Recovery of chemical components from Spondias mombin L. leaves using pressurized hot water, ultrasound and turbo-extraction techniques

Abstract

The fruit of Spondias mombin (cajazeira) is widely consumed in the northeast region of Brazil. In this work, three different extraction methods, namely ultrasound-assisted extraction (UAE), turbo-extraction (TE) and pressurized hot water extraction (PHWE), were evaluated in order to investigate the potential of cajazeira leaves as an alternative source of bioactive compounds. The extraction methods were compared in terms of yield, chemical composition and total phenolic content (TPC) of the extracts. The highest yields and TPC values were obtained by TE with a mixture ethanol: water (70:30%, v/v) as the solvent. PHWE has not yet been applied for the extraction of cajazeira leaves. Thus, it was evaluated as one alternative for the recovery of phenolic compounds under conditions of 80, 100 and 120 °C and 100 and 120 bar. A modeling study of PHWE kinetics was investigated at 80 °C and 200 bar. Furthermore, a biorefinery approach considering integrated processes to recover bioactive compounds was investigated and the results showed that combining processes may enhance the valorization of agricultural waste.

Key words
extraction; Spondias mombin; phenolic compounds; pressurized hot water

INTRODUCTION

The development of processes involving green chemistry, which aim to overcome the difficulties related to food and environmental issues, should receive even more attention in future industrial processes. The circularity and co-responsibility in food processing may correspond to the feasibility of using new sources of compounds of interest in food production chains (Wang et al. 2018). Thus, the development of processes and attention to food safety are crucial towards the use of economically viable and sustainable extraction technologies. High pressure extraction techniques, like carbon dioxide supercritical fluid extraction (CO₂-SFE) and pressurized hot water extraction (PHWE, also known as subcritical water extraction, SWE) are excellent alternatives, as they use non-toxic solvents, easily separated from products, and allow a selective recovery of different types of bioactive compounds (Herrero & Ibáñez 2016, Koyu et al. 2017). The use of CO₂-SFE has been extensively investigated in the last decades in the extraction, recovery and isolation of natural products, among various other applications, like textile dyeing, enzymatic reactions, impregnation, and chemical treatment of different materials (Brunner 1994, Pereira & Meireles 2010, Assis Filho et al. 2013, Cristofoli et al. 2019, Banožić et al. 2020, White et al. 2021, Torres et al. 2022). PHWE has been used to recover compounds of interest from different matrixes and agroindustrial residues, such as algae (Zakaria & Kamal 2016), green tea (Ko et al. 2014), carrot leaves (Song et al. 2018), kiwi (Guthrie et al. 2020), chaya (Rodrigues et al. 2020), soursop leaves (Santos et al. 2023), wakame seaweed (Gan & Baroutian 2022) and cotton flowers (Xu et al. 2021).

Water is a vital and widely available resource, also considered a universal solvent. Under room conditions, water has high polarity and a high dielectric constant (ɛ = 79). The variety of compounds extracted by water can increase with the manipulation of temperature and pressure in the extractive process. The pressure ensures that the water remains in the liquid phase, while the temperature above the normal boiling point (100 °C) and below the critical point (374 °C) enhances its extraction properties due to the temperature effect on its physicochemical properties. In this state, water changes its solubilization capacity, as a function of the change in the dielectric constant and may be able to reach values observed at room temperature for ethanol (ɛ = 25) and methanol (ɛ = 33) (Singh & Saldaña 2011, Mohsen-nia & Amiri 2013).

Spondias mombin L. (Anacardiaceae) is a native species of the Amazon region, which covers about eight countries in South America; the species also occurs in the West Indies (Adedokun et al. 2010). In Brazil it is popularly known as “cajazeira” and is especially cultivated in the northeast region. “Yellow mombin” or “hog plum” are additional denominations found in the literature (Silva et al. 2020). S. mombin fruits are widely consumed as juice. On the other hand, its leaves are only considered an agroindustrial waste in production areas.

The medicinal use of cajazeira leaf extracts in folk medicine is attributed to its anti-inflammatory, antiviral, and antioxidant activities (Silva et al. 2012, 2020). The extracts of this species also have been reported to present gastroprotective, anxiolytic, and antidepressant effects (Sampaio et al. 2018, Soares et al. 2019). Antimicrobial activities against bacteria, such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, are also related in the literature (Rey-Blanes et al. 2020).

All these benefits are usually attributed due to the presence of several phenols obtained in different extracts, including phenolic acids, flavonoids, and ellagitannins (Ishola et al. 2018, Silva et al. 2020). In order to recover these high-added value components from cajazeira leaves, a biorefinery approach may be considered as a promising alternative by using several extraction methods, like Soxhlet, hydrodistillation, maceration, and ultrasound. These methods may have some drawbacks, including the use of high temperatures, very long extraction times and the need for large amounts of potentially toxic solvents.

For these reasons, PHWE was investigated to recover target compounds from cajazeira leaves. So far, PHWE of S. mombin leaves has not yet been reported in the literature. Therefore, the present study compares the results obtained by use of PHWE with results from two extraction methods, turbo-extraction (TE) and ultrasound assisted extraction (UAE). The extracts were characterized and a mathematical modeling study was carried out to evaluate the PHWE kinetics using models presented in the literature. Additionally, solid residues (raffinate samples) obtained from PHWE experiments were collected and submitted to sequential TE and UAE experiments to investigate their potential in the recovery of high-added chemical compounds from cajazeira leaves.

MATERIALS AND METHODS

Materials

Cajazeira (Spondias mombin L.) leaves were collected in the municipality of Limoeiro, Pernambuco, Brazil (7°55’37.8”S, 35°30’18.2”W). A sample was identified and deposited in the herbarium of the Instituto Agronômico de Pernambuco (IPA-PE) under access number 90169. The access to the species was registered in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen) under the number A8C17C4.

Cajazeira leaves were dried using an air circulation oven (Lucadema, Model 82/480, Brazil) for 7 days under a temperature of 40 °C (up to a humidity of 10.5±0.4%). A knife mill (Marconi, Model MA340, Brazil) was used to crush the material, which was then packed in polyethylene pots and stored at room temperature. The particle size used to form the fixed bed of particles was determined by using the software ImageJ 1.8.0 (NIH, Maryland, USA). The moisture content was determined using an infrared balance (Marte Científica, Model ID-V.18, Brazil), using 1 g of the herbal material at a temperature of 130±2 °C until constant weight. The assay was performed in triplicate and the results were expressed in percentage (%).

All chemicals used were of analytical grade and purchased commercially from Sigma-Aldrich (Darmstadt, Germany). Experiments with water were conducted using ultra-pure water obtained from one Milli-Q system (Millipore Merck, Darmstadt, Germany).

Turbo-extraction (TE)

TE experiments were performed using water, ethanol and a mixture ethanol: water (70:30%, v/v). The methodology was the same as presented by Machado et al. (2021): four extraction cycles were carried out in an industrial blender (Metvisa, Model LAR.2, Brazil) using 15 g of solid residue and 300 mL of solvent, i.e., a solid:solvent ratio of 1:20 (w/v). Each TE cycle lasted 30 s at 18,000 rpm, followed by a “rest” period of 4 min. All experiments were carried out in triplicates. The extracts obtained were separated from the solid residue by vacuum filtration and stored in amber flasks at -18 °C until the analysis.

Ultrasound Assisted Extraction (UAE)

The UAE experiments were performed using an ultrasound probe (Ultronique, Model QR500, Brazil) with frequency and power of 18 kHz and 297 W, respectively. In each extraction, 5 g of solid material was used with 100 mL of solvent (water, ethanol and the ethanol: water mixture 70:30%, v/v) at room temperature (25±1 °C) for 15 min (Machado et al. 2021). All experiments were carried out in triplicates. Filtration of the extracts obtained took place under vacuum, and the extracts were stored in amber flasks at -18 °C before analysis.

Pressurized Hot Water Extraction (PHWE)

Pressurized hot water extractions (PHWE) were carried out in a self-assembled and customized experimental apparatus (Figure 1), described previously by Santos et al. (2023). Briefly, 10 g of cajazeira leaves were used to form the extraction bed, with the addition of glass beads to fill the extractor vessel (100 mL AISI 316 stainless steel, with internal dimensions of 25 mm in diameter and 180 mm in length). The solvent (water) was pumped at a rate of 3 mL/min, at temperatures of 80, 100 and 120 °C. Pressure levels of 100 and 200 bar, below the water critical pressure, were investigated and the solid/solvent ratio was held at 1:20 (m:v) during 60 min of extraction. All PHWE were conducted in duplicates and the experimental conditions evaluated were based on the work by Santos et al. (2023) and by equipment limitations (maximum solvent flow rate allowed). The extracts obtained were stored in amber flasks at (-18 °C) for later analysis.

Figure 1
Scheme of experimental apparatus for PHWE of cajazeira leaves: 1-Water supply; 2-Solvent pump; 3-Pressure gauge; 4-Water preheater; 5-Jacketed extractor vessel; 6-Temperature sensor; 7-Micrometering valve; 8-Sampling.

The remaining solid residues (raffinates) from PHWE experiments were dried following the same procedures used with the raw material and stored in amber flasks to avoid deterioration. The raffinates were then submitted to TE and UAE experiments using the ethanol: water mixture 70:30% (v/v) as solvent at room temperature (25±1 °C). The procedures for TE and UAE were the same described above for each extraction technique.

Extraction yields

The extraction yields were calculated as the percentage of mass of extract (m_Extract ) relative to total mass of raw material, on a wet basis (m_RM ). The m_Extract was determined, from triplicate values, by using an infrared scale (Marte Científica, Model ID-V.18, Brazil), with 5 mL of the extract solution at 130 ± 2 °C until constant weight.

Analytical methods

Total phenolic content (TPC)

The total phenolic content (TPC) was determined spectrophotometrically by the Folin-Ciocalteu method, according to Machado et al. (2021) methodology. Briefly, 20 μL of the sample solution, 5 mL of distilled water, and 0.5 mL of the Folin-Ciocalteu reagent (Sigma-Aldrich, USA) were mixed in a volumetric flask, and the volume was adjusted to 10 mL with a sodium carbonate (Na₂CO₃, 7.5% w/w) solution. The analyses were performed in triplicates, with distilled water used as blank. A 30 min reaction time was carried out at room temperature, in the absence of light. The absorbance of the solutions was measured at 760 nm in a spectrophotometer (Evolution 60S, Thermo Fisher Scientific, USA). The results were expressed in mg of gallic acid equivalent (GAE) per gram of sample (mg of GAE.g^-1).

High-performance liquid chromatography (HPLC)

The samples for HPLC analysis were used in a concentration of 1.2 mg/mL. The standards were used as follows: rutin and chlorogenic acid were prepared using methanol: water (1:1, v/v), ellagic acid in methanol: water 3:2 (v/v), and gallic acid in ultrapure water. All solutions were filtered into vials with 0.45 μm PVDF Chromafil filters. Identification and quantification were performed in a liquid chromatograph (Ultimate 3000, Thermo Fisher Scientific, USA) with a diode array detector (DAD, 3000RS), according to the methodology described by Machado et al. (2021). The separation was made using a C₁₈ column (250 mm × 4.6 mm, 5 μm; Dionex®) protected by a C₁₈ precolumn (3 mm × 4 mm, 3.9 μm, Phenomenex). The mobile phase was composed of ultrapure water as solvent A and HPLC grade methanol as solvent B. Both solvents were acidified with 0.05% trifluoroacetic acid. Elution was carried out in gradient mode, as follows: 0 to 10 min (20-30% B); 10 to 24 min (30-40% B); 24 to 30 min (40-75% B); 30 to 34 min (75-15% B) and 34 to 37 min (15% B), at a flow rate of 0.8 mL/min. The data were acquired by Chromeleon 6.0 software at 270 nm.

Extraction kinetics study for PHWE: extraction modeling

In order to obtain the overall extraction curve (OEC) of S. mombin leaves with pressurized hot water, two models reported in the literature were used: the models of Martínez et al. (2003) and Esquível et al. (1999). Martínez et al. (2003) presented an applied multicomponent solute model, which was used to evaluate the extraction process of ginger oleoresin. The mass transfer rate was characterized by a logistic equation. In their study, Esquível et al. (1999) assessed the supercritical CO₂ extraction of olive husk oil. The extraction curves were analyzed using an empirical model and the relationship between the yield and extraction time, as predicted by this model, exhibits a similar pattern to the variation of adsorbed mass with adsorbate pressure described by the Langmuir isotherm.

Statistical analysis

One-way analysis of variance (ANOVA) was performed using the Software Statistica (Statsoft Inc., USA). The Tukey test was used to assess significant differences (p < 0.05).

RESULTS AND DISCUSSION

Raw material

As described previously, the plant material (cajazeira leaves) was characterized in terms of moisture content and particle size. The results showed that the dried and ground leaves presented a moisture content of 10.56±0.40% and a mean particle diameter of 255 μm. According to Chemat & Vian (2014), the reduction of particle size can provide higher yields in extraction processes, but very small diameters may lead to an excessively compact bed of particles and, consequently, generate undesirable effects on the process. Values between 3 and 10% moisture generally present no negative impact on the mass transfer during extraction processes. In this moisture range, an expansion of the cellular structure of the material occurs, facilitating mass transfer between the solvent and the solid material.

Extraction yields and Total Phenolic Content (TPC)

The extraction yields and TPC values obtained from cajazeira leaves are presented in Table I. Turbo-extraction (TE), ultrasound-assisted extraction (UAE) and pressurized hot water extraction (PHWE) techniques were evaluated. As described previously, the solvents employed were ethanol (EtOH), water (H₂O) and an ethanol: water mixture (70:30%, v/v) (EtOH/H₂O). All TE and UAE experiments were conducted at room temperature and atmospheric pressure. PHWE was performed under six different temperature and pressure conditions.

Table I
Experimental conditions, yields and TPC results obtained from cajazeira leaves extracts and solid residues from PHWE experiments.

SampleA	MethodB	SolventC	Temperature (°C)	Pressure (bar)	Flow rate (mL-min-1)	Extraction timeD (min)	Extraction yieldE (%)	TPCE (mg GAE-g− 1)
	TE	H2O	-	-	-	2	0.76b ± 0.000	29.035c ± 0.003
	TE	EtOH	-	-	-	2	0.25ef ± 0.000	19.999d ± 0.041
	TE	EtOH/H2O	-	-	-	2	1.30a ± 0.042	79.298a ± 0.006
	UAE	H2O	-	-	-	15	0.26ef ± 0.064	12.196e ± 0.008
	UAE	EtOH	-	-	-	15	0.11f ± 0.028	7.712f ± 0.005
RM	UAE	EtOH/H2O	-	-	-	15	0.80b ± 0.014	50.421b ± 0.038
	PHWE 1	H2O	120	200	3	60	0.57cd ± 0.021	1.641gh ± 0.002
	PHWE 2	H2O	120	100	3	60	0.42de ± 0.007	1.382h ± 0.004
	PHWE 3	H2O	100	200	3	60	0.45d ± 0.028	1.372h ± 0.001
	PHWE 4	H2O	100	100	3	60	0.41de ± 0.099	1.536gh ± 0.001
	PHWE 5	H2O	80	200	3	60	0.64bc ± 0.021	1.977g ± 0.001
	PHWE 6	H2O	80	100	3	60	0.45d ± 0.049	1.371h ± 0.001
SR-HTE	TE	EtOH/H2O	-	-	-	2	-	2.047g ± 0.006
SR-LTE	TE	EtOH/H2O	-	-	-	2	-	1.700h ± 0.017
SR-HUA	UAE	EtOH/H2O	-	-	-	15	-	2.003g ± 0.006
SR-LUA	UAE	EtOH/H2O	-	-	-	15	-	1.663h ± 0.015

• ^A RM: raw material extractions; SR-HTE: solid residue of the highest TPC value obtained by PHWE experiments reprocessed by TE; SR-LTE: solid residue of the lower TPC value obtained by PHWE experiments reprocessed by TE; SR-HUA: solid residue of the highest TPC value obtained by PHWE experiments reprocessed by UAE; SR-LUA: solid residue of the lower TPC value obtained by PHWE experiments reprocessed by UAE.
  ^B TE: turbo-extraction; UAE: ultrasound assisted extraction; PHWE: pressurized hot water extraction.
  ^C EtOH/H₂O: ethanol: water mixture (70:30 %, v/v).
  ^D Extraction time in TE: 4 cycles of 30 s each with agitation and 4 min rest time.
  ^E Values sharing the same superscripts are not significantly different from each other at a confidence level of 95%.

Table I shows that the highest extraction yield was obtained by TE using the EtOH/H₂O mixture, 1.30±0.042%. This result occurred probably because TE is a technique with high stiring and grinding speed, where shear forces break the solid material cells, enhancing the dissolution of solutes into the solvent (Martins & Ferreira 2017). It is well known that ethanol presents amphiphilic properties, which results in a capability to dissolve simultaneously both hydrophobic and hydrophilic components. Thus, ethanol possesses the ability to extract bioactive compounds with a wide range of polarity. When using an hydroethanolic mixture, a change in the polarity of the system is suitable for extracting bioactive compounds with high polarities. This behavior was observed by Mazzutti et al. (2017) when obtaining bioactive extracts of Plantago major and Plantago lanceolata, using UAE with different solvents (hexane, methanol, ethanol, 70:30% (v/v) ethanol: water mixture). The ethanol: water mixture provided higher yields of extraction. Ahmed et al. (2021) extracted phenolic compounds from Moringa oleifera leaves; when using 80:20% (v/v) hydroethanolic mixture, the highest yield was obtained. They stated that the water-ethanol synergy effect had contributed to the higher solubilization of bioactive components.

The assays that obtained yields closer to the value obtained for TE EtOH/H₂O (1.30±0.042%) were TE H₂O, UAE EtOH/H₂O and PHWE 5. The values obtained by these three techniques showed no statistically significant difference (0.76, 0.80 and 0.64%, respectively). Comparing these techniques, it was noticed that the use of hydroethanolic solvent generated higher extraction results.

The TE EtOH, UAE EtOH and UAE H₂O experiments resulted in the lowest yields, respectively 0.25, 0.11 and 0.26%, with no significant difference among these values. The UAE technique, using ethanol and water as solvents, had the lowest extractive capacity in terms of yield. Similar results were obtained by Machado et al. (2017), where the recovery of anthocyanins from residues of Rubus fruticosus, Vaccinium myrtillus and Eugenia brasiliensis by UAE presented lower yields in comparison with the other extraction techniques (pressurized liquid and Soxhlet).

When considering only pressurized experiments (PHWE), it was observed that, at the lowest temperature and highest pressure (80 °C and 200 bar, i.e., experiment PHWE 5), the highest yield was obtained (0.64±0.021%). It was possible to observe that for the other temperature and pressure conditions, there was no significant difference in yields, ranging from 0.41 to 0.57%. Given the temperature and pressure ranges investigated in this work, PHWE did not provide high extraction yields. The factors that may have contributed to this result were the polarity of the solvent and the effect of the dielectric constant, which drops dramatically as the water is heated isobarically, reducing the intensity of the electric fields present in the medium, thus reducing the solvent dissolution capability. The ability of water to dissolve compounds present in S. mombin leaves may have been reduced because of the lower dielectric constant of water under the conditions investigated (Peterson et al. 2008).

The total phenolic contents (TPC) values varied from 1.371 to 79.298 mg GAE.g^-1. When comparing the TPC values with the extraction yields obtained for TE and UAE (low pressure experiments), a correlation between these two parameters can be observed, i.e., the higher the extraction yield, the higher the TPC obtained (Table I).

The TE EtOH/H₂O experiments obtained the highest TPC (mean value of 79.298±0.006 mg GAE.g^-1 extract), which was statistically different from the contents found for the other investigated extraction techniques. The second highest TPC was also found at room pressure (UAE EtOH/H₂O, mean value of 50.421±0.038 mg GAE.g^-1), also significantly different from the other listed values. The hydroethanolic solvent favored the extraction of phenolics present in cajazeira leaves, i.e., both the yield of dry extract and the extraction of phenolic compounds were higher when the mixed solvents were used. Similar results (higher TPCs) were observed by Dias et al. (2019) in the extraction of umbu (Spondias tuberosa) oil using a 70:30%(v/v) hydroethanolic mixture by UAE. The results were attributed to the polarity of the solvent, which provided the solubilization of a wider range of components from the herbal material.

The existence of a relationship between increasing TPC and increasing dry extract yields could be partially observed among PHWE experiments: PHWE 1, PHWE 4 and PHWE 5 showed the highest TPC contents, ranging from 1.37 to 1.97 mg GAE.g^-1 extract, not differing significantly from each other. Low values were obtained, possibly, because water as a high pressurized solvent was not suitable to extract the phenolic contents present in cajazeira leaves under the investigated operational conditions.

The use of PHWE in the extraction of cajazeira leaves and the recovery of bioactive compounds from extraction residues (PHWE raffinate samples) has not yet been reported in the literature. Cristofoli et al. (2019) evaluated the antioxidant and antimicrobial potential of cajazeira leaves by using supercritical CO₂ as solvent (ethanol as co-solvent) and Soxhlet and ultrasound techniques (room pressure) with hexane, ethanol and a hydroethanolic solution as solvents. The TPCs found varied between 12 and 410 mg GAE.g^-1 extract, with the highest value obtained for the Soxhlet technique with ethanol as solvent. Machado et al. (2021) performed the extraction of cajazeira leaves using the turbo-extraction technique with hydroethanolic solvent in different ratios, also varying the mass of plant material used. The highest TPC obtained was 204.8 mg GAE.g^-1 extract, using 15 g of the plant material and 80% hydroethanolic solvent. They observed that the content of polyphenols depended on the proportion of ethanol, i.e., the hydroethanolic mixture with the higher alcohol content favored the recovery of phenolic compounds from cajazeira leaves. The variations in the TPC values, however, may depend on numerous abiotic factors. Akula & Ravishankar (2011) stated in their study on the synthesis and accumulation of secondary metabolites in plants, that these variations are influenced by several abiotic factors, such as temperature, moisture, salinity, and alkalinity of the soil. The characteristics of each species can also strongly influence the chemical profiles of different extracts, and the comparison of quantitative results must always be made with caution.

Rufino et al. (2010) reported that the main bioactive compounds found in Brazilian fruit species are classified according to the content of phenols in three categories: low, for contents lower than 10 mg GAE.g^-1 extract; medium, for contents from 10 to 50 GAE.g¹ extract; and high, for contents higher than 50 GAE.g^-1 extract. According to this classification, TE EtOH/H₂O was the only technique that achieved high TPC. UAE H₂O, UAE EtOH/H₂O, TE H₂O, and TE EtOH provided medium TPC values. UAE EtOH, on the other hand, along with all PHWE assays, achieved low TPCs.

TPC analysis of the solid residue reprocessed from PHWE

TPC values of the solid residues (SR, also denoted as raffinate samples) reprocessed from the experiments with higher and lower TPCs (PHWE 5 and PHWE 6) are also presented in Table I. The SRs were submitted to treatments with turbo-extraction and ultrasound, using the hydroethanolic solution as the extraction solvent to recover compounds of interest that were not completely exhausted from the solid material (cajazeira leaves) under the experimental conditions investigated. The highest TPC value found was for SR-HTE, not statistically different from the residue reprocessed by UAE (SR-HUA).

The TPC value obtained was higher than that obtained in experiment PHWE 5 (2.047 mg GAE.g^-1 extract), indicating that TE (EtOH/H₂O) presented a higher extractive capacity for this herbal material. The use of hydroethanolic solvent in TE experiments favored the extraction of compounds not completely removed by hot water, indicating that the polarity of this solvent has considerable influence on the recovery of TPC from cajazeira leaves. Thus, the hydroethanolic mixture used as solvent can be considered a suitable alternative for the recovery of phenolic compounds.

Table I shows that SR-HUA experiments presented a mean TPC value of 2.003 mg GAE.g^-1 extract. This value was slightly lower that the obtained from the SR-HTE experiments. The SR reprocessing with UAE (EtOH/H₂O) provided an additional recovery of phenolic compounds, but not as efficient as the SR reprocessing with TE (EtOH/H₂O). The same trend could be observed when using the SR obtained from PHWE 6 experiments as starting material. Reprocessing of the residue provided higher TPC values for TE (EtOH/H₂O); and the application of UAE (EtOH/H₂O) over SR provided better results compared to PHWE 6 experiments. Thus, reprocessing of SR proved that only PHWE was not effective in the complete recovery of phenolics from cajazeira leaves at the evaluated experimental conditions.

Cristofoli et al. (2019) also investigated the reprocessing of SR derived from supercritical CO₂ extraction of cajazeira leaves. The authors obtained a high TPC content of the reprocessed residue, indicating that the residue from pressurized fluid extractions of cajazeira leaves still possessed compounds that could be extracted. The PHWE under the conditions studied in this work, followed by the reprocessing of raffinate samples (SR), proved to be an alternative in the recovery of the phenolic compounds contained in the plant material.

Extract composition profiles

The chemical components identified by HPLC as key-components of cajazeira leaves extracts and their respective concentrations for all extraction techniques are presented in Table II. The extract from cajazeira leaves is rich in phenolic acids, like gallic, ellagic, and chlorogenic acids, and also contains rutin, a very beneficial flavonoid. Similar results were obtained by Machado et al. (2021), who detected the presence of these same phytochemicals in extracts of cajazeira leaves. The same compounds were also identified by other authors (Sampaio et al. 2018, Aromolaran & Badejo 2014) through maceration with organic solvents (methanol, ethanol and acetone), water and ethanol: water mixtures.

Table II
Phenolic profiles of cajazeira leaves extracts (µg∙g−¹ of extract) obtained by TE, UAE and PHWE.

MethodA	SolventB	Gallic acidC (µg∙g− 1)	Ellagic acidC (µg∙g− 1)	Chlorogenic acidC (µg∙g− 1)	RutinC (µg∙g− 1)
TE	H2O	0.2735b ± 0.030	0.1410de ± 0.014	0.1050c ± 0.003	0.3245ab ± 0.016
TE	EtOH	0.0745de ± 0.001	0.1090ef ± 0.010	0.0320e ± 0.000	0.2625bc ± 0.002
TE	EtOH/H2O	0.1100d ± 0.000	0.4795b ± 0.026	0.1255b ± 0.006	0.3390a ± 0.014
UAE	H2O	0.2230c ± 0.017	0.1770d ± 0.020	0.0690d ± 0.001	0.1830d ± 0.052
UAE	EtOH	0.0705de ± 0.001	0.1340def ± 0.003	0.1875a ± 0.009	0.1935d ± 0.002
UAE	EtOH/H2O	0.0990de ± 0.001	0.4090c ± 0.013	0.0975c ± 0.001	0.2445d ± 0.008
PHWE 1	H2O	0.7490a ± 0.001	0.9560a ± 0.03	0.0300e ± 0.003	-
PHWE 2	H2O	0.0705de ± 0.001	0.0925f ± 0.001	0.0010f ± 0.000	0.0340e ± 0.000
PHWE 3	H2O	0.0745de ± 0.001	0.0995ef ± 0.001	0.0020f ± 0.000	0.0435e ± 0.001
PHWE 4	H2O	0.0695e ± 0.001	0.0930f ± 0.001	0.0010f ± 0.000	0.0385e ± 0.002
PHWE 5	H2O	0.0755de ± 0.002	0.0940f ± 0.001	0.0003f ± 0.000	0.0475e ± 0.001
PHWE 6	H2O	0.0700de ± 0.001	0.0940f ± 0.003	0.0010f ± 0.000	0.0355e ± 0.001

• ^A TE: turbo-extraction; UAE: ultrasound assisted extraction; PHWE: pressurized hot water extraction.
  ^B EtOH/H₂O: ethanol: water mixture (70:30 %, v/v). Also: see Table I for PHWE operational conditions.
  ^C Values sharing the same superscripts are not significantly different from each other at a confidence level of 95%.

Table II shows that the most effective technique for the extraction of gallic and ellagic acids was PHWE (0.7490 and 0.9560 μg/g extract, respectively) at the highest pressure and temperature levels investigated, i.e., 200 bar and 120 °C. For chlorogenic acid the highest mean value was obtained using UAE with ethanol as solvent (0.1875 μg/g extract). According to Wang et al. (2019), this compound is an ester of caffeic and quinic acids, with high molar mass, presenting lower affinity for solvents with high polarity, such as water, and higher solubility in ethanol. Besides this factor, the UAE technique possibly provided higher values due to the non-thermal effect of the acoustic cavitation bubbles of the ultrasonic bath in the solvent, which favored the formation of microscopic channels in the structure of the tissues of the leaves, facilitating the extraction (Alves Filho et al. 2020).

The highest rutin fractions were obtained using TE with the hydroethanolic mixture (EtOH/H₂O) as solvent, achieving a mean value of 0.3390 μg/g extract. In this work, TE was the technique that most favored the extraction of rutin. This may be attributed to the maximized shear forces present in this technique due to agitation, which favored the rapid dissolution of the component in the investigated solvents.

Manousi et al. (2019) stated that there is no single standard method for extraction of bioactive compounds from plants because the effectiveness of the techniques depends on parameters such as the amount of raw material used, the natural availability of the plant matrix, the type of chemical structure of the bioactive compounds and the solvents used. Besides presenting important bioactive actions, the extracted compounds also stand out for their high added value. This statement justifies the use of agroindustrial wastes, such as S. mombin leaves, and extraction process residues as starting materials in the recovery of important phenolic compounds.

PHWE kinetic analysis and mathematical modeling

The kinetic evaluation of PHWE was performed using the experimental data obtained at 80 °C and 200 bar (PHWE 5, Table I), where the highest TPC and extraction yield values were obtained (1.977 mg GAE.g^-1 extract and 0.64%, respectively).

Through the kinetic experimental curve of the cumulative TPC content (TPC_Cum) as a function of extraction time for PHWE of cajazeira leaves (Figure 2), it was possible to clearly identify two stages of the high-pressure extraction process. The constant extraction rate step (CER) was observed during approximately the first 20 minutes of extraction, obtaining a TPC_Cum content of 0.208 g, equivalent to 63.4% of the total extracted (0.328 g). This TPC content is in agreement with the literature, where 50 to 90% of the solute is usually obtained in this step (Pereira & Meireles 2010). The falling extraction rate step (FER) occurred then from 20 to 60 minutes of extraction; 0.119 g of TPC_Cum (equivalent to 36.54% of the total extracted) was obtained. This behavior was expected, considering that, with increasing extraction time, the solutes are being depleted from the solid matrix (cajazeira leaves). The diffusional period (the final extraction step) was not investigated because it would require a longer extraction time. According to Duba et al. (2015), a 60 min extraction time is considered satisfactory for an optimal recovery of phenolics and other components from PHWE experiments, avoiding unnecessary use of solvents and energy.

Figure 2
Kinetic experimental curve of cumulative TPC extraction yield as a function of time for PHWE of cajazeira leaves and modeled curves for 60 min extraction. Experimental conditions were 80 ^oC, 200 bar and flow rate of 3 mL/min.

Figure 2 also presents a comparison between the experimental OEC obtained and the curves obtained by fitting the Martínez et al. and Esquível et al. models. The parameters obtained for both models are shown in Table III.

Table III
Optimized parameters of the Martínez and Esquível models.

Model	ParameterA	Value
Martínez et al.	bi (min-1) tm (min) MSE	0.055 -28.330 4.136×10-5
Esquível et al.	b (min-1) MSE	11.390 7.882×10-4

• ^A b_i, t_m, b: adjustable parameters; MSE: mean square error.

Both models were able to represent the behavior of the extraction process in this work. The Martínez et al. model presented a better fit to the data, and the lowest mean square error (MSE) was obtained. The Martínez et al. model possesses two adjustable parameters, t_m and b_i , while the Esquível et al. model presents only one, b. This may have also contributed to the better adjustment of the Martínez et al. model. The literature presents several modeling studies, where the model of Martínez et al. has exhibited best fits compared to other models investigated for different plant materials and solvents (Dias et al. 2019, Nascimento et al. 2016, Tramontin et al. 2021, Alves et al. 2023).

CONCLUSIONS

In this study, three different methods were used to obtain extracts from S. mombin leaves. Evaluations of extraction yields, phenolic compounds chemical profiles and TPC were determined, with higher yields and TPC values obtained by TE using the hydroethanolic mixture (1.30% and 79.298 mg GAE.g−¹, respectively). It was possible to identify and quantify four bioactive compounds, namely rutin, gallic-, ellagic-, and chlorogenic acids, which are relevant because they present diverse medicinal uses.

PHWE was investigated as a potential application for the recovery of phenolics at different experimental levels. Although yields and TPC values for PHWE were lower than the values obtained for TE and UAE, PHWE experiments provided satisfactory results when evaluating the extract chemical profiles. The high selectivity of pressurized extraction methods corroborates the use of PHWE for the recovery of phenolic compounds from cajazeira leaves. The kinetic study of PHWE was performed for the experiment PHWE5 with the highest extraction yield and TPC value (80 °C and 200 bar), which were 1.977 mg GAE.g^-1 extract and 0.64%, respectively. The model of Martínez et al. presented the best kinetic data fit.

Finally, a biorefinery approach considering integrated processes to recover bioactive compounds has also been investigated. The reprocessing of SR presented a recovery of phenolic compounds that may be a potential option for the productive sector because it can add economic value to the complete processing chain. This can contribute to an environmentally sustainable process, which is expected and highly desirable.

ACKNOWLEDGMENTS

The authors wish to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, project number 404347/2016-9), Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE, project APQ-0219-3.06/19) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, finance code 001) for the financial support and fellowships. The English text of this paper has been revised by Sidney Pratt, Canadian, MAT (The Johns Hopkins University), RSAdip - TESL (Cambridge University).

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Publication Dates

• Publication in this collection
  25 Oct 2024
• Date of issue
  2024

History

• Received
  26 Feb 2024
• Accepted
  9 July 2024