High-Pressure Extraction Techniques for Efficient Recovery of Flavonoids and Coumarins from Flower Seeds

Abstract

The extraction of bioactive compounds, such as flavonoids and coumarins, from natural sources has gained significant attention due to their potential health benefits. This review aims to explore the application of high-pressure extraction processes, particularly supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), for obtaining flavonoids and coumarins from flower seeds. These techniques offer a greener, more efficient alternative to conventional extraction methods, minimizing the use of harmful solvents and improving the yield and purity of the target compounds. Flower seeds, a rich source of bioactive molecules, are an underutilized reservoir for these valuable compounds. For example, seeds from plants such as Calendula officinalis (calendula) and Helianthus annuus (sunflower) are rich in flavonoids and coumarins. The proposed review will examine the influence of extraction parameters—such as temperature, pressure, solvent choice, and extraction time—on the yield and quality of flavonoids and coumarins. This review aims to provide a comprehensive understanding of high-pressure extraction methods and optimize protocols for the efficient, sustainable extraction of flavonoids and coumarins from flower seeds.

1. Introduction

Flavonoids and coumarins, abundant in flower seeds, play a fundamental role in the physiological and morphological systems of plants [1]. Flavonoids constitute a group of polyphenolic compounds derived from the phenylpropanoid pathway and are characterized by a 15-carbon skeleton structured as C6-C3-C6 [2]. Flavonoids and coumarins are crucial secondary metabolites in plants. Derived from the shikimate and acetate pathways, flavonoids play roles in UV protection, pigmentation, and signaling processes essential for plant reproduction and defense. Coumarins, on the other hand, are involved in allelopathy, helping plants compete for resources by inhibiting the growth of neighboring plants. They also act as signaling molecules in plant–pathogen interactions and contribute to defense against microbial invasion. In a sense, they act as natural antioxidants and anti-inflammatory agents, protecting plants from environmental stresses, such as UV radiation and pathogens, while contributing to the characteristic coloration of flowers and seeds [3]. Contextually, coumarins are characterized as a class of benzopyrone derivatives, with a broad spectrum of bioactivities, including antioxidant, antimicrobial, and anticoagulant properties [4,5].

These compounds not only support plant development and defense mechanisms but are essential for pharmaceutical and industrial applications. In the food industry, flavonoids are incorporated as natural antioxidants in functional foods and beverages to improve health benefits and shelf life. In cosmetics, coumarins are used for their UV-protective and anti-aging properties, while flavonoids contribute to formulations with anti-inflammatory and skin-soothing effects. In pharmaceuticals, flavonoids are recognized for their cardiovascular benefits, while coumarins are employed as anticoagulants, with warfarin being a well-known derivative [6,7,8,9].

The bioactivities of flavonoids and coumarins make them valuable components for a significant industrial spectrum, mainly in the exploration of antioxidant and anti-inflammatory properties, inhibiting a series of complications in human health [10,11,12,13]. Furthermore, flavonoids also have neuroprotective and anti-aging effects, while coumarins contribute to anticoagulant therapies [14]. Also, the association of these compounds indicates the real importance of flower seeds as a resource for the development of therapeutic agents for industrial use.

Accordingly, natural sources, such as flower seeds, are rich in bioactive compounds, playing a key role in health and wellness applications [15]. These compounds, including polyphenols, flavonoids, alkaloids, and terpenoids, exhibit diverse biological activities, such as antioxidant, anti-inflammatory, antimicrobial, and anticancer properties [16]. Their extraction has gained attention in the scientific community due to a growing consumer preference for sustainable and natural products. Accordingly, innovative efficient extraction technologies, such as non-thermal and green technologies, have enabled the preservation of the activity of these compounds, making them valuable for enhancing the efficacy of pharmaceuticals and cosmetic formulations [17,18]. In the pharmaceutical industry, for example, bioactive compounds from seeds have shown promise in the treatment of chronic diseases, inflammation, and complications resulting from oxidative stress [19]. For example, a scientific study on Datura stramonium flower extracts highlighted potent anti-inflammatory agents, which can be used to treat inflammatory disorders [20]. Correspondingly, bioactive peptides and polysaccharides derived from seeds exhibit immunomodulatory and cardioprotective effects, indicating that these components may be interesting candidates for the development of new and effective drugs [21]. Therefore, the combination of these compounds is a potential enhancer of the emergence of eco-friendly and safer alternatives to synthetic drugs.

Recently, the exploration of innovative extraction methods for the recovery of bioactive compounds can overcome many challenges associated with conventional techniques, such as low extraction efficiency, high energy demand, high requirement of chemical solvents, and environmental concerns [22,23]. Traditional methods, such as Soxhlet extraction and maceration, rely heavily on highly toxic and expensive chemical solvents, in addition to requiring long processing and extraction times, which can degrade heat-sensitive bioactive compounds and lead to significant loss of the compounds of interest [17]. Nevertheless, emerging technologies, such as supercritical fluid extraction (SFE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), pressurized liquid extraction (PLE), and the use of natural deep eutectic solvents (NADESs), have shed light on the efficacy of recovering bioactive compounds from plants [24,25,26,27,28,29]. These methods indicate efficiency, productivity, and selectivity [22]. For example, SFE is highly effective in isolating nonpolar compounds using supercritical CO₂, while UAE and MAE accelerate extraction rates by disrupting cell walls using ultrasonic waves or microwave energy, respectively [26,30,31]. EAE exploits enzymes to disrupt plant material, promoting the selective extraction of compounds, such as polysaccharides and phenols [27]. These green techniques utilize optimized parameters to ensure high yields while minimizing environmental impact, making them favorable for sustainable applications [32]. Additionally, these techniques are highly selective and allow for the recovery of a range of target compounds, making them highly efficient for industrial-scale applications [23].

Nonetheless, advanced extraction technologies still present many challenges to be overcome, such as high initial investment costs, the requirement for skilled operators, and limitations in scaling up to industrial scale [23]. Optimizing parameters, such as temperature, pressure, and solvent quantification, can also be critical to achieving high yields and significant selectivity [33]. On the other hand, compared to conventional technologies, these methods still provide greater efficiency and productivity, even with high complexity in scaling up their exploration [34].

Accordingly, this review focuses on evaluating the potential of high-pressure extraction methods to isolate bioactive compounds from flower seeds. Furthermore, this comprehensive investigation aims to enhance an understanding of the sustainable and effective extraction of flavonoids and coumarins, shedding light on interesting insights into the potential of flower seeds as a resource for bioactive compounds. For the preparation of this review, the primary article search platforms were utilized, and studies containing relevant information on high-pressure extraction techniques and the recovery of flavonoids and coumarins from flower seeds were selected.

2. High-Pressure Extraction Techniques

2.1. Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction is a widely used technique for extracting compounds of interest from a solid matrix. In the process, the fluid used is submitted to conditions above its critical point of pressure and temperature in order to improve its physical–chemical properties and enhance the solubilization of predominantly hydrophobic and slightly polar compounds [35]. Figure 1 presents a pressure–temperature diagram of an arbitrary pure substance, showing the critical point (P_c—critical pressure; and T_c—critical temperature) of the distinction between the liquid and gaseous phases of the compound. Above this point, the fluid assumes so-called “supercritical” conditions, combining desirable properties of both phases, making it a “solvent with adjustable properties” to selectively extract different compounds according to variations in pressure and temperature imposed on the system [30].

Supercritical fluid extraction offers significant advantages over the “special state” of supercritical fluid, which combines advantageous properties of its liquid and gas phases. In the supercritical state, the fluid is in a condition with no distinction between liquid and vapor, enabling unique behaviors that maximize process efficiency and selectivity. An interesting enhanced characteristic regarding supercritical fluids is their high density, compared to liquids [24]. This property allows the dissolution of nonpolar and hydrophobic compounds efficiently, similar to what occurs in extraction with typical liquid solvents, allowing the extraction of a wide range of substances [36]. The high diffusivity associated with low viscosities of supercritical fluids, compared to gases, is also a relevant characteristic, as it allows the fluid to penetrate deeply into the matrix of the source material, reaching compounds in difficult-to-access regions, where a common liquid would not be able to penetrate [37]. Since the supercritical fluid moves faster, covering larger extractable areas, extraction time is reduced, improving process productivity [38].

Typically, carbon dioxide (CO₂) is the main compound used in supercritical fluid extraction processes [39,40,41]. With a critical point in relatively mild conditions (T_c = 31.1 °C, P_c = 74.8 bar), the use of CO₂ allows the process to occur under moderate conditions, minimizing the thermal degradation of thermosensitive compounds, such as flavonoids, as well as polyphenols and terpenes [36]. Moreover, supercritical CO₂ has a high density, similar to organic liquids, giving the compound a high solvation capacity for hydrophobic and slightly polar compounds [42]. Another advantage of using this compound is its easy recovery from the medium through a simple depressurization process, allowing its natural separation from the extracted compounds. It is important to emphasize that taking advantage of the selectivity of supercritical CO₂ depends on the control capacity of the operating system aiming to adjust the fluid conditions so that it interacts specifically with the compounds of interest in the biomass matrix. Adjusting the pressure and temperature of the system will directly influence the density and solubility of the fluid, making it more suitable for extracting nonpolar compounds (higher densities) or heavy and hydrophobic compounds (higher solubilities) (Figure 2).

Moreover, SFE is a technique that utilizes supercritical CO₂ to extract bioactive compounds. The process involves heating CO₂ above its critical temperature (31.1 °C) and pressurizing it beyond its critical pressure (73.8 bar), resulting in a supercritical state where it behaves as both a liquid and a gas. This allows CO₂ to penetrate plant material efficiently and dissolve lipophilic compounds (Figure 3).

CO₂ is the most widely used supercritical fluid due to its favorable properties. It is non-toxic, non-flammable, chemically inert, and environmentally friendly. Its moderate critical temperature and pressure make it ideal for preserving heat-sensitive compounds during extraction. Furthermore, CO₂ is readily available, cost-effective, and can be easily removed from the final product, ensuring high purity [43]. To overcome the limitations of CO₂ in solubilizing moderately polar compounds, co-solvents such as ethanol and methanol are commonly used. These co-solvents enhance the extraction efficiency by modifying the polarity of the supercritical CO₂, enabling it to dissolve a broader range of bioactive molecules, including flavonoids and coumarins. For instance, ethanol is frequently used as a green co-solvent due to its safety, compatibility, and effectiveness in extracting polar phenolic compounds [44,45,46].

Carbon dioxide in a supercritical state is a solvent with a nonpolar character. This fact makes it suitable for compounds with more hydrophobic characteristics, such as many coumarins [36,47]. However, it may have limitations in solubilizing moderately polar compounds, such as most flavonoids. Accordingly, the extraction efficiency of these compounds rich in polar hydroxyl groups via the supercritical CO₂ process is associated with the modification of the polarity of the fluid with the addition of co-solvents, such as methanol, ethanol, and water, expanding the range of compounds that can be dissolved [48]. An example can be verified in the work published by Do Dat et al. [49], which evaluated the use of ethanol as a co-solvent in the extraction of flavonoids from Celastrus hindsii leaves with supercritical CO₂. The authors reported an increase from 1.4 to 5.4 mg g⁻¹ in the flavonoid content when 10 mL of ethanol was added to the system. Under optimized conditions, the best yield for these bioactive compounds was obtained at 60 °C, 300 bar, 10 mL of ethanol, and 2 h, obtaining a flavonoid content of 6.51 mg quercetin equivalents per g dry matter. Similarly, Correa et al. [50] evaluated the extraction of flavonoids and phenolic compounds from blackberry seeds (Rubus spp. Xavante cultivar), obtaining at 70 °C, 250 bar, 2.00 g min⁻¹ of supercritical CO₂ plus ethanol, matrix to ethanol mass ratio of 1:1, and 60 min 24.05 wt.% of extract yield containing 411.13 mg (100 g)⁻¹ of rutin, 8.53 mg (100 g)⁻¹ of hesperidin, and 2.43 mg (100 g)⁻¹ of genistein.

2.2. Pressurized Liquid Extraction (PLE)

Under distinct thermodynamic conditions, pressurized liquid extraction is a technique that uses liquid solvents maintained under high pressure and moderate temperatures to extract compounds from solid matrices [51]. In this process, the solvent is kept in a liquid state above its normal boiling point (Figure 1) by pressurizing the system, increasing its capacity to solubilize compounds by improving its diffusivity [52]. Depending on the solvent selected, high pressures usually between 100 and 200 bar are selected, keeping the compound in a liquid state even at temperatures above its normal boiling point [53,54]. This control prevents the transition from fluid to gaseous phase, allowing the solvent to retain its high solvation capacity while benefiting from increased diffusivity at higher temperatures (ranging from 50 to 200 °C) [55].

The system (Figure 4) temperature is a crucial factor to be managed since, when it is controllably increased, it reduces the viscosity and surface tension of the solvent, facilitating penetration into solid matrices and solubility in target compounds [56,57]. The pressure, in turn, stabilizes the liquid phase of the solvent, optimizing extraction by maximizing molecular interactions between the solvent and the solutes of interest. Thus, the combination of pressure and temperature adjusted in the PLE process results in a selective and effective technique for compounds of different polarities.

The PLE process presents several advantages concerning its applicability for the extraction of flavonoids and coumarins due to the chemical and structural characteristics of these compounds [58,59]. As flavonoids and coumarins present a variety of functional groups that confer different polarities to these compounds, PLE allows that, through the selection of appropriate solvents, their solvation power can be adjusted regarding the process temperature, optimizing extraction performance. Thus, polar solvents, such as methanol or ethanol, could be used to improve the solubilization of flavonoids, while less polar solvents, such as acetone, ethyl ether, and toluene, may be more suitable for the extraction of coumarins. Another advantage is that the PLE process typically requires lower volumes of solvent compared to traditional methods [28,60]. High pressures allow more efficient penetrations of the solvent into the solid matrix, extracting desired compounds effectively and reducing the time required to reach solvent saturation. In other words, a smaller amount of solvent will be needed to extract the same amount of solute compared to traditional methods.

The selection of solvents for PLE is crucial. The most commonly used solvents in PLE, and the types of compounds they efficiently extract [28,61,62,63], are as follows: (i) water: effective for hydrophilic phenolic acids; (ii) ethanol: suitable for flavonoids due to moderate polarity; and (iii) ethanol-water mixtures: often used to balance polarity for mixed compounds, enhancing overall extraction yield.

In comparative terms, the PLE process has strengths and weaknesses in relation to supercritical fluid extraction [64,65,66]. Requiring that the solvent be subjected to conditions above its critical point, the supercritical fluid extraction process presents less versatility regarding solvents that can be considered since, usually, highly elevated pressures and temperatures may be necessary. On the other hand, under milder thermal conditions, PLE allows a wide range of solvents that can be adjusted in terms of their polarity as needed to extract specific compounds, such as flavonoids and coumarins. However, highly nonpolar compounds are theoretically easier to extract using supercritical CO₂, where the solvents considered in the PLE process usually have polar characteristics.

In conclusion, PLE uses liquid solvents at high pressures and moderate temperatures, allowing for efficient extraction with shorter times. However, it often requires more solvents and may not preserve heat-sensitive compounds as effectively as SFE. By contrast, SFE, typically using CO₂ as the solvent, operates at a low temperature with high pressure, preserving thermolabile compounds and ensuring solvent-free extracts. However, SFE involves higher operational costs due to the need for specialized equipment.

3. Flower Seeds as a Source of Bioactive Compounds

Plant seeds are a highly promising resource for obtaining large amounts of bioactive compounds, such as flavonoids, coumarins, phenolic acids, alkaloids, and polysaccharides [13]. Among the spectrum of flavonoids, compounds, such as quercetin, catechin, and kaempferol, stand out for their highly antioxidant, anti-inflammatory, and cardioprotective properties [67]. On the other hand, coumarins are also compounds of great interest, indicating antimicrobial, anticoagulant, and anticancer activities, making them valuable in pharmaceuticals and industrial segments [5]. Also, plant seeds have abundant concentrations of phenolic acids and other secondary metabolites that contribute to neuroprotective and human health actions [68].

Nonetheless, despite the high concentrations of bioactive compounds, plant seeds are still underutilized in extraction processes aimed at obtaining compounds of industrial interest [69]. The high resilience of biomaterials significantly affects the recovery and efficient isolation of specific compounds. Moreover, the variability in the composition of compounds and distinct species also slows down advances in the optimization and standardization of extraction procedures [70]. Therefore, emerging techniques, such as SFE and PLE, have shed light on the potential to overcome these barriers, providing higher yields and purity of target compounds compared to conventional processes [63,71]. The gap in the use of flower seeds is also influenced by the high level of exploitation of other more easily accessible plant matrices, such as leaves and fruits.

Flower seeds are an abundant resource of a diversity of bioactive compounds present in high concentrations, which enhance the pharmacological, nutraceutical, and industrial value of a range of plants. The main bioactive molecules in plant seeds include flavonoids, coumarins, phenolic acids, alkaloids, saponins, and polysaccharides. Flavonoids are antioxidants with anti-inflammatory, cardioprotective, and neuroprotective activities [72,73]. Coumarins, identified by their benzopyrone structure, indicate antimicrobial, anticoagulant, antifungal, antiviral, and anticancer properties (see for example [4]). Other abundant bioactive components include phenolic acids, alkaloids, polysaccharides, and carotenoids, such as β-carotene and lutein, contributing to antioxidant and anti-inflammatory activities [74,75,76,77]. Accordingly, harnessing the potential of plant seeds has led to the advancement of emerging eco-friendly extraction technologies with strong potential for industrial use, such as supercritical CO₂-based technologies and enzyme-assisted extraction (see for example [78,79]). These advanced technologies not only make the extraction process more efficient but drastically reduce the impact on the environment [80].

A study involving date (Phoenix dactylifera) seeds indicated high concentrations of phenolic compounds, flavonoids, carotenoids and anthocyanins, which indicated antioxidant and antiradical properties. Subcritical CO₂ extraction intensified the production of compounds, as well as stimulating their bioactivity [81]. Additionally, studies involving different citrus species (Citrus limonia, Citrus deliciosa, Citrus latifolia, Citrus sinensis) subjected to the SFE-CO₂ extraction process and extraction parameters, with pressures up to 250 bar and temperatures of 40–60 °C, indicated high concentrations of flavonoids such as nobiletin, sinensetin and tangerine, coumarin citropten. Yield performances varied by species, with C. sinensis reaching yields of up to 5.32% [36].

Furthermore, a study involving blackthorn fruits (Prunus spinosa) and the ethanol-based maceration process combined with reflux extraction, optimized in 50% ethanol, a temperature of 60 °C, and 30 min of extraction time reported significant amounts of phenolic and flavonoid compounds with strong antioxidant potential [82]. Additionally, an investigation involving Peucedanum decursivum (Miq.) Maxim (P. decursivum) indicated high concentrations of coumarins, such as umbelliferone and xanthotoxin [83]. The combined enzyme-assisted extraction (EAE) process with ultrasonic assistance and deep eutectic solvents (DES) achieved a yield of 2.65%. The optimized conditions included a liquid-to-solid ratio of 14:1 mL g⁻¹, ultrasonic temperature of 60 °C, and 50 min of sonication. Finally, a study involving aerial parts of Pterocaulon polystachyum for coumarin extraction, and optimized the SFE-CO₂ extraction process at 240 bar and 60 °C, reported high concentrations of coumarins with cytotoxic potential against tumor cells [84].

The exploration of plant species using advanced extraction techniques, such as pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE), is a promising area of research. Although remarkable results have been achieved using traditional extraction methods, the unique advantages offered by PLE and SFE—including enhanced efficiency, selectivity, and eco-friendliness—make them ideal candidates for the study of plant matrices. These techniques can be particularly valuable for unlocking the potential of plant seeds, which are known to be rich in bioactive compounds, such as flavonoids, coumarins, phenolics, and essential fatty acids.

Often considered by-products in various industries, plant seeds represent an underutilized source of high-value compounds with potential applications in food, cosmetics, and pharmaceuticals. The ability of PLE and SFE to target specific compounds while minimizing the use of harmful solvents aligns with the growing demand for sustainable and clean-label extraction methods.

Expanding the scope of research to include a broader range of plant species could unveil new sources of bioactive compounds, thus enhancing our understanding of their chemical diversity and bioactivity profiles. Future studies should prioritize the systematic evaluation of plant seeds using PLE and SFE, leveraging the strengths of these methods to optimize extraction yields and compound stability. Such efforts would not only contribute to the discovery of novel compounds but promote the sustainable utilization of plant resources in various industrial sectors.

For example, Nigella sativa is a renowned medicinal plant with a long-standing historical significance, and its black seeds are considered a highly valuable source of therapeutic agents. Their medicinal importance has even been highlighted in religious texts. The antimicrobial properties of N. sativa extracts have been demonstrated against several pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and the pathogenic fungus Candida albicans. Another study confirmed the antimicrobial activity of N. sativa extracts with high sensitivity against a broader range of pathogens, encompassing 11 gram-negative bacteria, 3 gram-positive bacteria, and C. albicans. Additionally, N. sativa oil and its green liqueur exhibit potent antioxidant properties, making them valuable in mitigating oxidative stress-related diseases. The aqueous seed extract of this plant contains a diverse array of phenolic compounds, most of which possess strong antioxidant activities. Key antioxidants identified in N. sativa black seeds include DL-α- and DL-γ-tocopherol, selenium, all-trans retinoids, and thymoquinone, further highlighting its potential as a natural source of bioactive compounds [85].

Nguyen et al. [86] reported the first investigation into the antioxidant properties (including total antioxidant content, reducing power, ferric ion-reducing antioxidant power, hydroxyl radical scavenging, and ferrous ion-chelating assays), anti-tyrosinase activity, anti-inflammatory effects, and hepatoprotective potential of Ochna integerrima (Loureiro) Merrill flowers and seeds in HepG2 cell lines. All extracts, except for n-hexane, exhibited notable antioxidant activity, characterized by high levels of tannins and proanthocyanidins. Using semi-preparative HPLC, four compounds were isolated: (1) luteolin, (2) 6-γ,γ-dimethylallylkaempferol-7-O-β-d-glucopyranoside, (3) 6-γ,γ-dimethylallylquercetin-7-O-β-d-glucopyranoside, and (4) 6-γ,γ-dimethylallyldihydrokaempferol-7-O-β-d-glucopyranoside. Among these, compounds 1–3 displayed significant anti-tyrosinase activity. The aqueous extracts exhibited the strongest hepatoprotective effects, while the flower extracts demonstrated superior anti-inflammatory properties, as evidenced by reduced NO production in RAW 264.7 cells and decreased bovine serum albumin protein. Notably, the n-hexane and EtOAc flower extracts showed promising anti-inflammatory activity, supported by in silico predictions of the activity of compounds 1–4. In conclusion, O. integerrima emerges as a promising natural source for applications in antioxidant, anti-tyrosinase, and anti-inflammatory therapies.

Sunflower seeds (Helianthus annuus) contain flavonoids, such as quercetin and phenolic acids, known for their antioxidant properties. In one study [87], the total phenolic content in sunflower seeds ranged from 99.64 mg GAE/100 g (Kalamis) to 130.75 mg GAE/100 g (Belfis), while the flavonoid content varied between 838.33 mg QE/100 g (Toramis) and 1455.67 mg QE/100 g (LG50.480). Antioxidant activity values were recorded between 1.27 mmol/kg (Pioner MM54) and 1.73 mmol/kg (excluding Bella). The quercetin and rutin contents were identified as 1.18 mg/100 g (Deray) to 28.72 mg/100 g (Toramis) and 0.97 mg/100 g (İsida) to 6.98 mg/100 g (Pioner MM54), respectively, with quercetin and rutin being the most abundant phenolic components. The linoleic acid content in sunflower seed oils ranged from 1.84% (Arsentik) to 69.56% (Jais), and oleic acid content varied from 15.72% (Jais) to 90.95% (Arsentik). Notably, the “Aromatic” and “Arsentik” varieties exhibited the lowest linoleic acid concentrations but the highest oleic acid levels.

Contextually, the extraction of bioactive compounds from plant seeds has been identified as a promising approach in the sustainable and industrial context. Despite significant advances in the parameterization and scale-up of emerging extraction technologies, significant gaps remain, posing several challenges for obtaining valuable compounds. Among the main recent challenges in the complex extraction and obtaining of components of interest from plant seeds, the complexity and resilience characteristic of plant seed matrices stand out, which indicate a dense network of lipids, fibers, and proteins that considerably hinder the recovery of bioactive compounds, such as flavonoids, phenolics, and alkaloids. Furthermore, the scalability of emerging techniques to a wide range of industrial applications remains an obstacle due to high costs and operational specificities. Another approach lies in the limited understanding of the direct association between extraction solvents and plant matrices. Although green processes, such as supercritical CO₂, are promising alternatives, their compatibility with different seed types and their specific characteristics can affect extraction selectivity. Accordingly, addressing these challenges promotes opportunities that are of great interest to the community, such as the integration of conventional and emerging technologies, boosting the potential effectiveness, scalability, and sustainability of extraction for broad industrial use.

4. Influence of Extraction Parameters

Table 1. Operating conditions typically employed for the extraction of flavonoids and coumarins in pressurized processes.

Parameter	Extraction Process		Reference
	Supercritical Fluid	Pressurized Liquid
Temperature (°C)	40–70	60–120	[17,24,28,48,51,57,58,88]
Pressure (MPa)	10–30	5–20
Vol. ratio solvent/matrix (vol vol−1)	10–20	20–30
Flow (mL min−1)	0.1–2.0	1.0–10.0
Time (min)	5–20	30–120

presents the process conditions typically considered for the supercritical fluid (CO₂) and the PLE extraction processes. The adjustment of these parameters must be performed considering the type of matrix used, where the influence of each of them on the yield of the target bioactive compound is influenced according to the discussions presented throughout Section 4.1, Section 4.2 and Section 4.3.

4.1. Temperature and Pressure

In supercritical fluid extraction, temperature plays a complex and crucial role, affecting both the solubility and diffusivity of solutes. Considering supercritical CO₂, the solubility of the solute in the fluid usually decreases with increasing temperature due to the reduction in the density of CO₂ [89]. However, higher temperatures can increase solute diffusivity and mass transfer rate, facilitating the extraction of target compounds from porous matrices. Another aspect to be considered is the antagonistic effect between temperature and pressure in the supercritical process. Higher pressures can compensate for the decrease in fluid density caused by the increase in system temperature, adjusting the phase equilibrium and maintaining the solvation capacity of the system [90]. This balance is essential, especially for thermosensitive compounds such as flavonoids and coumarins, avoiding degradation of the compounds during extraction. Accordingly, Fraguela-Meissimilly et al. [64], Rantaša et al. [91], and Uwineza et al. [92] describe that it is common to use temperatures ranging from 40 to 70 °C in these situations.

Another parameter that must be evaluated when varying the temperature of the supercritical fluid extraction process is the dielectric constant of the solvent. In general, an increase in temperature reduces the density of the supercritical fluid, reducing its dielectric constant and, consequently, the polarity of the solvent [93]. This decrease in dielectric constant reduces the fluid’s ability to solubilize polar compounds while improving extraction efficiency for nonpolar compounds. Considering flavonoids rich in hydroxyl groups and moderate polarity, the use of lower temperatures may be necessary to enhance the solubilization of these compounds, while increasing the system temperature and the consequent reduction of the fluid dielectric constant tends to favor the extraction of coumarins [36,94].

Concerning the PLE, pressure does not have as significant an influence as temperature, where a value is usually adopted that, considering the temperature range selected to carry out the study, under a given pressure, the fluid remains in a liquid state. Furthermore, since the liquids considered are typically incompressible, pressure variation will not substantially alter the solubility of the analyte, although considerably high pressures may force the fluid to penetrate the pores of the solid matrix, aiding in the desorption of the compound of interest [95]. On the other hand, the system temperature has a more significant impact on the process performance, since most compounds improve their solubility in solvents with increasing temperature, associated with a reduction in viscosity, surface tension, and dielectric constant of the fluids, leading to an improvement in the diffusion rate of the mass transfer process as well as modifying the polarity of the extractable compounds [96,97]. On the other hand, excessively high temperatures can accelerate the thermal degradation of thermosensitive compounds, in addition to catalyzing undesirable side reactions that result in the generation of undesirable co-products in the analyte [98].

4.2. Solvent Selection and Solvent-to-Matrix Ratio

As discussed throughout this study, the selection of the solvent to be used in extractions is closely associated with the selectivity of the process. Pure CO₂ is nonpolar and, therefore, highly efficient for the extraction of coumarins. The presence of polar functional groups in flavonoids, mainly hydroxyls and glycosides, can limit the solubility of these compounds in CO₂, thus requiring the use of a polar co-solvent to assist in the process. For the PLE process, the use of polar solvents, such as methanol or ethanol, is highly recommended for the extraction of flavonoids present in solid matrices, where the polarity of the solvent can be adjusted with temperature variation, especially in the case of subcritical water, whose dielectric constant decreases as the temperature increases, making it more nonpolar and suitable for the extraction of coumarins [99].

With solvent selection, defining the mass (or volume) ratio between the solvent and solid matrix is another crucial parameter to be evaluated. A solvent/matrix ratio that is too low may result in incomplete extraction, as the solvent may not be able to sufficiently saturate the matrix, leading to lower yields than expected. On the other hand, excess supercritical or pressurized fluid does not necessarily improve the process performance, as at a certain point, the solvents will reach saturation, especially for compounds with high solubility. Generally, in supercritical CO₂ extractions, typically volumetric ratios between solvent and matrix, ranging from 10 to 15 vol vol⁻¹, are applied for the solubilization of bioactive compounds [100,101]. For PLE, as the solvents considered usually present a lower diffusivity in relation to supercritical CO₂, a larger amount of fluid is necessary to ensure effective penetration into the solid matrix, dissolving the target compounds, where volumetric ratios between 20 and 30 vol vol ⁻¹ are applied [17,28].

4.3. Extraction Time

In supercritical fluid extraction processes, extraction time is another significant parameter to be considered to maximize the yield and selectivity of flavonoids and coumarins. A typical behavior to be observed in these systems is the high mass transfer rate in the initial moments of the process while the solvation capacity of the supercritical fluid is still high. Thus, more accessible compounds present in the substrate matrix are easily extracted [35]. As time progresses, it is typical to observe a drop in extraction rates, resulting from the depletion of these easily accessible compounds and the difficulty in solubilizing compounds present in more internal regions of the matrix or still linked to complex cellular structures. Flavonoids are usually found in conjugated forms—glycosides—or bound to cell wall structures of the material, where longer extraction times may be required to allow diffusion and rupture of these bonds [102]. Regarding coumarins, although these compounds have a higher affinity with solvents with more nonpolar characteristics, such as supercritical CO₂, it is expected that shorter extraction times would be sufficient to obtain satisfactory yields [36,103]. However, this is not always observed in practice because coumarins are typically located in denser and more internal regions of the plant matrix, such as cellulosic fibers and lignin, which are difficult for the fluid to access [102,104]. Thus, even if the solvent has high affinity, a prolonged contact time is necessary to allow complete penetration of the solvent and the gradual release of these compounds.

For PLE, similar behaviors are observed in the process in relation to the rapid extraction of more accessible compounds in the matrix during the initial extraction phase and a slower phase controlled by the diffusion of compounds located in the innermost regions of the solid matrix. Contextually, extending the extraction time can increase the process yield. However, the characteristics of the solvents lead to differences in the process time. Usually, pressurized liquid solvents have lower diffusivity and higher viscosity compared to supercritical CO₂ [105]. Therefore, solvent diffusion in the PLE may be limited, especially in slower matrices, and a longer extraction time could be required. Furthermore, the saturation dynamics between solvents are different, with the liquid compound typically saturating more slowly in PLE compared to the supercritical fluid, prolonging the process time [54]. However, delayed saturation in PLE may be advantageous in cases where gradual and selective extraction of compounds that require prolonged contact times for complete solubilization, such as conjugated flavonoids, is required [58].

5. Phenolic Compounds

Phenolic compounds are a diverse group of substances extensively produced by plants, with more than 200,000 compounds identified to date. These compounds, characterized by their aromatic structures, play pivotal roles in plant growth and reproduction, often acting as allelopathic agents. Among the various subclasses of phenolics, flavonoids stand out as the most abundant, comprising over 4000 naturally occurring types. These polyphenolic compounds are biosynthesized through the shikimate and acetate pathways and exhibit a wide range of biological activities [106,107,108,109].

Phenolic compounds are a diverse group of secondary metabolites widely distributed in plants, known for their potent bioactive properties. They include flavonoids, phenolic acids, tannins, lignans, and stilbenes, each contributing to a range of health-promoting effects. These compounds have garnered significant attention in nutritional science and medical research due to their antioxidant, anti-inflammatory, antimicrobial, and cardioprotective properties [110,111,112].

One of the primary health benefits of phenolic compounds lies in their antioxidant capacity. These molecules can neutralize reactive oxygen species (ROS) and free radicals, thereby reducing oxidative stress, which is implicated in aging and various chronic diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders. Phenolics, such as quercetin, catechins, and resveratrol, act by donating hydrogen atoms or electrons and chelating metal ions, preventing oxidative damage to cellular components like DNA, proteins, and lipids [113,114,115].

Several phenolic compounds exhibit antimicrobial activity against a broad spectrum of pathogens, including bacteria, viruses, and fungi. They disrupt microbial membranes, inhibit enzyme activity, and interfere with quorum sensing, which is critical for biofilm formation. These properties make phenolics potential candidates for natural food preservatives and therapeutic agents against antibiotic-resistant strains. Moreover, phenolics are recognized for their cardioprotective effects. They improve endothelial function, reduce blood pressure, and prevent platelet aggregation, thereby lowering the risk of atherosclerosis and thrombotic events. Flavonoids, such as anthocyanins, and phenolic acids, like caffeic acid, contribute to cholesterol regulation and the maintenance of vascular health through antioxidant and anti-inflammatory mechanisms [116,117,118,119].

Coumarins, another significant class of phenolic compounds, are low-molecular-weight benzo-α-pyrone derivatives with an isomeric chromone structure (benzo-γ-pyrone). These compounds, which can originate from natural sources, such as plants, fungi, and bacteria or be synthesized artificially, are notable for their stability and biological relevance. In plants, coumarins are secondary metabolites involved in various ecological interactions, including allelopathy, where they exhibit phytotoxic effects. For example, they are widely found in species belonging to the Umbelliferae, Rutaceae, Leguminosae, and Compositae families. These aromatic compounds are distributed throughout different plant parts, with the highest concentrations typically found in flowers and fruits, and smaller amounts in root exudates [120].

Research has shown that coumarins can act as signaling molecules, mediating interactions between plants and bacteria, whether symbiotic or pathogenic. Certain pathogenic bacteria, for instance, stimulate the accumulation of coumarin-derived compounds in plant roots and stems. These compounds subsequently serve as protective agents, enhancing plant immunity against bacterial invasion and propagation. Furthermore, coumarin has allelopathic properties that are concentration-dependent, with varying inhibitory effects on plant growth [121].

Quantitative studies on polyphenols typically focus on determining total phenolic content (TPC) and total flavonoid content in plant extracts and fractions. These are often reported as gallic acid equivalents (GAE) and quercetin equivalents (QE), respectively. However, the results vary significantly across studies, primarily due to differences in the plant species and parts analyzed, as well as variations in the extraction solvents and methodologies used.

In this sense, to analyze phenolic compounds and their metabolites, mass spectrometry-based metabolite profiling is considered the gold standard. Advanced techniques such as high-performance liquid chromatography–diode array detection (HPLC-DAD), ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS), and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) have emerged as powerful tools for detecting and characterizing these bioactive molecules [122].

6. Applications of Flavonoids and Coumarins

Reactive oxygen species (ROS) is a widely used term in biology to describe a group of unstable and highly reactive oxygen-containing chemicals found in mammalian cells. These include hydrogen peroxide, hydroxyl radicals, superoxide, peroxynitrite, lipid hydroperoxide, singlet oxygen, hypochlorous acid, ozone, alkoxyl radicals, and peroxyl radicals. ROS are primarily produced as physiological by-products of aerobic mitochondrial metabolism but can also be generated in response to immune activities targeting pathogens, xenobiotics, and cytokines. While ROS are essential for cellular signaling, their overproduction can disrupt cellular homeostasis, leading to damage to lipids, proteins, carbohydrates, and DNA, ultimately resulting in tissue and organ dysfunction [123,124,125].

To mitigate ROS-induced damage, antioxidants play a critical role by neutralizing free radicals through electron donation. These stable molecules can be either endogenously synthesized or acquired from exogenous sources. Endogenous antioxidants are further categorized into enzymatic and non-enzymatic types, produced through normal cellular metabolism. By contrast, exogenous antioxidants must be obtained through dietary intake or supplementation. Maintaining a balance between ROS production and antioxidant defense is essential for cellular health. Recent research has increasingly focused on nutrient-derived antioxidants due to the adverse effects associated with synthetic dietary supplements [126,127].

Plant-derived antioxidants are particularly significant, as they are abundant in herbs, fruits, seeds, spices, vegetables, and various food products. These sources contain a wide array of hydrophilic and lipophilic phytochemicals with antioxidant properties. Hydrophilic compounds include anthocyanins, phenolics, and ascorbic acid, while lipophilic antioxidants encompass chlorophylls, tocopherols, carotenoids, and unsaturated fatty acids (UFAs), including monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). Plant oils, for example, are rich in MUFAs and PUFAs, which offer health benefits, such as reducing insulin resistance, inflammation, oxidative stress, and skin aging (dermatoheliosis) [126].

PUFAs, known as essential fats, cannot be synthesized endogenously and must be acquired through diet. These amphipathic organic compounds, characterized by long hydrocarbon chains with two or more carbon–carbon double bonds, are primarily classified into omega-3 and omega-6 fatty acids. The distinction between omega-3 and omega-6 lies in the location of the final double bond within the carbon chain. Both MUFAs and PUFAs exhibit antioxidant activity by inhibiting inflammatory responses, preventing platelet aggregation, and protecting vascular endothelial cells, cardiac cells, and neurons from oxidative damage [128].

The therapeutic use of plants in traditional medicine is well-established across various diseases. The advantages of herbal medicine include lower costs, better patient tolerance, fewer side effects, sustainability, biodegradability, and widespread availability. Plants rich in secondary metabolites are particularly promising for developing alternative therapeutics to combat antimicrobial resistance. These metabolites have been shown to enhance drug delivery and act as resistance inhibitors against pathogens. Documented antimicrobial mechanisms include disruption of microbial membranes, inhibition of enzyme synthesis, and interference with quorum-sensing pathways to combat biofilm-associated infections [129].

Antioxidants also play diverse biological roles in the human body. These include inhibiting radical formation, reducing local oxygen concentrations, chelating metal ions, and converting peroxides into stable products. Antioxidant mechanisms are classified into three types: electron transfer (ET), proton transfer (HAT), and chelation. The ET mechanism involves direct electron donation, while the HAT mechanism, often occurring in aqueous environments, combines electron and proton transfer. Chelation, on the other hand, involves the transfer of an electron pair from a ligand to a metal ion [130].

Thus, flavonoids and coumarins have demonstrated significant potential for applications in the food, cosmetic, and pharmaceutical industries due to their remarkable bioactive properties. In the food sector, these compounds are valued for their antioxidant activities, which can extend product shelf life and promote consumer health by neutralizing free radicals and reducing inflammation. In cosmetics, flavonoids and coumarins are widely incorporated as active ingredients in formulations designed to protect the skin from UV-induced damage, while also offering anti-inflammatory and anti-aging benefits. In the pharmaceutical field, these molecules have gained attention for their antimicrobial, anticoagulant, anti-inflammatory, and cell-modulating properties, positioning them as promising candidates for the development of natural or complementary therapies for various health conditions.

Food additives are widely used to improve the sensory attributes of food, enhance nutritional value, optimize technical properties, and extend shelf life, thereby ensuring better protection against foodborne pathogens. These additives can be classified into various categories, including preservatives, flavor enhancers, colorants, sweeteners, and emulsifiers or stabilizers. Preservatives are further divided into antimicrobials and antioxidants. Common antimicrobial food additives include sulfites, nitrites, parabens, propionates, sorbates, and benzoates. Despite evidence linking these compounds to potential cytotoxic and carcinogenic effects in animal and human studies, they remain prevalent in food products. Similarly, synthetic antioxidants, such as propyl gallate, tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), are commonly added to food to prevent oxidation, but studies have highlighted their potential toxic and carcinogenic effects [131,132,133,134].

Consumer awareness of food additives has grown significantly, leading to a preference for natural over synthetic additives. Plants are rich sources of bioactive phytochemicals, including flavonoids, polyphenols, essential oils, terpenoid alkaloids, and organic acids, which exhibit antioxidant, antimicrobial, and pharmacological properties. These bioactive compounds not only enhance food preservation but contribute health benefits to consumers [129].

Among phytochemicals, flavonoids stand out as a key group. These plant-derived secondary metabolites play a crucial role in preventing lipid oxidation and protecting plant vitamins and enzymes. Although humans and animals cannot synthesize flavonoids, they are recognized for their antioxidant, antimicrobial, anti-inflammatory, and anticancer properties. Flavonoids are predominantly found in fruits and vegetables. Additionally, agro-industrial by-products serve as alternative sources of flavonoids, addressing the environmental issues posed by waste accumulation. Initiatives to recycle peels, seeds, and pulp from fruits, such as citrus, watermelon, and mango, have proven effective. These compounds exhibit significant biological, pharmacological, and therapeutic effects, particularly in combating oxidative stress and neutralizing free radicals.

The sustainable use of flower seeds as a source of flavonoids and coumarins not only adds value to underutilized by-products or raw materials but aligns with the growing demand for safer, more effective, and eco-friendly solutions. To expand their applications, further research is essential to optimize extraction efficiency, assess stability in different matrices, and elucidate the mechanisms of action of these bioactive compounds, alongside evaluations of their safety and efficacy in final formulations. By addressing these challenges, flavonoids and coumarins extracted from flower seeds have the potential to emerge as key ingredients driving innovation in food, cosmetic, and pharmaceutical product development.

7. Future Directions and Challenges

As the demand for natural, sustainable, and healthier alternatives to synthetic compounds continues to grow, the exploration and utilization of bioactive compounds from plants, such as flavonoids, coumarins, and phenolic compounds, hold significant promise. However, several future directions and challenges must be addressed to unlock their full potential in food, cosmetic, and pharmaceutical industries.

While methods like pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) have shown promise in extracting bioactive compounds, optimizing these techniques is crucial. The efficiency, selectivity, and scalability of these methods must be improved to ensure that the maximum yield of bioactive molecules is obtained while preserving their stability and potency.

Plants are a vast reservoir of bioactive compounds with potential as natural food preservatives. However, further research is needed to overcome the challenges associated with the extraction, stability, and application of natural phytochemical fractions.

One of the main challenges in incorporating plant-derived bioactive compounds into products is ensuring consistency in their composition and bioactivity. Natural sources exhibit significant variability depending on factors such as geographical location, climate conditions, and extraction techniques. Standardizing the levels of active ingredients in products and ensuring reproducibility across batches will require advanced quality control measures. Further investigation into the optimal conditions for growing, harvesting, and processing raw materials will be necessary.

Another significant challenge is enhancing the bioavailability of bioactive compounds, as many of these molecules have limited absorption or stability in the body. Developing delivery systems, such as nanoparticles or microencapsulations, to improve the solubility, stability, and absorption of these compounds in human and animal systems is critical. Research into how these compounds interact with the body at the cellular and molecular levels will help optimize their use in therapeutic applications. Many bioactive compounds from plants act synergistically, providing enhanced therapeutic effects when combined. Investigating the interactions between flavonoids, coumarins, phenolic compounds, and other phytochemicals will be crucial for developing effective product formulations. In addition, safety assessments are needed to evaluate the potential toxicity or adverse effects of long-term exposure to these compounds, especially when used in high concentrations in food, cosmetics, and medicines.

Appropriately, current research on bioactive compound extraction often lacks focus on long-term sustainability and economic viability. There is a significant gap in understanding the life cycle impact of advanced extraction methods, particularly their energy demands and waste generation. Furthermore, there is a dearth of scientific studies that address the variability in the properties of bioactive compounds across plant species and growing conditions, which can significantly influence extraction results. Further research is essential to adapt techniques to diverse plant sources and to characterize compounds, especially for plant wastes and agricultural by-products.

Exploring large-scale extraction systems capable of simultaneously processing compounds of different origins and properties can also improve the efficiency and cost-effectiveness of the process, enabling the advancement of these technologies worldwide.