Enzymatically derived oil-based L-ascorbyl esters: Synthesis, antioxidant properties and controlled release from cosmetic formulations

Sustainable Chemistry and Pharmacy 15 (2020) 100231

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Enzymatically derived oil-based L-ascorbyl esters: Synthesis, antioxidant properties and controlled release from cosmetic formulations � �c a, *, Ana Milivojevi�c b, Milica Simovi�c a, Katarina Banjanac b, Rada Pjanovi�c a, Marija Corovi Dejan Bezbradica a a b

Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000, Belgrade, Serbia Innovation Center, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000, Belgrade, Serbia

A R T I C L E I N F O

A B S T R A C T

Keywords: Vitamin C Natural triglycerides Lipase Transesterification Antioxidant Diffusion

Lipase-catalyzed transesterification of vitamin C by natural triglycerides is promising approach for cost-efficient synthesis of liposoluble food and cosmetic antioxidants. Nevertheless, application of these alternative acyl donors is insufficiently explored, despite of their low price, wide abundance and possibility for obtaining versatile products. Within current study, fatty acid ascorbyl esters were synthesized from vitamin C and natural tri­ glycerides (lard, sunflower, coconut and linseed oil) in a process catalyzed by immobilized lipase Novozym® 435 and their controlled release was examined. High conversions were achieved at optimized conditions, even at high substrate concentrations, thus high concentrations of acsorbyl-ester were synthesized using these natural lipids. All ester mixtures exhibited very high capacity for scavenging of DPPH radicals, among which linseed oil derived esters, with ascorbyl linolenate as prevailing compound, were the most efficient (IC50 0.663 μM), while coconut oil derived ester mixture (IC50 0.739 μM), composed predominantly of molecules with medium side chains, was second best. Coconut oil derived esters were successfully incorporated in typical cosmetic formulations for controlled release of bioactive compounds - O/W emulsion and gel-emulsion. Franz cell diffusion study demonstrated that esters release from mixture and two carrier systems was formulation dependent and revealed decrease of effective diffusivities with ester side acyl chain length increase. According to calculated effective diffusivity coefficients, faster trans-membrane delivery of the same molecule was achieved from gel-emulsion comparing to O/W emulsion. Applied approach enabled highly efficient cost-saving production of fatty acid ascorbyl esters, which could find direct application in various lipophilic products.

1. Introduction

review articles give valuable insight into the state of the art (Karmee, 2009; Bezbradica et al., 2017). It is established that in reactions cata­ lyzed with lipases (predominantly lipase from Candida antarctica type B, as well as lipase from Rhizomucor miehei and Thermomyces lanuginosus) ascorbyl esters can be synthesized at high conversion yields with wide range of acyl donors (Luhong et al., 2000; Reyes-Duarte et al., 2011; ~ o et al., Lerin et al., 2012; Jiang et al., 2016; Balen et al., 2017; Tufin 2019). Short, medium or long chain saturated fatty acids and long chain unsaturated fatty acids, have been used predominantly as acyl donors resulting with various type of esters differing not only in their structure, but also in their antioxidant activity and hydrophilic/hydrophobic properties (Karmee, 2009; Stojanovic et al., 2015; Bezbradica et al., 2017). Even higher esterification rates and yields have been achieved with vinyl esters as substrates, due to equilibrium shift towards synthesis caused by tautomerization of vinyl alcohol to acetaldehyde (Karmee,

Organic esters are chemicals widely used in the production of aromas (for food and perfumes), pharmaceuticals, fuels and fine chemicals. Ascorbyl esters are fat soluble forms of vitamin C used as antioxidant food additive or active substance in skin care products. Since they combine powerful bioactive properties of vitamin C and good solubility in lipophilic media due to presence of acyl moiety, they are adequate solution to rising market demand for liposoluble and environmentally acceptable antioxidants. Ascorbyl palmitate (E304) and ascorbyl stea­ rate (E305), only products of this group currently available on market, are produced by acid-catalyzed process (Nickels and Hackenberger, 1987), which is coupled with low selectivity and environmental con­ cerns (Karmee, 2009). Hence, enzymatic processes of ascorbyl ester preparation have been widely studied in last few decades and several * Corresponding author. � E-mail address: [email protected] (M. Corovi� c).

https://doi.org/10.1016/j.scp.2020.100231 Received 21 November 2019; Received in revised form 3 February 2020; Accepted 8 February 2020 Available online 14 February 2020 2352-5541/© 2020 Elsevier B.V. All rights reserved.

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2009). At current level of development in this field significant efforts are focused on making enzymatic process cost-efficient. Due to the fact that price of enzyme significantly participate in overall process costs, several reports have been dealing with attempts to increase productivity by using enzymatic reactors enabling prolonged use of immobilized bio­ � �c et al., 2017b), catalyst (Watanabe et al., 2003; Zhao et al., 2014; Corovi preparing novel immobilized biocatalyst (Moreno-Perez et al., 2013; � �c et al., 2017a) or optimization of reaction with cost function Corovi which includes enzyme consumption (Stojanovi�c et al., 2013). Other approach towards decrease of process costs could be use of less expensive substrates in comparison with pure fatty acids, among which animal fats and vegetable oils are the most interesting. Although they are pool of all fatty acid moieties necessary for synthesis of valuable esters, publications focused on enzymatic transesterification are very scarce and range of applied substrates is limited to olive oil (Mor­ eno-Perez et al., 2013), lard (Zhao et al., 2011, 2014), palm oil (Burham et al., 2009) and soybean oil (Hsieh et al., 2005). Also, these reports were prevalently organized with the aim to optimize reaction factors and maximize overall ester yield or triglyceride conversion, while composition of obtained ester mixtures or its effect on physiological activity was seldom analyzed (Hsieh et al., 2005; Burham et al., 2009; Zhao et al., 2014). Beside economic aspects, application of oils as acyl donors for acorbyl ester synthesis increases potential for their incorpo­ ration in different dermo-cosmetic formulations, since side reaction products (glycerol, mono- and diglycerides) are common skin care products ingredients, due to their emollient function. Namely, reaction mixtures could, without pricy and complicated purification procedures, be utilized after simple separation of immobilized biocatalyst and vac­ uum evaporation of solvent. Since oil derived esters include several molecules of different side alkyl chain properties, it is expected for products containing them to have multiple functionality as well as possibility for prolonged effect on skin since different release rates from carrier systems are expected. Furthermore, owing to emulsifying prop­ erties of unreacted oils as well as mono- and diglycerides, application of additional emulsifiers in such products could be avoided. The aim of this study was to evaluate natural lipids as acyl donors for lipase-catalyzed ascorbyl ester synthesis with respect to overall ester yield, but also composition of obtained ester mixture and its effect on antioxidant activity, as well as to evaluate possibility for incorporation in cosmetic formulations and examine their diffusion properties. Four lipids, covering wide range of fatty acids, were selected (Table 1). Co­ conut oil is used because it contains short and medium chain fatty acids, sunflower oil and lard contain longer chain saturated and unsaturated acids, while linseed oil was used because it is abundant source of lino­ lenic acid, valuable (omega-3) fatty acid with recognized physiological activity: cardiovascular-protective, anti-cancer, neuro-protective, antiosteoporotic, anti-inflammatory, and antioxidant (Kim et al., 2014). It is precursor of two important long chain omega-3 fatty acids, EPA (eico­ sapentaenoic acid, 20:3n-5) and DHA (docosahexaenoic acid, 22:3n-6), both of which have essential roles in brain development and cardio­ vascular health, possess anti-inflammatory effect, etc.(Leaf, 1992; Das, 2006; Wang et al., 2006). Antioxidant activities of obtained ester mix­ tures were compared with individual esters in order to evaluate their application prospects. Finally, possibility of ester mixture incorporation in cosmetic formulations was tested and their diffusion properties were examined.

2. Material and methods 2.1. Materials As a catalyst, immobilized lipase from Candida antarctica type B expressed in Aspergilus niger (Novozym® 435, Novozymes, Bagsvaerd, Denmark) was applied. L-ascorbic acid (>99%, TCI Europe N.V., Zwijndrecht, Belgium) was used as acyl acceptor, while coconut oil (Beyond, Ni�s, Serbia), sunflower oil (Vital, Vrbas, Serbia), linseed oil (Suncokret, Hajdukovo, Serbia) and lard (local market) were acyl do­ nors. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, analytical grade acetone and t-butanol and HPLC grade methanol, water, isopropanol and formic acid were purchased from Sigma–Aldrich Chemie Gmbh, Steinheim, Germany. For preparation of formulations Aristoflex® AVC polymer (Clariant International Ltd., Muttenz, Switzerland), Tegin® pellets (Evonik Industries, Essen, Germany) and Lanette® O (BASF Care Creations, Ludwigshafen, Germany) were used. 2.2. Ester synthesis Reactions were conducted in 100 ml sealed Erlenmeyer flasks, orbitally shaken at 150 rpm in a thermostat (IKA KS 4000i Control, Staufen, Germany) and 60–80 � C. Reaction mixtures consisted of vitamin C (0.135 M), acyl donors (0.405 M) whose compositions are given in Table 1, organic solvent and 1%(w/v) of biocatalyst. Reaction conditions including vitamin C concentration, substrates molar ratio, biocatalyst amount and shaking speed were chosen based on previously published work (Stojanovi�c et al., 2013). Reaction mixture volumes were 10 ml. Erlenmeyer flasks were briefly incubated at room temper­ ature in order to avoid solvent evaporation, and 50 μl of samples were taken by automatic micropipette for HPLC analysis during reaction time. Control samples (without lipase) were exposed to same treatment as all other samples, as well as samples containing acyl donor, solvent and lipase (without vitamin C), and no products were detected in them. All experiments were conducted in duplicates and average values are pre­ sented in graphs. Deviations were less than 5%. Related calculations are performed based on our previously published work (Stojanovi�c et al., 2013) with individual fatty acids as acyl donors and yields of different ascorbyl esters and conversion degrees were calculated based on following equations: Individual ester yieldðmMÞ ¼

Overall ester yieldðmMÞ ¼ Conversion degree ð%Þ ¼

peak area ⋅sample dilution factor 37 ð1=mMÞ

X Individual ester yieldsðmMÞ

overall ester yield ðmMÞ ⋅100 vitamin C initial concentrationðmMÞ

(1) (2) (3)

2.3. Kinetic study Time courses of ester syntheses were modeled using COPASI soft­ ware (version 4.16). For data fitting, reversible Michaelis-Menten ki­ netic mechanism was applied, whereas it was assumed that acyl residues are readily available in the reaction mixture and that their concentration could be considered constant, due to largely excessive amount

Table 1 Fatty acid composition of acyl donors. Acyl donor Coconut oil Linseed oil Sunflower oil Lard

Fatty acid composition, % C8

C10

C12

C14

C16

C18

C18/1

C18/2

C18/3

19.00 – – –

7.80 – – –

46.40 – – –

16.60 – – –

5.20 10.86 8.23 38.4

– 1.03 3.70 7.90

5.00 19.75 29.79 50.0

– 13.58 58.27 3.70

– 54.73 – –

2

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comparing to L-ascorbic acid. Equation (4) was used for that purpose: Vmf ⋅½S� Vmr ⋅½P� K K v ¼ ms ½S� mp½P� 1 þ Kms þ Kmp

� et al., 2006; Pjanovi�c et al., 2010). polar receptor mediums (Klimundova Additionally, application of synthetic membrane allowed us to obtain reproducible results since its physical and chemical properties are con­ stant. Before samples were placed at the donor compartment Franz cell was thermostated for 30 min at room temperature which was main­ tained at 25 � C. The samples were taken during 24 h from receptor chamber and analyzed by RP-HPLC.

(4)

where v (mM h 1), Vmf (mM h 1) and Vmr (mM h 1) represent overall, maximum forward and maximum backwards reaction rates, [S] and [P] (mM) are concentrations of substrate – vitamin C and product – ascorbyl esters, while Kms and Kmp (mM) are corresponding Michaelis-Menten constants. Kinetic parameters for the proposed model were deter­ mined by performing non-linear regression analysis using the genetic algorithm of the COPASI software package (version 4.16).

2.7. HPLC analysis Quantitative analyses of samples, properly diluted with methanol, were performed by HPLC on Dionex Ultimate 3000 Thermo Scientific (Waltham, USA) system and a reverse phase column (Hypersil GOLD C18, 150 mm � 4.6 mm, 5 μm). Gradient elution was applied, with mobile phase A (methanol:water 80:20%(v/v) with 0.1%(v/v) formic acid) and mobile phase B (100% isopropanol) in following mode: 0–10 min isocratic 0% B, 10–15 min gradient 0–50% B, 15–20 min isocratic 50% B, 20–20.1 min gradient 50-0% B, 20.1–25 min isocratic 0% B with a flow rate of 1 ml/min. Esters were detected at 235 nm by UV-VIS detector. Average retention times of detected compounds were as fol­ lows: caprylate – 2.657 min, caprinate – 3.750 min, laurate – 6.093 min, myristate – 10.957 min, linolenate – 10.067 min, linoleate – 13.643 min, palmitate – 15.160 min, oleate – 15.56 min and stearate – 16.473 min. Two representative chromatograms are given as a supplementary data (Fig. S.1.A and S.1.B).

2.4. Antioxidant activity assay Antioxidant activity of L-ascorbyl esters mixtures was measured spectrophotometrically at 517 nm by standard DPPH method based on ability of tested compounds to act as electron acceptors and to reduce stable DPPH radical, as described previously (Stojanovic et al., 2015). Samples with concentrations of 0–0.5 mg ml 1 at volumes of 0.2 ml were mixed with 0.6 ml of methanol and 0.2 ml of DPPH solution (0.15 mM in methanol). Control samples were composed of 0.8 ml of methanol and 0.2 ml of DPPH solution. Mixtures were vortexed for 2 min and left in dark at room temperature for 30 min. Based on absorbance at 517 nm, free radical scavenging capacity was calculated in percentages and final result was expressed IC50 (half minimal inhibitory concentration) value which represents concentration of antioxidant which lowers initial concentration of DPPH radicals by 50%.

2.8. Theory and calculation The diffusion coefficients were calculated using Fick’s second law, as described by Pjanovi�c et al. (2010) for ester mixtures: � � 1 CD CR D¼ ln 0 (5) β⋅t CD C0R

2.5. Donor formulations preparation and characterization As donor formulations, three different systems (ester mixture, gel emulsion and O/W emulsion) were used, all prepared by using mixture of esters obtained after 24 h of reaction at previously defined conditions and in a volume of 25 ml which was, after immobilized biocatalyst removal, subjected to vacuum evaporation to remove t-butanol. Pro­ cedures for formulation preparations, including operating conditions, type and concentration of encapsulation agent are previously optimized. Gel emulsion was prepared by using 1% solution of Aristoflex® AVC polymer for making gel which was mixed with esters in the ratio 90:10%. This polymer was used due its ability to emulsify up to 20% of oils. O/W emulsion was obtained by mixing 10% of ester mixture, 3% Tegin® pellets as emulsifier and 2% Lanette® O as co-emulsifier and consistency factor at 70 � C and water (preheated at 70 � C) was added at intense stirring (1500 rpm). Prepared formulations were subjected to analysis of zeta potential on a Malvern Zetasizer Nano ZS (Malvern In­ struments, Worcestershire, UK). For that matter, samples were diluted 1000 times with deionised water. Microscopic analysis was performed on a light microscope (Zeiss, Axio Imager. A1) under magnification of 100X. Images were recorded with the AxioCamMRC Camera and Axio Vision Software (Zeiss).

And it’s linearized form � 0 � C C0R ln D ¼ D ⋅ β⋅t CD CR

(6)

Where β (24900 min/m2) is geometric constant of cell, t (min) is time, C (g/l) is concentration, subscripts R and D refer to receptor and donor compartment, respectively and superscript 0 refers to initial conditions (at zero time). For emulsions, Higuchi equation derived from Fick’s second low which describes diffusion of components from system with controlled release is commonly applied: Mt ¼ k*t1=2 M∞

(7)

Where Mt and M∞ are masses of bioactive compounds in receptor compartment at time t and ∞, k is kinetic constant which includes diffusion coefficient and t is time. Due to complexity of systems for controlled release and dependence of diffusion properties on gel hy­ dration and formation, as well as polymer chain relaxation during incorporation of bioactive compound, Korsmeyer-Pepas modification of basic Higuchi equation was applied, as well:

2.6. Diffusion study Diffusion studies were conducted using Franz diffusion cell with Oring joint (20 ml volume and 25 mm orifice diameter, PermeGear, Inc., USA) and cellulose acetate membrane (pore size of 0.2 μm). Experiments were carried out at 25 � C. In the donor compartment, approximately 2 g of ester mixtures or prepared formulations were placed. The receptor compartment was filled with 50% ethanol and continuously stirred at � et al., 2006; Balan�c et al., 400 rpm using magnetic bead (Klimundova 2016). Used stirring rate of 400 rpm was previously established as optimal since it enabled receptor medium homogeneity. Regarding membrane and receptor fluid choice, it was based on previously pub­ lished works in which this hydrophilic membrane was used with such

Mt ¼ k*tn M∞

(8)

Where all symbols remained previous meaning, while coefficient n could have theoretical values from 0.5 to 1 depending on transport mechanism – n ¼ 0.5 for diffusion, n ¼ 1 for polymer relaxation and 0.5 < n < 1 for their combined effect. Both equations refer to (Dozie-Nwachukwu et al., 2017) and (Jayanudin et al., 2019). If influence of lag phase occurring after experiment setting up and during which esters do not diffuse is considered, following forms of Eq. 3

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(5) are used: Mt ¼ k* t M∞

3.2. The effect of lipid on overall ester yield and product composition tlag

�n

(9)

In various studies of lipase-catalyzed ascorbyl ester synthesis, wide range of temperature optimums was observed, but in vast majority of studies temperature optimums between 55 � C and 65 � C were observed (Karmee, 2009; Bezbradica et al., 2017). Hereby, temperature was varied within range 60–80 � C. It is obvious that reaction temperature did not affect final product yields, but had significant influence on initial reaction rate in range 60–70 � C (Table 2). Due to significantly higher initial reaction rates at 70 � C, maximum ester yields were achieved after only 24 h with all four lipids. On the other hand, at 60 � C maximum was achieved after 24 h only with linseed oil, but with lard after 48 h, with coconut oil after 72 h and with sunflower oil after 91 h. Further increase of temperature to 80 � C did not bring any improvement in terms of initial reaction rates, conversion degrees and overall ester yields, while energy consumption increased significantly. Since the same process productivity was accomplished at 70 � C and 80 � C, but cost effectiveness was lower at 80 � C, this temperature was not further applied. Temper­ ature of 70 � C was, therefore, considered as optimum and used within subsequent part of the study. From Table 2, it can be seen that conversion degrees above 65% (more than 90 mM of esters) were achieved with all applied lipids. The highest overall yield (85.7% or 115.69 mM) was achieved with coconut oil, while other reaction courses were similar (Fig. 2) and resulted with final yields between 66.76 and 72.55%. Obtained results could be an indicator of higher transesterification activity of immobilized CALB to­ wards saturated medium chain fatty acids since these are abundant in coconut oil (Table 1). These results are in line with our previously published findings in which pure carboxylic acids of various chain length were applied and high initial reaction rates and ester yields were accomplished with medium chain fatty acids as substrates (Stojanovic et al., 2015). With the respect to general affinity of enzyme towards triglyceride substrates, it can be concluded that all applied lipids were very good substrates for ester synthesis. Along with the high conversion achieved, it’s worth mentioning that initial vitamin C concentration was rather high comparing to many other publications and it was chosen because in our previous study on optimization of acsorbyl oleate syn­ thesis had been found as optimal (Stojanovi�c et al., 2013). When palm oil was previously used as a substrate for vitamin C esterification, final product was composed of oleate (61%), palmitate (30%) and stearate (9%) and overall conversion of 70–75% was accomplished in t-amyl alcohol with Novozym® 435 as a biocatalyst (Burham et al., 2009). Different product composition (45.9% palmitate, 42.6% oleate and 10.1% linoleate) was obtained by Hsieh et al. and conversion of 62% was achieved when methyl esters of palm oil were used as acyl donors, acetone as a solvent and Novozym® 435 as a catalyst (Hsieh et al., 2005). Within same study, significantly lower conversion degree – 17% was accomplished with soybean oil methyl esters. In Table 3, compositions of final products obtained with four lipids are presented, while corresponding time courses of individual ester synthesis are given in Fig. 3. By using lard (Fig. 3A), mixture of oleate, linoleate, palmitate and stearate was synthesized with oleate as major product (~59 mM) and overall conversion of 69.26% was accomplished. Similar limiting substrate conversion degree was achieved (69.18%) when methyl esters from lard were used as substrates in co-solvent mixtures, but at much lower initial concentrations of vitamin C, hence approximately 7 mM of esters was produced (Zhao et al., 2011). Ester mixture obtained with sunflower oil (Fig. 3B) contained 47 mM of ascorbyl linoleate, valuable ester. Song et al., for example obtained higher concentrations - 65.4 mM of ester with 0.3 M methyl linoleate (21.8% conversion). However, methyl linoleate is far more expensive comparing to sunflower oil and overall ester yield obtained within current study was 90.13 mM implying that cost-efficiency of process is even higher when natural triglycerides are applied instead of pure PUFAs. Linseed oil derived esters were rich in ascorbyl linolenate (Fig. 3C), which was previously synthesized from pricey acyl donor -

Where tlag is time without detected diffusion. MATLAB software was used for fitting of results (R2 higher than 0.95) and calculation of coefficients values. Effective diffusion co­ efficients (Deff) were finally calculated as follows: Deff ¼

π⋅δ2 ⋅k2 4

(10)

Where δ represents sum of membrane and donor formulation thicknesses. 3. Results and discussion 3.1. Selection of solvent Lipases are important industrial enzymes which catalyze different reactions in aqueous and non-aqueous environments (Pfluck et al., 2018; Machado et al., 2019). Lipase-catalyzed ester syntheses are commonly conducted in organic solvents (Bezbradica et al., 2017). In general, due to hydrophilic nature of ascorbic acid solvents with logP values between 1 and 1 are used in synthesis of ascorbyl esters since they enable good solubility of both substrates and does not show harmful effect on lipase catalytic conformation (Karmee, 2009; Bezbradica et al., 2017). Since in our previous studies t-butanol and acetone had been observed as favorable solvents for ascorbyl ester syntheses, these two solvents were applied in this study. Obtained results (Fig. 1) indicate that t-butanol is by far more convenient solvent for transesterification of all analyzed triglycerides comparing to acetone, since obtained ester yields were 3–6 times higher. Although t-butanol or t-pentanol were identified as better solvents than more polar acetone or acetonitrile in several studies with fatty acid or their vinyl esters as acyl donors, differences were not so large as in this study. It is plausible that in this case solubility of fats and oils becomes limited, hence t-butanol is significantly better solvent being more hydrophobic (logP 0.58) than acetone (logP 0.21). It is further corroborated by the fact that the lowest discrepancy (around three times higher ester yield) was observed with coconut oil (Fig. 1), which is better soluble in polar organic solvents because it contains medium chain fatty acids. Hence, all further experiments were conducted in t-butanol.

Fig. 1. Influence of organic solvent on transesterification yield. Reactions were performed at 60 � C, stirring rate 150 rpm, for 91 h, at 0.135 M of vitamin C and 3-fold excess of oil. 4

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Table 2 Influence of reaction temperature on transesterification of vitamin C. Acyl donor

Reaction rate, mM h

Coconut oil Sunflower oil Linseed oil Lard

1

Conversion, %

Yield, mM

60 C

70 C

80 C

60 C

70 C

80 C

60 � C

70 � C

80 � C

20.83 5.99 15.00 12.25

26.60 18.06 21.65 15.92

26.32 18.34 20.96 15.01

82.89 66.73 71.17 63.84

85.70 66.76 72.55 69.26

84.53 66.13 70.57 68.44

115.29 90.09 96.08 86.18

115.69 90.13 97.94 93.51

114.11 89.28 95.27 92.40

�

�

�

�

�

�

linolenic acid, which is in line with previous observations. At the end of this part of the study, operational stability of immo­ bilized lipase was tested by its application in consecutive reaction cycles. As it can be seen from Fig. 4, biocatalyst could be used 3–4 times with activity loss of only ~10%, after which gradual decrease of accom­ plished yields was observed. After ten consecutive reuses, achieved yields were 73.5, 70.1, 70.0 and 68.3% of initial (achieved in first re­ action cycle) for lard, coconut, sunflower and linseed oil, respectively. Similar trend was observed when we previously used oleic acid as acyl donor for ascorbic acid esterification catalyzed by the same lipase preparation in acetone. Slightly higher stability of Novozym® 435 in current system could be attributed to lower polarity of t-butanol comparing to acetone, which can lower potential for disruption of essential water layer which surrounds lipase molecules and maintain its active conformation. 3.3. Antioxidant capacity of ascorbyl esters mixtures Fatty acid ascorbyl esters are primarily produced as shelf life extending additives for food, cosmetics and personal care products. Their antioxidant properties are therefore crucial parameter during characterization, since lower amount of additive is needed if its free radical scavenging capacity is higher. Obtained ester mixtures were therefore subjected to DPPH analyses for their antioxidant potencies. As shown in Table 3, all synthesized mixtures possessed very high DPPH radical scavenging capacity according to calculated IC50 values. Namely, when compared to commercially available L-ascorbyl palmitate (IC50 3.73) and parent molecule (IC50 4.60) – vitamin C, these IC50 values are significantly lower indicating that same inhibition degree of DPPH radical could be achieved with lower amounts of synthesized ester mixtures. Statistical analysis showed significant difference between four synthesized reaction mixtures antioxidant capacity, as well (single factor ANOVA p ¼ 0.033). It was observed that among mixtures with pre­ vailing ascorbyl esters with unsaturated acyl residues, ones with more double bonds showed higher antioxidant efficiency (IC50 linseed ˂ IC50 sunflower ˂ IC50 lard). Such results were previously reported by other researches and are in line with our results obtained for single esters (Viklund et al., 2003; Stojanovic et al., 2015). Furthermore, coconut oil derived esters were second best, which confirm that medium length side chained ascorbyl esters are potent antioxidants, as well. Namely, our previously published results of antioxidant capacity determination for single esters, obtained from pure carboxylic acids, proved that among fatty acid ascorbyl esters with saturated acyl residue, ones with shorter side chain are more effective comparing to long chained fatty acid esters (Stojanovic et al., 2015). In addition, it seems that antioxidant activity of coconut oil derived ascorbyl esters is even higher than that of

Fig. 2. Overall ester yields of transesterification reactions at 70 � C.

pure alpha-linolenic acid in concentrations ~30 mM (60% conversion) in acetone. It seems that application of linseed oil in t-butanol provided adequate environment for high catalytic activity of lipase, since >50 mM of ascorbyl linolenate was produced and overall ester concentration was 97.94 mM (72.55% conversion). Ascorbyl esters with saturated medium side acyl chains, with laurate as prevailing product (~40%), were synthesized in a reaction with coconut oil. Considering above­ mentioned literature examples, it’s evident that natural triglycerides are highly suitable substrates for lipase-catalyzed L-ascorbyl esters synthesis. Data obtained at 70 � C were modeled using COPASI software, in order to determine kinetic model which would be adequate for describing reaction systems and for determination of relevant kinetic constants. It has been shown that, by using reversible Michaelis-Menten kinetic mechanism good data fitting could be achieved (Fig. S2), since standard deviations below 5% were observed for all four acyl donors. Estimated kinetic constants are shown in Table 4. In all systems, reversible reaction of ester hydrolysis was included in the model and calculated constants were significantly lower comparing to forward re­ actions of esters syntheses. Furthermore, it has been shown that Vm values were higher for coconut and linseed oil comparing to sunflower oil and lard, indicating higher affinity of lipase towards fatty acid resi­ dues which are abundant in these oils – medium chain fatty acids and Table 3 Antioxidant properties of ascorbyl ester mixtures. Acyl donor Coconut oil Linseed oil Sunflower oil Lard

Product composition, % of ascorbyl ester

IC50 (μM)

C8

C10

C12

C14

C16

C18

C18/1

C18/2

C18/3

18.25 – – –

9.71 – – –

39.93 – – –

18.15 – – –

11.03 5.26 2.99 20.20

– – 4.39 16.97

2.90 25.23 36.81 47.15

– 13.81 55.81 15.68

– 55.70 – –

5

0.739 0.663 0.771 0.838

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Fig. 3. Time course of vitamin C esterification with lard (A), sunflower oil (B), linseed oil (C) and coconut oil (D) at 70 � C. Table 4 Estimated values of kinetic constants. Parameter Vmf (mM h 1) Vmr (mM h 1) Kms (mM) Kmp (mM) SD (%)

Acyl donor Coconut oil

Sunflower oil

Lard

Linseed oil

48.56 8.41 9.55 6.57 2.97

28.50 15.76 9.95 10.08 1.59

22.20 10.45 9.57 9.61 2.08

45.73 6.44 3.28 1.06 4.70

SD - standard deviation.

corresponding single esters, since notably lower IC50 values were ob­ tained. This observation is in line with results obtained previously with soybean oil as acyl donor for ascorbyl esters synthesis, whereby obtained ester mixtures demonstrated higher –OH radical scavenging capacity and reducing power comparing to individual esters (Liu et al., 2011). Interestingly, superoxide scavenging activity of vitamin C and ascorbyl palmitate were, on the other hand, significantly higher comparing to soybean oil derived ester mixture, as well as oleate and linoleate. Additional investigations of the mechanisms of action which would explain these results, are still needed in future.

Fig. 4. Operational stability study.

incorporation and release from different cosmetic formulations as car­ rier systems. Coconut oil was chosen as a substrate for this part of the study, due to high yields and good antioxidant properties of synthesized ascorbyl esters, as well as its importance for dermo-cosmetic industry. It is applied in a wide range of cosmetic products as a cleanser, foaming

3.4. Preparation and characterization of cosmetic formulations Since oil-derived ascorbyl esters are attractive for application in cosmetic and pharmaceutical topical products as emollients and vitamin C/fatty acid sources, further focus was to examine potential for their 6

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Sustainable Chemistry and Pharmacy 15 (2020) 100231

agent or stabilizer at concentrations up to 50%. It is widely recognized as efficient skin-conditioning agent, since it acts as emollient. Due to antimicrobial properties, it is used as a constituent of products for skin infections treatment (Aburjai and Natsheh, 2003). Two commonly applied formulation types – O/W emulsion and gelemulsion were prepared and characterized in terms of zeta potential as relevant parameter for system stability. Zeta potential of both formu­ lations was determined immediately after preparation and it had nega­ tive value - 34.5 mV for O/W emulsion and 72.9 mV for gelemulsion. Same parameter was measured after 14 days long storage at 4 � C and zeta potential values were almost unchanged ( 34.1 mV and 73.8 mV for O/W and gel-emulsion, respectively). Values of measured zeta potentials indicate satisfying stability of two preparations where, according to Riddick’s classification (Riddick and Zeta-Meter, 1968), O/W emulsion was moderately stable while gel-emulsion was in a range of systems with very good stability. Moreover, the light microscopy was employed for determination of particle size and images obtained at 100 times magnification are presented in Fig. 5. Both emulsions were visu­ ally characterized as polydisperse systems with particle size between 1 and 5 μm. According to results of applied tests, prepared formulations could be examined as carrier system for controlled release of incorpo­ rated biomolecules.

biocatalyst and solvent. In Fig. 6, resulting overall diffusion profiles of esters obtained from experiments conducted in a Franz cell are pre­ sented. Portion of esters which diffused from donor compartment and were detected in receptor chamber was highest for ester mixture. Fatty acid ascorbyl esters incorporated in two formulations showed different diffusion profiles comparing to ester mixture and to each other. Apparently, gel type emulsion as a carrier system provided higher diffusion rate and approximately 2.3 times higher amounts of delivered active compounds relative to classical O/W emulsion after 24 h. These results indicated significant influence of formulation type on transmembrane diffusion of fatty acid ascorbyl esters and substantiated earlier findings that L-ascorbyl palmitate release rate from cosmetic preparations is formulation dependent (Gosenca and Ga�sperlin, 2011; Gosenca et al., 2013). Amounts of individual esters released from three examined systems which were detected in receptor chamber after 24 h are presented in Fig. 7. Significant effect of side alkyl chain properties was observed, since the increase of side acyl chain length led to decrease in portions of esters which diffused from all analyzed samples (Fig. 7). Therefore, by comparing percentage of esters which diffused from all tested prepara­ tions to receptor chamber, it could be noticed that caprylate diffused in the highest extent, then caprinate, laurate and myristate, respectively, while palmitate, oleate and stearate were detected in traces. This implies that, depending on molecular size and hidrophobicity, different trans­ port mechanisms could be responsible for transfer of different ascorbyl esters, which should be subject of more detailed examinations. Low diffusion rate of esters of longer chain fatty acids is not surprising, because when ascorbyl palmitate diffusion using different carrier sys­ tems was previously examined in Franz cell with pig ear skin membrane, compound was also not detected in receptor fluid and it was mainly delivered to epidermis (Gosenca and Ga�sperlin, 2011). For diffusion coefficients calculation from ester mixtures, equation derived from second Fick’s low, commonly used for diffusion from so­ lutions in diaphragm cell, was employed (Eq. (6), section 3) and resulting diffusion coefficients are shown in Table 5. For formulations, fitting of experimental results was done using Higuchi and KorsmeyerPeppas equation (Eqs. (7)–(9), section 3) and, based on obtained values of constants, effective diffusivity coefficients were calculated (Eq. (10), section 3). Fitting of results revealed that 1–2 h long initial lagphase was characteristic of all esters for all formulations, hence lagphase effect was included in models describing diffusion from O/W emulsion and gel-emulsion. Determined values of all corresponding coefficients are presented in Table 6. In accordance with observed

3.5. Diffusion study Experiments in Franz diffusion cell are commonly applied for revealing mechanism of release of different active ingredients and as an indicator for the potential of particular carrier system for their transmembrane delivery. Diffusion of coconut oil derived fatty acid ascorbyl esters from O/W emulsion and gel-emulsion was tested and compared with ester mixture obtained after simple separation of

Fig. 6. Franz cell diffusion profiles of coconut oil derived ascorbyl esters from mixture, gel-emulsion and emulsion.

Fig. 5. Light microscopy images of O/W emulsion (A) i gel-emulsion (B). 7

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Sustainable Chemistry and Pharmacy 15 (2020) 100231

Table 6 Effective diffusion coefficients of ascorbyl esters from different formulations. Formulation

Ascorbyl ester

tlag, min

k⋅104, min 0.5

Deff⋅1012, m2 min 1

O/W emulsion

Ascorbyl caprylate Ascorbyl caprinate Ascorbyl laurate Ascorbyl myristate

88

7.2

0.47

119

6

0.33

120 122

3.8 2.4

0.13 0.05

Ascorbyl caprylate Ascorbyl caprinate Ascorbyl laurate Ascorbyl myristate

79

24.5

3.63

116

17.3

1.81

119 120

12 7.3

0.87 0.32

Gel-emulsion

revealed that release mechanisms of different esters were independent of carrier system and were attributed entirely to diffusion process. On the other hand, their effective diffusion coefficients were dependant on their properties as well as formulation structure, providing possibility for targeted delivery of these bioactive molecules and versatile functionality of final products by choosing adequate formulation type.

Fig. 7. Ascorbyl esters detected in receptor chamber after 24 h of diffusion from different donor systems. Table 5 Diffusion coefficients of ascorbyl esters from ester mixture. Ascorbyl ester

log P

D⋅108, m2 min

Ascorbyl caprylate Ascorbyl caprinate Ascorbyl laurate Acorbyl myristate

2.37 3.25 4.38 5.27

7.63 4.40 2.58 1.00

Funding

1

This work was supported by the Serbian Ministry of Education, Sci­ ence and Technological Development [projects III 46010 and TR 31035]. Declaration of competing interest

diffusion profiles of individual esters, calculated diffusivities (Table 5) and effective diffusivity coefficients (Table 6) were higher for ascorbyl esters with shorter side alkyl chains. Furthermore, as it can be seen from Table 6, adequate fitting of experimental results for emulsion and gelemulsion was accomplished when transfer of components was attrib­ uted entirely to diffusion process (n ¼ 0.5 in Eq. (9)) and no significant effect of polymer relaxation was observed. All determined effective diffusivity coefficients of single esters were higher for gel-emulsion comparing to O/W emulsion indicating that gel-based carrier systems could enable delivery of higher concentrations of coconut oil derived Lascorbyl esters in a shorter time and are more suitable for further testing and application.

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4. Conclusions Lipophilic derivatives of L-ascorbic acid obtained from natural tri­ glycerides are desirable constituents of food, cosmetics and pharma­ ceutics. Plant and animal fat were, to the best of our knowledge, for the first time examined as substrates for the lipase-catalyzed trans­ esterification of vitamin C and evaluated as antioxidants and constitu­ ents of dermo-cosmetic preparations. High conversions were accomplished and, depending on lipid fatty acid composition, ester mixtures of different lipophilicities and functional properties were ob­ tained. Operational stability study revealed that approximately 70% of initial activity of the lipase could be maintained after ten consecutive reuses. All products possessed high DPPH radical scavenging capacity, particularly ones rich in ascorbyl esters with long polyunsaturated (linseed oil derived esters) and medium length saturated (coconut oil derived esters) side acyl chains. Coconut oil derived esters, which are of particular interest for cosmetic industry, were successfully incorporated into two common cosmetic formulations – O/W emulsion and gel emulsion, which were tested as systems for their controlled delivery. Esters with saturated medium side alkyl chain diffused more rapidly than long-chained ones, regardless of carrier system. Diffusion studies 8

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Sustainable Chemistry and Pharmacy 15 (2020) 100231

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