Lipophilization of ascorbic acid: A monolayer study and biological and antileishmanial activities

Article abstract of DOI:10.1021/jf5029398

Ascorbyl lipophilic derivatives (Asc-C₂ to Asc-C_18:1) were synthesized in a good yield using lipase from Staphylococcus xylosus produced in our laboratory and immobilized onto silica aerogel. Results showed that esterification had little effect on radical-scavenging capacity of purified ascorbyl esters using DPPH assay in ethanol. However, long chain fatty acid esters displayed higher protection of target lipids from oxidation. Moreover, compared to ascorbic acid, synthesized derivatives exhibited an antibacterial effect. Furthermore, ascorbyl derivatives were evaluated, for the first time, for their antileishmanial effects against visceral (Leishmania infantum) and cutaneous parasites (Leishmania major). Among all the tested compounds, only Asc-C₁₀, Asc-C₁₂, and Asc-C_18:1 exhibited antileishmanial activities. The interaction of ascorbyl esters with a phospholipid monolayer showed that only medium and unsaturated long chain (Asc-C₁₀ to Asc-C_18:1) derivative esters were found to interact efficiently with mimetic membrane of leishmania. These properties would make ascorbyl derivatives good candidates to be used in cosmetic and pharmaceutical lipophilic formulations.

Full text of DOI:10.1021/jf5029398

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Article
Lipophilisation of ascorbic acid: A monolayer
study, Biological and Antileishmanial activities
Nadia Kharrat, Imen Aissa, Manel Sghaier, Mohamed Bouaziz,
Mohamed Sellami, Dhafer Laouini, and youssef Gargouri
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5029398 • Publication Date (Web): 22 Aug 2014
Downloaded from http://pubs.acs.org on August 22, 2014
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Title: Lipophilisation of ascorbic acid: A monolayer study, Biological and Antileishmanial
activities
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Authors list: Nadia Kharrata, Imen Aissaa, Manel Sghaierb, Mohamed Bouazizc, Mohamed
Sellamia, Dhafer Laouinib and Youssef Gargouria*
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Affiliation List:
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a Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, Route de Soukra,
BPW 1173ꢀ 3038 SfaxꢀTunisie.
b
Groupe Immunobiologie des Leishmanioses, Laboratoire de Transmission, Contrôle et
Immunobiologie des Infections (LTCII), Institut Pasteur de Tunis, 13, Place Pasteur,
BP 74 ,1002 TunisꢀBelvédère, Tunisie.
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c Laboratoire d’Electrochimie et Environnement, ENIS, Route de Soukra, BPW 1173ꢀ 3038,
Université de Sfaxꢀ Tunisie.
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* Corresponding author: Prof. Youssef Gargouri,
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Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, Université de Sfax,
Route de Soukra, 1173 SfaxꢀTunisie.
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Tel/ Fax: + 21674675055,
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Eꢀmail : ytgargouri@yahoo.fr
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24 Abstract
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Ascorbyl lipophilic derivatives (AscꢀC2 to AscꢀC18:1), were synthesized in a good
yield using lipase from Staphylococcus xylosus produced in our laboratory and immobilized
onto silica aerogel. Results showed that esterification had little effect on radicalꢀscavenging
capacity of purified ascorbyl esters using DPPH assay in ethanol. However, long chain fatty
acids esters displayed higher protection of targetꢀlipids from oxidation. Moreover, compared
to ascorbic acid, synthesized derivatives exhibited an antibacterial effect. Furthermore,
ascorbyl derivatives were evaluated, for the first time, for their antileishmanial effects against
visceral (Leishmania infantum) and cutaneous parasites (Leishmania major). Among all the
tested compounds, only AscꢀC10, AscꢀC12 and AscꢀC18:1 exhibited antileishmanial activities.
The interaction of ascorbyl esters with a phospholipid monolayer showed that only medium
and unsaturated long chain (AscꢀC10 to AscꢀC18:1) derivative esters were found to interact
efficiently with mimetic membrane of leishmania. These properties would make ascorbyl
derivatives good candidates to be used in cosmetic and pharmaceutical lipophilic
formulations.
Keywords: Ascorbic acid; lipophilic derivatives; antimicrobial activity;
leishmanicidal activity; phospholipid monolayers
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1. Introduction
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Lipid deterioration increases in edible and inedible fatty products through processing,
heating, storage and culinary practices. As a result, the oxidative processes generate offꢀflavor
compounds such as aldehydes, peroxides, ketones and oxyacids.1 These compounds cause a
decrease in food nutritive value and sensorial quality. To prevent food oxidation and extend
its shelf life, they should be stored under proper conditions, e.g. at low temperature in an inert
atmosphere and suitable packaging. In this sense, antioxidants are widely recommended as
additives which have the potential to control the oxidation process, thereby stabilizing fatꢀ
containing foodꢀstuffs.2 Synthetic antioxidants such as butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), tertꢀbutylhydroquinone (TBHQ) and propyl gallates were
widely used as scavenging free radicals to prevent the formation of initiating lipid radicals.
Yet, according to toxicologists and nutritionists, the application of these synthetic
antioxidants in food materials is a matter of concern related to the formation of toxic
compounds. These substances can show possible sideꢀeffects such as carcinogenicity in living
organisms.3 Lanigan and Yamarik3 showed that only 50 % of BHT can be eliminated by urine
within the first 24 hours and the other parts are accumulated in adipose tissue. Natural
compounds with better antioxidant capacity and less toxicity have acquired major interest for
their implication as prophylactic and therapeutic agents in many diseases and for their better
food preservation. Currently, ascorbic acid is a labile molecule and is wellꢀknown by virtue of
its reducing property. Moreover, it can prevent chronic diseases due to oxidative stress,
including cancer, hypertension, cardiovascular disease and stroke.4 Ascorbic acid, which
cannot be synthesized by humans, was identified at a high level ranging from 162 to 135mg
per 100g in piper and kiwi to shield them against peroxidizing factors. Some fruits, like
oranges, contain in their peels a rather higher level of ascorbic acid than in their juice. An
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amount of 100g of fresh orangeꢀpeel provides 136mg of ascorbic acid, while its flesh contains
just about 71mg/100 g.
Ascorbic acid is also available in a wide range of supplements such as tablets, capsules
and crystalline powder. Unfortunately, it is unstable when exposed to air, light, heat and is
rapidly oxidized and irreversibly decomposed upon entering the body, thus exhibiting low
solubility and stability in the lipophilic media.5 This behavior can limit its uses efficiently.
Being fat soluble, lipophilic ascorbyl derivatives exhibit a better affinity with lipophilic
membrane constituents. They can be easily absorbed and retained into the lipid domain of
biological system for a longer period of time.5 The enzymatic synthesis catalyzed by
commercial lipases to produce some ascorbyl esters via esterification in organic solvents has
been emphasized in several works.6,7 Amphiphilic ascorbyl derivatives display particularly
interesting characteristics resulting from the modification of molecular flexibility. Hence,
lipophilic derivatives of ascorbic acid and, in particular, long acyl chain esters, would exhibit
a better affinity with lipophilic membrane constituents of drug target cells.8 This property
would make from ascorbyl derivatives good candidates to be used in pharmaceutical and
cosmetic lipophilic formulations.9 The interest in new compounds with antimicrobial
properties has been revived because of current problems associated with the use of
antibiotics.10 Aissa et al.11 have synthesized a large series of tyrosyl esters with increasing
lipophilicity using lipase of Candida antarctica. Authors showed that the parent tyrosol does
not have any effect on various pathogenic bacteria. However, tyrosyl esters and especially
medium chain tyrosyl derivatives exhibited an antimicrobial activity against staphylococcus
strains.
In the wake of resistance to pentavalent antimonial drugs, new therapeutic alternatives
are desirable.12 Hence, it is of interest to analyze the performance of the amphiphilic ascorbyl
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derivatives and study the relationship between their structure and their antileishmanial
activity.
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Based on the above mentioned information, a large series of ascorbyl fatty acid esters
with increasing lipophilicity was synthesized by the direct esterification of ascorbic acid with
various fatty acids using Staphylococcus xylosus immobilized onto silica aerogel produced in
our laboratory as catalyst and evaluated for their antioxidant, emulsifying and antimicrobial
activity against several pathogenic strains and their antileishmanial effects on both cutaneous
(Leishmania major) and visceral parasites (Leishmania infantum). Finally, the interaction of
the ester derivatives with a mimetic phospholipid film mixture was evaluated using
monomolecular film technique.
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2. Materials and Methods
2.1. Materials
Ascorbic acid was purchased from Bio Basic Inc (Switzerland); chloroform and
methanol from Scharlau (Spain); acetonitrile and acetic acid from Pharmacia (Uppsala,
Sweden); caprylic, capric, lauric, palmitic, stearic, oleic acids and 2ꢀmethylꢀ2ꢀpropanol from
Fluka (Germany); 4,5ꢀdimethylꢀthiazolꢀ2ꢀylꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) and 2,2ꢀ
diphenylꢀ1ꢀpicrylhydrazyle (DPPH) were purchased from Fluka (Suisse); BHT (purity ≥
99%), αꢀtocopherol (purity ≥ 96%) and Arabic Gum (purity 99%) from Sigma and soya
lecithin (verolec Fꢀ62) was purchased from LASENOR. EggPC, cholesterol,
phosphatidylethanolamin (PE), phosphatidylglycerol (PG) and sphingolipid (SL) were
purchased from Avanti. The eggꢀyolk lecithin was extracted from fresh eggꢀyolks using a
modified method described by Palacios and Wang.13 The purity of eggꢀyolk lecithin was
checked by TLC. Staphylococcus xylosus lipase was produced as described by Mosbah et al.14
Enzyme immobilization was made onto silica aerogel as previously described by Kharrat et
al. 15
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2.2. Methods
2.2.1. Esterification reactions
Ascorbyl lipophilic esters (AscꢀC2 to AscꢀC18:1) were prepared by direct esterification
between ascorbic acid (Asc) and various fatty acids in screwꢀcapped flasks. Asc (8 mg/ml)
was dissolved in 2ꢀmethylꢀ2ꢀpropanol to an acetonitrile volume ratio of 0.2 (3mL of total
volume). The fatty acid concentration was adjusted to obtain an ascorbic acid/fatty acid molar
ratio of eight. The mixture was stirred at 45 °C in an orbital shaker at 220 rpm and in the
presence of Staphylococcus xylosus lipase immobilized onto silica aerogel (600 IU). Control
reactions in the absence of lipase were also carried out parallelly. Aliquots from the mixture
reaction were withdrawn after 72 h of incubation and filtered to be used for HPLC analysis.
The conversion yield of ascorbyl derivatives was calculated as the ratio of the number of
moles of ascorbic acid converted per the total number of moles of ascorbic acid. All
experiments were performed in triplicate. Study has been selected from five microorganism
lipases produced in our laboratory. This enzyme presented the higher yield conversion (data
not shown).
2.2.2. Purification and identification of ascorbyl esters
The reaction mixture resulting from the esterification of ascorbic acid with different
fatty acids contains a mixture of ascorbic acid ester and residual substrates. After enzyme
removal by centrifugation at 8000 rpm for 15min, the solvent (2ꢀmethylꢀ2ꢀpropanol) was
evaporated at 45°C under vacuum and the synthesized ester was purified as follows: 100 mg
of target product was taken up in 2 mL chloroform. The purification of esters was achieved by
chromatography on a silica gel 60 column (Merck) (25 cm × 2 cm) according to Aissa et al.16.
The column was previously equilibrated in chloroform. Elution was carried out using
chloroform/ methanol/ acetic acid mixtures (78:18:4). The collected solvent fractions were
analyzed by TLC. The spots were identified under evaporated iodine. Purified fractions were
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pooled and solvents evaporated at 45°C under vacuum. Final purity of the products obtained
was checked using LC/MS analysis.
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2.2.3. HPLC Analysis
The identification and conversion yields of ascorbyl derivatives were carried out by
HPLC analysis (Ultimate 3000, Dionex, Germany). The HPLC system was equipped with a
pump (LPGꢀ3400SD), column oven and diodeꢀarray UV–vis detector (DADꢀ 3000RS). The
output signal of the detector was recorded using Dionex ChromeleonTM chromatography Data
System. Separation was executed on an Inertsil ODSꢀ4 Cꢀ18 column (5 µm, 4.6 mm×250
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mm; Shimpack) maintained at 30 °C. The flow rate used was 1.5 mL/min and the detection
UV wavelength was set at 254 nm. The used mobile phase was 0.05% acetic acid in water (A)
versus 0.1% acetic acid in acetonitrile (B) for a total running time of 12 min and the following
proportions of solvent (B) were used for elution: 0ꢀ3 min: 10ꢀ30% at; 3ꢀ5 min: 30ꢀ90%; 5ꢀ10
min: 90% and 10ꢀ12 min: 90ꢀ10%.
2.2.4. LC-MS/MS Analysis
An analytical LC–UV–MS/MS analysis was performed on a Phenomenex Luna C18
(2) column (150 × 4.6 mm i.d., 5 µm particle size), using a 1 ml/ min linear mobile phase
gradient of 20–50% aq. MeOH (containing 1% HOAc) in 30 min, and mass spectra were
recorded by a Thermo Scientific ‘LCQ Classic’ ion trap mass spectrometer fitted with an ESI
source. Accurate mass measurements were performed on Finnigan MAT900 XLT or Thermo
Scientific LTQ Orbitrap XL mass spectrometers in negative and positive ESI mode.
2.2.5. NMR and FT-IR experiments
1H and 13C NMR spectra were recorded in deuterated chloroform (CDCl3) on a Bruker
TOPSPIN spectrometer operating at 400 MHz. IR spectra were recorded on FT/IRꢀ410
(JASCO).
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AscꢀC10: 1H NMR (400 MHz, CDCl3): 4.25 (2H (H1’), d); 3.85 (1H (H2’), dd); 4.35 (1H
(H3’), d); 4.27 (1H (H7’), s); 4.9 (1H (H8’), s); 5.19 (1H (H9’), s); 2.3 (2H (H2), t, ꢀCH2CO); 1.6
(2H (H3), m); 1.3 (nH (H4ꢀH9), m, (CH2)6); 0.9 (3H (H10), t, CH3).
13C NMR (400 MHz, CDCl3): 14.08 (C10), 22.68 (C3), 24.74ꢀ 33.81 (C4ꢀC9), 34.11
(C2), 64.16 (C1’), 67.47 (C2’), 130.16 (C4’), 130.88 (C5’), 153.05 (C3’), 172.48 (C6’, C=O);
178.35 (C1, C=O).
IR (liquid) cm−1: 3108ꢀ3023 (HOꢀØ), 2905 (CꢀH), 1700 (C=O), 1200ꢀ 1350 (CH2)
stretching, 1100 (CꢀO).
2.2.6. Antioxidant activity
The free radical scavenging activity of Asc and its derivatives was determined using
the method of BrandꢀWilliams and al. with a little modifications.17 An aliquot of ethanol
absolute solution (0.1 mL) containing different concentrations (1:2 serial dilutions from the
initial sample) of Asc and its esters was added to 3.9 mL of DPPH solution (0.06 mM in
ethanol). The mixture was vortexed vigorously and incubated at room temperature (25°C) in
darkness for 60 min then the absorbance was read at 517 nm against ethanol blank using a
spectrophotometer (Uvi Light XT5).
The IC50 values denote the concentration of tested compounds, which is required to
scavenge 50% of DPPH free radicals. The corresponding inhibition percentages were
calculated according to the following equation:
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(Ablank − Asample
)
Radical scavenging activity (%)
=
×100
Asample
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Where, Ablank is the absorbance of the control (prepared in the same manner without test
compound), and Asample is the absorbance of the test compound. Ascorbic acid, butylated
hydroxytoluene (BHT) and αꢀtocopherol were used as standard control. The values are
presented as the means of triplicate analysis.
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2.2.7. Lipophilicity evaluation
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The partition coefficients (miLogP) values and the molecular weight of ascorbyl esters
were calculated using Molinspiration software, (available online at www.molinspiration.com).
The lipophilic character of ascorbyl esters was evaluated as described by Viskupicova et al.18
2.2.8. Measurement of conjugated diene (CD) and triene (CT)
The effect of ascorbyl esters on the oxidative state of oil was evaluated by the
measuring of conjugated diene (CD) and conjugated triene (CT) as previously described by
Della et al.19 Oxidation was induced during 20 days by storing soya oil at 70°C. Every 2 days,
the absorbance was measured at 232 nm (for CD) and 270 nm (for CT), using hexane as a
blank. All measurements were performed in triplicate. BHT and αꢀtocopherol were used as
standard control.
2.2.9. Emulsifying activity
The ability of the purified ascorbyl esters to form oilꢀinꢀwater (o/w) emulsions against
soya oil was determined using the method described by Gutierrez et al.20. Activities were
compared under neutral (0.1 M phosphateꢀbuffered saline, pH 7), acidic (0.1 M sodium
acetate buffer, pH 4) and basic (0.1 M TrisꢀHCl buffer, pH 8.5) conditions. Five mL of buffer
were vortexed for 2 min at 2200 rpm with 0.8 mL of soya oil containing 0.02 % (w/v) of each
emulsifier. The turbidity of the lower aqueous layer was measured using a spectrophotometer
at 540 nm. All measurements were carried out in triplicates and the mean values of the
triplicates were reported with standard error. Eggꢀyolk lecithin, soya lecithin and Arabic gum
were used as commercial emulsifiers.
2.2.10. Determination of the minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC)
The antibacterial activities of Asc, Asc + FAs (separated substrates taken together at
the same concentration) and ascorbyl derivatives were tested against several bacteria strains
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using LuriaꢀBroth (LB) medium. The minimum inhibitory concentration (MIC) values, which
correspond to the lowest compound concentration that completely inhibits the growth of
microorganisms, were determined by a microꢀwell dilution method as previously described by
Eloff21 using 3ꢀ(4,5ꢀdimethylꢀthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT). The
inoculum of each bacterium was prepared and the suspensions were adjusted to 107 CFU/mL.
All the compounds were dissolved in 100% ethanol, and then dilutions series were prepared
in a 96ꢀwell plate, ranging from 3.125µg/mL to 4mg/mL. Each well of the microplate
contained 175µL of the growth medium, 5µL of inoculum and 20µL of the diluted sample
extract. Ethanol was used as a negative control. The plates were incubated at 37°C for 24 h,
then 40µL of MTT, at a final concentration of 0.5mg/mL freshly prepared in sterile water,
was added to each well and incubated for 30min. The change to purple color indicated that the
bacteria were biologically active. The MIC was taken where no change of MTT colour was
observed in the well.
To determine the minimum bactericidal concentration (MBC), a liquid portion from
each well that showed no change in color will be placed on solid LB and incubated at 37°C
for 24h. The lowest concentration that yielded no growth after this subꢀculturing will be taken
as the MBC.22 All experiments were made in duplicate. Several bacterial strains were used:
i.e., Bacillus cereus (BC), Bacillus subtilis (BS), Staphyloccocus aureus (Sa), Staphyloccocus
epidermidis (SEp), Enterococcus faecalis (EF), Enterococcus faecium (EFa), Enterobacter
cloacae (EC), Micrococcus Luteus (ML), Brevibacterium flavum (BF), Pseudomonas
Aeruginosa (Ps), Salmonella Typhimurium (STyphi), Klebsielle pneumonia (KP), Echerichia
coli (Ecoli) and Staphyloccocus xylosus (Sx).
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2.2.11.2. Leishmanicidal activity
(These experiments have been performed in the Pasteur Institute of Tunis (Tunisia))
L. major (MHOM/TN/95/GLC94)23 and L. infantum (MHOM/TN/94/LV50)24 strains
isolated from Tunisian patients were used within this study. Promastigotes were cultured in
solid medium at 26°C during 6 days. They must be in their infective metacyclic forms as
mentioned by Aissa et al.11. The effects of ascorbyl derivatives on Leishmania promastigotes
were evaluated by the MTT assay as described by Dutta et al. . Parasites (107parasites/well
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in the complete medium) were incubated for 24 h in the presence of serially diluted
concentrations of ascorbyl derivatives (ranging from 25 to 400µg/ml).11 After this treatment,
microtitre plates were centrifuged at 1700g for 10 min and supernatants were removed and
replaced with the same volume of 1mg/ml of MTT freshly dissolved in PBS. Plates were then
incubated overnight at room temperature. After centrifugation at 2500g, formazan salt formed
inside the parasite mitochondries was solubilized by discarding supernatants and adding SDS
10% for 2h at 37°C in the dark. Absorbance was measured at 540 nm using an ELISA plate
reader. Negative controls were conducted without addition of ascorbyl derivative solutions.
Each assay was performed in duplicate.
2.2.12. Monolayer study of the ascorbyl derivatives
Experiments were carried out on KSV 2200 Baroꢀstat equipment as described by Aissa
et al.11 Teflon trough equipped with two hydrophilic Delrin barriers (symmetric compression)
and a Wilhelmy plate as a surfaceꢀpressure sensor. Software KSV 2200 was used to control
the experiments. Before each utilisation, the Teflon trough was cleaned and brushed in the
presence of distilled ethanol, subsequently washed again with tap water, and finally rinsed
with doubleꢀdistilled water. The aqueous subphase (buffer A) was composed of 10 mM Tris–
HCl, pH 8, 100 mM NaCl, 21 mM CaCl2, and 1 mM EDTA. Buffer was prepared with
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doubleꢀdistilled water and filtered through a 0.45 µM Millipore filter. Residual surface active
impurities were removed before each assay by sweeping and suction of the surface.26
2.2.13. Measurement of the ascorbyl derivatives penetration into mixture film monolayer
The surface pressure increase, due to the penetration of the ascorbyl derivatives into a
mixture film monolayer, was measured in a cylindrical trough drilled into a Teflon block
(surface area 17.42 cm2, total volume 15 mL). The aqueous subphase (buffer A) was stirred
continuously at 250 rpm with a magnetic rod. The critical surface pressures were determined
as described previously.27 A sample of Asc or synthesized ester solution (0.5 µM) was
injected with Hamilton microsyringe, under a monomolecular film of mixture film spreads at
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an initial surface pressure (πi) ranging from 4 to 37 mN /m. To mimic the lipidic components
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of leishmania membrane, a mixture of phospholipids was used, containing 40% EggPC, 30%
cholesterol, 10% phosphatidylethanolamin (PE), 10% phosphatidylglycerol (PG) and 10%
sphingolipid (SL).28
2.2.14. Statistical analysis
All analysis were carried out in triplicates. Results were expressed as mean values ±
standard deviation (SD) (n = 3). The differences were calculated using oneꢀway analysis of
variance (ANOVA), and statistically significant differences were reported at P < 0.05. Data
analysis was carried out using the SPSS 10.0 software.
3. Results and discussion
3.1. Enzymatic synthesis and Characterization of Ascorbyl fatty acid Esters
A chemoselective enzymatic esterification was used to synthesize lipophilic ascorbyl
esters derivatives (AscꢀC2 to AscꢀC18:1). Several solvents such as hexane, chloroform, acetone
and toluene were used as reaction media. However, among all these organic solvents tested,
only coꢀsolvent 2ꢀmethylꢀ2ꢀpropanol/acetonitrile was suitable for this esterification reaction.
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The conversion yields calculated after 72h of incubation using lipase from
staphylococcus xylosus immobilized onto silica aerogel as a catalyst are presented in Table 1
.
As it can be seen, the highest ester synthesis yield was obtained when using short acyl chains
ester AscꢀC2. For medium and long chain esters (AscꢀC8 to AscꢀC18:1), the conversion yield
decreased progressively with the increase of the acyl chain length (Table 1) although
Staphylococcus xylosus lipase hydrolyses triacylglycerols without significant chain length
preference. 14
These results are in line with those observed previously by Aissa et al.16. The fatty
acid unsaturation seems to affect the synthesis yield. In fact, the ester conversion yield
reached 52.86% with ascorbyl palmitate and decreased to 40.96% of ascorbyl oleate when
using oleic acid as acyl donor. Our observations are in agreement with those described by
Selmi et al.29. They concluded that the increase of the unsaturation number is responsible for
the lower rate of triacylglycerols synthesis using immobilized Rhizomucor miehei lipase.
These results are however in contradiction with those described by Song et al.30 which
showed that C18 unsaturated FAs gave better conversion yield as compared to C18 saturated
FAs using Novozym 435 as catalyst. These contradictions between these findings could be
attributed to the nature of the lipase used and the composition of the reaction medium
(solvent, water content, substrate chemistry) or the operating conditions.31,32
3.2. Purification and identification of ascorbyl fatty acid esters
The purification of each ascorbyl ester was achieved by chromatography on a silica
gel 60 column according to Aissa et al.16 as described above in Materials and Methods. The
purity of these products was then checked via HPLC analysis (Fig. 1A); their retention time is
mentioned in Table 1. RMN, MS1 and IR analysis were used to identify the purified products.
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As shown in Fig. 1B, MS1 analysis in the negative mode of pure AscꢀC10, taken as a typical
compound, exhibited a molecular ion at m/z = 329.34 [MꢀH]ꢀ attributed to the molecular
weight of calculated ester using Molinspiration software (330.37 g/mol) shown in Table 1
.
The MS2 experiments focusing on the fragment generated from the peak in m/z = 329.34 [Mꢀ
H]ꢀ revealed a fragment corresponding to the pseudo molecular ion at m/z = 171.1 attributed
to capric acid and m/z = 175.0 attributed to ascorbic acid ion. The ion at m/z = 157 was
formed by the cleavage of the capric acid and neutral losses of H2O linked to the ascorbic acid
(Fig. 1C)
3.3. Antioxidant activity
Ascorbic acid exhibits low solubility and stability in the lipophilic media, which can
.
limit its efficient use in these conditions. Hence, the lipophilic derivatives of Asc and, in
particular, long acyl chain esters, would exhibit a better affinity with lipophilic matrices.
Being a lipophilic compound may improve its solubility as well as maintain or enhance its
radical scavenging capacity.16,32 This property would make ascorbyl derivatives strong
candidates for pharmaceutical and cosmetic lipophilic formulation.
The antioxidant efficiency of ascorbic acid was checked after lipophilisation using
DPPH in an ethanolic medium, a rapid and sensitive scavenging test. As indicated in Table 1
,
Ascorbic acid and its synthesized esters were shown to be strong DPPH radical scavengers.
Esterification of AscꢀC2 to AscꢀC18:0 seems to slightly affect the radicalꢀscavenging potential.
All ascorbyl derivatives exhibited a high antioxidant activity with an IC50 varying between 3
and 3.5 µg/mL which are near the IC50 value of ascorbic acid (2.01 µg/mL). Moreover, the
antioxidant effect of ascorbic acid and its derivatives was twice as efficient as the known
synthetic antioxidants such as BHT and αꢀtocopherol. These results are, however, in
contradiction with those described by Burham et al.33 who showed that ascorbic acid
exhibited a higher antioxidant activity than ascorbyl palmitate and pure palmꢀbased ascorbyl
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esters. The radicalꢀscavenging capacity of ascorbic acid and its derivatives can be explained
by the number and position of hydrogenꢀdonating groups (OH) in hydroxyl head group.32
3.4. Ascorbyl ester derivatives effect on oil oxidative stability
The effect of lipophilic ascorbyl derivatives on the oxidative stability of oils was
determined by the measurement of conjugated diene (CD) and triene (CT). Ascorbic acid and
its acyl esters (AscꢀC2 to AscꢀC18:1) were added to refined soy oil. CD and CT of the different
preparations were measured upon 20 days storage at 70°C in the open air (Figs. 2A and B).
The CD and CT determine the primary products of oil oxidation.34 A remarkable increase in
the CD and CT of refined soya oil was observed. As shown in Figs. 2A and B, the addition of
ascorbyl derivatives, as antioxidants, prevented oil oxidation as well as BHT and αꢀ
tocopherol. Interestingly, the protective effect against oxidation by ascorbyl derivatives was
higher than that of Asc and increased with the fatty acid chain length. However, Asc, AscꢀC2
and AscꢀC8 derivatives displayed the lowest protective effect of soya oil against oxidation.
Based on the increasing trend of miLog P values (Table 1), it seems that Ascorbyl esters
having a high lipophilicity (miLog P higher than 3.2) display the best antioxidant capacities.
Ascorbyl derivatives, the amphiphilic natural antioxidants, could be used for increasing
protection of targetꢀlipids from oxidation such as skin creams, facial treatments (acne or
wrinkle removers), bath oils, sunscreens, preꢀshave and makeꢀup.
3.5. Emulsifying activity
Today, more than ever, a large amount of surfactant is required for a microemulsion
system used for pharmaceutical/cosmetic purposes.35 However, a surfactant may exhibit
undesirable effects like skin irritation, allergy, etc. when used excessively. Therefore, one
must choose materials that are biocompatible, nonꢀtoxic, clinically acceptable, and use
emulsifiers in an appropriate concentration range that will result in mild and nonꢀaggressive
microemulsions. 35 The excipients generally regarded as safe (GRAS) are being increasingly
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preferred. For these reasons, the ability of purified ascorbic acid derivatives to emulsify soya
oil in water was tested. The emulsifying capacity of the different ascorbyl derivatives was
determined under acidic, neutral and basic conditions. Arabic gum, soya lecithin and eggꢀ
yolkꢀlecithin were taken as control emulsifiers since they are the most important hydrocolloid
ingredients in the formulation of many food products. Based on average results provided in
acidic, neutral and basic pH conditions presented in Fig.3, the ascorbyl derivatives produced
emulsifying activities that were significantly different from those of control. In fact, apart
from Asc and AscꢀC2, all ascorbyl derivatives (AscꢀC8 to AscꢀC18:1) produced a significantly
higher emulsifing activity than arabic gum and soya lecithin, but lower than eggꢀyolkꢀlecithin.
Comparable results were obtained by Kuwabara et al. 36 who demonstrated that caproyl and
lauroyl lꢀascorbates are useful emulsifiers for emulsions. In addition, under acidic conditions,
the emulsifying activities of all ascorbyl esters were significantly higher compared to those
measured under basic or neutral conditions (Fig.3). Among all the tested derivatives, Ascꢀ
C18:1 was found to be the most potent as emulsifier.
3.6. Determination of the minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC)
The antibacterial activity of Asc, FAs, Asc + FAs and the synthesized esters (AscꢀC2
to AscꢀC18:1) was checked against Gramꢀpositive (Bacillus cereus (BC), Bacillus subtilis (BS),
Brevibacterium flavum (BF), Micrococcus Luteus (ML), Staphyloccocus xylosus (Sx),
Staphyloccocus aureus (Sa), Staphyloccocus epidermidis (SEp), Enterococcus faecalis (EF)
and Enterococcus faecium (EFa)) and Gramꢀnegative (Enterobacter cloacae (EC), Pseudomonas
Aeruginosa (Ps), Salmonella Typhimurium (STyphi), Klebsielle pneumonia (KP) and Echerichia
coli (Ecoli)) bacteria by the determination of MIC and MBC values. As can be seen from
Tables 2A and B, ascorbic acid does not show any inhibitory or bactericidal effect up to a
concentration of 4 mg/mL. Compared to Asc and mixed solution of Asc + FAs, synthesized
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ester exhibits the most effect against the bacteria tested, in particular Gram (+) ones. In fact,
with Gram+ bacteria, MIC values ranged from 0.0625 to 2 mg/mL and the MBC values are
between 0.500 and 4 mg/mL, while Gram (ꢀ) bacteria appear to be less sensitive to the tested
compounds. Previous studies reported that Gram (ꢀ) bacteria presented lower sensitivity than
Gram (+) bacteria to various polyphenols.37 This higher resistance may be related to minor
differences present in the outer membrane in the cell wall composition. 37 As it can be seen
from Table 2A, MIC and MBC values showed that ascorbyl derivatives exhibit a similar or a
slightly higher inhibitory activity and more bactericidal effect against Gram (+) bacteria than
the separated substrates (Asc + FAs). These results indicate that we have significantly
enhanced the antimicrobial activity of the ascorbic acid after esterification. These findings are
in agreement with those of Aissa et al.11 who showed that only medium chain tyrosyl esters
had an antibacterial activity especially against Staphylococcus strains.
These results could be explained by their hydrophobicity which may allow them to
partition the lipids of the bacterial cell membrane, making them more permeable and leading
to ions leakage and other cell constituents.38 The authors suggested that these compounds
could be able to infiltrate the cell and interact with some metabolic mechanisms, which
allowed us to show their bacteriostatic effect.
3.7. Antileishmanial activity
Ascorbic acid and its lipophilic derivatives were screened for their leishmanicidal
23
activity. Screening was carried out using two Leishmania species: L. major GLC94 and L.
infantum LV50.24 As shown in Table 3, only AscꢀC10
, AscꢀC12 and AscꢀC18:1 ascorbyl
derivatives were effective against both Leishmania species, while either ascorbic acid or Ascꢀ
C2, AscꢀC8, AscꢀC16 and AscꢀC18:0 derivatives had no leishmanicidal activity up to 400
µg/mL. Interestingly, the three effective derivatives showed a higher activity against L. major
promastigotes compared to that obtained against L. infantum promastigote. Indeed, IC50
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values were approximately two times higher against the former than the latter. This indicates
that L. major parasites are more sensitive to these compounds than L. infantum ones. Ascꢀ
C18:1, the most effective derivative, showed an IC50 of 154.23 µg/mL and 205.81 µg/mL
against L. major and L. infantum, respectively. Also, AscꢀC12 showed a moderate activity of
196.262 µg/mL and 284.8 µg/mL against L. major and L. infantum, respectively. Finally,
AscꢀC10 was the less active compound of the three derivatives which showed an IC50 of
217.74 µg/mL and 347.12 µg/mL against the dermotropic and the visceraotropic strain,
respectively. These results are in line with those observed previously by Aissa et al.11 who
showed that the medium chain tyrosyl esters had an antileishmanial effect. However, for our
case, the most effective derivative was ascorbyl oleate (AscꢀC18:1). These results could be
attributed to the nature of the hydroxyl head group of ascorbic acid and besides their
amphiphilicity degree.
3.8. Interactions of ascorbyl derivatives with mixture film monolayer
In order to study the influence of the length and the nature of the ascorbyl derivatives
426
acyl chain on the esters adsorption properties, their critical surface pressures (πc) were
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428
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430
compared using a mixture film monolayer, containing the principal components of leishmania
membranes: 40% EggPC, 30% cholesterol, 10% phosphatidylethanolamin (PE), 10%
phosphatidylglycerol (PG) and 10% sphingolipid (SL).28
The maximum surface pressure increase was determined at different initial pressures
431
432
433
434
435
436
(
π
i) of mixture film ranging from 5 to 40 mN /m (Fig. 4). The critical surface pressure (
each ester was estimated by the linear extrapolation of the experimental curves at zero surface
pressure increase. A critical pressure for penetration ( c) may thus be defined; it corresponds
to the extrapolated value of initial beyond which there is no increase in surface pressure. The
value of the maximum surface pressure increase (∆ max) reached at equilibrium (around 40
πc) for
π
π
min after the injection of the ascorbyl derivative in the aqueous subphase) was determined
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and plotted as a function of πi (Fig. 4). For all ascorbyl derivatives, a general trend was
observed: higher was
π
i, lower was the interaction of the ascorbyl derivatives with the
AscꢀC2 AscꢀC8 cannot
439
440
441
monolayer because of the higher packing of phospholipids. Asc
,
,
interact with the mixture film (data not shown). It can be seen from Table 4, that all ascorbyl
esters (AscꢀC10 to AscꢀC18:1) could interact with the mimetic membrane of leishmania. The
442
critical surface pressures (πc) of these esters on mixture film monolayer are ranged from
443
444
445
446
447
448
449
450
451
452
453
454
36.96 mN /m to 50.74 mN /m (Table 4). Mottola et al.39 showed that the AscꢀC16 is an
anionic amphiphilic molecule which allowed them to penetrate phospholipid monolayers.
These results are in line with those observed previously by Aissa et al.16 who showed that the
medium and long chain tyrosyl esters could interact with biological membrane. We can also
notice that unsaturation in the acyl chain (AscꢀC18:1) seems to strengthen its interaction of
with the mixture film. In fact, the critical surface pressure of AscꢀC18:1 and AscꢀC18:0 are
50.74 mN /m and 41.30 mN /m, respectively (Table 4). Makyla and Paluch40 demonstrated
that the presence of the unsaturated fatty acid in cholesterol/DPPC mixed monolayer makes
the membrane more fluid. AscꢀC16:0 and AscꢀC18:0 showed an interaction with mimetic
membrane model but not an antileishmanial activity. This result could be explained by the
selfꢀaggregation and the reduced mobility of long saturated ascorbyl derivatives in the
aqueous phase of the parasite culture medium as it was hypothesized by Laguerre et al.41 In
455
light of these results, if one takes the πc as a threshold value to appreciate the capacity of the
456
457
458
459
460
461
ascorbyl esters to penetrate mixture films monolayers, we can tentatively conclude that the
medium and long chain ascorbyl esters (AscꢀC10 to AscꢀC18:1) could interact efficiently with a
biological membrane, characterized by a surface pressure between 25 and 35 mN /m.42
In conclusion, from all the tested derivatives, only medium and unsaturated ascorbyl
derivatives (AscꢀC10, AscꢀC12 and AscꢀC18:1) exhibited a potent antimicrobial and
antileishmanial activities
.
These amphiphilic antioxidants could be used as membraneꢀ
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perturbing surfactants in clinical applications such as for the dermatological and
pharmacological uses.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NK carried out all the studies, analyzed the data and drafted the manuscript. IA helped with
the discussion of the data and the correction of the manuscript. RMS carried out the
antileishmanial activity. MB helped with the NMR, IR and LCꢀMS analysis. MS and DL
helped with discussion of the data. YG participated in the study design and helped to draft the
471
manuscript. All authors have read and approved the final manuscript.
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Acknowledgements
This work represents a part of the thesis of Mrs Nadia Kharrat. It received financial
support from the Ministry of Higher Education and Scientific Research in Tunisia. The
authors would like to thank Pr. Sofiane Bezzine (ENIS, SfaxꢀTunisia) for his generous gift of
bacterial strains. We are grateful to Pr. Verger Robert and Pr. Pierre Villeneuve for their
fruitful discussion. We are grateful to Pr. Bejaoui Hafedh (FSS, SfaxꢀTunisia) for his help
with English. Parasite experiments were partially supported by NIH/NIAID/DMID Grant
Number 5P50AI074178 to LTCII.
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Biochim Biophys Acta. 2013, 1828, 2496ꢀ2505.
40. Makyla, K.; Paluch, M. The linoleic acid influence on molecular interactions in the model
of biological membrane. Colloid surface B. 2009, 71, 59ꢀ66.
41. Laguerre, M.; Bayrasy, C.; Lecomte, J.; Chabi, B.; Decker, E.A.; WrutniakꢀCabello, C.;
Cabello, G.; Villeneuve, P. How to boost antioxidants by lipophilization? Biochimie.
2013, 95, 20ꢀ26.
42. SanchezꢀMartin, M.J.; Haro, I.; Alsina, M.A.; Busquets, M.A.; Pujol, M. A Langmuir
monolayer study of the interaction of E1 (145–162) Hepatitis G virus peptide with
592
593
phospholipid membranes. J. Phys. Chem. B. 2010, 114, 448ꢀ456.
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594
Figure Captions:
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
Fig.1. (A) HPLC profile of ascorbyl caprate (AscꢀC10) after biosynthesis and purification. The
biosynthesis reaction of ascorbic acid with capric acid ( ascorbic acid/fatty acid molar ratio of
eight) was catalyzed by Staphylococcus xylosus lipase immobilized onto silica aerogel at
45°C and 220 rpm in 2ꢀmethylꢀ2ꢀpropanol to acetonitrile volume ratio of 0.2 (72 h). HPLC
analysis was carried out on a Cꢀ18 column by gradient elution using acidified water and
acetonitrile at a flow rate of 1.5 mL/min.
(B) MS1 spectra of purified ascorbyl caprate (AscꢀC10).
(C) MS2 spectra of purified ascorbyl caprate (AscꢀC10).
Fig.2. Antioxidant activities of Ascorbyl ester derivatives (AscꢀC2 to AscꢀC18:1) tested by the
measurement of conjugated diene (A) and conjugated triene (B) of refined soy oil during
storage at 70°C. For each test, negative and positive controls using BHT and αꢀ Tocopherol
were run in parallel. Values are mean ± SD (n = 3) of three determinations
.
Fig.3. Emulsifying activity at pH 4 ( ); pH 7 ( ) and pH 8.5 ( ) of ascorbic acid and its
derivatives (AscꢀC2 to AscꢀC18:1). The activity reflects the capacity of the emulsifier to form
soya oilꢀinꢀwater emulsions. Eggꢀyolkꢀlecithin, Arabic Gum and Soya lecithin were used as
control emulsifiers. Values are mean ± SD (n = 3) of three determinations
.
Fig.4. Maximal increase in surface pressure after ascorbyl derivatives injection with respect to
the initial surface pressure of mixture films (40% PC, 30% cholesterol, 10% SL, 10% PG,
10% PE) spread in a cylindrical Teflon trough (volume, 15 mL; surface, 17.42 cm2). Final
ascorbyl derivatives concentration, 0.5 µM. Buffer, 10 mM Tris–HCl, pH 8.0, 100 mM NaCl,
21 mM CaCl2, and 1 mM EDTA.
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Table 1
The conversion yields of the ascorbyl derivatives and physicoꢀchemical parameters of ascorbic acid and its esters related to their lipophilicity.
Conversion
yield (%)
ꢀ
82.57 ± 2.32 Yellow slimy oil
68.21 ± 2.75 Yellow slimy oil
59.39 ± 2.30 White amorphous solid
58.24 ± 2.72 White amorphous solid
52.86 ± 2.71 White amorphous solid
42.08 ± 1.62 White amorphous solid
40.96 ± 1.07 Yellow oil
MW
miLog
Pa
Rt
IC 50
Compounds
Ascorbic acid
Abbreviations Radicals
Characteristics
White solid
(g/mol)a
176.124
218.161
302.323
330.377
358.431
414.539
442.593
440.577
430.717
220.356
(min)b
1.990
3.540
6.627
7.807
8.372
9.537
(µg/mL)c
C6H8O6
ꢀCH3
ꢀC7H15
ꢀ 1.402
ꢀ 0.698
2.243
3.253
4.263
6.284
7.295
6.809
2.010 ± 0.085
3.435 ± 0.091
3.515 ± 0.084
3.215 ± 0.078
3.355 ± 0.092
3.205 ± 0.088
Asc
Ascorbyl acetate
Ascorbyl caprylate
Ascorbyl caprate
Ascorbyl laurate
Ascorbyl palmitate
Ascorbyl stearate
Ascorbyl oleate
αꢀtocopherol
AscꢀC2
AscꢀC8
AscꢀC10
AscꢀC12
AscꢀC16
AscꢀC18:0
AscꢀC18:1
ꢀ
ꢀC9H19
ꢀC11H23
ꢀC15H31O
ꢀC17H35O
ꢀC17H33O
C29H50O2
C15H24O
11.392 3.310 ± 0.099
9.743 2.980 ± 0.085
5.760 ± 0.028
7.265 ± 0.064
ꢀ
ꢀ
Yellow viscous liquid
White solid
9.043
ꢀ
ꢀ
BHT
ꢀ
5.435
a miLog P and MW values calculated using Molinspiration program
b Retention time from HPLC
c Antioxidant activity of ascorbic acid and their fatty acid esters determined by DPPH method.
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Table 2
Minimum inhibitory concentration and Minimum Bactericidal concentration of Ascorbyl ester derivatives on bacteria.
Bacteria strain
Gram
MIC (mg/ml)
A
a
Asc
(Asc + C8)a AscꢀC8 (Asc + C10)a AscꢀC10 (Asc + C12)a AscꢀC12 (Asc + C18:1
)
AscꢀC18:1
> 4
2
2
2
2
2
2
2
2
4
1
1
2
2
2
2
2
2
2
2
4
0.5
0.25
0.25
0.25
0.25
0.5
1
1
2
2
2
2
2
2
2
0.125
0.125
0.062
0.062
0.062
0.25
2
1
1
1
1
2
2
2
2
1
+
+
+
+
+
ꢀ
Bacillus cereus
Bacillus subtilis
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
0.5
0.25
0.25
0.25
0.5
0.5
0.5
0.5
1
Staphylococcus aureus
Staphylococcus xylosus
Staphylococcus epidermidis
Enterobacter cloacae
Salmonella Typhimurium
Klebsielle pneumoniae
Escherchia coli
1
0.25
0.5
0.25
0.5
ꢀ
1
0.125
0.5
1
ꢀ
2
1
0.5
ꢀ
Bacteria strain
Gram
MBC (mg/ml)
B
a
Asc (Asc + C8)a AscꢀC8 (Asc + C10)a AscꢀC10 (Asc + C12)a AscꢀC12 (Asc + C18:1
)
AscꢀC18:1
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
> 4
4
4
2
2
2
4
4
4
4
2
2
1
1
1
2
2
2
2
4
4
2
2
4
4
4
4
4
0.5
0.5
0.25
0.25
0.5
0.5
0.5
1
2
2
1
1
1
2
4
4
4
0.25
0.25
0.125
0.125
0.125
0.5
4
4
2
2
+
+
+
+
+
ꢀ
Bacillus cereus
Bacillus subtilis
2
0.5
0.5
0.5
1
Staphylococcus aureus
Staphylococcus xylosus
Staphylococcus epidermidis
Enterobacter cloacae
Salmonella Typhimurium
Klebsielle pneumoniae
2
2
> 4
> 4
> 4
> 4
0.25
0.25
0.5
1
ꢀ
2
ꢀ
1
2
ꢀ
Escherchia coli
a : Taken together at the same concentration.
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Table 3
IC50 activities of ascorbic acid and its acyl chain derivatives against L. major and L. infantum parasite species evaluated
by the MTT assay.
Compounds
IC50 (µg/mL)
L. major
Asc
AscꢀC2 AscꢀC8
AscꢀC10
AscꢀC12
AscꢀC16 AscꢀC18:0
AscꢀC18:1
a
a
a
a
a
217.74 ± 13.33 196.26 ± 10.53
347.12 ± 12.32 284.80 ± 20.33
a
a
a
a
154.23 ± 12.41
IC50 (µg/mL)
L. infantum
a
205.81± 14.35
a : Without effect up to 400 µg/ml.
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