From Waste to Value: Phenolic Content and Antioxidant Potential in Cistus ladanifer Residues via Solid–Liquid and Subcritical Water Extraction ^†

Abstract

The aim of this work was to extract phenolic compounds (PCs) from Cistus ladanifer L. post-distillation residues using two different methods (solid–liquid extraction (SLE) and subcritical water extraction (SWE)) and to compare the extracts’ total phenolic content (TPC) and antioxidant activity (AA) by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^•+) scavenging activities, as well as by the ferric-reducing antioxidant power (FRAP) assay. SWE extraction displayed a higher TPC value (increased from 146.53 ± 11.68 to 276.37 ± 20.59 mg gallic acid equivalents (GAEs)/g extract dry weight (dw)) and higher AA in the DPPH^• (increased from 334.27 ± 36.06 to 532.17 ± 66.38 mg Trolox equivalents (TEs)/g extract dw), ABTS^•+ (increased from 438.07 ± 77.22 to 594.08 ± 33.57 mg TEs/g extract dw), and FRAP (increased from 10.91 ± 2.03 to 170.26 ± 25.36 mg ascorbic acid equivalents (AAEs)/g extract dw) assays. These results demonstrate the importance of the extraction method in PC extraction and the antioxidant power of the extracts produced. These results provide critical insights into the potential application of C. ladanifer post-distillation residues and the production of polyphenol rich extracts that might be useful in the food, cosmetic, and pharmaceutical sectors.

1. Introduction

Cistus ladanifer L. is a shrub belonging to the Cistaceae family that is widely distributed in the Mediterranean region [1]. C. ladanifer can grow in a wide range of climates, as it is very resistant to cold, dryness, and high temperatures [2]. Its leaves are coated with a highly aromatic resin, called labdanum, which is also one of the common names given to this shrub. Both labdanum and the essential oil have high importance in the perfume industry, being present in about 30% of modern perfumes [1]. C. ladanifer is used in traditional medicine as an anti-inflammatory, an antiulcerogenic, an antimicrobial, a wound healing, a cytotoxic, or a vasodilation agent [3]. Typically, essential oils are extracted by steam distillation, and approximately 100 tons of C. ladanifer essential oil are produced annually [4]. Its post-distillation residues are, however, underutilized.

In this study, the total phenolic content (TPC) and antioxidant activity (AA) of C. ladanifer extracts were evaluated. The C. ladanifer post-distillation residues were used for extraction by two different methods: solid–liquid extraction (SLE) and subcritical water extraction (SWE). SLE relies on the use of expensive, volatile, flammable, and often toxic organic solvents [5]. SWE is an alternative technique, where water, an environmentally friendly solvent, is used. SWE utilizes water at temperatures and pressures below its critical point (Tc = 374.15 °C, Pc = 22.1 MPa) [6,7]. As the water temperature increases, its dielectric constant drops, weakening hydrogen bonds and giving it properties closer to those of lower-polarity solvents such as methanol or ethanol. This allows SWE to dissolve both polar and less-polar compounds, improving extraction efficiency when compared to SLE [6,7]. Moreover, SWE is faster and more selective, as temperature and pressure adjustments allow for the precise extraction of compounds based on their polarity [6]. Research indicates that SWE yields higher TPC and stronger antioxidant activity against DPPH^• for extracts derived from spent coffee grounds and winery waste compared to conventional techniques [8,9].

In a previous study, we investigated the TPC and AA of several agrifood wastes, and C. ladanifer displayed promising results [10]. This study provides the first direct comparison between SLE and SWE for the extraction of PCs from C. ladanifer post-distillation residues and the AA of the produced extracts. It will provide information on the influence of the extraction method on the production of phenolic-rich extracts from C. ladanifer. These may have potential applications for value-added compounds in the food, pharmaceutical, and cosmetic industries.

2. Materials and Methods

2.1. Samples and Extractions

C. ladanifer post-distillation residues (Figure 1) were kindly donated by Naturalness Essential Oil Distillery (Louriçal do Campo, Portugal). C. ladanifer leaves were dried in a dehydrator under 41 °C until less than 10% moisture remained and stored in the dark until further use. Ultrapure water (with a resistivity of 18.2 MΩ/cm) was acquired using a Milli-Q water purification system from Millipore (Molsheim, France).

Solid–liquid extractions (SLEs) were performed at two different conditions: (1) SLE40 °C—a biomass/solvent ratio of 1 g:50 mL of 50:50 H₂O:MeOH (v/v), T = 40 °C, and t = 1 h; (2) SLE60 °C—a ratio of 1 g:100 mL solvent (50:50 H₂O:MeOH (v/v)), T = 60 °C, and t = 1 h. The extracts were filtered using paper filter (FILTER-LAB^®, Barcelona, Spain), and the solvent mixture was evaporated in a rotary evaporator. For further assays, the samples were redissolved in 50:50 H₂O:MeOH (v/v). The temperatures for extraction were chosen based on a previous study [10].

Subcritical water extractions (SWEs) were carried out using a Parr Series 4560 Reactor controlled by a Parr 4848 Reactor Controller (Moline, IL, USA). Two sets of extraction conditions were tested based on the range of pressures and temperatures used previously [11]: (1) SWE at 100 °C with a biomass-to-solvent ratio of 2 g to 200 mL ultrapure water, operating at 60 bar; (2) SWE at 150 °C with the same biomass-to-solvent ratio of 2 g to 200 mL, also at 60 bar. Following extraction, the resulting extracts were filtered through a paper filter (FILTER-LAB^®, Barcelona, Spain) and subsequently lyophilized. The dried samples were then reconstituted in water.

2.2. Total Phenolic Content (TPC) and Antioxidant Activity

TPC was determined using the Folin–Ciocalteu method, following the procedure described by Macedo et al. [12], with minor modifications. A calibration curve was prepared using gallic acid (10 to 200 mg/mL). Measurements were carried out in triplicate using a microplate reader (Synergy HT, Biotek Instruments, Winooski, VT, USA). The results were expressed as mg gallic acid equivalents (GAEs)/g extract dw.

The antioxidant capacity was evaluated using DPPH^• scavenging, ABTS^•+ scavenging, and FRAP assays, adapted from Macedo et al. [12], with slight adjustments. For the DPPH^• assay, samples or standards (25 µL) were mixed with 200 µL of DPPH^• solution and incubated for 30 min in the dark. Absorbance was read at 517 nm. The results were expressed as mg Trolox equivalents per gram (mg TEs/g dw). For the ABTS^•+ assay, samples or standards (20 µL) were combined with 180 µL of ABTS^•+ solution and incubated for 6 min. Absorbance was measured at 734 nm. The results were also reported as mg TEs/g dw. For the FRAP assay, samples or standards (20 µL) were added to 180 µL of FRAP reagent and incubated for 4 min at 37 °C. Absorbance was read at 593 nm. The results were expressed as mg ascorbic acid equivalents per gram (mg AAEs/g dw). All measurements were performed in triplicate using a Synergy HT microplate reader (Biotek Instruments, Winooski, VT, USA).

2.3. Statistical Analysis

The data were expressed as the mean ± standard deviation based on at least three independent replicates. To compare the TPC and AA, a one-way ANOVA, followed by Tukey’s post hoc test, was performed using GraphPad Prism (version 8.0.1). Differences were considered statistically significant when the p value was less than 0.05.

3. Results

Total Phenolic Content and Antioxidant Activity

The results obtained for the TPC are presented in

Table 1. The TPC values and antioxidant activity of the C. ladanifer extracts.

Sample	TPC (mg GAEs/g dw)	DPPH• (mg TEs/g dw)	ABTS•+ (mg TEs/g dw)	FRAP (mg AAEs/g dw)
SLE40 °C	175.24 ± 21.82 b	334.27 ± 36.06 b	438.07 ± 77.22 c	10.91 ± 2.03 c
SLE60 °C	146.53 ± 11.68 b	381.02 ± 85.18 b	453.80 ± 41.23 c	11.64 ± 2.96 c
SWE100 °C	276.37 ± 20.59 a	532.17 ± 66.38 a	594.08 ± 33.57 a	170.26 ± 25.36 a
SWE150 °C	256.05 ± 32.61 a	475.73 ± 50.42 a	520.42 ± 17.50 b	132.39 ± 27.96 b

Abbreviations: dw—dry weight; GAEs—gallic acid equivalents AAEs—ascorbic acid equivalents; TEs—Trolox equivalents. Different superscript lowercase letters correspond to statistically significant differences at p < 0.05.

. The SWE displayed a higher TPC value than SLE (p < 0.0001). This demonstrates that SWE is a better extraction method than SLE. SWE100 °C displayed a higher numerical value than SWE150 °C, with no statistically significant difference (p = 0.2974). SLE40 °C displayed a higher numerical value than SLE60 °C, with no statistically significant difference (p = 0.1885).

The results obtained for the antioxidant activity assays are presented in Table 1. SWE displayed higher antioxidant activity in every assay, particularly in FRAP (p < 0.0001). SWE100 °C displayed better results than SWE150 °C in every assay, although the DPPH^• scavenging did not show a significant difference between both SWE conditions. In the ABTS^•+ (p = 0.0217) and FRAP (p = 0.0004) assays, SWE100 °C displayed significantly higher activity than SWE150 °C.

4. Discussion

The use of SWE resulted in higher amounts of PCs being extracted and increased AA in all assays. The different results obtained can be explained by a combination of processes. For one side, the efficiency of SWE goes beyond that of conventional solvent extraction at room temperature. As the temperature increases, water’s dielectric constant, viscosity, and surface tension decrease, while its diffusivity improves. These changes enhance the ability of hot pressurized water to break solute–matrix interactions, thereby promoting the release and transfer of solutes. Furthermore, at elevated temperatures and pressures, water exhibits properties like those of organic solvents, enabling the extraction of not only polar compounds but also medium-polar, low-polar, and non-polar substances. High pressure also allows water to penetrate the micro-pores of the sample matrix—something not possible under atmospheric conditions [7]. Another aspect is related to the drying method used for SLE and SWE extraction, which may impact PC stability. Studies have reported that PCs can be impacted by light, and the exposure to pressure and heat in a rotary evaporator can cause the modification and/or degradation of PCs [13]. Although, in the current study, heat exposure and light exposure have been minimized in the solvent evaporation process (40 °C and protected from light), freeze-drying is regarded as the process of choice to ensure high bioactive ingredient stability. This is because it is carried out in negative-temperature conditions and at a significantly reduced pressure (1–50 Pa) [14]. Moreover, research studies have demonstrated that the Folin–Ciocalteu reagent interacts with other compounds besides phenolic compounds, overestimating TPC values. Elevated temperatures in SWE can trigger Maillard and caramelization reactions, leading to the formation of numerous reaction products, including melanoidins, which have been found to have antioxidant properties [15]. This may account for the observed increase in AA in the SWE, although the highest temperature tested was lower than some used in previous studies in order to minimize the Maillard reaction [11]. Tavares et al. [16] performed extractions from distillation by-products of C. ladanifer using ethanol and 70% acetone and achieved TPC values of 177.5 ± 0.2 and 275.6 ± 0.0 mg GAEs/g extract, respectively. These extracts displayed scavenging activity for ABTS^•+ of 1.8 ± 0.1 and 4.1 ± 0.0 mmol TEs/g extract. El Karkouri et al. [2] produced extracts from the whole plant (leaves and flowers) by various methods, with values ranging between 3.85 and 182.00 mg GAEs/g extract, with the highest value obtained for a hydromethanolic extraction. Bouothomany et al. [17] produced extracts from C. ladanifer leaves with four different solvents (ethyl acetate, ethanol, dichloromethane, n-hexane). The TPC ranged from 67.366 ± 5.745 to 76.066 ± 9.978 µg GAEs/mg dry extract, with the highest value obtained for the dichloromethane extract. The authors also assessed the AA of the extracts and reported IC₅₀ values in the DPPH^• ranging from 825.17 ± 11.18 to 266.6 ± 0.8288 µg/mL, with the lowest value obtained for the ethanolic extract. Abdelfattah et al. [3] produced aqueous and methanolic extracts from C. ladanifer leaves and obtained TPCs of 21.18 ± 2.1 and 69.08 ± 1.69 mg GAEs/g dw, respectively. The authors also assessed the AA and obtained higher results for the methanolic extract (IC₅₀ of 0.115 ± 0.003 mg/mL, compared to 0.149 ± 0.004 mg/mL in the DPPH^• assay; IC₅₀ of 0.251 ± 0.006 and 0.517 ± 0.04 mg/mL in the ABTS^•+ assay; 62.74 ± 0.07 and 44.66 ± 0.08 mg TEs/g dw in the FRAP assay for the methanolic and aqueous extracts, respectively). As can be seen from different studies, different extraction methods and solvents can produce extracts with significant differences in PC content and antioxidant activity. Furthermore, the climatic conditions in which C. ladanifer plants are grown may influence the secondary metabolites produced.

5. Conclusions

Two different methods (SLE and SWE) were used to produce extracts from C. ladanifer post-distillation residues. A direct comparison of the extracts’ TPC and AA, assessed by DPPH^• and ABTS^•+ scavenging activities and ferric-reducing power, was established between SLE and SWE. SWE displayed higher TPC and AA than SLE in all assays. Using a temperature of 100 °C displayed better results than using 150 °C for the ABTS^•+ and FRAP assays, with statistically significant differences. These results offer valuable insight into the valorization of C. ladanifer wastes. The extraction method and the extraction conditions used can greatly impact the obtained extracts and can be tailored to specific applications. Further research is necessary to optimize the extractions, characterize the phenolic profile by LC-MS, and understand the influence of the conditions in which the plants are grown, which influence the quantity of secondary metabolites produced. This knowledge may enhance the use of phenolic-rich C. ladanifer wastes in industries such as food, pharmaceuticals, and cosmetics.