Microemulsions Based on a Sunflower Lecithin–Tween 20 Blend

Article pubs.acs.org/JAFC

Microemulsions Based on a Sunflower Lecithin−Tween 20 Blend Have High Capacity for Dissolving Peppermint Oil and Stabilizing Coenzyme Q10 Huaiqiong Chen, Yongguang Guan, and Qixin Zhong* Department of Food Science and Technology, University of Tennessee, 2510 River Drive, Knoxville, Tennessee 37996, United States ABSTRACT: The objectives of the present study were to improve the capability of microemulsions to dissolve peppermint oil by blending sunflower lecithin with Tween 20 and to study the possibility of codelivering lipophilic bioactive compounds. The oil loading in microemulsions with 20% (w/w) Tween 20 increased from 3% (w/w) to 20% (w/w) upon gradual supplementation of 6% (w/w) lecithin. All microemulsions had particles of 0.05) was observed for all treatments. a

isotropic transparent dispersions after confirming Newtonian viscosity and stable and small monodispersed droplets, as presented in the following sections, are treated as microemulsions and summarized in Figure 2B, and those with the highest peppermint oil content at different lecithin concentrations are listed in Table 1. The hydrophile−lipophile balance (HLB) is commonly used to indicate the properties of surfactants emulsifying oils. Theoretically, surfactants with higher HLB values have better solubility in water,24 and surfactants with HLB values higher and lower than 7 are usually used to form O/W and W/O emulsions, respectively.24 The HLB of a surfactant mixture (HLBmix) can be estimated using the summation of the product of HLB and mass percentage of individual surfactants.25 When the HLB of a surfactant or surfactant mixture is similar to the required HLB of the oil phase, the system provides the minimum energy condition for microemulsion formation.26 Tween 20 and soybean lecithin have respective HLB values of 16.7 and 8.27 Assuming similar HLB of sunflower and soybean lecithin, HLBmix decreases from 16.7 (of Tween 20) to 14.69 when the lecithin content increases from 0% to 6% (w/w). Therefore, the addition of lecithin decreased HLBmix to be closer to 12.3, which was found to be the most appropriate HLB value to form O/W emulsions of peppermint oil.28 Additionally, two long hydrocarbon chains of lecithin facilitate the dissolving of oil in the dispersed phase of O/W microemulsions.29 These two factors may have resulted in a high loading of peppermint oil in the microemulsions of the present study. Particle Size and Stability of Selected Microemulsions. Microemulsions are thermodynamically stable systems that typically contain particles with a dimension in the range from 2 to 50 nm.30 The particle sizes of selected microemulsions containing 1−6% lecithin and 5−20% peppermint oil (Table 1) were tested. No significant differences (P > 0.05) were observed from the values of d4,3 and d3,2 of all samples before and after storage for 30 and 70 d at room temperature (21 °C, Table 2). Particle dimensions ranging from ∼7 to ∼11 nm enabled their transparent appearance. A single narrow peak was observed for all the measured transparent samples (data not shown). The nanoemulsions of peppermint oil typically show the growth of particle dimensions during storage due to Ostwald ripening.31 Because a stable particle dimension and visible appearance were always observed for all transparent dispersions, it can be concluded that they are microemulsions. Viscosity of Selected Microemulsions. Shear rate ramps of microemulsions in Table 1 are presented in Figure 3. Linear correlations between shear rate and shear stress, with R2 values all greater than 0.99 (Table 3), and an intercept of zero indicate

cooling and turbidity changes after heating were used to determine the PIT. Figure 1 demonstrates changes of the viscosities of the coarse emulsion during cooling from 95 to 25 °C. During cooling, the coarse emulsion can change from a water (W)/oil (O) emulsion to a W/O/W emulsion, with partial W/O emulsion, and finally to an O/W emulsion.21 The PIT can be determined as the lowest temperature at which the emulsion converts from a fine W/O emulsion to a W/O/W emulsion.21 On the basis of the viscosity data, the PIT was determined to be 60.7 °C (Figure 1). When the turbidity data were compared, samples heated at a higher temperature had lower turbidity (Figure 1). The sample heated at 60 °C showed some degree of phase inversion when compared to the coarse emulsion but had a higher absorbance than samples heated at 80 °C or higher, corresponding to turbid and transparent dispersions, respectively. Since the turbidity was similar at 80 °C and above, heating treatment at 80 °C for 5 min was chosen for further study. Influence of the Lecithin Content on Microemulsion Formation. The impacts of the lecithin content on the phase behavior of oil/water/surfactant mixtures with a base composition of 20% (w/w) Tween 20 were studied after the samples were heated at 80 °C for 5 min. The sample appearance after ambient incubation overnight is shown in Figure 2A. Emulsions prepared with 1−6% (w/w) lecithin alone were turbid even with only 1% (w/w) peppermint oil, and treatments prepared with 1−3% (w/w) lecithin showed some creaming after overnight storage (data not shown). Tween 20 (20%, w/w) alone was able to dissolve 3% (w/w) peppermint oil. The combinations of 20% (w/w) Tween 20 and 1%, 2%, and 3% (w/w) lecithin increased the amount of dissolved peppermint oil to 5%, 7%, and 9% (w/w), respectively. With 4% (w/w) lecithin, the treatments with 1% (w/w) and 7−12% (w/w) peppermint oil were transparent, while the ones with 2−6% (w/w) oil showed phase separation. Similar observations were noted for treatments with 5% (w/w) lecithin, with higher peppermint oil levels in transparent samples (10−16%, w/w). At the highest lecithin content (6%, w/w) studied, 14−20% (w/w) peppermint oil was dissolved as transparent dispersions. For mixtures with 4−6% (w/w) lecithin, samples separated into a clear lower phase (O/W microemulsion) and a turbid upper phase (with excess lecithin) after storage have the characteristics of Winsor-I phase behavior.22 When transparent samples were examined for sample homogeneity and birefringence using a polarized light microscope, all samples appeared completely dark (data not shown). Therefore, these samples were isotropic, which is a distinct feature of microemulsions.23 Formulations resulting in 986

DOI: 10.1021/jf504146t J. Agric. Food Chem. 2015, 63, 983−989

Article

Journal of Agricultural and Food Chemistry

Figure 4. Absorbance of microemulsions S1−S6 at 240 nm (the maximum absorbance of peppermint oil) after 200-fold dilution and incubation at 21 °C for up to 72 h. The sample compositions are listed in Table 1.

Figure 3. Rheogram of freshly prepared microemulsions S1−S6 with the compositions listed in Table 1 at 25 °C.

Table 3. Summary of Fitting Shear Rate−Shear Stress Data into the Newtonian Model for the Microemulsions in Table 1 at 25 °Ca sample S1 S2 S3 S4 S5 S6

R2

Newtonian viscosity (Pa s) 0.0085 0.0122 0.0187 0.0334 0.0781 0.1969

± ± ± ± ± ±

0.0001 0.0003 0.0008 0.0012 0.0025 0.0011

f e d c b a

dilutability of microemulsions is an important feature for practical applications. Photostability of CoQ10 in Microemulsions. Microemulsions after dissolving CoQ10 had a d3,2 smaller than 7 nm (Table 4), and there was no significant (P > 0.05) difference for

0.9978 0.9991 0.9995 0.9999 0.9997 0.9999

± ± ± ± ± ±

0.0009 0.0002 0.0001 0.0001 0.0003 0.0000

Table 4. Area-Volume Mean Diameters (d3,2) of Microemulsions with 20% (w/w) Tween 20, 3% (w/w) Peppermint Oil, 0.1% (w/w) CoQ10, and 0% or 3% (w/w) Lecithin before and after Treatment at 302 nm and Ambient Conditions (21 °C) for up to 32 ha

Numbers are the mean ± standard deviation from three independent measurements. Different online Roman letters indicate the statistical difference in the mean (P < 0.05). a

d3,2 (nm) UV treatment duration (h) 0 2 4 8 16 32

these microemulsions are Newtonian fluids. The Newtonian viscosity ranged from 0.0085 Pa s for the sample with 1% lecithin and 5% peppermint oil to 0.1969 Pa s for that with 6% lecithin and 20% peppermint oil (Table 3). A higher viscosity of microemulsions with higher contents of peppermint oil and lecithin is expected. The trend agreed with the literature microemulsion systems with isopropyl myristate dissolved by a soybean lecithin and Tween 80 mixture and orange oil by a sucrose laurate/soybean lecithin mixture.29,32 Stability of Microemulsions after Dilution. The stability of microemulsions diluted 50-, 100-, and 200-fold using diwater was monitored at 240 and 600 nm to indicate the chemical stability of peppermint oil and physical stability (turbidity), respectively, at 21 °C for up to 72 h. The wavelength of 240 nm, corresponding to the maximum absorbance of peppermint oil in the UV/vis regime, was used to monitor the amount of peppermint oil. All diluted samples showed no significant change in the absorbance at 240 nm (presented in Figure 4 for microemulsions after 200-fold dilution), indicating good chemical stability of peppermint oil in microemulsions after dilution. The absorbance at 600 nm of all detected samples after 50-, 100-, and 200-fold dilution using diwater was smaller than 0.1. The excellent dilution stability of microemulsions may be due to the close HLB values of surfactant mixtures and peppermint oil, as discussed above. Because flavor oils such as peppermint oil are used at low concentrations, the excellent

with 0% lecithin 5.98 4.78 6.09 6.85 5.57 6.45

± ± ± ± ± ±

1.47 0.79 1.13 0.69 0.67 1.56

with 3% lecithin 5.24 6.05 4.53 4.69 4.85 4.29

± ± ± ± ± ±

0.87 1.30 0.25 0.55 0.80 0.68

Numbers are the mean ± standard deviation from two measurements from two independent repetitions. No significance difference (P > 0.05) was observed for all treatments.

a

the treatments with 0% or 3% (w/w) lecithin. These dimensions were similar (P > 0.05) to those of microemulsions without CoQ10 (Table 2). d3,2 did not change significantly (P > 0.05) after UV radiation at 302 nm for up to 32 h (Table 4). The UV stability of CoQ10 was studied at 254 and 302 nm. As demonstrated in Figure 5, no significant degradation of CoQ10 in the control solution was observed after radiation at 254 nm for 32 h (P > 0.05), which was the same as previously reported.14 However, significant (P < 0.05) degradation of CoQ10 in the control solution was observed after radiation at 302 nm, decreasing to less than 25% of the initial concentration after 8 h of radiation. The energy of photons is proportional to the frequency of electromagnetic waves, and UV 254 nm, with a higher frequency (shorter wavelength) than UV 302 nm, is expected to cause a greater extent of structural degradation, including the mechanism related to heat after energy conversion.33,34 Conversely, the electromagnetic energy (P) at 987

DOI: 10.1021/jf504146t J. Agric. Food Chem. 2015, 63, 983−989

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UV 302 nm has a longer penetration depth36 and therefore has a greater impact on reactions induced by radiation than UV 254 nm, despite the lower photon energy.37 Therefore, a quicker degradation of CoQ10 at 302 nm than at 254 nm was observed in the present study, as well as others.14,38 The stability of CoQ10 in microemulsions was therefore only tested for 302 nm treatments. The appearance and residual CoQ10 concentration after UV 302 nm treatments are shown in Figure 5, and the estimated parameters according to eqs 3 and 4 are compiled in Table 5. The microemulsions improved the photostability of CoQ10, and the improvement was more apparent for the treatment with lecithin. The first-order kinetic model fits the degradation data well, with coefficients of determination above 0.8, which agrees with previous reports.17,18 The degradation rate constant decreased from 0.167 h−1 in the control solution to 0.0448 and 0.00825 h−1 in the microemulsion without and with lecithin, respectively. Accordingly, the half-life increased 3.7and 20.3-fold, respectively, for CoQ10 in the microemulsion without and with lecithin. It is well-established that Tween 20 does not have antioxidant capacity, while lecithin is a good antioxidant.39 The improved photostability of CoQ10 in the microemulsion without lecithin likely resulted from the antioxidant capacity of peppermint oil.40 The combination of antioxidant properties of lecithin and peppermint oil apparently improved the stability of CoQ10 dissolved in the microemulsion with lecithin. On the basis of a previous report,39 Tween 20 at the O/W interface cannot prevent the diffusion of peroxyl radicals into the oil body, while the opposite is the case for lecithin. Because Tween 20 is present at a higher content than lecithin in the microemulsion (20% vs 3%), the much improved stability of CoQ10 in the microemulsion with lecithin further indicates that the two surfactants synergistically dissolved peppermint oil, as discussed previously. Overall, the combination of sunflower lecithin and Tween 20 enabled the formation of stable microemulsions capable of dissolving high levels of peppermint oil, increasing from 3% to 20% when the lecithin content increased from 0% to 6%. Microemulsions were isotropic and transparent with particle sizes smaller than 12 nm and had relatively low Newtonian viscosity and excellent water-dilution and storage stability at ambient conditions. Additionally, microemulsions had the capability of dissolving CoQ10 and reducing its degradation under UV radiation at 302 nm, which likely resulted from the presence of both peppermint oil and lecithin. Our findings indicate that the microemulsions in the present study are promising systems to deliver both flavor oils and lipophilic bioactive compounds in transparent products such as beverages.

Figure 5. Appearance (A) and normalized residual concentrations (B) of 0.1% (w/w) CoQ10 dissolved in microemulsions with 20% (w/w) Tween 20, 3% (w/w) peppermint oil, and 0% or 3% (w/w) lecithin after radiation at UV 302 nm under ambient conditions in the dark for up to 32 h. The control solution contained 0.1% (w/w) CoQ10 dissolved in ethyl acetate and was treated at both 254 and 302 nm. Error bars are standard deviations (n = 4).

a depth z decays exponentially from the energy on the surface (P0), as expressed in the well-known Beer−Lambert law: P = P0e−αz

(5)

where α is a constant and equals 1/dp, with dp being the penetration depth that corresponds to a z value where P becomes 1/e of P0.35

Table 5. Degradation Rate Constant (k), Half-Life (t1/2), and R2 When Fitting the Degradation Data at 302 nm (Figure 5) to the First-Order Kinetics Modela sample

k (h−1)

R2

t1/2 (h)

control solution microemulsion with 0% lecithin microemulsion with 3% lecithin

0.167 ± 0.00150 a 0.0448 ± 0.00293 b 0.00825 ± 0.000785 c

0.905 ± 0.0249 0.828 ± 0.0693 0.953 ± 0.0180

4.15 ± 0.0380 c 15.5 ± 0.975 b 84.6 ± 8.08 a

a The control solution had 0.1% (w/w) coenzyme Q10 dissolved in ethyl acetate. Microemulsions contained 20% (w/w) Tween 20, 3% (w/w) peppermint oil, 0.1% (w/w) CoQ10, and 0% or 3% (w/w) lecithin. Numbers are the mean ± standard deviation (n = 4). Different online Roman letters indicate the statistical difference (P < 0.05).

988

DOI: 10.1021/jf504146t J. Agric. Food Chem. 2015, 63, 983−989

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(20) Rao, J.; McClements, D. J. Stabilization of phase inversion temperature nanoemulsions by surfactant displacement. J. Agric. Food Chem. 2010, 58, 7059−7066. (21) Allouche, J.; Tyrode, E.; Sadtler, V.; Choplin, L.; Salager, J. L. Simultaneous conductivity and viscosity measurements as a technique to track emulsion inversion by the phase-inversion-temperature method. Langmuir 2004, 20, 2134−2140. (22) Flanagan, J.; Singh, H. Microemulsions: a potential delivery system for bioactives in food. Crit. Rev. Food Sci. 2006, 46 (3), 221− 237. (23) Patel, R. B.; Patel, M. R.; Bhatt, K. K.; Patel, B. G. Formulation consideration and characterization of microemulsion drug delivery system for transnasal administration of carbamazepine. Bull. Fac. Sci., Cairo Univ. 2013, 51, 243−253. (24) Walstra, P. Surface phenomena. In Physical Chemistry of Foods; CRC Press: New York, Basel, 2002; p 348. (25) Yu, L.; Li, C.; Xu, J.; Hao, J.; Sun, D. Highly stable concentrated nanoemulsions by the phase inversion composition method at elevated temperature. Langmuir 2012, 28, 14547−14552. (26) Pestana, K. C.; Formariz, T. P.; Franzini, C. M.; Sarmento, V. H. V.; Chiavacci, L. A.; Scarpa, M. V.; Egito, E. S. T.; Oliveira, A. G. Oilin-water lecithin-based microemulsions as a potential delivery system for amphotericin B. Colloids Surf., B 2008, 66, 253−259. (27) Rodriguez, M.; Oses, J.; Ziani, K.; Mate, J. I. Combined effect of plasticizers and surfactants on the physical properties of starch based edible films. Food Res. Int. 2006, 39, 840−846. (28) Orafidiya, L. O.; Oladimeji, F. A. Determination of the required HLB values of some essential oils. Int. J. Pharm. 2002, 237, 241−249. (29) Moreno, M. A.; Ballesteros, M. P.; Frutos, P. Lecithin-based oilin-water microemulsions for parenteral use: pseudoternary phase diagrams, characterization and toxicity studies. J. Pharm. Sci. 2003, 92, 1428−1437. (30) McClements, D. J.; Rao, J. Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity. Crit. Rev. Food Sci. 2011, 51, 285−330. (31) Liang, R.; Xu, S.; Shoemaker, C. F.; Li, Y.; Zhong, F.; Huang, Q. Physical and antimicrobial properties of peppermint oil nanoemulsions. J. Agric. Food Chem. 2012, 60, 7548−7555. (32) Tchakalova, V.; Bailly, C.; Fieber, W. Food-grade bicontinuous microemulsions. Flavour Fragrance J. 2014, 29, 67−74. (33) Derossi, A.; De Pilli, T.; Fiore, A. G. Vitamin C kinetic degradation of strawberry juice stored under non-isothermal conditions. LWTFood Sci. Technol. 2010, 43, 590−595. (34) Zhao, M.; Li, Y.; Xu, X.; Wu, J.; Liao, X.; Chen, F. Degradation kinetics of malvidin-3-glucoside and malvidin-3,5-diglucoside exposed to microwave treatment. J. Agric. Food Chem. 2013, 61, 373−8. (35) Larsen, M. L.; Clark, A. S. On the link between particle size and deviations from the Beer−Lambert−Bouguer law for direct transmission. J. Quant. Spectrosc. Radiat. Transfer 2014, 133, 646−651. (36) Vincent, W. F.; Roy, S. Solar ultraviolet-B radiation and aquatic primary production: damage, protection, and recovery. Environ. Rev. 1993, 1, 1−12. (37) Rodil, R.; Moeder, M.; Altenburger, R.; Schmitt-Jansen, M. Photostability and phytotoxicity of selected sunscreen agents and their degradation mixtures in water. Anal. Bioanal. Chem. 2009, 395, 1513− 24. (38) Kwon, S. S.; Nam, Y. S.; Lee, J. S.; Ku, B. S.; Han, S. H.; Lee, J. Y.; Chang, I. S. Preparation and characterization of coenzyme Q10loaded PMMA nanoparticles by a new emulsification process based on microfluidization. Colloids Surf., A 2002, 210, 95−104. (39) Pan, Y.; Tikekar, R. V.; Nitin, N. Effect of antioxidant properties of lecithin emulsifier on oxidative stability of encapsulated bioactive compounds. Int. J. Pharm. 2013, 450, 129−137. (40) Fatemi, F.; Dini, S.; Rezaei, M. B.; Dadkhah, A.; Dabbagh, R.; Naij, S. The effect of γ-irradiation on the chemical composition and antioxidant activities of peppermint essential oil and extract. J. Essent. Oil Res. 2014, 26, 97−104.

AUTHOR INFORMATION

Corresponding Author

*Phone: 865-974-6196. Fax: 865-974-7332. E-mail: qzhong@ utk.edu. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Ziani, K.; Fang, Y.; McClements, D. J. Fabrication and stability of colloidal delivery systems for flavor oils: effect of composition and storage conditions. Food Res. Int. 2012, 46, 209−216. (2) Klossek, M. L.; Marcus, J.; Touraud, D.; Kunz, W. Highly water dilutable green microemulsions. Colloids Surf., A 2014, 442, 105−110. (3) Yaghmur, A.; Aserin, A.; Garti, N. Phase behavior of microemulsions based on food-grade nonionic surfactants: effect of polyols and short-chains alcohols. Colloids Surf., A 2002, 209, 71−81. (4) Artman, N. R. Safety of emulsifiers in fats and oils. J. Am. Oil Chem. Soc. 1975, 52, 49−52. (5) Csaki, K. F. Synthetic surfactant food additives can cause intestinal barrier dysfunction. Med. Hypotheses 2011, 76, 676−681. (6) Ema, M.; Hara, H.; Matsumoto, M.; Hirata-Koizumi, M.; Hirose, A.; Kamata, E. Evaluation of developmental neurotoxicity of polysorbate 80 in rats. Reprod. Toxicol. 2008, 25, 89−99. (7) Cilek, A.; Celebi, N.; Tirnaksiz, F. Lecithin-based microemulsion of a peptide for oral administration: preparation, characterization, and physical stability of the formulation. Drug Delivery 2006, 13, 19−24. (8) Gosenca, M.; Bester-Rogac, M.; Gasperlin, M. Lecithin based lamellar liquid crystals as a physiologically acceptable dermal delivery system for ascorbyl palmitate. Eur. J. Pharm. Sci. 2013, 50, 114−122. (9) Arnold, G.; Schuldt, S.; Schneider, Y.; Friedrichs, J.; Babick, F.; Werner, C.; Rohm, H. The impact of lecithin on rheology, sedimentation and particle interactions in oil-based dispersions. Colloids Surf., A 2013, 418, 147−156. (10) Hanning, S. M.; Yu, T.; Jones, D. S.; Andrews, G. P.; Kieser, J. A.; Medlicott, N. J. Lecithin-based emulsions for potential use as saliva substitutes in patients with xerostomia-viscoelastic properties. Int. J. Pharm. 2013, 456, 560−568. (11) Sheweita, S. A.; Tilmisany, A. M.; Al-Sawaf, H. Mechanisms of male infertility: role of antioxidants. Curr. Drug Metab. 2005, 6, 495− 501. (12) Flint Beal, M. Coenzyme Q 10 as a possible treatment for neurodegenerative diseases. Free Radical Res. 2002, 36, 455−460. (13) Mortensen, S. A.; Kumar, A.; Dolliner, P.; Filipiak, K. J.; Pella, D.; Alehagen, U.; Steurer, G.; Ping, C. S.; Littarru, G. P.; Rosenfeldt, F. ASSA13-13-1 long-term results with coenzyme Q10 as adjunctive therapy in chronic heart failure. Heart 2013, 99, 55−56. (14) Fir, M. M.; Smidovnik, A.; Milivojevic, L.; Zmitek, J.; Prosek, M. Studies of CoQ10 and cyclodextrin complexes: solubility, thermo- and photo-stability. J. Inclusion Phenom. Macrocyclic Chem. 2009, 64, 225− 232. (15) Kommuru, T. R.; Ashraf, M.; Khan, M. A.; Reddy, I. K. Stability and bioequivalence studies of two marketed formulations of coenzyme Q10 in beagle dogs. Chem. Pharm. Bull. 1999, 47, 1024−1048. (16) Zhang, L.; Hayes, D. G.; Chen, G.; Zhong, Q. Transparent dispersions of milk-fat-based nanostructured lipid carriers for delivery of β-carotene. J. Agric. Food Chem. 2013, 61, 9435−9443. (17) Zou, J.; Yu, B.; Song, Y.; Zhang, L.; Wang, F.; Deng, Y. Effects of particle size and concentration on photodegradation kinetics of coenzyme Q10 liposomes. J. Shenyang Pharm. Univ. 2010, 5. (18) Onoue, S.; Uchida, A.; Kuriyama, K.; Nakamura, T.; Seto, Y.; Kato, M.; Hatanaka, J.; Tanaka, T.; Miyoshi, H.; Yamada, S. Novel solid self-emulsifying drug delivery system of coenzyme Q10 with improved photochemical and pharmacokinetic behaviors. Eur. J. Pharm. Sci. 2012, 46, 492−499. (19) Souza, V. B.; Spinelli, L. S.; Gonzalez, G.; Mansur, C. R. E. Determination of the phase inversion temperature of orange oil/water emulsions by rheology and microcalorimetry. Anal. Lett. 2009, 42, 2864−2878. 989

DOI: 10.1021/jf504146t J. Agric. Food Chem. 2015, 63, 983−989