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Synthesis and Surface Active Properties of Sodium N-Acyl Phenylalanines and their Cytotoxicity Madhumanchi Sreenu, R.B.N. Prasad, Pombala Sujitha, and C.Ganesh Kumar Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/ie503764v • Publication Date (Web): 30 Jan 2015 Downloaded from http://pubs.acs.org on February 3, 2015

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Industrial & Engineering Chemistry Research

Synthesis and Surface Active Properties of Sodium N-Acyl Phenylalanines and their Cytotoxicity Madhumanchi Sreenu,a Rachapudi Badari Narayana Prasad*,a Pombala Sujitha,b and Chityal Ganesh Kumarb a

Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology (CSIR-

IICT), Hyderabad 500007, India b

Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of

Chemical Technology (CSIR-IICT), Hyderabad 500 007, India

*Correspondence to: Rachapudi Badari Narayana Prasad, Telephone: +91-4027193179, Fax: +91-40-27193370; E-mail: [email protected]

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Abstract: Sodium N-acyl phenylalanines (NaNAPhe) were synthesized using mixture of fatty acids obtained from coconut, palm, karanja, Sterculia foetida and high oleic sunflower oils via Schotten-Baumann reaction in 60-78% yields to see the influence of hydrophobic group of fatty acyl group functionality with head group phenylalanine on their surface active properties. The products were characterized by chromatographic (TLC, column, GC) and spectral techniques (IR, NMR, Mass). The synthesized products were evaluated for their surface active properties such as surface tension, wetting power, foaming characteristics, emulsion stability, calcium tolerance, critical micelle concentration (CMC) and thermodynamic properties. The results showed that all the products exhibited superior surface active properties like critical micelle concentration (CMC, 0.018– 0.00041 mmol/L), calcium tolerance (26.5-65.8 ppm) and emulsion stability (262844 s) compared to reference sodium lauryl sulphate (SLS). All the sodium Nacyl phenylalanines except sodium N-coconut fatty acyl phenylalanines exhibited promising cytotoxicity against human cancer cell lines. These new vegetable oilbased surfactants have potential in personal care, pharmaceutical and industrial applications.

Keywords: Cytotoxicity, mixture of fatty acids, phenylalanine, surface active properties, emulsion stability.

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1. INTRODUCTION Nowadays, the eco-friendly surfactants obtained from the renewable molecules of oils and fats, carbohydrates and proteins have attracted tremendous attention due to their high biodegradability, low toxicity compared to synthetic petroleum-based surfactants.1

Amino acid-based surfactants were

prepared by the reaction of acylation, esterification, amidification and alkylation of the amino acids/peptides with individual fatty alkyl or mixture of alkyl substrates.2,3

Infante et al.4-7 reported the synthesis of amino acid/peptide

surfactants employing chemo-enzymatic methods and characterized their surface active

properties,

self-assembly

properties,

biodegradability,

cytotoxicity,

hemolytic activity, antimicrobial properties etc. Mhaskar et al.8-10 reported the preparation of the amino acid surfactants using pure fatty alkyl substrates and pure amino acids and were characterized for their surface active properties like calcium tolerance, wetting power, foaming power, emulsion stability, surface tension, CMC and detergency and also antibacterial properties. Xia et al.11,12 studied the synthesis and structure activity relationship of surface and antimicrobial properties of N-acyl amino acid surfactants using pure amino acids/waste proteins and alkyl substrates. Some chiral sodium N-palmitoyl amino acids and N-oleoyl amino acids were prepared using pure amino acids and fatty acids and evaluated their surface active and self-assembly properties and antimicrobial activities.13-15 In addition to surface and biological studies, another application of this class of compounds is low molecular weight gelator and the gelation ability due to their chirality and hydrogen bonds was also studied. These gels are potential in personal care products, pharmaceutical and industrial applications.16,17 Amino acid derived surfactants were also used in the anticorrosive applications and have the better extraction ability of membrane proteins of various topologies compared to conventional SLS.18,19 The reported literature was mostly based on the pure fatty acids and its derivatives even though mixed fatty acid-based surfactants are economically more beneficial. Reports are available on the CMC and biological properties of Nacyl amino acid-derivatives of fatty acid mixtures of coconut and palm oil along

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with single fatty chain counterparts.20-22 The vegetable oil-based surfactants as mixture of sodium N-acyl isoleucines were also synthesized using mixture of fatty acids obtained from coconut, palm kernel, palm, jatropha, karanja, Sterculia foetida, castor and high oleic sunflower oils.23 These products exhibited better biological properties and superior surface active properties like calcium tolerance, critical micelle concentration (CMC) and emulsion stability compared to the reference surfactant, sodium lauryl sulphate (SLS). In the present study, sodium N-acyl phenylalanines were prepared from the mixture of fatty acids obtained from the vegetable oils. The study was also aimed to elucidate the effects of the chain length and functionalities and also the synergetic effect varied with the type of vegetable oil and aromatic phenylalanine head group of these new surfactants on their surface active properties and their cytotoxicity. These new vegetable oil based surfactants have potential in many industrial, household applications and skin care formulations. 2. EXPERIMENTAL 2.1. Materials and Methods. Phenylalanine, sodium lauryl sulphate (SLS), other analytical and laboratory grade solvents and chemicals were purchased from M/s SD Fine Chemicals, Mumbai, India. Karanja and Sterculia foetida (Java Olive) oils were extracted from their oilseeds with hexane using soxhlet method. All other vegetable oils were procured from local market, Hyderabad, India. Oxalyl chloride was purchased from Sigma-Aldrich, St. Louis, MO, USA. Silica gel (60-120 mesh) for column chromatography was purchased from M/s. Acme Synthetic Chemicals, Mumbai, India and pre-coated TLC plates (silica gel F254) were purchased from Merck, Darmstadt, Germany. AOCS official method Cc 1-25, 1997 was used for the determination of slip melting points (SMP) of the synthesized compounds. A Lambda 25 UV-visible spectrophotometer (Perkin-Elmer, Shelton, CT, USA) was used to measure the absorbance. IR spectra were recorded on a (Perkin-Elmer, Shelton, CT, USA)

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FT-IR

spectrophotometer

(Model:

Spectrum

BX)

as

thin

film

using

dichloromethane solvent. Proton NMR spectra were recorded on 400 MHz (Varian, Palo Alto, CA, USA) spectrophotometer (Model: INOVA 400) using TMS as internal standard. The vegetable oils fatty acid compositions are given as their corresponding

fatty

acid

methyl

esters

(FAME).24

Agilent

6890

Gas

chromatograph unit equipped with FID was used for the analysis of FAME’s. Similarly column purified mixture of N-acyl phenylalanines (NAPhe) were converted into their methyl esters using diazomethane for the analysis of GC-MS. The GC-MS analysis was performed with Agilent 6890 (Philadelphia, PA, USA) gas chromatograph connected to Agilent 5973 mass spectrophotometer at 70 eV (m/z 50-600; source at 230 °C and quadruple at 150 °C) in the EI mode with a HP-1 MS capillary column (30 m × 0.25 mm × 0.5 µm). 2.2. Synthesis of Sodium N-Acyl Phenylalanines. 2.2.1. Typical Procedure for the Preparation of Mixed Fatty Acids from Coconut Oil (RCOOH). Coconut fatty acids mixture was prepared using a method reported in the literarure.23 Briefly, coconut oil (27.0 g, 40.0 mmol) was taken into of three neck round bottom flask (1 lit) and aqueous NaOH solution (0.3 mol, 100 mL) was added to coconut oil over a period of 5 min with constant stirring. The contents were stirred for 4 h at 90 °C and the reaction was monitored by micro TLC (hexane: ethyl acetate, 90:10, v/v). After completion of reaction, the contents were neutralized using diluted HCl. The organic layer was separated and washed with distilled water till to neutral and coconut fatty acid mixtures were obtained after vacuum drying (24.9 g, 98.1% yield). IR (νmax, cm-1, CH2Cl2): 2926 (br) and 1710 (s). Similarly, fatty acid mixtures of palm, karanja, high oleic sunflower, and Sterculia foetida oils were prepared in 96-98% yields. 2.2.2. Typical Procedure for the Preparation of Mixed Coconut Fatty Acyl Chlorides (RCOCl). Mixed fatty acyl chlorides were prepared according method described in the literature.23 Coconut fatty acid mixtures (15.0 g, 0.07 mol) was taken in a two necked round bottomed flask and oxalyl chloride (10.68 g, 0.084 mol) was slowly added at 30 °C from the dropping funnel. The reaction mixture was stirred for 3 h at 30 °C and IR spectroscopy was used for the confirmation of

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completion of reaction. Mixed fatty acid chlorides (15.9 g, 99% yield) were obtained by distilling out of excess oxalyl chloride under vacuum. IR (νmax, cm-1, CH2Cl2): 2926 (s) and 1800 (s). Similarly fatty acyl chlorides of palm, karanja, high oleic sunflower and Sterculia foetida fatty acid mixtures were also prepared in 98-99% yields. 2.2.3. Typical Procedure for the Preparation of Coconut N-Acyl Phenylalanines (CNAPhe). N-Acyl phenylalanines were prepared by SchttoenBaumann

reaction

according

to

method

reported

in

the

literature.8,23

Phenylalanine (8.25 g, 50 mmol) was dissolved in 10% aqueous NaOH solution (150 mL) at 30 °C and coconut fatty acyl chlorides (11.65 g, 50 mmol) were slowly added from dropping funnel at pH 10. The reaction mixture was further stirred for 2 h at 30 °C. After 2 h, the contents were acidulated (pH 5) with diluted sulfuric acid. The coconut N-acyl phenylalanines were extracted with ethyl acetate (3 × 50 mL) and the extracts were washed with brine solution and distilled water and then dried over anhydrous Na2SO4. The crude product was obtained by evaporation of solvent and purified with silica gel column chromatography using hexane/ethyl acetate (90:10, v/v) as eluent to obtain the pure product (13.34 g, 78.2% yield). Similarly N-acyl phenylalanines of karanja, palm, high oleic sunflower and Sterculia foetida fatty acid mixtures were prepared and characterized by spectral and chromatographic techniques. Coconut N-Acyl Phenylalanines (CNAPhe). SMP 58.5 °C, IR (νmax, cm-1, CH2Cl2): 3300, 2924, 2854, 1713, 1633, 1537, 1455 and 1218. 1H NMR (CDCl3, δ ppm): 7.1-7.3 ( m, ArCH-), 5.2-5.3 (m, -CH=CH-), 4.5-4.7 (m, α-CHR-), 3.1-3.2 (m, βCH2-), 2.1-2.2 (m, -CH2-CH=CH-, -CO-CH2-), 1.5-1.6 (m, -CO-CH2-CH2-), 1.21.4 (m, alkyl -CH2-) and 0.8-0.9 (t, -CH3, J = 7.0 Hz). GC-MS (EI, 70eV): m/z C8:0NAPhe: 305 (M+), C10:0-NAPhe: 333 (M+), C12:0-NAPhe: 361 (M+), C14:0-NAPhe: 389 (M+), C16:0-NAPhe: 417 (M+), C18:0-NAPhe: 445 (M+) and C18:1-NAPhe: 443 (M+). Palm Fatty N-Acyl Phenylalanines (PNAPhe). SMP 51.6°C, Yield, 63.4%. IR (νmax, cm-1, CH2Cl2): 3297, 2928, 2850, 1719, 1650, 1545, 1439 and 1202. 1H NMR (CDCl3, δ ppm): 6.9-7.2 (m, ArCH-), 5.9-6.2 (brs, -NH-), 5.2-5.3 (m, -

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CH=CH-), 4.8-4.9 (m, α-CHR-), 3.0-3.2 (m, β-CH2-), 1.9-2.2 (m, -CH2-CH=CH-, CO-CH2-), 1.4-1.6 (m, -CO-CH2-CH2-),1.0-1.2 (m, alkyl -CH2-) and 0.8-0.9 (t, CH3, J = 6.79 Hz). GC-MS (EI, 70 eV): m/z C14:0-NAPhe: 389 (M+), C16:0-NAPhe: 417 (M+), C18:0-NAPhe: 445 (M+) and C18:1-NAPhe: 443 (M+) and C18:2-NAPhe: 441 (M+). Karanja Fatty N-Acyl Phenylalanines (KNAPhe). SMP 49.6°C, Yield 71.2%. IR (νmax, cm-1, CH2Cl2): 3307, 2930, 2852, 1718, 1618, 1542, 1449 and 1202. 1H NMR (CDCl3, δ ppm): 8.2- 8.6 (brs, -NH-), 7.1-7.3 (m, ArCH-), 5.3- 5.4 (m, -CH=CH-), 4.8-4.9 (m, α-CHR-), 3.1-3.3 (m, β-CH2), 2.0-2.3 (m, -CH2CH=CH-, -CO-CH2-), 1.5-1.7 (m, -CO-CH2-CH2-), 1.2-1.4 (m, alkyl -CH2-) and 0.8-0.9 (t, -CH3, J = 6.62 Hz). GC-MS (EI, 70 eV): m/z C16:0-NAPhe: 417 (M+), C18:0-NAPhe 445 (M+), C18:1-NAPhe: 443 (M+), C18:2-NAPhe: 441 (M+), C20:0NAPhe: 473 (M+), C20:1-NAPhe: 471 (M+), C22:0-NAPhe: 501 (M+), and C24:0NAPhe: 549 (M+). Sterculia Fatty N-Acyl Phenylalanines (SNAPhe). SMP 38.5°C, Yield 62.4%. IR (νmax, cm-1, CH2Cl2): 3412, 2919, 1716, 1610, 1449 and 1191. 1H NMR (CDCl3, δ ppm): 7.1-7.3 (m, Ar-CH-), 5.2-5.4 (m, -CH=CH-), 4.8-4.9 (m, α-CH-), 3.5-3.6 (m, β-CH2-), 2.0-2.3 (m, -CH2-CH=CH-, -CO-CH2), 1.5-1.7 (m, -CO-CH2CH2), 1.0-1.2- (m, alkyl-CH2-) and 0.8-0.9 (t, -CH3, J = 6.79 Hz). GC-MS (EI, 70 eV): m/z C16:0-NAPhe: 417 (M+), C18:0-NAPhe: 445 (M+), C18:1-NAPhe: 443 (M+), C18:2-NAPhe: 441 (M+), CMA-NAPhe: 441 (M+) and CSA-NAPhe: 455 (M+). High Oleic Sunflower Fatty N-Acyl Phenylalanines (HNAPhe). SMP 28.7°C, Yield 60.1%. IR (νmax, cm-1, CH2Cl2): 3411, 2917, 2852, 1712, 1604, 1449 and 1202. 1H NMR (CDCl3, δ ppm): 8.9-9.2 (brs, -NH-), 7.2-7.4 (m, ArCH-), 5.2-5.3 (m, -CH=CH-), 4.5-4.6 (m, α-CH-), 3.3-3.5 (m, β-CH2-), 2.0-2.3 (m, COCH2-, -CH2-CH=CH-), 1.5-1.7 (m, -CO-CH2-CH2-), 1.0-1.2 (m, alkyl -CH2-) and 0.8-0.9 (t, -CH3, J = 6.79 Hz). GC-MS (EI, 70 eV): m/z C16:0-NAPhe: 417 (M+), C18:0-NAPhe: 445 (M+), C18:1-NAPhe: 443 (M+) and C18:2-NAPhe: 441 (M+). 2.2.4. Preparation of Sodium N-Acyl Phenylalanines of Coconut Fatty Acids (NaNAPhe). Sodium N-acyl phenylalanines were prepared using a method described in the literature.23 Briefly, ethanol which contains coconut N-acyl

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phenylalanine (5.0 g, 14.65 mmol) and aqueous NaOH (0.587 g, 14.65 mmol) solution were taken in a round bottom flask. The reaction mixture were stirred for 2 h at 50 °C, and then filtered. The sodium N-acyl phenylalanines were obtained by evaporation of solvent under vacuum in quantitative yield. Sodium N-acyl phenylalanines of karanja, palm, high oleic sunflower and Sterculia foetida fatty acid mixtures were prepared similarly in quantitative yields. These sodium salts were dissolved in deionized water and evaluated for their surface active properties and their cytotoxicity. 2.3. Surface Active Properties of Sodium N-Acyl Phenylalanines. 2.3.1. Wetting Power. The Draves-Clarkson method as described in Indian Standard specification (BIS-1185, Bureau of Indian Standards, New Delhi, 1957) was used for estimating the wetting power of sodium N-acyl phenylalanines.14 2.3.2.

Calcium

Tolerance.

Calcium

tolerance

of

sodium

N-acyl

phenylalanines was determined by modified Hart’s method reported in the literature.25,23 2.3.3. Foaming Properties. Foaming properties were determined using pour foam apparatus at ambient temperature according to reported method.26, 14 2.3.4.

Emulsion

Stability.

Emulsion

stability

of

sodium

N-acyl

phenylalanines was determined according to a method described in the literature.27,23 2.3.5. Surface Tension and CMC Determination using Kruss Tensiometer. Surface tension and CMC values were determined using K100MK2 Processor Tensiometer (Krüss, GmbH, Hamburg, Germany) as reported method in the literature.15 2.4. In Vitro Cytotoxicity of Sodium N-Acyl Phenylalanines. The cytotoxicity of the sodium N-acyl phenylalanines was assessed by using the MTT assay28 against a panel of five different human tumor cell lines23. All these cell lines were maintained in a customized DMEM medium supplemented with fetal bovine serum (10%), non-essential amino acids without L-glutamine (1%), antibiotic mixture (1%, 10000 units penicillin and streptomycin, 10 mg/mL), sodium hydrogen carbonate (0.2%) and sodium pyruvate (1%). The tumor cell

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lines were washed and re-suspended in the above medium and this suspension (100 µL) was seeded in 96 well plates. These tumor cells were incubated in a humidified 5% CO2 incubator at 37 °C (Model 2406 Shellab CO2 incubator, Sheldon, Cornelius, OR) for 24 h. Then the cells were treated with sodium N-acyl phenylalanines at concentrations ranging from 0.1 to 100 µM in methanol (1%) for 2 days. Each assay was performed at the end of the second day with two internal controls, one is with cells (IC0) and other one is with media (IC100). The MTT colorimetric assay (5 mg/mL) was performed after incubation of 24 h. The effect of the synthesized compounds on the feasibility of the cancer cell lines was measured on a multimode reader (Infinite® M200, Tecan, Switzerland) at 540 nm. The 50% inhibitory concentration (IC50) values were determined from the plotted absorbance data for the dose-response curves. In this assay, doxorubicin was used as standard and 1% methanol as a vehicle control. To avoid methanol toxicity, the values obtained for the methanol control were subtracted from those of the synthesized compounds. IC50 values (in µg/mL) are presented as the mean of two independent experiments. 3. RESULTS AND DISCUSSION 3.1. Synthesis of Sodium N-Acyl Phenylalanines. Reported results revealed that the N-acyl condensates with mixture of coconut fatty acids using arginine which exhibited low irritational effects on eye and skin as compared to the synthetic commercial surfactant, SLS.21,22 Mixture of fatty acids of coconut, palm, high oleic sunflower, karanja, and sterculia oils were prepared using alkaline hydrolysis in 96-98% yields (Scheme 1). These oils represent saturated, unsaturated, and cyclopropene-rich fatty acids. The fatty acid composition of five oils was analyzed by GC (Table 1). Coconut oil fatty acids are of medium and short chain with 90% saturation. Palm oil fatty acids are long chain fatty acids with about 50% unsaturation. Karanja oil and high oleic sunflower oil fatty acids contain long chain fatty acids with 78 and 90% unsaturation respectively. Sterculia fatty acids comprised of 50% long chain fatty acids with cyclopropene

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functionality. In the second step, all the fatty acid mixtures were converted into fatty acid chlorides with oxalyl chloride in 98-99% yields. The conversion was monitored by IR spectroscopy. N-Acyl phenylalanines were prepared by reacting fatty acid chlorides with phenylalanine at pH 10. All the products were characterized using chromatographic techniques namely TLC, column, GC-MS, and spectral methods like IR, 1H-NMR and Mass (supplementary material). The physical state of the products of all the N-acyl phenylalanines is semi-solid except high oleic fatty acyl derivatives which was a viscous liquid at room temperature due to unsaturation. The slip melting points (SMP) of N-acyl phenylalanines were found in the range of 28.7-58.5 °C. The GC-MS analysis of methyl esters of N-acyl phenylalanines revealed that there was no change much in the fatty acid composition observed before and after the N-acylation. In the final step of Scheme 1, the sodium N-acyl phenylalanines were prepared in quantitatively and confirmed by IR spectroscopy. The IR spectra of all the sodium N-acyl phenylalanines showed the appearance of two strong -CO- bands near 1600 and 1450 cm-1 corresponding to the carboxylate group of sodium salt and the disappearance of the carboxylic -CO- band around 1710 cm-1. Based on the GC-MS compositions, average molecular weights of the sodium N-acyl phenylalanines surfactants (NaNAPhe) are given in Table 2 and evaluated their surface active properties.

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3.5 M Aq. NaOH 90 °C, 4 h

OCOR OCOR

dil. HCl - Glycerol

OCOR

1.2 eq. (COCl)2 RCOCl

3 RCOOH 30 °C, 3 h

Mixture of fatty acids

Triglyceride

O

Fatty acid chlorides

Phenylalanine Aq. NaOH pH = 10.0

H N

Aq. NaOH 50 °C, 2 h

30 °C, 2 h dil. H 2SO 4

H N

O

R

R +NaO

HO

O

Sodium N-acyl phenylalanines

O

N-Acyl phenylalanines

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


* *

* * * * * * Fatty acid side chains R = * R = Mixture of fatty acids from coconut, palm, karanja, sterculia and high oleic sunflower oils

Scheme 1. Synthesis of sodium N-acyl phenylalanines 3.2. Surface Active Properties of Sodium N-Acyl Phenylalanines. From a commercial point of view, all the surface active properties namely calcium tolerance, wetting power, emulsion stability and foaming properties are very important for any newly synthesized amphiphiles. Aqueous solutions (0.1 wt %) of sodium N-acyl phenylalanines were used for the evaluation of surface active properties at ambient temperature. The surface active properties were evaluated using standard methods in comparison to SLS and the results are mean of the three independent measurements and given in Table 2. 3.2.1. Calcium Tolerance. Calcium tolerance property reveals the efficiency of surfactant towards hard water and acidic/alkali solutions. In general,

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anionic surfactants are more sensitive to hard water and acidic solutions compared to cationic and nonionic surfactants solutions.29 All the phenylalaninebased compounds (26.5-65.8 ppm) exhibited better calcium tolerance compared to SLS (8.8 ppm) and also superior compared to reported mixture of isoleucinebased surfactants of coconut (2.5 ppm), palm kernel (2.6 ppm), palm (24 ppm) and sterculia fatty N-acyl isoleucines (16.4 ppm).23 For phenylalanine-based derivatives, the calcium tolerance order was found to be CNAPhe < PNAPhe < KNAPhe < HNAPhe < SNAPhe. The calcium tolerance of short and medium chain fatty acid-based compounds was lower than the cyclopropene and long chain unsaturated fatty acids. 3.2.2. Wetting Power. Wetting property is most important in both industrial and house hold applications. Especially anionic surfactants are good wetting agents in textile industry.30 Generally, medium chain fatty acid (C12 and C14) derivatives exhibits better wetting ability compared to short and long chain fatty acid-based surfactants and wetting power decreases with increasing the chain length from C14 fatty acid whereas wetting power increases with increasing the unsaturation.8 The phenylalanine-based surfactants prepared in this study exhibited inferior wetting ability compared to SLS (Table 2). However, the wetting ability of coconut fatty acid derivatives (6.2 s) was almost closer to SLS (4.5 s). The wetting ability was superior in case of short and medium chain saturated coconut fatty acids-based product. Karanja fatty acid derivatives exhibited inferior wetting ability compared to other phenylalanine-based compounds due to long chain fatty acid derivatives. The wetting power of cyclopropene fatty acids-based product was superior compared to long chain unsaturated fatty acids and inferior to saturated medium chain fatty acids. The phenylalanine-based products exhibited their wetting power as follows: SLS (4.5 s) > CNAPhe (6.2 s) > HNAPhe (15.8 s) > SNAPhe (16.2 s) > PNAPhe (28.7 s) > KNAPhe (45.8 s). Phenylalanine-based surfactants (6.2-45.8 s) exhibited inferior wetting power compared to reported isoleucine-based compounds (5-22 s).23 Generally, the wetting ability increases by increasing hydrophobicity of amino acid head group.26 The wetting ability was reduced by changing the amino acid head group

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isoleucine with phenylalanine which is higher hydrophobic group than isoleucine. Similar trend was also observed in the sodium N-oleoyl amino acids15 and is reverse trend to the conventional amino acid surfactants series.31 3.2.3. Surface Tension. Usually surfactants detergency action is good at the micelle concentration. Detergency mainly depends on the monomeric surfactant concentration and it is generally unaffected by micelle.14 Based on surface tension values, the compounds can be predicted for different applications. For example, solutions having less than 40 mN/m surface tension can be exploited as wetting agents.30 In the present study, surface tension was measured at 0.1 wt% solutions and these values were less than 40 mN/m (Table 2). Surface tension of each surfactant solution was measured using Kruss K100MK2 tensiometer and the results are represented as average of five measurements in Table 2. PNAPhe (27.6 mN/m) exhibited superior surface activity compared to SLS (28.9 mN/m) and KNAPhe (28.9 mN/m) exhibited similar surface activity. Surface tension reduction ability of the remaining products solution is found to be inferior compared to SLS. In general, the surface activity increased with the increase in the length of fatty alkyl group of the same analogue. 3.2.4. Foaming Characteristics. The foaming characteristics of all the sodium N-acyl phenylalanines are inferior compared to SLS (Table 2). Foaming power and foam stability of phenylalanine-based compounds are inferior compared to reported isoleucine derivatives.23 With increase in the polarity of the amino acid head group, foaming characteristics are enhanced. The foaming power of phenylalanine-based products is as follows: CNAPhe (9.5 cm) < KNAPhe (11.5 cm) < PNAPhe (12.5 cm) ~ HNAPhe (12.5 cm) < SNAPhe (14.0 cm). The cyclopropene fatty acid-based phenylalanines exhibited better foaming characteristics. Usually, foaming power increases with the increase in the alkyl chain length of same analogue.32 Short and medium chain saturated fatty acyl amino acid derivatives exhibited inferior foaming power and foam stability compared to cyclopropene, unsaturated long chain fatty acid derivatives. These

13



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Page 14 of 29

low foaming surfactants are useful in oil extraction, waste water treatment, surface coating, automatic dish washing, textile and paper making industries.33 3.2.5. Emulsion Stability. Surfactants are very important for enhancing the emulsion property in cosmetics, agriculture, food, photography, leather, drugdelivery systems, etc.34 In general, the emulsifying power and stability of surfactant increases with an increase in the length of fatty alkyl chain of same analogue due to more solubilization of hydrophobic group in oil.30,35 The emulsion stability of all the sodium N-acyl phenylalanines (Table 2) was superior compared to SLS (216 s) and therefore, all these products can find application as good long term stable emulsifying agents. The emulsion stability time for 10 mL and 20 mL separation of the sodium N-acyl phenylalanines is varied from 243 to 844 s and 403 to 1419 s respectively. The increasing order of the emulsion stability with respect to fatty acid alkyl chain is as follows: cyclopropene fatty acids < short and medium chain fatty acids < unsaturated long chain fatty acids. The emulsion stability of phenylalanine-based products is as follows: SNAPhe < HNAPhe < CNAPhe < PNAPhe < KNAPhe < SLS. The data revealed that the sterculia fatty acid-based surfactants are weak emulsifying surfactants compared to others. 3.2.6. CMC and Thermodynamic Properties by Kruss Tensiometer. One of the very important characteristic of surface property is the CMC which can define the surfactant efficiency. Surfactants are more efficient with the lower values of the CMC. CMC values of these products were determined as shown in Fig. 1 from intersection points of average surface tension (SFT) vs. log concentration (ln C) values and represented in Table 3. Lower CMC values are observed with increase in the longer unsaturated alkyl chain derivatives and all the products are found to be lower CMC values compared to SLS (8.1 mmol/L)36

and pure

sodium N-lauroyl phenylalanine (0.7 mmol/L) and N-lauroyl (0.92 mmol/L), Npalmitoyl (0.08 mmol/L) and N-oleoyl leucine (0.21 mmol/L).8 Due to higher hydrophobicity of phenylalanine head group compared to isoleucine, sodium Nacyl phenylalanines exhibited lower CMC values compared to the reported mixture of sodium N-acyl isoleucines surfactants obtained from vegetable oils.23 CMC value of CNAPhe comprised of 50% of C12 fatty acyl chains is 2 fold order

14


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lower compared to SLS which is also C12 alkyl compound due to mixture of long chain fatty acid (C14, C16, and C18) derivatives. Generally, an increase in the hydrophobicity of amino acid or fatty acid decreases the CMC value. Three fold orders of magnitude of CMC value of HNAPhe (0.00146 mmol/L) is lower compared to SLS. Four fold orders of magnitude of CMC values of PNAPhe (0.0008 mmol/L), KNAPhe (0.00041 mmol/L) and SNAPhe (0.00063 mmol/L) are lower as compared to SLS (8.1 mmol/L). All these compounds except coconut fatty acid derivatives possess similar order CMC values compared to pure sodium N-oleoyl phenylalanine (0.0007 mmol/L).15 Surface tension reduction efficiency (pC20 = Negative logarithm of surfactant concentration is required to reduce the surface tension of pure water by 20 units) values were also measured from the surface tension plots in Fig. 1. Palm, karanja, high oleic sunflower, sterculia fatty acid surfactants having higher pC20 values are most efficient compared to coconut fatty acid-based products having lower pC20 value. Literature data also indicated that the surfactants having more than 3 pC20 value are efficient surfactants.37 Thermodynamic properties of these sodium N-acyl phenylalanines including effectiveness of surface tension reduction (Πcmc), maximum surface excess concentration (Γmax), minimum surface area per molecule (Amin) at airwater interfacial tension were determined using following equations. Πcmc = γ0 – γ;

(1)

where γ0 is pure water surface tension, γ is surface tension at CMC.

Γmax = − (1/nRT) (dγ/dlnC)

(2)

Amin = 1/N. Γmax

(3)

where T is absolute temperature, R is the gas constant (8.314 J/mol.K), C is surfactant concentration, γ is surface tension and N is Avogadro’s number. The value of n is taken to be 2 as there is one counter ion associated with one ionic head group.37 The surfactant adsorption and micellization are observed at the airwater interface and in the bulk aqueous solution occurs respectively. The standard free energy of adsorption (∆G°ads) and standard free energy of

15



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Page 16 of 29

micellization (∆G°mic) of the surfactant at the air-water interface can be evaluated by use of the following equations 4 and 5.38,39 ∆G°mic = RT ln CMC

(4)

where R is gas constant 8.314 J/mol.K and T is absolute temperature. ∆G°ads = ∆G°mic – (Πcmc / Γmax)

(5)

The most efficient surfactant reduces the maximum surface tension of water at their critical micelle concentration. Higher the Πcmc value surfactant is the most effective. The Πcmc values of these products solutions are found in the range of 31.1-39.4 mN/m (Table 3) and lower compared to the reported mixture of

sodium N-acyl isoleucines (40-43 mN/m) which are having lower

hydrophobicity head group.23 Among these SNAPhe exhibited lower efficacy may be due to the presence of higher hydrophobic cyclopropene fatty acid derivatives. The same trend was also reported by Sakamoto et al31 in the neutral amino acid surfactant series and also reverse linear correlation between surface tension and CMC. Surface excess concentration (Γmax) value of the coconut fatty acids-based surfactant is lower than the other fatty acyl phenylalanine-based surfactants. All the sodium N-acyl phenylalanines except coconut fatty acid derivatives exhibited higher Πcmc values compared to isoleucine derivatives. Phenylalanine head group surfactant molecules excess concentration (9.71 – 20.2 mol/cm2) was higher than the isoleucine head group surfactants (2.6 – 6.07 mol/cm2).23 The head group effect was observed in the surface excess concentration except coconut fatty acid derivatives at air-water interface. The minimum area (Amin) per molecule at the air-water interface reveals the information of the degree of packing and the orientation of the adsorbed surfactant molecule. Higher the surface excess concentration (Γmax) of a surfactant results lower minimum area per molecule at air-water interface. Amin values decreased with an increase in the chain length of unsaturated fatty alkyl group of phenylalanine-based surfactants. This indicates that the saturated molecules were not tightly packed at the air/water interface compared to the unsaturated alkyl surfactants. Thermodynamic properties

16


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(∆G°mic and ∆G°ads) exhibited large negative values for the synthesized surfactants, representing that adsorption and micellization processes are spontaneous at 27 °C. ∆G°mic is lower negative than ∆G°ads indicating that adsorption is more spontaneous compared to the micellization due to lesser repulsions between water and hydrophobic group interactions at air/water interface. The increasing order of the spontaneous micellization of phenylalaninebased surfactants is as follows: CNAPhe < HNAPhe < PNAPhe < SNAPhe < KNAPhe (Table 3). Karanja fatty acid-based compounds are most efficient compounds compared to other phenylalanine-based surfactants due to long chain fatty acid derivatives (C20:0, C20:1, C22:0, and C24:0). Medium chain saturated coconut fatty acids-based surfactant exhibited lower efficiency compared to the long chain unsaturated fatty acids-based products towards micellization. Phenylalanine-based compounds are the most efficient surfactants compared to isoleucine-based products towards micellization.23

Figure 1. Plots of γ vs. ln C (mol/L) of sodium N-acyl phenylalanines 3.3.

In

Vitro

Cytotoxicity

Assessment

of

Sodium

N-Acyl

Phenylalanines. The in vitro study results showed that the lysine-based anionic

17



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surfactants with different counter ions exhibited lower irritancy than that of conventional surfactants SLS, HTAB and TGB.40 Lysine-based surfactants analogues to the lecithin molecules were evaluated for the skin irritation test employing cell culture test alternative to in vivo test. The irritancy of the surfactants depends on the size of the counter ion size. Irritancy of lighter size ion surfactant is higher than the heavier size ion surfactant.41 Due to lower irritancy potential, high biodegradability of amino acid-based surfactants may offer as pharmaceutical, cosmetic and cleaning applications as compared to conventional SLS.13,42,43 Lipoamino acids were also used as potential antiinflammatory agents.44 In the present study, cytotoxicity of the sodium N-acyl phenylalanines was determined on the basis of measurement of in vitro growth inhibition of tumor cell lines up to a concentration of 50 µg/mL. 50% of the cell growth inhibition concentration of compounds (IC50) was calculated and presented in Table 4. PNAPhe exhibited promising cytotoxicity towards MDAMB-231 (4.63 µg/mL) and MCF-7 (6.95 µg/mL) and moderate cytotoxicity towards HeLa (15.35 µg/mL) cell lines. KNAPhe exhibited the promising cytotoxicity towards MCF-7 (7.61 µg/mL) and moderate cytotoxicity towards A549 (20.4 µg/mL) and MDA-MB-231 (11.17 µg/mL) cell lines. SNAPhe exhibited promising towards all (except Neuro2a) tested cell lines and similar activity compared to standard doxorubicin (0.58 µg/mL) against human breast adenocarcinoma cells cell lines. HNAPhe exhibited promising cytotoxicity towards HeLa (1.69 µg/mL) cell line only. CNAPhe did not exhibit cytotoxicity at tested concentrations. Among these, sterculia (cyclopropene) fatty acid-based products are most cytotoxic towards human cancer cell lines. Sodium N-acyl phenylalanines except SNAPhe exhibited inferior cytotoxicity compared to the reported mixture of sodium N-acyl isoleucines may be due to less polar phenylalanine head group compared to the isoleucine.23 Gopal et al.45 reported the cytotoxicity of series of pure N-sapienoyl (C16:1) amino acids and among those N-sapienoyl leucine exhibited the higher activity towards only selective tumor cell lines. N-Sapienoyl phenylalanine did not show the cytotoxicity but mixture of palm oil based N-acyl phenylalanines (PNAPhe) which is rich in C16:0

18


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fatty acids exhibited the cytotoxicity may be due to presence of mixture of fatty acids. Compound SNAPhe exhibited superior activity towards all tested cell lines compared to other N-acyl phenylalanines and mixture of N-acyl isoleucines and also N-sapienoyl (C16:1) amino acids. 4. CONCLUSIONS In the present study, phenylalanine-based surfactants were synthesized from mixture of fatty acids obtained from vegetable oils and pure phenylalanine in 60-78% yields and characterized by spectral and chromatographic techniques. All the products were evaluated for surface active properties and cytotoxicity and interpreted with respective of change in the functionalities in the fatty acids. All surfactants exhibited better calcium tolerance and emulsifying power compared to SLS. CNAPhe exhibited almost similar wetting ability compared to SLS. Lower CMC values (0.0004 - 0.018 mmol/L) and higher pC20 values (5.8-6.5) showed the efficiency of sodium N-acyl phenylalanines. Foaming properties, surface tension and wetting ability of phenylalanine-based surfactants were inferior compared to SLS. These low foaming surfactants along with lower CMC values can be used as alternative to petroleum based surfactants and also as stable emulsifiers in many industrial process and cleansing applications. All surfactants except CNAPhe exhibited promising cytotoxicity towards the tested tumor cell lines. These new vegetable oil-based surfactants have economical potential in pharmaceutical, skin care and cosmetic applications. Acknowledgements M. Sreenu acknowledges to Department of Science and Technology, Government of India, New Delhi, for providing the research fellowship. P. Sujitha is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for the award of SRF. The authors have declared no conflict of interest.

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Abbreviations: FA: Fatty acid; MA: Malvalic acid, (8-9- cycylopropene octadecanoic acid); SA: Sterculic acid, (9-10-cyclopropene nonadecanoic acid); Phe: phenylalanine; NAPhe: N-acyl phenylalanine; C8:0-NAPhe: N-octanoyl phenylalanine; C10:0-NAPhe: N-decanoyl phenylalanine C12:0-NAPhe: N-lauroyl phenylalanine; C14:0-NAPhe: N-myristoyl phenylalanine C16:0-NAPhe: N-palmitoyl phenylalanine; C18:0-NAPhe: N-stearic phenylalanine C18:1-NAPhe: N-oleic phenylalanine; C18:2-NAPhe: N-linoleic phenylalanine C20:0-NAPhe: N-cosanoic phenylalanine; C20:1-NAPhe: N-cosenoic phenylalanine C22:0-NAPhe: N-docosanoic phenylalanine.

phenylalanine;

24

C24:0-NAPhe:


N-tetracosanoic

Page 25 of 29

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Table 1. Fatty Acid Composition of Seed Oils Used in this Study Vegetable oil

Fatty acid (wt %) C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C20:0 C20:1 C22:0 C22:1 C24:0

Coconut

6.5

5.8

47.5

19.7

8.9

3.0

6.7

1.9

-

-

-

-

-

Palm

-

-

-

1.9

44.3

4.6

38.7

10.5

-

-

-

-

-

Karanja

-

-

-

-

9.8

7.8

54.5

17.0

1.9

1.1

1.2

5.1

1.6

Sterculia*

-

-

-

-

25.4

4.7

12.8

7.7

-

-

High oleic sunflower

-

-

-

-

3.2

5.1

81.9

9.8

-

-

-

-

-

*Sterculia oil also contains malvalic acid (5.5%) and sterculic acid (43.9%)

25



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Page 26 of 29

Table 2. Surface Active Properties of Sodium N-Acyl Phenylalanines* Surfactant

Average

Calcium

Wetting

Surface

MW of

tolerance

time

tension

SNAAA

(ppm)

(s)

(mN/m)

Foam height (cm) Initial

After 5 min

Emulsion stability time (s) 10 mL

20 mL

Separation

Separation

CNAPhe

363.2

31.6

6.2

30.0

9.5

7.0

473

922

PNAPhe

419.5

26.5

28.7

27.6

12.5

10.5

603

1050

KNAPhe

435.7

30.4

45.8

28.9

11.5

8.0

844

1419

SNAPhe

406.2

65.8

16.2

34.7

14.0

9.0

243

403

HNAPhe

431.7

40.5

15.8

31.5

12.5

10.0

262

475

288

8.8

4.5

28.9

16.0

15.0

216

438

SLS

* 0.1 wt % surfactant solution at 27 °C; MW = Molecular Weight

26


Page 27 of 29

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Table 3. CMC and Thermodynamic Properties of Sodium N-Acyl Phenylalanines* Surfactant CNAPhe

Πcmc

Γmax×1010

Amin

∆G°mic

∆G°ads

(mN/m)

(mol/cm2)

(Å2)

(kJ/mol)

(kJ/mol)

5.83

38.8

2.96

56.11

-27.14

-40.24

6.27

39.4

19.08

8.7

-35.01

-37.07

pC20

γcmc

CMC

(mN/m)

(mmol/L)

33.1

1.88×10-2 -4

PNAPhe

32.5

8.0×10

KNAPhe

36.8

4.15×10-4

6.51

35.1

20.2

8.2

-36.65

-38.39

SNAPhe

40.8

6.32×10-4

6.35

31.1

12.14

13.6

-35.6

-38.16

HNAPhe

37.9

1.46×10-3

6.08

34.0

9.71

17.0

-33.51

-37.01

*Surface tension measured at 27°C; γcmc, surface tension value at the CMC; CMC, critical micelle concentration; Πcmc, effectiveness of surface tension reduction; Γmax, maximum surface excess; pC20, Negative logarithm of surfactant concentration is required to reduce the surface tension of pure water by 20 units; Amin, minimum surface area per molecule; ∆G°mic, standard free energy of micellization; ∆G°ads, standard free energy of adsorption

27



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Page 28 of 29

Table 4. In Vitro Cytotoxicity of the Sodium N-Acyl Phenylalanines Compound

IC50 (µg/mL) A549

MDA-MB-231

MCF-7

HeLa

Neuro2a

PNAPhe

-

4.63

6.95

15.35

-

KNAPhe

20.4

11.17

7.61

-

-

SNAPhe

2.66

0.58

4.26

4.29

-

HNAPhe

-

-

-

1.93

-

0.58

Synthesis and Surface-Active Properties of Sodium ... - ACS Publications

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