Structure-Property Influence on the Amphiphilicity of Phenolipids ^†

Abstract

In recent years, increasing interest has been observed in phenolipids used for enhancing the quality of products containing lipids in the food, pharmaceutical and cosmetic industries. A better understanding of the physicochemical properties of these amphiphilic compounds is crucial to maximizing their antioxidant and antiproliferation properties. Therefore, certain p-hydroxycinnamic acid derivatives were synthesized and their lipophilicity expressed as a partition coefficient (Log P) was measured using the shake-flask method. Additionally, the obtained results were compared with the calculated data in ALOGPS 2.1. An increase in lipophilicity was observed along with an increased alkyl chain length. Moreover, hydrophilic/hydrophobic properties are closely related with the number of substituents, especially the hydroxyl group, in aromatic rings.

1. Introduction

Phenolipids are amphiphilic antioxidants with both hydrophilic phenolic moiety and hydrophobic molecules. These compounds should maintain the original functional properties of their parent compounds such as antioxidant, chelating, free radical scavenging, antiallergic, anti-inflammatory, antimicrobial, antiviral and anticarcinogenic properties. These properties, especially antioxidant properties, are mainly associated with the number and distribution of hydroxyl groups in the aromatic ring of phenolic acid [1]. Alkyl esters of phenolic acids are the main known phenolipids. These compounds can be considered as potential replacements for synthetic antioxidants such as butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) commonly used in the fat and cosmetic industry [2]. Therefore, it is desirable to gain a better understanding of the physicochemical properties of phenolic acid esters.

One of the most important parameters used to predict information regarding physicochemical properties is the partition coefficient (log P). This parameter allows us to predict lipophilicity and the quantitative structure–activity relationships (SAR) of bioactive compounds [3,4]. Therefore, log P is widely used in the pharmaceutical industry to check the efficiency of the proposed drug such as by measuring the achievement of the target and binding at the target [5]. The most popular method for experimental determination of log P is the shake-flask method, in which the sample is partitioned between organic (n-octanol) and water (or aqueous buffer) phases in a flask. Next, the concentration of analyte in both the organic and water phases is quantified by UV–VIS spectroscopy or a different method of spectrometric detection, high-performance liquid chromatography (HPLC) or gas chromatography (GC) [3]. However, to avoid time-consuming and labor-intensive experimental methods, computer programs were developed to estimate log P.

The aim of this study was to estimate the lipophilicity of certain phenolipids, p-hydroxycinnamic acid (HCA) derivatives, especially sinapic acid alkyl esters. Lipophilicity was expressed as a log P and measured using the shaking-flask method. Additionally, the obtained results were compared with calculated data in ALOGPS 2.1.

2. Materials and Methods

2.1. Reagents

All reagents, reactants, and solvents were purchased from Merck (Warsaw, Poland).

2.2. Synthesis of Phenolic Acid Esters

Synthesis procedures of esters: ethyl sinapate (ESA), octyl sinapate (OSA), cetyl sinapate (CSA), octyl caffeate (OCA) and octyl ferulate (OFA) were described in our previous studies [2].

2.3. Calculation of Partition Coefficients (log P) for Phenolic Compounds

2.3.1. Shake-Flask Method

Phenolic antioxidants: SA, ESA, OSA, CSA, CA, OCA, FA, OFA and BHA were diluted in the octanol and water phase. The prepared solutions were placed in an ultrasonic cleaner bath (Sono Swiss, SW 6H, Labo Plus, Warsaw, Poland) with ultrasound input power of 180 kW for 15 min due to enhanced solubility. The UV spectra of the analyzed phenolic compounds were recorded using a Hitachi U-2900 spectrophotometer (Tokyo, Japan) in a 1 cm quartz cell in the octanol and water phases in order to find the characteristic band of the studied compounds. Next, calibration curves were constructed by plotting the concentrations as a function of UV absorbance values in the ranges for organic phase: 2.97 × 10⁻²–2.23 × 10⁻¹ µmol/mL, 4.71 × 10⁻²–2.35 × 10⁻¹ µmol/mL, 2.05 × 10⁻²–1.74 × 10⁻¹ µmol/mL, 2.00 × 10⁻²–1.70 × 10⁻¹ µmol/mL, 3.50 × 10⁻²–2.97 × 10⁻¹ µmol/mL, 2.55 × 10⁻²–2.55 × 10⁻¹ µmol/mL, 4.08 × 10⁻²–2.04 × 10⁻¹ µmol/mL, 4.72 × 10⁻²–4.01 × 10⁻¹ µmol/mL, 3.75 × 10⁻¹–1.88 µmol/mL and for water phase: 3.37 × 10⁻²–2.36 × 10⁻¹ µmol/mL, 3.59 × 10⁻²–2.15 × 10⁻¹ µmol/mL, 2.94 × 10⁻²–1.96 × 10⁻¹ µmol/mL, 2.12 × 10⁻²–1.42 × 10⁻¹ µmol/mL, 1.28 × 10⁻²–1.03 × 10⁻¹ µmol/mL, 1.30 × 10⁻¹–2.60 × 10⁻¹ µmol/mL, 1.18 × 10⁻²–4.71 × 10⁻² µmol/mL, 1.46 × 10⁻¹–2.63 × 10⁻¹ µmol/mL, 2.52 × 10⁻²–2.52 × 10⁻¹ µmol/mL SA, ESA, OSA, CSA, CA, OCA, FA, OFA and BHA, respectively.

1-Octanol–water distribution coefficients were determined using the shake flask method according to the OECD Guideline for the Testing of Chemicals [6]. Briefly, selected phenolic compounds were weighed at a concentration within the calibration curve and dissolved in previously saturated 2-phase solutions in a 50 mL conical flask. Then, the prepared solutions were shaken 250 cycles/min for 6 h using an orbital shaker (SHKA25081 CE, Labo Plus, Warsaw, Poland) and then the mixtures were left to stand for 6 h to be partitioned between 2 phases. The absorbance of both phases was measured. The patriation coefficients were calculated according to Equation (1)

$${\mathrm{P}}_{\mathrm{o}/\mathrm{w}}=\frac{{\mathrm{c}}_{\mathrm{n}-\mathrm{octanol}}}{{\mathrm{c}}_{\mathrm{n}}}$$

(1)

in which:

C_n-octanol—concentration of phenolic compounds in n-octanol,

C_n—concentration of phenolic compounds in water [6].

2.3.2. Theoretical Calculation

Log P values were also calculated using the ALOGPS 2.1 online program at the Virtual Computational Chemistry Laboratory accessed on 18 August 2022 [http://www.vcclab.org/lab/alogps/]. This program simulates participation in an n-octanol–water system.

2.4. Statistical Analysis

The log P values were determined three times within one day using the shake-flask method. The obtained results were presented as mean (c) ± standard deviation (SD).

3. Results

The lipophilicity of selected phenolic compounds are presented in

Table 1. Log P_exp and calculated log P_ALOGPs values for HCA derivatives.

Phenolic Compound	Log Pexp ± SD	Log PALOGPs
SA	0.98 ± 0.05	1.26
ESA	3.87 ± 0.13	2.60
OSA	5.20 ± 0.24	5.34
CSA	7.63 ± 0.39	8.87
CA	0.99 ± 0.12	0.94
OCA	4.75 ± 0.05	5.02
FA	1.12 ± 0.07	1.25
OFA	5.72 ± 0.04	5.32
BHA	3.64 ± 0.05	3.15

Values are means (n = 3) ± standard deviations (SD).

.

It is noteworthy that log P obtained for the HCA derivatives both by calculation (Log P_ALOGPs) and those experimentally determined using the shake-flask method (log P_exp) are similar. As can be seen in Figure 1, the relationship between Log P_exp and Log P_ALOGPs can be expressed using the following linear regression Equation (2):

Log P_ALOGPs = 1.0773 Log P_exp

(2)

The correlation coefficient r was 0.9678 and r² = 0.9367

In general, the esterification of phenolic acids with alkyl alcohols increased the lipophilicity of the synthesized phenolipids (log P_ALOGPs = 0.94–1.26; log P_exp = 0.98–1.12 and log P_ALOGPs = 2.60–8.87; log P_exp = 3.87–7.63 for phenolic acids and their alkyl esters, respectively). As seen in Table 1, the lipophilicity of the HCA derivatives are well correlated with their structural features. The log P values increased along with the elongation of the alkyl ester side-chain. The same tendency was observed by Gaspar et al. [7] for SA derivatives and by Garrido et al. [8] for FA and CA derivatives with different acyl donor chain lengths from C1 to C4, and in our previous work. Additionally, the obtained log P values for tested HCA demonstrated that lipophilicity is closely related with the number of hydroxyl group substitutions in aromatic rings. The Log P_ALOGPs values for CA was the lowest (Log P_ALOGPs = 0.94) in comparison with FA (log P_ALOGPs = 1.25) and SA (log P_ALOGPs = 1.26). The same tendency was observed in the tested phenolic acid octyl ester—log P_ALOGPs were 5.02, 5.32 and 5.34 for OCA, OFA and OSA, respectively. Surprisingly, there were no differences between log P_exp for CA and SA (Δlog P_exp = 0.01). Moreover, the obtained data demonstrated for both series (phenolic acids and octyl esters) slight differences in lipophilicity (log P_ALOGPs differences between SA and FA 0.01 and log P_ALOGPs differences between OSA and OFA 0.02).

Food additives such as BHA had log P_exp = 3.64 and Log P_ALOGPs = 3.15. Therefore, there is more hydrophobic than phenolic acid but less than in the obtained HCA derivatives, except for ESA.

4. Conclusions

The obtained data in this study proves that the computational method of log P is convenient for estimating the lipophilicity of HCA derivatives. The esterification of phenolic acid with alcohol effectively increased their lipophilicity. Furthermore, the log P values of phenolipids depend on the length of the alkyl-ester side-chain and the number of hydroxyl groups in aromatic rings.

Log P allows us to predict the overall physicochemical parameters of new functionalized compounds such as phenolipids due to its correlation with antioxidant and cytotoxic activates [4,7]. The higher lipophilicity of antioxidants is often desired in fat-based products because it changes the absorption and distribution properties of HCA derivatives. Increasing lipophilicity increased the ability of phenolipids to achieve local concentration at the water–lipid interface, where lipid oxidation started, through free radical attack from the aqueous phase [4,7]. Therefore, phenolipids can be used as an effective additive to food-based products to prevent oxidation.