Lipophilization of Ascorbic Acid - American Chemical Society

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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

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Authors list: Nadia Kharrata, Imen Aissaa, Manel Sghaierb, Mohamed Bouazizc, Mohamed

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Sellamia, Dhafer Laouinib and Youssef Gargouria*

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Affiliation List:

6

a

7

BPW 1173- 3038 Sfax-Tunisie.

8

b

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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

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BP 74 ,1002 Tunis-Belvédère, Tunisie.

11

c

12

Université de Sfax- 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,

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Route de Soukra, 1173 Sfax-Tunisie.

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Tel/ Fax: + 21674675055,

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E-mail : [email protected]

Laboratoire d’Electrochimie et Environnement, ENIS, Route de Soukra, BPW 1173- 3038,

18 19 20 21 22 23 1 ACS Paragon Plus Environment


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Abstract

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Ascorbyl lipophilic derivatives (Asc-C2 to Asc-C18:1), were synthesized in a good

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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,

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ascorbyl derivatives were evaluated, for the first time, for their antileishmanial effects against

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visceral (Leishmania infantum) and cutaneous parasites (Leishmania major). Among all the

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tested compounds, only Asc-C10, Asc-C12 and Asc-C18:1 exhibited antileishmanial activities.

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The interaction of ascorbyl esters with a phospholipid monolayer showed that only medium

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and unsaturated long chain (Asc-C10 to Asc-C18:1) derivative esters were found to interact

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efficiently with mimetic membrane of leishmania. These properties would make ascorbyl

37

derivatives good candidates to be used in cosmetic and pharmaceutical lipophilic

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formulations.

39 40

Keywords:

Ascorbic

acid;

lipophilic

derivatives;

antimicrobial

activity;

leishmanicidal activity; phospholipid monolayers

41 42 43

2 ACS Paragon Plus Environment

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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

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compounds such as aldehydes, peroxides, ketones and oxyacids.1 These compounds cause a

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decrease in food nutritive value and sensorial quality. To prevent food oxidation and extend

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its shelf life, they should be stored under proper conditions, e.g. at low temperature in an inert

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atmosphere and suitable packaging. In this sense, antioxidants are widely recommended as

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additives which have the potential to control the oxidation process, thereby stabilizing fat-

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containing food-stuffs.2 Synthetic antioxidants such as butylated hydroxyanisole (BHA),

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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

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antioxidants in food materials is a matter of concern related to the formation of toxic

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compounds. These substances can show possible side-effects such as carcinogenicity in living

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organisms.3 Lanigan and Yamarik3 showed that only 50 % of BHT can be eliminated by urine

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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

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their implication as prophylactic and therapeutic agents in many diseases and for their better

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food preservation. Currently, ascorbic acid is a labile molecule and is well-known by virtue of

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its reducing property. Moreover, it can prevent chronic diseases due to oxidative stress,

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including cancer, hypertension, cardiovascular disease and stroke.4 Ascorbic acid, which

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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

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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

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and crystalline powder. Unfortunately, it is unstable when exposed to air, light, heat and is

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rapidly oxidized and irreversibly decomposed upon entering the body, thus exhibiting low

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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

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membrane constituents. They can be easily absorbed and retained into the lipid domain of

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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

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been emphasized in several works.6,7 Amphiphilic ascorbyl derivatives display particularly

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interesting characteristics resulting from the modification of molecular flexibility. Hence,

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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

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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

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not have any effect on various pathogenic bacteria. However, tyrosyl esters and especially

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medium chain tyrosyl derivatives exhibited an antimicrobial activity against staphylococcus

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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

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our laboratory as catalyst and evaluated for their antioxidant, emulsifying and antimicrobial

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activity against several pathogenic strains and their antileishmanial effects on both cutaneous

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(Leishmania major) and visceral parasites (Leishmania infantum). Finally, the interaction of

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the ester derivatives with a mimetic phospholipid film mixture was evaluated using

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monomolecular film technique.

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2. Materials and Methods

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2.1. Materials

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Ascorbic acid was purchased from Bio Basic Inc (Switzerland); chloroform and

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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-

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diphenyl-1-picrylhydrazyle (DPPH) were purchased from Fluka (Suisse); BHT (purity ≥

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99%), α-tocopherol (purity ≥ 96%) and Arabic Gum (purity 99%) from Sigma and soya

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lecithin

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phosphatidylethanolamin (PE), phosphatidylglycerol (PG) and sphingolipid (SL) were

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purchased from Avanti. The egg-yolk lecithin was extracted from fresh egg-yolks using a

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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

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Enzyme immobilization was made onto silica aerogel as previously described by Kharrat et

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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

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volume). The fatty acid concentration was adjusted to obtain an ascorbic acid/fatty acid molar

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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

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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

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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

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lipases produced in our laboratory. This enzyme presented the higher yield conversion (data

131

not shown).

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2.2.2. Purification and identification of ascorbyl esters

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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

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evaporated at 45°C under vacuum and the synthesized ester was purified as follows: 100 mg

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of target product was taken up in 2 mL chloroform. The purification of esters was achieved by

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chromatography on a silica gel 60 column (Merck) (25 cm × 2 cm) according to Aissa et al.16.

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The column was previously equilibrated in chloroform. Elution was carried out using

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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

143

was checked using LC/MS analysis.

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2.2.3. HPLC Analysis

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The identification and conversion yields of ascorbyl derivatives were carried out by

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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

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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

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mm; Shimpack) maintained at 30 °C. The flow rate used was 1.5 mL/min and the detection

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UV wavelength was set at 254 nm. The used mobile phase was 0.05% acetic acid in water (A)

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versus 0.1% acetic acid in acetonitrile (B) for a total running time of 12 min and the following

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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

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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

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gradient of 20–50% aq. MeOH (containing 1% HOAc) in 30 min, and mass spectra were

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recorded by a Thermo Scientific ‘LCQ Classic’ ion trap mass spectrometer fitted with an ESI

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source. Accurate mass measurements were performed on Finnigan MAT900 XLT or Thermo

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Scientific LTQ Orbitrap XL mass spectrometers in negative and positive ESI mode.

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2.2.5. NMR and FT-IR experiments

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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

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(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

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(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);

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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)

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stretching, 1100 (C-O).

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2.2.6. Antioxidant activity

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The free radical scavenging activity of Asc and its derivatives was determined using

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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

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initial sample) of Asc and its esters was added to 3.9 mL of DPPH solution (0.06 mM in

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ethanol). The mixture was vortexed vigorously and incubated at room temperature (25°C) in

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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 )

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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

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presented as the means of triplicate analysis.

Asample

× 100


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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

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were calculated using Molinspiration software, (available online at www.molinspiration.com).

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The lipophilic character of ascorbyl esters was evaluated as described by Viskupicova et al.18

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2.2.8. Measurement of conjugated diene (CD) and triene (CT)

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The effect of ascorbyl esters on the oxidative state of oil was evaluated by the

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measuring of conjugated diene (CD) and conjugated triene (CT) as previously described by

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Della et al.19 Oxidation was induced during 20 days by storing soya oil at 70°C. Every 2 days,

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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

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standard control.

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2.2.9. Emulsifying activity

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The ability of the purified ascorbyl esters to form oil-in-water (o/w) emulsions against

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soya oil was determined using the method described by Gutierrez et al.20. Activities were

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compared under neutral (0.1 M phosphate-buffered saline, pH 7), acidic (0.1 M sodium

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acetate buffer, pH 4) and basic (0.1 M Tris-HCl buffer, pH 8.5) conditions. Five mL of buffer

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were vortexed for 2 min at 2200 rpm with 0.8 mL of soya oil containing 0.02 % (w/v) of each

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emulsifier. The turbidity of the lower aqueous layer was measured using a spectrophotometer

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at 540 nm. All measurements were carried out in triplicates and the mean values of the

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triplicates were reported with standard error. Egg-yolk lecithin, soya lecithin and Arabic gum

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were used as commercial emulsifiers.

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2.2.10. Determination of the minimum inhibitory concentration (MIC) and the minimum

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bactericidal concentration (MBC)

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The antibacterial activities of Asc, Asc + FAs (separated substrates taken together at

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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

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correspond to the lowest compound concentration that completely inhibits the growth of

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microorganisms, were determined by a micro-well dilution method as previously described by

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Eloff21 using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The

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inoculum of each bacterium was prepared and the suspensions were adjusted to 107 CFU/mL.

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All the compounds were dissolved in 100% ethanol, and then dilutions series were prepared

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in a 96-well plate, ranging from 3.125µg/mL to 4mg/mL. Each well of the microplate

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contained 175µL of the growth medium, 5µL of inoculum and 20µL of the diluted sample

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extract. Ethanol was used as a negative control. The plates were incubated at 37°C for 24 h,

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then 40µL of MTT, at a final concentration of 0.5mg/mL freshly prepared in sterile water,

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was added to each well and incubated for 30min. The change to purple color indicated that the

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bacteria were biologically active. The MIC was taken where no change of MTT colour was

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observed in the well.

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To determine the minimum bactericidal concentration (MBC), a liquid portion from

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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

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as the MBC.22 All experiments were made in duplicate. Several bacterial strains were used:

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i.e., Bacillus cereus (BC), Bacillus subtilis (BS), Staphyloccocus aureus (Sa), Staphyloccocus

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epidermidis (SEp), Enterococcus faecalis (EF), Enterococcus faecium (EFa), Enterobacter

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cloacae (EC), Micrococcus Luteus (ML), Brevibacterium flavum (BF), Pseudomonas

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Aeruginosa (Ps), Salmonella Typhimurium (STyphi), Klebsielle pneumonia (KP), Echerichia

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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))

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L. major (MHOM/TN/95/GLC94)23 and L. infantum (MHOM/TN/94/LV50)24 strains

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isolated from Tunisian patients were used within this study. Promastigotes were cultured in

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solid medium at 26°C during 6 days. They must be in their infective metacyclic forms as

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mentioned by Aissa et al.11. The effects of ascorbyl derivatives on Leishmania promastigotes

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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,

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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

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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

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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.

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Each assay was performed in duplicate.

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2.2.12. Monolayer study of the ascorbyl derivatives

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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

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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

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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

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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

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2.2.14. Statistical analysis

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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.

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3. Results and discussion

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3.1. Enzymatic synthesis and Characterization of Ascorbyl fatty acid Esters

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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


Page 15 of 35


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



Page 16 of 35

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


Page 17 of 35


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



Page 18 of 35

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

Page 19 of 35


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


Page 20 of 35

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.


Page 21 of 35


480

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Candida antarctica lipase B in ionic liquids and water activity control by salt hydrates.

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free medium. Enz Microb Technol. 1998, 23, 182-186.

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Am. Oil Chem. Soc. 2010, 87, 615-620.

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32. Laguerre, M.; López Giraldo, L.J.; Lecomte, J.; Figueroa-Espinoza, M.C.; Baréa, B.;

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Weiss, J.; Decker, E.A.; Villeneuve, P. Chain length affects antioxidant properties of

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chlorogenate esters in emulsion: the cutoff theory behind the polar paradox. J. Agric. Food

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33. Burham, H.; Abdul Rasheed, R.; Ainaa, G.; Noorullhamezon, M.N.; Suzaini, B.; Sidek, H.

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35. Azeem, A.; Rizwan, M.; Farhan, J.A.; Khan Zeenat, I.; Khar Roop, K.; Aqil, M.;

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Talegaonkar, S. Emerging Role of Microemulsions in Cosmetics. Recent Pat Drug Deliv

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Formul. 2008, 2, 275-289.

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36. Kuwabara, K.O.; Watanabe, Y.; Adachi, S.; Nakanishi, K.; Matsuno, R. Emulsifier

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properties of saturated acyl l-ascorbates for preparation of O/W emulsions. Food Chem.

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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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38. Ultee, A.; Bennik, M.H.J.; Moezelaar, R. The phenolic hydroxyl group of carvacrol is

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essential for action against the food-borne pathogen Bacillus cereus. Appl. Environ.

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Microbiol. 2002, 68, 1561-1568.

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39. Mottola, M.; Wilke, N.; Benedini, L.; Oliveira, R.G.; Fanani, M.L. Ascorbyl palmitate

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interaction with phospholipid monolayers: electrostatic and rheological preponderancy

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Biochim Biophys Acta. 2013, 1828, 2496-2505.

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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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monolayer study of the interaction of E1 (145–162) Hepatitis G virus peptide with

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593



Page 26 of 35

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


Page 27 of 35


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.

27

ACS Paragon Plus Environment

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


Page 28 of 35

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.

28


Page 29 of 35


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.

29



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


Page 30 of 35

Page 31 of 35


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



Page 32 of 35

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)


20

22

Page 33 of 35


Fig.3.



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)


Page 34 of 35

Page 35 of 35


TOC Graphic :


Lipophilization of Ascorbic Acid - American Chemical Society

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