages of HS15, the shells of emulsion droplets were displayed in various shapes and oil droplets were pressed into different shapes.
3.6 Emulsion droplet morphology on IFM IFM examination of emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 was shown in Figure 7. Under blue fluorescence excitation, the emulsion droplets presented green fluorescence. With the dosage of HS15 increased, the shape and size of emulsion droplets were different in IFM. At 0.2 % or 1.8 % HS15 as co-emulsifier, the emulsion droplets were rounder than those of other dosages. The emulsion droplets of emulsion containing 1.8 % HS15 were bigger than those containing 0.2 % HS15.
4 Discussions So far, O/W emulsions had been so ubiquitous that it was hard to imagine life without them. And the stability of O/W emulsion was worthy of close attention. Because the stability of emulsion was closely related to the microscopic structure of emulsion membrane. The arrangement of emulsifier and co-emulsifier at oil-water interface might directly influence the stability of emulsion. In order to study this viewpoint, we firstly used average particle size, centrifugal stability constant and oil-water interfacial tension measure methods to study the stability
13
of emulsions. In Figure 2, three measurement results showed that they had the same variation trend. Keeping the dosage of egg yolk lecithin constant, average particle size and centrifugal stability constant of emulsion, as well as interfacial tension between oil and water phase showed a wavy shape variation, which firstly decreased then increased and decreased again with increasing dosage of oleic acid or HS15. In this wavy variation trend, two low points appeared, at which the emulsions had better stability. As was shown in Figure 2, the average particle size of point A was smaller than that of point B, but the centrifugal stability constant and oil-water interfacial tension of point A were bigger than that of point B, which illustrated that the stability of emulsions was better at point B. So we surmised that emulsifier and co-emulsifier might arrange into stable monolayer and bilayer emulsion membrane at point A and point B respectively. What’s more, the emulsion with bilayer emulsion membrane had larger particle size, but smaller oil-water interfacial tension and centrifugal stability constant, indicating a better stability. In order to study whether the arrangement of emulsifier and co-emulsifier at oil-water interface would change with increasing dosage of co-emulsifier, 1H NMR and 13
C NMR were used. For emulsions stabilized with egg yolk lecithin and HS15, the
characteristic signal of trimethyl ammonium group of egg yolk lecithin disappeared at 0.2 % and 1.8 % HS15, but the characteristic signal of polyoxyethylene chain of Solutol HS15 never disappeared (as shown in Figure 4-I) in 1H NMR. So, it could be speculated that the trimethyl ammonium group of egg yolk lecithin should tend toward lipophilic core, while the polyoxyethylene chain of Solutol HS15 should tend to aqueous phase (as
14
shown in Figure 8). For emulsions stabilized by egg yolk lecithin and oleic acid, the signal of carboxyl group of oleic acid would be observed in 13C NMR. But the signal of carboxyl group of oleic acid appeared only when NaOH wasn’t used to adjust the pH value of emulsion (as shown in Figure 5-II/III). This meant that carboxyl group of oleic acid would react with NaOH to produce ester group (-COONa). As NaOH just exist in aqueous phase, it suggested that the carboxyl group should also tend toward aqueous phase (as shown in Figure 8). Combining the analysis results above with the amphiphilic of surfactants, the directional diagram of emulsifier and co-emulsifier molecules at oil-water interface was deduced as Figure 8. And then, the arrangement of emulsifier and co-emulsifiers at oil-water interface would also be inferred through 1H NMR and 13C NMR.
As was shown in Figure 4, similar with the results of average particle size, centrifugal stability constant and oil-water interfacial tension, the signal intensity of trimethyl ammonium group of egg yolk lecithin had the same wavy variation trend in 1
H NMR of emulsions containing 1.2 % egg yolk lecithin and increasing dosage of
different co-emulsifier (HS15 or oleic acid). And the signal of trimethyl ammonium group disappeared at two low points (point A: 0.2 % HS15/oleic acid; point B: 1.8 % HS15 /0.6 % oleic acid). As was proved above, the trimethyl ammonium group of egg yolk lecithin should tend to lipophilic core. So at the two low points, the dosage of co-emulsifier should be appropriate, which could guarantee the egg yolk lecithin arranging around oil droplet in a closer and more ordered array. As a result, the signal of trimethyl ammonium group tending to lipophilic core disappeared. Furthermore, as the average particle size of
15
emulsions at point A was smaller than that at point B, it could be speculated that monolayer and bilayer emulsion membrane respectively formed at point A and B. In other cases, the signal of trimethyl ammonium group could be detected, which demonstrated that the dosage of co-emulsifier was inappropriate, leading to the increase of free egg yolk lecithin. Two reasons might account for why the dosage of free egg yolk lecithin increased. When co-emulsifier was at low concentration, such as 0.1 % HS15 or oleic acid, the emulsion membrane was loose and egg yolk lecithin could escape from emulsion membrane. When co-emulsifier was at high concentration, such as at 0.8/2.0 % HS15 or 0.4/0.8 % oleic acid, co-emulsifier competed against egg yolk lecithin at oil-water interface and some of egg yolk lecithin squeezed out from emulsion membrane. After egg yolk lecithin escaping or squeezing out from emulsion membrane, the trimethyl ammonium group would be exposed and its signal could be detected. The signal intensity of polyoxyethylene chain of HS15 had some reduction at 1.8 % HS15 rather than continuously increased with increasing dosage of HS15 in 1H NMR of emulsion. According to the above explanation, 1.8 % HS15 formed bilayer emulsion membrane with egg yolk lecithin. Furthermore, the polyoxyethylene chain has been proved to tend to aqueous phase, so polyoxyethylene chains which located in inner layer of emulsion membrane were hard to avoid being covered by outer molecules. When monolayer emulsion membrane formed by 0.2 % HS15 and egg yolk lecithin, polyoxyethylene chains were still exposed. So the signal intensity of polyoxyethylene chain became stronger from 0.1 % to 0.2 % HS15. This result supported the viewpoint about monolayer and bilayer emulsion membrane. The signal intensity of carboxyl group of oleic acid decreased at 0.6 % oleic acid rather than continuously increased with increasing dosage of oleic acid in 13C NMR of
16
emulsion. According to the above explanation, 0.6 % oleic acid formed bilayer emulsion membrane with egg yolk lecithin. Similarly, the carboxyl group was tended to aqueous phase, so carboxyl groups locating in inner layer of emulsion membrane were hard to avoid being covered by outer molecules. When monolayer emulsion membrane formed by 0.2 % oleic acid and egg yolk lecithin, carboxyl groups were exposed. So the signal intensity of carboxyl group didn’t decrease at 0.2% oleic acid. The viewpoint of monolayer and bilayer emulsion membrane had been verified again. On the base of the analysis above, it could be concluded that both the stability of emulsion and arrangement of emulsifier and co-emulsifier molecules at oil-water interface would change with the dosage of co-emulsifier increased. And these changes could be visually presented with the help of TEM and IFM. In TEM, the shell of emulsion droplet observed was the emulsion membrane which was formed by emulsifier and co-emulsifier. As for 0.2 % and 1.8 % HS15, emulsion membranes were more continuous and neater, and the oil droplets were rounder in TEM photomicrographs. In addition, the emulsion droplets were also rounder in IFM photomicrographs. So in the two cases, the arrangements of emulsifier and co-emulsifier were in order. What’s more, the emulsion droplets of emulsion containing 1.8 % HS15 were bigger than those containing 0.2 % HS15 both in TEM and IFM photomicrographs, which was consistent with the result of average particle size. Thereout, 0.2 %/1.8 % HS15 and egg yolk lecithin respectively arranged into monolayer and bilayer emulsion membrane. While in other dosages of HS15, the thickness of emulsion membranes were uneven and oil droplets had edges and corners in shape in TEM photomicrographs. In addition, the shapes of emulsion droplets weren’t round in IFM photomicrographs. So in
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these cases, the arrangements of emulsifier and co-emulsifier were in disorder. These phenomena in TEM and IFM all reflected arrangement of emulsifier and co-emulsifier molecules at oil-water interface and approved the view about monolayer and bilayer emulsion membrane. 5 Conclusions The microscopic structure of emulsion membrane was inherently connected to the stability of emulsion. When emulsifier and co-emulsifier arranged orderly into monolayer or bilayer emulsion membrane, the stability of emulsions would enhance. When emulsifier and co-emulsifier arranged in disorder at oil-water interface, the stability of emulsions would decrease. This discovery had an important significance for increasing efficiency and minimizing cost during the development of stable emulsions.
Acknowledgements The project were supported by Major Scientific and Technological Project of Guangzhou technology bureau (Item number: 201300000046) and Natural Sciences Fund of The First Affiliated Hospital of Guangdong Pharmaceutical University (Item number: GYFYLH201310).
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Figure Captions
Figure 1 The structure of fluorescent lipid analogue BODIPY-PC.
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(I)
(II)
(III) Figure 2
(I) Average particle size of O/W emulsions, (II) Centrifugal stability constant of
O/W emulsions, (III) Interfacial tension between oil and water phase that were prepared
24
according to the prescriptions showed in Table I: with egg yolk lecithin (1.2 %, w/v) and increasing dosage of (left) HS15; (right) oleic acid.
25
(I)
(II)
(III)
(IV)
26
Figure 3
(I) The structure of Egg yolk lecithin; (II) The structure of Oleic acid; (III) The
structure of HS15; (IV) 1H NMR of emulsifier and co-emulsifiers.
27
(I)
(II)
Figure 4 1H NMR of emulsions: (I) emulsified with egg yolk lecithin and HS15; (II) emulsified with egg yolk lecithin and oleic acid.
28
(I)
(II)
(III) Figure 5
13
C NMR of (I) egg yolk lecithin and oleic acid; (II) emulsions emulsified with egg
29
yolk lecithin and oleic acid and its amplified spectra (right); (III) emulsions emulsified with egg yolk lecithin and oleic acid and its amplified spectra (right), but hadn’t its pH value adjusted.
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Figure 6 TEM photomicrographs of the emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 at magnifications of 200,000×. The dosage of HS15 was: (A) 0.1 %; (B) 0.2 %; (C) 0.8 %; (D) 1.8 %; (E) 2.0 % (w/v).
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Figure 7 IFM photomicrographs of the emulsions emulsified with egg yolk lecithin (1.2 %, w/v) and increasing dosage of HS15 at magnifications of 400×. The dosage of HS15 was: (A) 0.1 %; (B) 0.2 %; (C) 0.8 %; (D) 1.8 %; (E) 2.0 % (w/v).
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Figure 8 Directional diagram of emulsifier and co-emulsifiers at oil-water interface of O/W emulsion.
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Tables Table Ⅰ Composition of O/W emulsions Excipients (%, w/v)
Formulation 1
Formulation 2
Soybean oil
10
10
Egg yolk lecithin
1.2
1.2
Oleic acid
—
0.1/0.2/0.4/0.6/0.8
HS15
0.1/0.2/0.8/1.8/2.0
—
Glycerol
2.25
2.25
Distilled water
86.45/86.35/85.75/84.75/84.55
86.45/86.35/86.15/85.95/85.75
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Table Ⅱ Preparation of test solutions for NMR Nuclear sequence Test objects
Test project
magnetism
number reagent 1
Egg yolk lecithin
1
H NMR/13C NMR
CDCl3
2
Oleic acid
1
H NMR/13C NMR
CDCl3
3
HS15
1
H NMR
D2O
H NMR/13C NMR
D2O
Emulsion stabilized by egg yolk lecithin 4
1
(1.2 %, w/v) and oleic acid (0.1/0.2/0.4/0.6/0.8 %, w/v) Emulsion stabilized by egg yolk lecithin
5
(1.2 %, w/v) and Solutol HS15 (0.1/0.2/0.8/1.8/2.0 %, w/v)
35
1
H NMR
D2O