Novel Aromatic Estolide Esters from Biobased Resources by a Green Synthetic Approach

Abstract

The use of vegetable oils and their derivatives for polymer synthesis has been a major focus in recent years due to their universal availability, low production costs and biodegradability. In this study, the enzymatic synthesis of oligoesters of ricinoleic acid obtained from castor oil combined with three aromatic natural derivatives (cinnamyl alcohol, sinapic acid, and caffeic acid) was investigated. The formation of the reaction products was demonstrated by FT-IR, MALDI-TOF MS and NMR spectroscopy and for the oligo (ricinoleyl)-caffeate the thermal properties and biodegradability in sweet water were analyzed and a rheological characterization was performed. The successful enzymatic synthesis of oligoesters from ricinoleic acid and aromatic monomers using lipases not only highlights the potential of biocatalysis in green chemistry but also contributes to the development of sustainable and biodegradable methods for synthesizing products with potential applications as cosmetic ingredients.

1. Introduction

Oils and fats of vegetal and animal origin are considered essential renewable feedstocks, particularly for the chemical industry, showing a remarkable increase in the past decade. The annual worldwide production of the major vegetable oils increased by 47.8% between 2009 and 2019 [1]. Applications that use vegetable oils as raw materials include surfactants, cosmetics and lubricants [2,3]. Efficient synthetic transformations of triglycerides and fatty acids represent an essential part of oleochemistry, a domain with increasing industrial importance which allows the sustainable valorization of easily accessible and cheap resources. A multitude of chemical modifications of carboxylic acids and their esters are possible, including transformations of the carboxylic group leading to alcohols, amines, amides, nitriles, or oxidations of the unsaturated acids and esters, yielding high value products by epoxidation, allylic oxidation, or oxidative cleavage [4]. Due to their relatively low costs, vegetable oils as well as saturated and unsaturated fatty acids derived from them are considered among the most important renewable raw materials for obtaining functional biopolymers and polymeric materials [5,6].

Hydroxy-fatty acids (HFAs) and their derivatives represent a class of compounds of great interest. In addition to being essential molecules in biological systems, they are used in a wide variety of industrial processes and, more recently, in the field of materials chemistry. Structurally, hydroxy fatty acids may be saturated or unsaturated and may contain one or more hydroxyl groups and a long, branched chain with a terminal carboxyl group. They occur in nature as components of the cerebrosides of triacylglycerols, waxes, and other lipids in animals, plants, and microorganisms [7]. Unlike non-hydroxylated fatty acids, HFAs have reactivity, miscibility in organic solvents, stability and high viscosity due to the presence of the hydroxyl group in the molecule [8]. Thus, they have utilizations in the chemical, food and cosmetic industries as raw materials for the manufacture of lubricants, emulsifiers and stabilizers [9]. HFAs also have pharmaceutical potential, including anti-bacterial, anti-fungal and anti-diabetic activity [10,11]. Polymers synthesized from HFAs have several advantages over petroleum-derived polymers, such as higher heat resistance, resistance to chemicals, high flexibility, high biocompatibility, and virtually no toxicity [12].

The synthesis of estolides, oligomers of fatty acids, belongs to this sustained effort of oleochemistry development and can be achieved by (i) the addition of a fatty acid to an unsaturated one; (ii) epoxidation and epoxide ring opening; and (iii) polymerization of a hydroxy fatty acid, caused by the addition of water to the C = C double bond through enzymatic catalysis using fatty acid hydratases [13]. The interest for estolides of hydroxy fatty acids has continuously grown in the past decade, mainly due to their applications as biolubricants, but their utilizations as food additives, cosmetic ingredients or sources of polyols for polyurethanes cannot be neglected [14]. Estolide esters are synthesized mostly by chemical catalysis from ricinoleic acid (RCA), a natural hydroxy acid present in castor oil and various carboxylic acids like capric, lauric, myristic or stearic acid [15], although oleic acid can also be the starting material [16]. At the same time, the harsh reaction conditions can result in colored products and undesired side reactions, requiring purification. To avoid these drawbacks, the enzymatic synthesis of estolides and estolide esters has been successfully investigated by several research groups [17,18,19,20]. Although estolides are valuable products by themselves, additional capping of the free hydroxy and carboxyl groups by reaction with an aliphatic acid or alcohol, respectively, can add new functionalities and improved properties, as was also revealed by a recent white paper of the company Seqens [21]. However, the utilization of an aromatic alcohol or acid in these reactions is difficult to accomplish. In fact, only one report was published in this topic until now, addressing the synthesis of cinnamate estolides from milkweed seed oil. Heterogeneous chemical catalysts have been used for the epoxidation of unsaturated groups of this oil, followed by epoxy ring opening to yield polyhydroxy triacylglycerols, which reacted with ferulic acid [22]. The antioxidant properties of these compounds were also demonstrated, as they prevented lipid peroxidation in phospholipid vesicles [23]. Copolymers with alternating RCA and 4-hydroxycinnamic acid derivatives were obtained by pre-coupling the monomer molecules, followed by self-condensation of the resulting hetero-dimers using various chemical catalysts [24].

Lipases are extremely versatile enzymes, being able to catalyze a wide range of reactions including the synthesis of oligomers (estolides) [25] or co-oligomers [26] of HFAs, particularly ricinoleic acid. RCA is a biobased hydroxy acid with great potential for sustainable manufacturing of green chemicals, although in some countries its source, castor bean (Ricinus communis), is considered an invasive plant [27]. Greco-Duarte et al. studied the estolide size profile and the evolution of the polymerization degree of RCA over a 24 h reaction time [28]. Arslan et al. obtained RCA trimers and tetramers directly from castor oil by transesterification with lipase A from Candida antarctica [17]. The estolide esters were also obtained with lipases, in both native or immobilized form. Ortega-Requena et al. used lipase B from Candida antarctica immobilized by adsorption to yield polyglycerol polyricinoleate, an important food emulsifier [29]. Another naturally occurring hydroxy acid, lesquerollic acid from lesquerella oil, was also investigated for the lipase-catalyzed synthesis of estolide esters by endcapping with oleic, stearic or 2-ethylhexanoic acids [30].

The synthesis of aromatic esters catalyzed by lipases is more difficult compared to aliphatic esters, due to the lower substrate specificity towards aromatic substrates, as demonstrated by our earlier works [31,32]. The selection of an appropriate enzyme is essential since this substrate specificity is strongly influenced by the structure of the binding site of the enzyme [33].

The objective of the present work was the valorization of RCA, a biomass-based unsaturated hydroxy fatty acid, for the green synthesis of new high added-value oligomers, using a cascade approach involving hydrolysis of castor oil, enzymatic synthesis of oligo-ricinoleates and endcapping with an aromatic acid or alcohol. Alongside RCA, the aromatic acids and aromatic alcohols can also be obtained from renewable resources. 4-Hydroxycinnamic acids can be recovered from agricultural by-products and also synthesized by engineered microorganisms [34]. 4-Hydroxycinnamyl alcohols have been produced from inexpensive phenylpropanoic acids by a three-step biocatalytic cascade reaction using genetically engineered Escherichia coli strains [35]. Enzymatic synthesis of such aromatic estolide derivatives has not yet been reported. Since the estolides still hold a hydroxy and a carboxy group available for functionalization, the selection of either an aromatic acid or an aromatic alcohol is possible for the synthesis of the end capped oligomeric esters, using the suitable biocatalyst. The aim of this first study was to demonstrate the possibility of synthesizing estolides of ricinoleic acid endcapped by an aromatic functional group at either the carboxy or hydroxy end. The obtained estolide esters are biobased compounds, synthesized from a vegetable oil-derived hydroxy acid and aromatic acids or alcohols also selected based on their availability from renewable raw materials by enzymatic or chemoenzymatic pathways. Advanced techniques were employed for the characterization of the obtained aromatic oligoesters, highlighting the rheological properties as being essential for their possible utilizations. The biodegradability of a selected product was assessed as another application-related essential requirement. The improved functionality of these compounds could pave the way for a new class of cosmetic ingredients.

2. Materials and Methods

2.1. Materials

Lipase B from Candida antarctica, recombinant, expressed in Aspergillus niger, immobilized on acrylic resin (Novozyme 435), with specific activity of 5000 U/g (assayed by propyl laurate hydrolysis), caffeic acid, cinnamyl alcohol, sinapic acid, trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene] malononitrile (DCTB) and potassium trifluoroacetate were all Sigma-Aldrich products (Merck, Darmstadt, Germany). The solvents toluene (~99%), acetone (~99%) and tetrahydrofuran (~99%) were purchased from Merck (Darmstadt, Germany). Cold-pressed castor oil was bought from Herbavit (Oradea, Romania).

2.2. Chemical Synthesis of Ricinoleic-Based Oligoesters

To prepare ricinoleic acid (RCA), 250 g of castor oil was weighed and hydrolyzed by refluxing with alcoholic KOH solution. Ethanol was evaporated in a water bath and the residue was dissolved in 1.2 L of distilled water and acidified with HCl at pH = 1. Free fatty acids were extracted with 600 mL of ethyl acetate. The organic layer was dried with magnesium sulfate, decolored with Nurit and the filtrate and ethyl acetate were evaporated in a water bath at a temperature of 50 °C.

The determination of the fatty acid distribution in the obtained RCA was carried out by gas chromatographic analysis of methyl esters obtained from alcoholysis with methanol in the presence of BF₃/CH₃OH, using a Varian 450 Chromatograph (Varian Inc., Utrecht, The Netherlands) equipped with a flame ionization detector (FID), using a 15 m × 0.25 mm VF-1ms non-polar capillary column with a 0.25 μm film thickness of dimethylpolysiloxane. The analysis conditions were the oven temperature, which ranged from 150 to 250 °C with a heating rate of 10 °C/min; the injector temperature was 300 °C, the detector temperature was 350 °C and the carrier gas flow (hydrogen) was 2.0 mL/min.

The calculated percentage of ricinoleic acid in the obtained product was 88%.

2.3. Enzymatic Synthesis of the Aromatic Oligoesters

The reactions were carried out in 2 mL Eppendorf tubes, at 80 °C, 1200 rpm, using an Eppendorf Thermomixer Comfort heating shaker (Eppendorf, Hamburg, Germany) at a 1:2 molar ratio of the reagents (RCA/aromatic co-substrate), in 1 mL toluene, by using 50 U/mmole lipase Novozyme 435 as catalysts. After 72 h, the samples were dissolved in 3 mL tetrahydrofuran and the immobilized enzyme was removed by filtration. The reactions were performed in duplicate. The product was obtained by evaporating the solvent and drying overnight under vacuum at 60 °C. A control reaction was carried out using only RCA as a starting material, in the same reaction conditions, to synthesize the RCA estolides. Product yields were determined after each synthesis. The values were all above 85%. The yield values were as follows: oligo (ricinoleyl) cinnamate 86.8%, oligo (ricinoleyl) caffeate 94.8%, oligo (ricinoleyl) sinapate 92.6%.

2.4. Structural Analysis of the Reaction Products

The reaction products were analyzed by MALDI TOF-MS spectrometry, performed with a Bruker Autoflex Speed mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), equipped with a time-of-flight (TOF) mass analyzer as previously described [26]. In all cases, 21 kV and 9.55 kV were applied as reflector voltage 1 and reflector voltage 2, respectively. The matrix used was trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene] malononitrile (DCTB) and sodium trifluoroacetate. Calibration of the system was conducted with 600, 1000 and 2000 Da polyethylene glycol (PEG) solutions. In total, 10 μL of sample (10 mg/mL) with 10 μL of DCTB solution (40 mg/mL) and 3 μL of NaTFA solution (5 mg/mL) were mixed. Approximately 1 μL of this mixture was deposited onto the sample and the MS spectra were acquired in the positive ion mode. The MS spectra were processed and evaluated, using the FlexControl 3.3 version and FlexAnalysis 3.4. version software packages from Bruker (Bruker Daltonik GmbH, Bremen, Germany).

Fourier transform infrared (ATR FT-IR) spectra were recorded using a Bruker Vertex 70 spectrometer (Bruker Daltonik GmbH, Germany) equipped with a Platinium ATR, Bruker Diamond Type A225/Q.I. A total of 128 Co-added scans were performed in the range 4000–400 cm⁻¹, with a resolution of 4 cm⁻¹.

NMR spectra were recorded on a BrukerAvance III spectrometer operating at 500 MHz (1H) and 125 MHz (13C). Samples were dissolved in THF-d8, and chemical shifts δ are given in ppm, relative to TMS.

2.5. Viscosity Measurements

The kinematic viscosity of the samples at 40 °C and 100 °C was determined according to ASTM D445 (https://www.astm.org/d0445-24.html (accessed on 2 August 2024)). These two kinematic viscosity values were also used to determine the viscosity index (VI), for which an online calculator [36,37] was used.

The rheological characterization of the raw material (RCA) and of the synthesized estolide esters with caffeic acid in toluene was carried out using a Brookfield Ametek DV2 rheometer (AMETEC Brookfield, Middleboro, MA, USA), with the outer cylinder fixed and the inner cylinder (SC4-21) rotating at a known speed, under temperature-controlled conditions. The device measures the torque generated by the annular layer of material introduced between the two cylinders. Experimental determinations were carried out in the temperature range of 40–70 °C, at shear rates between 0.93 s⁻¹ and 65.1 s⁻¹.

2.6. DSC Analysis

The thermal behavior of the synthesized reaction products and starting materials (as reference) was characterized by differential scanning calorimetry (DSC). DSC analyses were performed using the DSC 204 F1 Phoenix differential scanning calorimeter (Netzsch, Germany), in a nitrogen atmosphere, in the temperature range −70 °C to 200 °C, with a heating rate of 10 °C/min. Samples were weighed in sealed crucibles, and the lids were pierced so no pressure built up inside the crucible during the potential evaporation process of the samples.

2.7. Biodegradation Studies

Biodegradation studies of the oligoester were carried out in accordance with measurements of the OECD 306 protocols and the OxiTop^® system equipped with measuring units (amber glass bottles (510 mL) and self-check measuring units), an inductive stirring platform and magnetic stirrer bars. In each bottle, there was 327.5 mL of the salt solution (prepared in accordance with OECD 306); 1 mL of DMSO or sample dissolved in DMSO; and 36.5 mL of sea water (inoculum) to reach a final concentration of 100 mg/L. The BOD measurements were performed at 21 ± 1 °C. The BOD and Dt (biodegradability) were calculated as previously reported [26,38].

Biodegradability was assessed through the biochemical oxygen demand (BOD) consumed by aerobic microorganism in order to metabolize the oligomers containing ricinoleic acid and caffeic, determined using an OxiTop^® system from Xylem Analytics, Weilheim, Germany. This system is equipped with a sensor to measure the BOD. The biodegradability assessment for the synthesized oligomers was performed based on their solubility in DMSO, following the standardized protocol ISO 17556:2019 [39]. The measurements were taken in an aqueous culture medium using an inoculum sourced from natural river water, specifically the Bega River in Timisoara, Romania. The methodology described by Zappaterra et al. [40] was used in the preparation of the aqueous culture medium, which included a salt solution and liquid inoculum. The sample was dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 100 mg/L in the liquid medium and was tested in duplicate. The sample was then incubated at 21 °C under magnetic stirring for 21 days and compared against a control sample that contained the same amount of DMSO but did not include the oligomeric material. Measurements of the dissolved oxygen were taken every 24 h.

The BOD values for the oligoester sample (BOD_s) and the control samples (BOD_c) were initially expressed in milligrams per liter (mg/L) and later converted to a weight-based measure in the mg/mg unit (BOD_d) for the biodegradability analysis, as defined in Equation (1):

$${BOD}_d={\displaystyle \frac{{BOD}_s-{BOD}_c}{c}}(\mathrm{mg}/\mathrm{mg})$$

(1)

In this equation, BOD_d represents the biochemical oxygen demand required to degrade the sample over a specified number of days (d) in mg/mg; BOD_s is the biochemical oxygen demand measured for the OxiTop^® bottle containing the sample in mg/L; BOD_c is the biochemical oxygen demand measured for the control bottle without the sample in mg/L; and c is the sample concentration in the aqueous medium in mg/L.

The BOD_d values were then compared with the theoretical oxygen consumption (TOD) for each sample, calculated using Equation (2):

$$TOD={\displaystyle \frac{16·\left(2C+0.5H-O\right)}{M_n}}(\mathrm{mg}/\mathrm{mg})$$

(2)

In this equation, 16 represents the molecular weight of oxygen in mg/mmol, while C, H, and O denote the weight fractions of carbon, hydrogen, and oxygen present in the chemical structure of the sample, M_n is the molecular weight of the sample expressed in mg/mmol.

Finally, the ultimate degree of biodegradability (D_t) was expressed as a percentage using Equation (3):

$$D_t={\displaystyle \frac{{BOD}_d}{TOD}}·100(\%)$$

(3)

3. Results and Discussion

3.1. Synthesis of Ricinoleic Acid by Saponification of Castor Oil

The ricinoleic acid (RCA) used as bio-based monomer was obtained by a well-known chemical method, saponification of castor oil followed by acidification with concentrated HCl. The fatty acid composition (as methyl esters) of the purified product was assayed by gas chromatography (Figure S1) and the calculated RA content was 88%, similar to that of most commercial products.

The physico-chemical characteristics of the obtained RCA were determined by standard methods [41], as presented below:

• Color: intense brown-yellow;
• Melting point: 5.5–6 °C;
• Boiling point: 246 °C;
• Density at 20 °C: 940 kg/m³;
• Acid index: 186.34 mgKOH/g;
• Iodine index: 85.23 gI₂/100 g;
• Refractive index at 20 °C: 1.4694.

These values of the physico-chemical properties are close to those reported in the literature for RCA obtained by a similar procedure or calculated for pure ricinoleic acid [42]. A more advanced purification of RCA by vacuum distillation (4–5 mm Hg) could not be achieved because of the small differences in volatility compared with the accompanying fatty acids and the ease to undergo, when heated, secondary reactions to form oligoesters (estolides) due to the carboxyl and hydroxyl functional groups and the C = C double bond present in its structure.

3.2. Enzymatic Synthesis of Oligo (Ricinoleate) Aromatic Esters

The enzymatic synthesis of ricinoleic acid-based aromatic oligoesters provides a sustainable and efficient approach to oligomeric products with unique properties. Using lipase as a biocatalyst, the synthesis process involves the esterification of ricinoleic acid under mild conditions, which not only preserves the integrity of the monomer structure but also minimizes the environmental impact compared to traditional chemical methods.

In this study, three different aromatic compounds (caffeic acid, cinnamyl alcohol and synapic acid) were investigated as substrates for the synthesis of aromatic oligoesters of ricinoleic acid. The reaction conditions (biocatalyst, molar ratio, temperature, solvent, reaction time) were selected based on our previous results concerning oligoesters of hydroxy acids [20] and some preliminary experiments. These preliminary experiments evidenced that the immobilized lipase from Pseudomonas stutzeri, the most efficient for the oligomerization of ricinoleic acid [20], was not effective for the endcapping esterification with an aromatic acid, unlike the immobilized lipase from Candida antarctica B (Novozyme 435), which was consequently selected for these studies.

A general reaction scheme is presented in Figure 1.

The reactions were performed in toluene for 72 h, as described in the Methods Section. Monoesters of RCA with the aromatic alcohol or acid used in the reaction are also possible to be synthesized, as well as homo-oligomers of RCA. The products were not purified for the removal of the RCA estolide.

3.3. Structural Characterization of the Estolide Ester Products

3.3.1. FT-IR Analysis

FT-IR spectrometry can provide structural information that is needed to certify the formation of the ester bonds, particularly of the aromatic esters at the carboxy or hydroxy end of the ricinoleic acid oligomer. Figure 2 presents the superimposed FT-IR spectra of the reaction product of RCA with caffeic acid and of the starting materials. The FT-IR spectra of the other two ricinoleic estolide aromatic esters, with sinapic acid and cinnamyl alcohol, respectively, are shown in the Supplementary Materials (Figures S2 and S3), together with the assignment of the relevant absorption bands.

The broad ν_(O-H) stretching bands confirm the presence of aliphatic and aromatic hydroxyl groups in the product. The phenolic OH stretch is usually recorded at higher wavenumbers compared to the OH stretching vibration of aliphatic alcohols. The two rounded shape peaks in the 3200–3400 cm⁻¹ region of the product can be assigned to the aromatic OH group of the caffeic acid moiety, demonstrating the esterification with this acid. In the same time, the strong ν_(C-H) stretching bands at 2923.8 cm⁻¹ in both the RCA and product spectra indicate the presence of the aliphatic chains of the ricinoleic acid moiety also in the product. The shift of the ν_ (C = O) stretching vibration band to 1731.9 cm⁻¹ from 1708.7 cm⁻¹ of the carboxyl group in the RCA spectrum and 1641.2 cm⁻¹ of the carboxyl group in the caffeic acid spectrum confirms the formation of an ester product. The bands in the 1450–1600 cm⁻¹ region can be assigned to the aromatic ν_(Ar) and olefinic ν_(C = C) stretching vibrations as also being identified in the FT-IR spectrum of caffeic acid. The ν_(C-C-O) stretching vibrations in the ester linkages were identified at 1211.17 cm⁻¹ in the product`s spectrum.

The FT-IR spectra demonstrate the enzymatic synthesis of oligoesters from ricinoleic acid and caffeic acid. The changes in the spectra, such as the shift in the carbonyl stretching, the decrease in hydroxyl group intensity and the appearance of new ester C-O stretching peaks provide clear evidence of esterification. This transformation suggests that the product contains ester linkages, confirming the successful formation of oligoesters.

3.3.2. MALDI-TOF MS Analysis

The formation of reaction products was monitored by MALDI-TOF MS spectrometry. Examples of MALDI-TOF MS spectra are represented in Figure 3, Figure 4, Figure 5 and Figure 6, for the products obtained in toluene, using commercial Candida antarctica B immobilized lipase (Novozyme 435). In all spectra, the identified products were linear copolymers. Thus, the series of peaks attributed to the potassium adducts of the oligomer series ([M + K]⁺) indicate the formation of linear copolymers containing RCA and aromatic moieties. Being new products, the spectra of all three oligoricinoleate esters will be presented and discussed. A table with the selected molecular weights of the reaction products identified in the MALDI-TOF MS spectrum and the theoretical values of the mases are included in Table S1.

As a control reaction, ricinoleic acid was used as monomer in the absence of aromatic moieties, in similar reaction conditions. The resulting oligoesters exhibited a degree of polymerization ranging from 2 to 8 units, demonstrating the successful synthesis of linear copolymers. The correspondent MALDI TOF MS spectrum is presented in Figure 3. As an example, the peaks with m/z 937.2, 1217.4, 1498.4 correspond to the 2K⁺ adducts of linear estolides with 3 ÷ 5 RCA units.

The MALDI-TOF MS spectra of the reaction products obtained from caffeic acid and RCA is presented in Figure 4. The peaks with m/z 818.4, 1099.4, 1380.7 correspond to the 2K⁺ adducts of linear oligoesters with 1 ÷ 3 RCA units and one caffeic acid unit. The highest polymerization degree for these products was 6.

The MALDI-TOF MS spectra of the reaction products obtained from sinapic acid and RCA are presented in Figure 5. The peaks with m/z 583.0, 862.7, 1144.2 correspond to the 2K⁺ adducts of linear oligoesters with 1 ÷ 3 RCA units and one sinapic acid unit. The maximum polymerization degree for these products was 3. The spectrum also shows visible adducts with 1K⁺.

The MALDI-TOF MS spectra of the reaction products obtained from cinnamyl alcohol and RCA is shown in Figure 6. The peaks with m/z 735.0, 1015.4, 1296.6 correspond to the 2K⁺ adducts of linear co-oligoesters with 1 ÷ 3 RCA units and one cinnamyl alcohol unit. The maximum polymerization degree for these products was 4. Interestingly, the formation of estolides was also observed in this case.

The medium molecular weights and dispersity values are presented in

Table 1. The average molecular weights and dispersity values calculated based on the MALDI-TOF MS analysis of the oligoricinoleate esters.

Endcapping Compound	Mn [g/mole]	Mw [g/mole]	ĐM
-	1104	1258	1.14
Caffeic acid	1003	1184	1.18
Sinapic acid	821	898	1.09
Cinnamyl alcohol	965	1035	1.07

. Compared to the estolides, the values obtained for the aromatic oligoesters were lower. The M_n of the oligoester with caffeic acid was about 9.2% lower compared to the simple estolides, while the M_w was about 5.9% lower and the dispersity (Đ_M) was slightly higher by 3.5%. The polymer with sinapic acid had an M_n that was 25.6% lower and an M_w that was 28.6% lower than the estolide, with a Đ_M that was also 4.4% lower, indicating the most significant reduction in molecular weights associated with the most uniform distribution.

The polymer with cinnamyl alcohol showed a 12.6% lower M_n and a 17.7% lower M_w than the estolide, with a 6.1% lower Đ_M. Overall, the use of the endcapping aromatic co-monomers reduces the M_n and M_w of the polymers, with sinapic acid and cinnamyl alcohol producing more uniform distribution of the oligomeric species (lower ĐM), whereas caffeic acid introduces greater variability in chain lengths.

3.3.3. Characterization of the Estolide Ester Products by NMR Spectroscopy

¹H NMR spectroscopy has been utilized for the structural analysis of the reaction products of ricinoleic acid with the aromatic acids and alcohol, respectively, to demonstrate the formation of the targeted aromatic estolide esters. As new compounds, the most significant signals that enable the structural identification will be discussed below for each of the three product types.

In the ¹H NMR spectrum of oligo (ricinoleyl) caffeate shown in Figure 7, the most relevant signals were attributed as follows: δ (ppm): 4.30–4.00 ppm (m) to the methylene protons (COO-CH₂) adjacent to the ester linkage; aromatic protons of the caffeic acid moiety are seen at δ (ppm): 7.00–6.80 (m) and δ (ppm): 6.70–6.50 (m); olefinic protons in the caffeic acid moiety are at δ (ppm): 6.50–6.20 (m); olefinic protons of the ricinoleic acid moiety are found at δ (ppm): 5.50–5.30 (m). In addition, the hydroxymethyl protons (CH₂OH) in the ricinoleic acid moiety correspond to the signals at δ (ppm): 3.60–3.40 (m), while the methine proton (CH-OH) resonates at δ (ppm): 3.40–3.20 (m). The signals at δ (ppm): 2.60–2.20 (m) are assigned to the allylic protons (CH₂) in the ricinoleic acid chain.

The assignment of the most relevant NMR signals for oligo (ricinoleyl)-cinnamate depicted in Figure S4 is δ (ppm): 7.46–7.22 ppm (m) and corresponds to the five aromatic protons on the benzene ring from cinnamyl alcohol; δ (ppm): 6.59 ppm (d) is attributed to the olefinic proton adjacent to the phenyl group; δ (ppm): 6.39–6.41 ppm (d) represents the olefinic proton near the ester linkage; δ (ppm): 5.38–5.30 ppm covers the olefinic protons in the ricinoleic chain; δ (ppm): 4.78–4.69 ppm (t) relates to the methylene protons next to the ester linkage; and δ (ppm): 2.78–2.69 ppm is the allylic protons adjacent to the double bond in the ricinoleic chain.

The ¹H NMR spectrum of the reaction product of ricinoleic acid and sinapic acid, Figure S5, and the most relevant signals are δ (ppm): 6.90–6.80, attributed to the aromatic protons from sinapic acid; δ (ppm): 6.60–6.50 ppm, attributed to the olefinic protons in the sinapic acid moiety; δ (ppm): 5.50–5.30, attributed to the olefinic protons in the ricinoleic acid moiety (shifted compared to the previous spectra); δ (ppm): 4.20–4.00, attributed to the methylene protons (CH₂) adjacent to the ester linkage; δ (ppm): 3.60–3.40 ppm, attributed to the hydroxymethyl protons (CH₂OH) from ricinoleic acid; δ (ppm): 3.40–3.20, attributed to the methine proton (CH) adjacent to the hydroxyl group in the ricinoleic acid moiety; δ (ppm): and 2.50–2.20, attributed to the allylic protons adjacent to the double bond in the ricinoleic chain.

3.3.4. Thermal Analysis by Differential Scanning Calorimetry (DSC)

The thermograms of the same reaction product of RCA with caffeic acid were recorded from −70 °C up to 200 °C, in two cycles (Figure 8). The DSC thermogram does not show any melting or crystallization processes. Both the heating (purple line) and cooling (blue line) curves show a slight drift in the baseline, reflecting the change in specific heat, most likely due to the temperature change. On the heating thermogram, a weak endothermic process can be observed at around −40 °C, probably due to some rearrangements in the solid structure with changes in intermolecular forces. These processes take place in a temperature range due to the different lengths of the oligoester molecules, which allow different spatial arrangements and implicitly different intermolecular cohesion forces. These thermal properties provide important insights into the stability and potential applications of the synthesized ester product.

3.3.5. Rheological Characterization

Rheological characterization of the synthesized estolide ester products is helpful for the future formulation of cosmetic products that could include these oligoesters. The study was accomplished for the caffeic acid ester of the RCA estolide. The rheological behavior of the ricinoleic acid monomer was also studied, for comparison. The rheological properties are essential for understanding the flow properties necessary for different unit operations, as well as for blending with other constituents of possible cosmetic compositions.

The kinematic viscosity and viscosity index values of the starting ricinoleic acid and the obtained oligomerization product are presented in

Table 2. Rheological properties of the estolide ester product synthesized from ricinoleic acid and caffeic acid, compared to the ricinoleic acid used as a raw material.

Sample	Kinematic Viscosity, cSt		Viscosity Index
	40 °C	100 °C
RCA	142.5	10.6	29
Oligo(ricinoleyl)-caffeate	200.6	14.7	61

.

It is observed that both samples fall into the category of oils with a medium viscosity index [43].

For ricinoleic acid, the relationships between shear stress τ and shear rate $\dot{\mathit{\gamma }}$ at three temperature values, shown in Figure S6, are linear. This behavior is characteristic of Newtonian fluids, which are fluids in which viscosity is independent of the shear rate [44]. The rheological equations obtained using the Table Curve 2D program are presented in

Table 3. Rheological equations of ricinoleic acid.

Temperature, °C	$\mathbf{Herschel}–\mathbf{Bulkley} \mathbf{Model}: \mathit{\tau }={\mathit{\tau }}_0+\mathbf{k}·{\dot{\mathit{\gamma }}}^{\mathit{n}}$	R2
40	$\tau =0.215+0.128·{\dot{\gamma }}^{1.009}$	0.999975
55	$\tau =0.22+0.062·{\dot{\gamma }}^{1.003}$	0.999908
70	$\tau =0.24+0.033·{\dot{\gamma }}^{1.002}$	0.99961

. However, from the expression of these equations, it can be seen that the dependence τ = f($\dot{\mathit{\gamma }}$) is better represented by the Herschel–Bulkley model, where τ₀ is the yield stress below which there is no flow, k is the consistency index and n is the power index [45].

It can be observed that increasing the temperature leads to a slight increase in the yield stress and a decrease in the consistency index without changing the nature of the rheological behavior. The values of power index, slightly higher than 1, suggest that the flow is shear thickening [45], which means that the apparent viscosity increases with the increase in shear rate, after the yield stress value has been exceeded.

For ricinoleic acid, the variation in the apparent viscosity with the shear rate, at three temperature values, is depicted in Figure 9.

It is found that the thickening effect is slightly more visible at lower temperatures (40 °C), and as the temperature increases, the apparent viscosity values remain approximately constant (Newtonian behavior, the power index n is very close to 1).

The τ = f($\dot{\mathit{\gamma }}$) dependences, at 40 °C and 70 °C, for ricinoleic acid and the estolide ester synthesized with caffeic acid in toluene (RCN-CT) are shown in Figure 10.

As results from Figure 9, in the case of estolides the dependencies are linear, and a small yield shear stress is present, which places them in the class of Herschel–Bulkley fluids. It can also be observed that, at the same temperature, the estolides synthesized in toluene have higher shear stress values compared to ricinoleic acid. This increase in viscosity is the result of the significant increase in molecular mass, along with the increase in the degree of oligomerization, as well as of structural changes due to the introduction of benzene rings in the structure of estolides. The characteristic rheological equations of the two types of estolides are given in

Table 4. Rheological equations of the ricinoleic estolide esters with caffeic acid in toluene.

Temperature, °C	$\mathbf{Herschel}–\mathbf{Bulkley} \mathbf{Model}: \mathit{\tau }={\mathit{\tau }}_0+\mathbf{k}·{\dot{\mathit{\gamma }}}^{\mathit{n}}$	R2
40	$\tau =0.2+0.177·{\dot{\gamma }}^{1.015}$	0.999987
55	$\tau =0.2+0.0865·{\dot{\gamma }}^{1.009}$	0.999945
70	$\tau =0.19+0.0495·{\dot{\gamma }}^{0.985}$	0.99982

.

From the characteristic rheological equations of estolides, it can be seen that, in their case, an increase in temperature leads to a decrease in the consistency index, while the values of the yield shear stress remain approximately equal.

At 40 °C, the variation in apparent viscosity with the shear rate for the analyzed samples, after the yield stress value has been exceeded, is shown in Figure 11. It is found that at the same temperature, the thickening effect is more pronounced to estolides than to the ricinoleic acid.

3.4. Biodegradability Study

The biodegradability of the oligoesters synthesized with ricinoleic acid and caffeic acid was evaluated by monitoring the biochemical oxygen consumption (BOD) in an aqueous environment at 24 h intervals over a period of 21 days after exposure of the sample to the metabolic activity of an inoculum collected from the Bega River in Timisoara, Romania. The BOD of the sample was measured against a control sample without the oligoester using an OxiTop^® control system and following the standardized protocol of ISO 17556:2019. Both the theoretical oxygen demand (TOD) and the percentage biodegradation relative to TOD were calculated. This respirometry method allowed a direct correlation between the BOD consumption over time and the biodegradability percentage of the sample, since aerobic microorganisms consume a certain amount of oxygen when they break down the polymer into different metabolites. The BOD is one of the key parameters for assessing biodegradability, reflecting the extent of decomposition of organic matter in the environment.

The degree of biodegradability (Dt) was expressed as a percentage of the degradability measured at intervals of 5, 10 and 21 days, as shown in

Table 5. The biodegradability degree D_t calculated for the oligoesters of ricinoleic acid and caffeic acid, after 5, 10 and 21 days of incubation.

ThOD a [mg/mg]	BOD5 [mg/mg]	Dt5 [%]	BOD10 [mg/mg]	Dt10 [%]	BOD21 [mg/mg]	Dt21 [%]
2.156	0.276	13	0.432	20	0.652	30

^a ThOD—the percentage composition of C, H, N and O elements was calculated considering 3 ricinoleic acid molecules and 1 caffeic acid molecule.

. After an incubation period of 21 days, the OxiTop^® system measurements showed that the ricinoleic acid oligomer with caffeic acid exhibited a good degree of biodegradability, reaching up to 30% biodegradation with a recorded BOD of 64.6 mg/L. The biodegradation process starts rapidly, with 13% of the oligoester degraded after only 5 days of incubation, while 20% of the sample underwent a biodegradation process after 10 days. This result may be related to the presence of ester linkages in the oligomeric structure, as biodegradation of polymeric materials is more likely to occur in compounds containing ester groups in their chemical architecture due to their high susceptibility to hydrolysis in an aqueous environment [46]. In addition, average molecular weight plays an important role in the biodegradation process, so lower molecular weight oligomers are generally more susceptible to metabolism by microbial enzymes. This increased susceptibility increases the rate of microbial degradation, resulting in faster degradation.

Therefore, the efficient biodegradation of these oligoesters is essential because of its implications for the creation of ecofriendly materials. Compounds that rapidly degrade in the environment can help reduce the accumulation of nonbiodegradable waste and alleviate harmful effects on ecosystems. Monitoring the biochemical oxygen consumption, analyzing molecular mass and examining the chemical structure of the estolide esters offer important insights into their environmental degradation processes, thereby supporting the efforts toward environmental protection and sustainability.

4. Conclusions

The results contribute to the development of sustainable and biodegradable methods for synthesizing cosmetic ingredients. The enzymatic approach aligns with the increasing demand for green and natural products, offering an eco-friendly alternative to traditional chemical synthesis. In this respect, the present work demonstrated the possibility of synthesizing new estolide esters with new functionalities by capping the hydroxy or carboxy end groups with aromatic acids or alcohols, using a commercially available immobilized lipase. These findings were validated by the structural characterization of the new oligomers using FT-IT, NMR and MALDI-TOF MS, as well as by the investigation of their thermal, rheological and biodegradability properties.

The successful enzymatic synthesis of oligoesters from ricinoleic acid and aromatic acids or alcohols using lipases not only highlights the potential of biocatalysis in green chemistry but also paves the way for the development of innovative, sustainable materials for the cosmetic industry. Since estolides are already products with industrial interests as lubricants, plasticizers, emulsifiers or moisturizers, with applications in the automotive, cosmetic and food industries [21], we presume that the new aromatic functionalities could provide a wider practical utilization range. This study underscores the importance of integrating biotechnological advances with environmental stewardship, contributing to the broader goal of sustainable development.