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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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Journal of Agricultural and Food Chemistry
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Title: Lipophilisation of ascorbic acid: A monolayer study, Biological and Antileishmanial
2
activities
3
Authors list: Nadia Kharrata, Imen Aissaa, Manel Sghaierb, Mohamed Bouazizc, Mohamed
4
Sellamia, Dhafer Laouinib and Youssef Gargouria*
5
Affiliation List:
6
a
7
BPW 1173- 3038 Sfax-Tunisie.
8
b
9
Immunobiologie des Infections (LTCII), Institut Pasteur de Tunis, 13, Place Pasteur,
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, Route de Soukra,
Groupe Immunobiologie des Leishmanioses, Laboratoire de Transmission, Contrôle et
10
BP 74 ,1002 Tunis-Belvédère, Tunisie.
11
c
12
Université de Sfax- Tunisie.
13
* Corresponding author: Prof. Youssef Gargouri,
14
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, Université de Sfax,
15
Route de Soukra, 1173 Sfax-Tunisie.
16
Tel/ Fax: + 21674675055,
17
E-mail :
[email protected] Laboratoire d’Electrochimie et Environnement, ENIS, Route de Soukra, BPW 1173- 3038,
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Abstract
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Ascorbyl lipophilic derivatives (Asc-C2 to Asc-C18:1), were synthesized in a good
26
yield using lipase from Staphylococcus xylosus produced in our laboratory and immobilized
27
onto silica aerogel. Results showed that esterification had little effect on radical-scavenging
28
capacity of purified ascorbyl esters using DPPH assay in ethanol. However, long chain fatty
29
acids esters displayed higher protection of target-lipids from oxidation. Moreover, compared
30
to ascorbic acid, synthesized derivatives exhibited an antibacterial effect. Furthermore,
31
ascorbyl derivatives were evaluated, for the first time, for their antileishmanial effects against
32
visceral (Leishmania infantum) and cutaneous parasites (Leishmania major). Among all the
33
tested compounds, only Asc-C10, Asc-C12 and Asc-C18:1 exhibited antileishmanial activities.
34
The interaction of ascorbyl esters with a phospholipid monolayer showed that only medium
35
and unsaturated long chain (Asc-C10 to Asc-C18:1) derivative esters were found to interact
36
efficiently with mimetic membrane of leishmania. These properties would make ascorbyl
37
derivatives good candidates to be used in cosmetic and pharmaceutical lipophilic
38
formulations.
39 40
Keywords:
Ascorbic
acid;
lipophilic
derivatives;
antimicrobial
activity;
leishmanicidal activity; phospholipid monolayers
41 42 43
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Journal of Agricultural and Food Chemistry
1. Introduction
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Lipid deterioration increases in edible and inedible fatty products through processing,
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heating, storage and culinary practices. As a result, the oxidative processes generate off-flavor
47
compounds such as aldehydes, peroxides, ketones and oxyacids.1 These compounds cause a
48
decrease in food nutritive value and sensorial quality. To prevent food oxidation and extend
49
its shelf life, they should be stored under proper conditions, e.g. at low temperature in an inert
50
atmosphere and suitable packaging. In this sense, antioxidants are widely recommended as
51
additives which have the potential to control the oxidation process, thereby stabilizing fat-
52
containing food-stuffs.2 Synthetic antioxidants such as butylated hydroxyanisole (BHA),
53
butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallates were
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widely used as scavenging free radicals to prevent the formation of initiating lipid radicals.
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Yet, according to toxicologists and nutritionists, the application of these synthetic
56
antioxidants in food materials is a matter of concern related to the formation of toxic
57
compounds. These substances can show possible side-effects such as carcinogenicity in living
58
organisms.3 Lanigan and Yamarik3 showed that only 50 % of BHT can be eliminated by urine
59
within the first 24 hours and the other parts are accumulated in adipose tissue. Natural
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compounds with better antioxidant capacity and less toxicity have acquired major interest for
61
their implication as prophylactic and therapeutic agents in many diseases and for their better
62
food preservation. Currently, ascorbic acid is a labile molecule and is well-known by virtue of
63
its reducing property. Moreover, it can prevent chronic diseases due to oxidative stress,
64
including cancer, hypertension, cardiovascular disease and stroke.4 Ascorbic acid, which
65
cannot be synthesized by humans, was identified at a high level ranging from 162 to 135mg
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per 100g in piper and kiwi to shield them against peroxidizing factors. Some fruits, like
67
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 orange-peel provides 136mg of ascorbic acid, while its flesh contains
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just about 71mg/100 g.
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Ascorbic acid is also available in a wide range of supplements such as tablets, capsules
71
and crystalline powder. Unfortunately, it is unstable when exposed to air, light, heat and is
72
rapidly oxidized and irreversibly decomposed upon entering the body, thus exhibiting low
73
solubility and stability in the lipophilic media.5 This behavior can limit its uses efficiently.
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Being fat soluble, lipophilic ascorbyl derivatives exhibit a better affinity with lipophilic
75
membrane constituents. They can be easily absorbed and retained into the lipid domain of
76
biological system for a longer period of time.5 The enzymatic synthesis catalyzed by
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commercial lipases to produce some ascorbyl esters via esterification in organic solvents has
78
been emphasized in several works.6,7 Amphiphilic ascorbyl derivatives display particularly
79
interesting characteristics resulting from the modification of molecular flexibility. Hence,
80
lipophilic derivatives of ascorbic acid and, in particular, long acyl chain esters, would exhibit
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a better affinity with lipophilic membrane constituents of drug target cells.8 This property
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would make from ascorbyl derivatives good candidates to be used in pharmaceutical and
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cosmetic lipophilic formulations.9 The interest in new compounds with antimicrobial
84
properties has been revived because of current problems associated with the use of
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antibiotics.10 Aissa et al.11 have synthesized a large series of tyrosyl esters with increasing
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lipophilicity using lipase of Candida antarctica. Authors showed that the parent tyrosol does
87
not have any effect on various pathogenic bacteria. However, tyrosyl esters and especially
88
medium chain tyrosyl derivatives exhibited an antimicrobial activity against staphylococcus
89
strains.
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In the wake of resistance to pentavalent antimonial drugs, new therapeutic alternatives
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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
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activity.
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Based on the above mentioned information, a large series of ascorbyl fatty acid esters
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with increasing lipophilicity was synthesized by the direct esterification of ascorbic acid with
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various fatty acids using Staphylococcus xylosus immobilized onto silica aerogel produced in
97
our laboratory as catalyst and evaluated for their antioxidant, emulsifying and antimicrobial
98
activity against several pathogenic strains and their antileishmanial effects on both cutaneous
99
(Leishmania major) and visceral parasites (Leishmania infantum). Finally, the interaction of
100
the ester derivatives with a mimetic phospholipid film mixture was evaluated using
101
monomolecular film technique.
102
2. Materials and Methods
103
2.1. Materials
104
Ascorbic acid was purchased from Bio Basic Inc (Switzerland); chloroform and
105
methanol from Scharlau (Spain); acetonitrile and acetic acid from Pharmacia (Uppsala,
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Sweden); caprylic, capric, lauric, palmitic, stearic, oleic acids and 2-methyl-2-propanol from
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Fluka (Germany); 4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) and 2,2-
108
diphenyl-1-picrylhydrazyle (DPPH) were purchased from Fluka (Suisse); BHT (purity ≥
109
99%), α-tocopherol (purity ≥ 96%) and Arabic Gum (purity 99%) from Sigma and soya
110
lecithin
111
phosphatidylethanolamin (PE), phosphatidylglycerol (PG) and sphingolipid (SL) were
112
purchased from Avanti. The egg-yolk lecithin was extracted from fresh egg-yolks using a
113
modified method described by Palacios and Wang.13 The purity of egg-yolk lecithin was
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checked by TLC. Staphylococcus xylosus lipase was produced as described by Mosbah et al.14
115
Enzyme immobilization was made onto silica aerogel as previously described by Kharrat et
116
al. 15
(verolec
F-62)
was
purchased
from
LASENOR.
EggPC,
cholesterol,
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2.2. Methods
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2.2.1. Esterification reactions
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Ascorbyl lipophilic esters (Asc-C2 to Asc-C18:1) were prepared by direct esterification
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between ascorbic acid (Asc) and various fatty acids in screw-capped flasks. Asc (8 mg/ml)
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was dissolved in 2-methyl-2-propanol to an acetonitrile volume ratio of 0.2 (3mL of total
122
volume). The fatty acid concentration was adjusted to obtain an ascorbic acid/fatty acid molar
123
ratio of eight. The mixture was stirred at 45 °C in an orbital shaker at 220 rpm and in the
124
presence of Staphylococcus xylosus lipase immobilized onto silica aerogel (600 IU). Control
125
reactions in the absence of lipase were also carried out parallelly. Aliquots from the mixture
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reaction were withdrawn after 72 h of incubation and filtered to be used for HPLC analysis.
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The conversion yield of ascorbyl derivatives was calculated as the ratio of the number of
128
moles of ascorbic acid converted per the total number of moles of ascorbic acid. All
129
experiments were performed in triplicate. Study has been selected from five microorganism
130
lipases produced in our laboratory. This enzyme presented the higher yield conversion (data
131
not shown).
132
2.2.2. Purification and identification of ascorbyl esters
133
The reaction mixture resulting from the esterification of ascorbic acid with different
134
fatty acids contains a mixture of ascorbic acid ester and residual substrates. After enzyme
135
removal by centrifugation at 8000 rpm for 15min, the solvent (2-methyl-2-propanol) was
136
evaporated at 45°C under vacuum and the synthesized ester was purified as follows: 100 mg
137
of target product was taken up in 2 mL chloroform. The purification of esters was achieved by
138
chromatography on a silica gel 60 column (Merck) (25 cm × 2 cm) according to Aissa et al.16.
139
The column was previously equilibrated in chloroform. Elution was carried out using
140
chloroform/ methanol/ acetic acid mixtures (78:18:4). The collected solvent fractions were
141
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
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was checked using LC/MS analysis.
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2.2.3. HPLC Analysis
145
The identification and conversion yields of ascorbyl derivatives were carried out by
146
HPLC analysis (Ultimate 3000, Dionex, Germany). The HPLC system was equipped with a
147
pump (LPG-3400SD), column oven and diode-array UV–vis detector (DAD- 3000RS). The
148
output signal of the detector was recorded using Dionex ChromeleonTM chromatography Data
149
System. Separation was executed on an Inertsil ODS-4 C-18 column (5 µm, 4.6 mm×250
150
mm; Shimpack) maintained at 30 °C. The flow rate used was 1.5 mL/min and the detection
151
UV wavelength was set at 254 nm. The used mobile phase was 0.05% acetic acid in water (A)
152
versus 0.1% acetic acid in acetonitrile (B) for a total running time of 12 min and the following
153
proportions of solvent (B) were used for elution: 0-3 min: 10-30% at; 3-5 min: 30-90%; 5-10
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min: 90% and 10-12 min: 90-10%.
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2.2.4. LC-MS/MS Analysis
156
An analytical LC–UV–MS/MS analysis was performed on a Phenomenex Luna C18
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(2) column (150 × 4.6 mm i.d., 5 µm particle size), using a 1 ml/ min linear mobile phase
158
gradient of 20–50% aq. MeOH (containing 1% HOAc) in 30 min, and mass spectra were
159
recorded by a Thermo Scientific ‘LCQ Classic’ ion trap mass spectrometer fitted with an ESI
160
source. Accurate mass measurements were performed on Finnigan MAT900 XLT or Thermo
161
Scientific LTQ Orbitrap XL mass spectrometers in negative and positive ESI mode.
162
2.2.5. NMR and FT-IR experiments
163
1
H and 13C NMR spectra were recorded in deuterated chloroform (CDCl3) on a Bruker
164
TOPSPIN spectrometer operating at 400 MHz. IR spectra were recorded on FT/IR-410
165
(JASCO).
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Asc-C10: 1H NMR (400 MHz, CDCl3): 4.25 (2H (H1’), d); 3.85 (1H (H2’), dd); 4.35 (1H
167
(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
168
(2H (H3), m); 1.3 (nH (H4-H9), m, (CH2)6); 0.9 (3H (H10), t, CH3).
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13C NMR (400 MHz, CDCl3): 14.08 (C10), 22.68 (C3), 24.74- 33.81 (C4-C9), 34.11
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(C2), 64.16 (C1’), 67.47 (C2’), 130.16 (C4’), 130.88 (C5’), 153.05 (C3’), 172.48 (C6’, C=O);
171
178.35 (C1, C=O).
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IR (liquid) cm−1: 3108-3023 (HO-Ø), 2905 (C-H), 1700 (C=O), 1200- 1350 (CH2)
173
stretching, 1100 (C-O).
174
2.2.6. Antioxidant activity
175
The free radical scavenging activity of Asc and its derivatives was determined using
176
the method of Brand-Williams and al. with a little modifications.17 An aliquot of ethanol
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absolute solution (0.1 mL) containing different concentrations (1:2 serial dilutions from the
178
initial sample) of Asc and its esters was added to 3.9 mL of DPPH solution (0.06 mM in
179
ethanol). The mixture was vortexed vigorously and incubated at room temperature (25°C) in
180
darkness for 60 min then the absorbance was read at 517 nm against ethanol blank using a
181
spectrophotometer (Uvi Light XT5).
182
The IC50 values denote the concentration of tested compounds, which is required to
183
scavenge 50% of DPPH free radicals. The corresponding inhibition percentages were
184
calculated according to the following equation:
185 ( Ablank − Asample )
186
Radical scavenging activity (%) =
187
Where, Ablank is the absorbance of the control (prepared in the same manner without test
188
compound), and Asample is the absorbance of the test compound. Ascorbic acid, butylated
189
hydroxytoluene (BHT) and α-tocopherol were used as standard control. The values are
190
presented as the means of triplicate analysis.
Asample
× 100
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Journal of Agricultural and Food Chemistry
2.2.7. Lipophilicity evaluation
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The partition coefficients (miLogP) values and the molecular weight of ascorbyl esters
193
were calculated using Molinspiration software, (available online at www.molinspiration.com).
194
The lipophilic character of ascorbyl esters was evaluated as described by Viskupicova et al.18
195
2.2.8. Measurement of conjugated diene (CD) and triene (CT)
196
The effect of ascorbyl esters on the oxidative state of oil was evaluated by the
197
measuring of conjugated diene (CD) and conjugated triene (CT) as previously described by
198
Della et al.19 Oxidation was induced during 20 days by storing soya oil at 70°C. Every 2 days,
199
the absorbance was measured at 232 nm (for CD) and 270 nm (for CT), using hexane as a
200
blank. All measurements were performed in triplicate. BHT and α-tocopherol were used as
201
standard control.
202
2.2.9. Emulsifying activity
203
The ability of the purified ascorbyl esters to form oil-in-water (o/w) emulsions against
204
soya oil was determined using the method described by Gutierrez et al.20. Activities were
205
compared under neutral (0.1 M phosphate-buffered saline, pH 7), acidic (0.1 M sodium
206
acetate buffer, pH 4) and basic (0.1 M Tris-HCl buffer, pH 8.5) conditions. Five mL of buffer
207
were vortexed for 2 min at 2200 rpm with 0.8 mL of soya oil containing 0.02 % (w/v) of each
208
emulsifier. The turbidity of the lower aqueous layer was measured using a spectrophotometer
209
at 540 nm. All measurements were carried out in triplicates and the mean values of the
210
triplicates were reported with standard error. Egg-yolk lecithin, soya lecithin and Arabic gum
211
were used as commercial emulsifiers.
212
2.2.10. Determination of the minimum inhibitory concentration (MIC) and the minimum
213
bactericidal concentration (MBC)
214
The antibacterial activities of Asc, Asc + FAs (separated substrates taken together at
215
the same concentration) and ascorbyl derivatives were tested against several bacteria strains
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using Luria-Broth (LB) medium. The minimum inhibitory concentration (MIC) values, which
217
correspond to the lowest compound concentration that completely inhibits the growth of
218
microorganisms, were determined by a micro-well dilution method as previously described by
219
Eloff21 using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The
220
inoculum of each bacterium was prepared and the suspensions were adjusted to 107 CFU/mL.
221
All the compounds were dissolved in 100% ethanol, and then dilutions series were prepared
222
in a 96-well plate, ranging from 3.125µg/mL to 4mg/mL. Each well of the microplate
223
contained 175µL of the growth medium, 5µL of inoculum and 20µL of the diluted sample
224
extract. Ethanol was used as a negative control. The plates were incubated at 37°C for 24 h,
225
then 40µL of MTT, at a final concentration of 0.5mg/mL freshly prepared in sterile water,
226
was added to each well and incubated for 30min. The change to purple color indicated that the
227
bacteria were biologically active. The MIC was taken where no change of MTT colour was
228
observed in the well.
229
To determine the minimum bactericidal concentration (MBC), a liquid portion from
230
each well that showed no change in color will be placed on solid LB and incubated at 37°C
231
for 24h. The lowest concentration that yielded no growth after this sub-culturing will be taken
232
as the MBC.22 All experiments were made in duplicate. Several bacterial strains were used:
233
i.e., Bacillus cereus (BC), Bacillus subtilis (BS), Staphyloccocus aureus (Sa), Staphyloccocus
234
epidermidis (SEp), Enterococcus faecalis (EF), Enterococcus faecium (EFa), Enterobacter
235
cloacae (EC), Micrococcus Luteus (ML), Brevibacterium flavum (BF), Pseudomonas
236
Aeruginosa (Ps), Salmonella Typhimurium (STyphi), Klebsielle pneumonia (KP), Echerichia
237
coli (Ecoli) and Staphyloccocus xylosus (Sx).
238 239 240
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2.2.11.2. Leishmanicidal activity
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(These experiments have been performed in the Pasteur Institute of Tunis (Tunisia))
243
L. major (MHOM/TN/95/GLC94)23 and L. infantum (MHOM/TN/94/LV50)24 strains
244
isolated from Tunisian patients were used within this study. Promastigotes were cultured in
245
solid medium at 26°C during 6 days. They must be in their infective metacyclic forms as
246
mentioned by Aissa et al.11. The effects of ascorbyl derivatives on Leishmania promastigotes
247
were evaluated by the MTT assay as described by Dutta et al. 25. Parasites (107parasites/well
248
in the complete medium) were incubated for 24 h in the presence of serially diluted
249
concentrations of ascorbyl derivatives (ranging from 25 to 400µg/ml).11 After this treatment,
250
microtitre plates were centrifuged at 1700g for 10 min and supernatants were removed and
251
replaced with the same volume of 1mg/ml of MTT freshly dissolved in PBS. Plates were then
252
incubated overnight at room temperature. After centrifugation at 2500g, formazan salt formed
253
inside the parasite mitochondries was solubilized by discarding supernatants and adding SDS
254
10% for 2h at 37°C in the dark. Absorbance was measured at 540 nm using an ELISA plate
255
reader. Negative controls were conducted without addition of ascorbyl derivative solutions.
256
Each assay was performed in duplicate.
257
2.2.12. Monolayer study of the ascorbyl derivatives
258
Experiments were carried out on KSV 2200 Baro-stat equipment as described by Aissa
259
et al.11 Teflon trough equipped with two hydrophilic Delrin barriers (symmetric compression)
260
and a Wilhelmy plate as a surface-pressure sensor. Software KSV 2200 was used to control
261
the experiments. Before each utilisation, the Teflon trough was cleaned and brushed in the
262
presence of distilled ethanol, subsequently washed again with tap water, and finally rinsed
263
with double-distilled water. The aqueous subphase (buffer A) was composed of 10 mM Tris–
264
HCl, pH 8, 100 mM NaCl, 21 mM CaCl2, and 1 mM EDTA. Buffer was prepared with
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double-distilled water and filtered through a 0.45 µM Millipore filter. Residual surface active
266
impurities were removed before each assay by sweeping and suction of the surface.26
267
2.2.13. Measurement of the ascorbyl derivatives penetration into mixture film monolayer
268
The surface pressure increase, due to the penetration of the ascorbyl derivatives into a
269
mixture film monolayer, was measured in a cylindrical trough drilled into a Teflon block
270
(surface area 17.42 cm2, total volume 15 mL). The aqueous subphase (buffer A) was stirred
271
continuously at 250 rpm with a magnetic rod. The critical surface pressures were determined
272
as described previously.27 A sample of Asc or synthesized ester solution (0.5 µM) was
273
injected with Hamilton microsyringe, under a monomolecular film of mixture film spreads at
274
an initial surface pressure (πi) ranging from 4 to 37 mN /m. To mimic the lipidic components
275
of leishmania membrane, a mixture of phospholipids was used, containing 40% EggPC, 30%
276
cholesterol, 10% phosphatidylethanolamin (PE), 10% phosphatidylglycerol (PG) and 10%
277
sphingolipid (SL).28
278
2.2.14. Statistical analysis
279
All analysis were carried out in triplicates. Results were expressed as mean values ±
280
standard deviation (SD) (n = 3). The differences were calculated using one-way analysis of
281
variance (ANOVA), and statistically significant differences were reported at P < 0.05. Data
282
analysis was carried out using the SPSS 10.0 software.
283
3. Results and discussion
284
3.1. Enzymatic synthesis and Characterization of Ascorbyl fatty acid Esters
285
A chemoselective enzymatic esterification was used to synthesize lipophilic ascorbyl
286
esters derivatives (Asc-C2 to Asc-C18:1). Several solvents such as hexane, chloroform, acetone
287
and toluene were used as reaction media. However, among all these organic solvents tested,
288
only co-solvent 2-methyl-2-propanol/acetonitrile was suitable for this esterification reaction.
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The conversion yields calculated after 72h of incubation using lipase from
290
staphylococcus xylosus immobilized onto silica aerogel as a catalyst are presented in Table 1.
291
As it can be seen, the highest ester synthesis yield was obtained when using short acyl chains
292
ester Asc-C2. For medium and long chain esters (Asc-C8 to Asc-C18:1), the conversion yield
293
decreased progressively with the increase of the acyl chain length (Table 1) although
294
Staphylococcus xylosus lipase hydrolyses triacylglycerols without significant chain length
295
preference. 14
296
These results are in line with those observed previously by Aissa et al.16. The fatty
297
acid unsaturation seems to affect the synthesis yield. In fact, the ester conversion yield
298
reached 52.86% with ascorbyl palmitate and decreased to 40.96% of ascorbyl oleate when
299
using oleic acid as acyl donor. Our observations are in agreement with those described by
300
Selmi et al.29. They concluded that the increase of the unsaturation number is responsible for
301
the lower rate of triacylglycerols synthesis using immobilized Rhizomucor miehei lipase.
302
These results are however in contradiction with those described by Song et al.30 which
303
showed that C18 unsaturated FAs gave better conversion yield as compared to C18 saturated
304
FAs using Novozym 435 as catalyst. These contradictions between these findings could be
305
attributed to the nature of the lipase used and the composition of the reaction medium
306
(solvent, water content, substrate chemistry) or the operating conditions.31,32
307
3.2. Purification and identification of ascorbyl fatty acid esters
308
The purification of each ascorbyl ester was achieved by chromatography on a silica
309
gel 60 column according to Aissa et al.16 as described above in Materials and Methods. The
310
purity of these products was then checked via HPLC analysis (Fig. 1A); their retention time is
311
mentioned in Table 1. RMN, MS1 and IR analysis were used to identify the purified products.
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312
As shown in Fig. 1B, MS1 analysis in the negative mode of pure Asc-C10, taken as a typical
313
compound, exhibited a molecular ion at m/z = 329.34 [M-H]- attributed to the molecular
314
weight of calculated ester using Molinspiration software (330.37 g/mol) shown in Table 1.
315
The MS2 experiments focusing on the fragment generated from the peak in m/z = 329.34 [M-
316
H]- revealed a fragment corresponding to the pseudo molecular ion at m/z = 171.1 attributed
317
to capric acid and m/z = 175.0 attributed to ascorbic acid ion. The ion at m/z = 157 was
318
formed by the cleavage of the capric acid and neutral losses of H2O linked to the ascorbic acid
319
(Fig. 1C).
320
3.3. Antioxidant activity
321
Ascorbic acid exhibits low solubility and stability in the lipophilic media, which can
322
limit its efficient use in these conditions. Hence, the lipophilic derivatives of Asc and, in
323
particular, long acyl chain esters, would exhibit a better affinity with lipophilic matrices.
324
Being a lipophilic compound may improve its solubility as well as maintain or enhance its
325
radical scavenging capacity.16,32 This property would make ascorbyl derivatives strong
326
candidates for pharmaceutical and cosmetic lipophilic formulation.
327
The antioxidant efficiency of ascorbic acid was checked after lipophilisation using
328
DPPH in an ethanolic medium, a rapid and sensitive scavenging test. As indicated in Table 1,
329
Ascorbic acid and its synthesized esters were shown to be strong DPPH radical scavengers.
330
Esterification of Asc-C2 to Asc-C18:0 seems to slightly affect the radical-scavenging potential.
331
All ascorbyl derivatives exhibited a high antioxidant activity with an IC50 varying between 3
332
and 3.5 µg/mL which are near the IC50 value of ascorbic acid (2.01 µg/mL). Moreover, the
333
antioxidant effect of ascorbic acid and its derivatives was twice as efficient as the known
334
synthetic antioxidants such as BHT and α-tocopherol. These results are, however, in
335
contradiction with those described by Burham et al.33 who showed that ascorbic acid
336
exhibited a higher antioxidant activity than ascorbyl palmitate and pure palm-based ascorbyl
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Journal of Agricultural and Food Chemistry
337
esters. The radical-scavenging capacity of ascorbic acid and its derivatives can be explained
338
by the number and position of hydrogen-donating groups (OH) in hydroxyl head group.32
339
3.4. Ascorbyl ester derivatives effect on oil oxidative stability
340
The effect of lipophilic ascorbyl derivatives on the oxidative stability of oils was
341
determined by the measurement of conjugated diene (CD) and triene (CT). Ascorbic acid and
342
its acyl esters (Asc-C2 to Asc-C18:1) were added to refined soy oil. CD and CT of the different
343
preparations were measured upon 20 days storage at 70°C in the open air (Figs. 2A and B).
344
The CD and CT determine the primary products of oil oxidation.34 A remarkable increase in
345
the CD and CT of refined soya oil was observed. As shown in Figs. 2A and B, the addition of
346
ascorbyl derivatives, as antioxidants, prevented oil oxidation as well as BHT and α-
347
tocopherol. Interestingly, the protective effect against oxidation by ascorbyl derivatives was
348
higher than that of Asc and increased with the fatty acid chain length. However, Asc, Asc-C2
349
and Asc-C8 derivatives displayed the lowest protective effect of soya oil against oxidation.
350
Based on the increasing trend of miLog P values (Table 1), it seems that Ascorbyl esters
351
having a high lipophilicity (miLog P higher than 3.2) display the best antioxidant capacities.
352
Ascorbyl derivatives, the amphiphilic natural antioxidants, could be used for increasing
353
protection of target-lipids from oxidation such as skin creams, facial treatments (acne or
354
wrinkle removers), bath oils, sunscreens, pre-shave and make-up.
355
3.5. Emulsifying activity
356
Today, more than ever, a large amount of surfactant is required for a microemulsion
357
system used for pharmaceutical/cosmetic purposes.35 However, a surfactant may exhibit
358
undesirable effects like skin irritation, allergy, etc. when used excessively. Therefore, one
359
must choose materials that are biocompatible, non-toxic, clinically acceptable, and use
360
emulsifiers in an appropriate concentration range that will result in mild and non-aggressive
361
microemulsions. 35 The excipients generally regarded as safe (GRAS) are being increasingly
15 ACS Paragon Plus Environment
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362
preferred. For these reasons, the ability of purified ascorbic acid derivatives to emulsify soya
363
oil in water was tested. The emulsifying capacity of the different ascorbyl derivatives was
364
determined under acidic, neutral and basic conditions. Arabic gum, soya lecithin and egg-
365
yolk-lecithin were taken as control emulsifiers since they are the most important hydrocolloid
366
ingredients in the formulation of many food products. Based on average results provided in
367
acidic, neutral and basic pH conditions presented in Fig.3, the ascorbyl derivatives produced
368
emulsifying activities that were significantly different from those of control. In fact, apart
369
from Asc and Asc-C2, all ascorbyl derivatives (Asc-C8 to Asc-C18:1) produced a significantly
370
higher emulsifing activity than arabic gum and soya lecithin, but lower than egg-yolk-lecithin.
371
Comparable results were obtained by Kuwabara et al. 36 who demonstrated that caproyl and
372
lauroyl l-ascorbates are useful emulsifiers for emulsions. In addition, under acidic conditions,
373
the emulsifying activities of all ascorbyl esters were significantly higher compared to those
374
measured under basic or neutral conditions (Fig.3). Among all the tested derivatives, Asc-
375
C18:1 was found to be the most potent as emulsifier.
376
3.6. Determination of the minimum inhibitory concentration (MIC) and the minimum
377
bactericidal concentration (MBC)
378
The antibacterial activity of Asc, FAs, Asc + FAs and the synthesized esters (Asc-C2
379
to Asc-C18:1) was checked against Gram-positive (Bacillus cereus (BC), Bacillus subtilis (BS),
380
Brevibacterium flavum (BF), Micrococcus Luteus (ML), Staphyloccocus xylosus (Sx),
381
Staphyloccocus aureus (Sa), Staphyloccocus epidermidis (SEp), Enterococcus faecalis (EF)
382
and Enterococcus faecium (EFa)) and Gram-negative (Enterobacter cloacae (EC), Pseudomonas
383
Aeruginosa (Ps), Salmonella Typhimurium (STyphi), Klebsielle pneumonia (KP) and Echerichia
384
coli (Ecoli)) bacteria by the determination of MIC and MBC values. As can be seen from
385
Tables 2A and B, ascorbic acid does not show any inhibitory or bactericidal effect up to a
386
concentration of 4 mg/mL. Compared to Asc and mixed solution of Asc + FAs, synthesized
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Journal of Agricultural and Food Chemistry
387
ester exhibits the most effect against the bacteria tested, in particular Gram (+) ones. In fact,
388
with Gram+ bacteria, MIC values ranged from 0.0625 to 2 mg/mL and the MBC values are
389
between 0.500 and 4 mg/mL, while Gram (-) bacteria appear to be less sensitive to the tested
390
compounds. Previous studies reported that Gram (-) bacteria presented lower sensitivity than
391
Gram (+) bacteria to various polyphenols.37 This higher resistance may be related to minor
392
differences present in the outer membrane in the cell wall composition. 37 As it can be seen
393
from Table 2A, MIC and MBC values showed that ascorbyl derivatives exhibit a similar or a
394
slightly higher inhibitory activity and more bactericidal effect against Gram (+) bacteria than
395
the separated substrates (Asc + FAs). These results indicate that we have significantly
396
enhanced the antimicrobial activity of the ascorbic acid after esterification. These findings are
397
in agreement with those of Aissa et al.11 who showed that only medium chain tyrosyl esters
398
had an antibacterial activity especially against Staphylococcus strains.
399
These results could be explained by their hydrophobicity which may allow them to
400
partition the lipids of the bacterial cell membrane, making them more permeable and leading
401
to ions leakage and other cell constituents.38 The authors suggested that these compounds
402
could be able to infiltrate the cell and interact with some metabolic mechanisms, which
403
allowed us to show their bacteriostatic effect.
404
3.7. Antileishmanial activity
405
Ascorbic acid and its lipophilic derivatives were screened for their leishmanicidal 23
406
activity. Screening was carried out using two Leishmania species: L. major GLC94
and L.
407
infantum LV50.24 As shown in Table 3, only Asc-C10, Asc-C12 and Asc-C18:1 ascorbyl
408
derivatives were effective against both Leishmania species, while either ascorbic acid or Asc-
409
C2, Asc-C8, Asc-C16 and Asc-C18:0 derivatives had no leishmanicidal activity up to 400
410
µg/mL. Interestingly, the three effective derivatives showed a higher activity against L. major
411
promastigotes compared to that obtained against L. infantum promastigote. Indeed, IC50
17 ACS Paragon Plus Environment
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412
values were approximately two times higher against the former than the latter. This indicates
413
that L. major parasites are more sensitive to these compounds than L. infantum ones. Asc-
414
C18:1, the most effective derivative, showed an IC50 of 154.23 µg/mL and 205.81 µg/mL
415
against L. major and L. infantum, respectively. Also, Asc-C12 showed a moderate activity of
416
196.262 µg/mL and 284.8 µg/mL against L. major and L. infantum, respectively. Finally,
417
Asc-C10 was the less active compound of the three derivatives which showed an IC50 of
418
217.74 µg/mL and 347.12 µg/mL against the dermotropic and the visceraotropic strain,
419
respectively. These results are in line with those observed previously by Aissa et al.11 who
420
showed that the medium chain tyrosyl esters had an antileishmanial effect. However, for our
421
case, the most effective derivative was ascorbyl oleate (Asc-C18:1). These results could be
422
attributed to the nature of the hydroxyl head group of ascorbic acid and besides their
423
amphiphilicity degree.
424
3.8. Interactions of ascorbyl derivatives with mixture film monolayer
425
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
427
compared using a mixture film monolayer, containing the principal components of leishmania
428
membranes: 40% EggPC, 30% cholesterol, 10% phosphatidylethanolamin (PE), 10%
429
phosphatidylglycerol (PG) and 10% sphingolipid (SL).28
430
The maximum surface pressure increase was determined at different initial pressures
431
(πi) of mixture film ranging from 5 to 40 mN /m (Fig. 4). The critical surface pressure (πc) for
432
each ester was estimated by the linear extrapolation of the experimental curves at zero surface
433
pressure increase. A critical pressure for penetration (πc) may thus be defined; it corresponds
434
to the extrapolated value of initial beyond which there is no increase in surface pressure. The
435
value of the maximum surface pressure increase (∆πmax) reached at equilibrium (around 40
436
min after the injection of the ascorbyl derivative in the aqueous subphase) was determined 18 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
437
and plotted as a function of πi (Fig. 4). For all ascorbyl derivatives, a general trend was
438
observed: higher was πi, lower was the interaction of the ascorbyl derivatives with the
439
monolayer because of the higher packing of phospholipids. Asc, Asc-C2, Asc-C8 cannot
440
interact with the mixture film (data not shown). It can be seen from Table 4, that all ascorbyl
441
esters (Asc-C10 to Asc-C18:1) 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
36.96 mN /m to 50.74 mN /m (Table 4). Mottola et al.39 showed that the Asc-C16 is an
444
anionic amphiphilic molecule which allowed them to penetrate phospholipid monolayers.
445
These results are in line with those observed previously by Aissa et al.16 who showed that the
446
medium and long chain tyrosyl esters could interact with biological membrane. We can also
447
notice that unsaturation in the acyl chain (Asc-C18:1) seems to strengthen its interaction of
448
with the mixture film. In fact, the critical surface pressure of Asc-C18:1 and Asc-C18:0 are
449
50.74 mN /m and 41.30 mN /m, respectively (Table 4). Makyla and Paluch40 demonstrated
450
that the presence of the unsaturated fatty acid in cholesterol/DPPC mixed monolayer makes
451
the membrane more fluid. Asc-C16:0 and Asc-C18:0 showed an interaction with mimetic
452
membrane model but not an antileishmanial activity. This result could be explained by the
453
self-aggregation and the reduced mobility of long saturated ascorbyl derivatives in the
454
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
ascorbyl esters to penetrate mixture films monolayers, we can tentatively conclude that the
457
medium and long chain ascorbyl esters (Asc-C10 to Asc-C18:1) could interact efficiently with a
458
biological membrane, characterized by a surface pressure between 25 and 35 mN /m.42
459
In conclusion, from all the tested derivatives, only medium and unsaturated ascorbyl
460
derivatives (Asc-C10, Asc-C12 and Asc-C18:1) exhibited a potent antimicrobial and
461
antileishmanial activities. These amphiphilic antioxidants could be used as membrane19 ACS Paragon Plus Environment
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462
perturbing surfactants in clinical applications such as for the dermatological and
463
pharmacological uses.
464
Competing interests
465
The authors declare that they have no competing interests.
466
Authors' contributions
467
NK carried out all the studies, analyzed the data and drafted the manuscript. IA helped with
468
the discussion of the data and the correction of the manuscript. RMS carried out the
469
antileishmanial activity. MB helped with the NMR, IR and LC-MS analysis. MS and DL
470
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.
472
Acknowledgements
473
This work represents a part of the thesis of Mrs Nadia Kharrat. It received financial
474
support from the Ministry of Higher Education and Scientific Research in Tunisia. The
475
authors would like to thank Pr. Sofiane Bezzine (ENIS, Sfax-Tunisia) for his generous gift of
476
bacterial strains. We are grateful to Pr. Verger Robert and Pr. Pierre Villeneuve for their
477
fruitful discussion. We are grateful to Pr. Bejaoui Hafedh (FSS, Sfax-Tunisia) for his help
478
with English. Parasite experiments were partially supported by NIH/NIAID/DMID Grant
479
Number 5P50AI074178 to LTCII.
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480
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37. Taguri, T.; Tanaka, T.; Kouno, I. Antimicrobial activity of 10 different plant polyphenols against bacteria causing food-borne disease. Biol. Pharm. Bull. 2004, 27, 1965.
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39. Mottola, M.; Wilke, N.; Benedini, L.; Oliveira, R.G.; Fanani, M.L. Ascorbyl palmitate
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40. Makyla, K.; Paluch, M. The linoleic acid influence on molecular interactions in the model of biological membrane. Colloid surface B. 2009, 71, 59-66.
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41. Laguerre, M.; Bayrasy, C.; Lecomte, J.; Chabi, B.; Decker, E.A.; Wrutniak-Cabello, C.;
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42. Sanchez-Martin, M.J.; Haro, I.; Alsina, M.A.; Busquets, M.A.; Pujol, M. A Langmuir
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592
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593
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594
Figure Captions:
595
Fig.1. (A) HPLC profile of ascorbyl caprate (Asc-C10) after biosynthesis and purification. The
596
biosynthesis reaction of ascorbic acid with capric acid ( ascorbic acid/fatty acid molar ratio of
597
eight) was catalyzed by Staphylococcus xylosus lipase immobilized onto silica aerogel at
598
45°C and 220 rpm in 2-methyl-2-propanol to acetonitrile volume ratio of 0.2 (72 h). HPLC
599
analysis was carried out on a C-18 column by gradient elution using acidified water and
600
acetonitrile at a flow rate of 1.5 mL/min.
601
(B) MS1 spectra of purified ascorbyl caprate (Asc-C10).
602
(C) MS2 spectra of purified ascorbyl caprate (Asc-C10).
603
Fig.2. Antioxidant activities of Ascorbyl ester derivatives (Asc-C2 to Asc-C18:1) tested by the
604
measurement of conjugated diene (A) and conjugated triene (B) of refined soy oil during
605
storage at 70°C. For each test, negative and positive controls using BHT and α- Tocopherol
606
were run in parallel. Values are mean ± SD (n = 3) of three determinations.
607
Fig.3. Emulsifying activity at pH 4 (
608
derivatives (Asc-C2 to Asc-C18:1). The activity reflects the capacity of the emulsifier to form
609
soya oil-in-water emulsions. Egg-yolk-lecithin, Arabic Gum and Soya lecithin were used as
610
control emulsifiers. Values are mean ± SD (n = 3) of three determinations.
611
Fig.4. Maximal increase in surface pressure after ascorbyl derivatives injection with respect to
612
the initial surface pressure of mixture films (40% PC, 30% cholesterol, 10% SL, 10% PG,
613
10% PE) spread in a cylindrical Teflon trough (volume, 15 mL; surface, 17.42 cm2). Final
614
ascorbyl derivatives concentration, 0.5 µM. Buffer, 10 mM Tris–HCl, pH 8.0, 100 mM NaCl,
615
21 mM CaCl2, and 1 mM EDTA.
); pH 7 (
) and pH 8.5 (
) of ascorbic acid and its
26 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
Table 1 The conversion yields of the ascorbyl derivatives and physico-chemical parameters of ascorbic acid and its esters related to their lipophilicity. Compounds Ascorbic acid Ascorbyl acetate Ascorbyl caprylate Ascorbyl caprate Ascorbyl laurate Ascorbyl palmitate Ascorbyl stearate Ascorbyl oleate
α-tocopherol BHT
Abbreviations
Radicals
Asc Asc-C2 Asc-C8 Asc-C10 Asc-C12 Asc-C16 Asc-C18:0 Asc-C18:1 -
C6H8O6 -CH3 -C7H15 -C9H19 -C11H23 -C15H31O -C17H35O -C17H33O
C29H50O2 C15H24O
Conversion yield (%) 82.57 ± 2.32 68.21 ± 2.75 59.39 ± 2.30 58.24 ± 2.72 52.86 ± 2.71 42.08 ± 1.62 40.96 ± 1.07 -
Characteristics White solid Yellow slimy oil Yellow slimy oil White amorphous solid White amorphous solid White amorphous solid White amorphous solid Yellow oil Yellow viscous liquid White solid
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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MW (g/mol)a 176.124 218.161 302.323 330.377 358.431 414.539 442.593 440.577 430.717 220.356
miLog Pa - 1.402 - 0.698 2.243 3.253 4.263 6.284 7.295 6.809 9.043 5.435
Rt (min)b 1.990 3.540 6.627 7.807 8.372 9.537 11.392 9.743 -
IC 50 (µg/mL)c 2.010 ± 0.085 3.435 ± 0.091 3.515 ± 0.084 3.215 ± 0.078 3.355 ± 0.092 3.205 ± 0.088 3.310 ± 0.099 2.980 ± 0.085 5.760 ± 0.028 7.265 ± 0.064
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Table 2 Minimum inhibitory concentration and Minimum Bactericidal concentration of Ascorbyl ester derivatives on bacteria.
A
Bacteria strain
Bacillus cereus Bacillus subtilis Staphylococcus aureus Staphylococcus xylosus Staphylococcus epidermidis Enterobacter cloacae Salmonella Typhimurium Klebsielle pneumoniae Escherchia coli
B
Bacteria strain
Bacillus cereus Bacillus subtilis Staphylococcus aureus Staphylococcus xylosus Staphylococcus epidermidis Enterobacter cloacae Salmonella Typhimurium Klebsielle pneumoniae Escherchia coli
Gram Asc >4 >4 >4 >4 >4 >4 >4 >4 >4
+ + + + + -
(Asc + C8)a 2 2 2 2 2 2 2 2 4
MIC (mg/ml) (Asc + C10)a Asc-C10 (Asc + C12)a 2 0.5 1 2 0.25 1 2 0.25 2 2 0.25 2 2 0.25 2 2 0.5 2 2 0.25 2 2 0.5 2 4 1 2
Asc-C8 1 1 0.5 0.5 0.5 1 1 1 2
Gram Asc
(Asc + C8)a Asc-C8 (Asc + C10)a
MBC (mg/ml) Asc-C10 (Asc + C12)a
Asc-C12 (Asc + C18:1)a 0.125 2 0.125 1 0.062 1 0.062 1 0.062 1 0.25 2 0.25 2 0.125 2 0.5 2
Asc-C12
(Asc + C18:1)a
Asc-C18:1 1 0.5 0.25 0.25 0.25 0.5 0.5 1 0.5
Asc-C18:1
+
>4
4
2
4
0.5
2
0.25
4
2
+
>4
4
2
4
0.5
2
0.25
4
2
+
>4
2
1
2
0.25
1
0.125
2
0.5
+
>4
2
1
2
0.25
1
0.125
2
0.5
+ -
>4 >4
2 4
1 2
4 4
0.5 0.5
1 2
0.125 0.5
2 >4
0.5 1
-
>4 >4
4 4
2 2
4 4
0.5 1
4 4
0.25 0.25
>4 >4
1 2
-
>4
4
2
4
1
4
0.5
>4
2
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 Asc Asc-C2 Asc-C8 Asc-C10 Asc-C12 Asc-C16 Asc-C18:0 Asc-C18:1 IC50 (µg/mL) a a a 217.74 ± 13.33 196.26 ± 10.53 a a 154.23 ± 12.41 L. major IC50 (µg/mL) a a a 347.12 ± 12.32 284.80 ± 20.33 a a 205.81± 14.35 L. infantum a : Without effect up to 400 µg/ml.
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Journal of Agricultural and Food Chemistry
Table 4 Critical surface pressure (πc) of the ascorbic acid and ascorbyl acyl esters. Compounds
Asc Asc-C2 Asc-C8 Asc- C10 Asc- C12 Asc-C16 Asc-C18 :0 Asc-C18 :1
πc (Mixture film) (mN /m)
0 0 0 36.96 42.07 50.06 41.30 50.74
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Fig.1. A
Relative absorbance (%)
O 6'
O
O 9
5
7
3
1' 1
10
8
6
2
4
O
5'
OH
3' 2'
(9') 4'
OH OH
(8')
(7')
Asc-C10
157.0
Relative absorbance (%)
B
[M-H]-
MS1
MS2
C
171.1
175.0
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Fig.2.
70 60 Control BHT α- Tocopherol Asc Asc-C2 Asc-C8 Asc-C10 Asc-C12 Asc-C16 Asc-C18:0 Asc-C18:1
K232 (10-3)
50 40 30 20 10 0 0
2
4
6
8
10
12
14
16
18
20
22
Time (Days) 10 9 8
Control BHT α- Tocopherol Asc Asc-C2 Asc-C8 Asc-C10 Asc-C12 Asc-C16 Asc-C18:0 Asc-C18:1
K270 (10-3 )
7 6 5 4 3 2 1 0 0
2
4
6
8
10
12
14
16
18
Time (Days)
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20
22
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Journal of Agricultural and Food Chemistry
Fig.3.
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Fig.4.
Surface pressure increase (mN /m)
25
Mixture Film/Water 20 Asc-C10 15
Asc-C12 Asc-C16 Asc-C18:0
10
Asc-C18:1 5
0 0
10
20
30
40
50
60
Initial surface pressure (mN /m)
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TOC Graphic :
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