Subcritical Water Extraction as a Green Technology for the Development of Standardized Plant Extracts for Food and Pharmaceutical Uses

Abstract

The increasing global demand for natural bioactive compounds in the food, nutraceutical, and pharmaceutical sectors highlights the need for sustainable extraction technologies capable not only of efficiently valorizing crop biomass and agro-waste but also of producing reproducible and standardized botanical extracts. Subcritical water extraction (SWE), which utilizes pressurized hot water at temperatures between 100 °C and 374 °C to modify solvent properties, has emerged as a promising green alternative to conventional organic solvent-based extraction methods. Despite its advantages in terms of environmental compatibility, extraction efficiency and tunable selectivity, the industrial application of SWE remains limited, and strategies for obtaining standardized extracts using this technology are still insufficiently explored. This review provides a comprehensive overview of SWE in the context of natural product extraction and the development of standardized plant extracts. The fundamental principles of SWE are discussed, including temperature-dependent changes in water polarity, solvent–solute interactions, and the influence of key process parameters such as temperature, pressure, extraction time, and particle size. Particular emphasis is placed on how these factors affect extraction selectivity, phytochemical composition, and reproducibility, which are critical aspects for extract standardization. Mechanistic insights into plant cell disruption, compound stability, and hydrothermal transformations under SWE conditions are also examined. Recent applications of SWE for the extraction of phenolics, flavonoids, terpenoids, alkaloids, and other pharmacologically relevant compounds are reviewed, highlighting the relationship between extraction conditions and extract quality. Finally, current challenges and future perspectives for integrating SWE into the production of standardized botanical extracts suitable for food, nutraceutical, and pharmaceutical applications are discussed, paving the way for the wider industrial adoption of this environmentally friendly technology.

1. Introduction

Plants represent an abundant source of structurally diverse bioactive compounds that have attracted increasing attention in the food, nutraceutical, and pharmaceutical industries. These compounds are commonly classified into primary and secondary metabolites. While primary metabolites such as carbohydrates, proteins and amino acids, and lipids are essential for plant growth and metabolism, secondary metabolites play crucial ecological roles in plant defense, signaling and adaptation to environmental stress [1,2,3]. Among these, phenolic compounds, flavonoids, alkaloids, terpenoids, glycosides, and tannins constitute major groups of phytochemicals exhibiting a wide range of biological activities, including antioxidant, anti-inflammatory, antimicrobial, anticancer and cardioprotective effects [4,5,6]. Secondary metabolites are particularly important because of their contribution to the therapeutic properties of medicinal plants and plant-derived preparations. Numerous studies have demonstrated that plant extracts rich in polyphenols and other phytochemicals possess significant pharmacological potential and are therefore widely incorporated into functional foods, dietary supplements and pharmaceutical formulations [7,8,9]. However, the concentration and composition of these compounds may vary significantly depending on plant species, cultivation conditions, harvest time, and processing methods. Such variability often leads to inconsistencies in biological activity and product quality, emphasizing the need for reliable strategies to ensure reproducible phytochemical composition and efficacy, such as standardization [10,11]. Standardized botanical extracts are defined as plant extracts that contain a specified amount of one or more marker compounds or groups of bioactive constituents within a defined range, ensuring batch-to-batch consistency and predictable biological activity [12,13,14]. Standardization represents a key aspect of quality control in herbal medicines and plant-based products, allowing manufacturers and researchers to ensure identity, purity, potency and safety [15,16]. However, achieving reliable standardization remains a significant challenge, especially in the pharmaceutical industry. One of the major obstacles is the inherent variability of plant raw materials, which is influenced by genetic, environmental, agronomic and post-harvest factors, leading to substantial fluctuations in phytochemical composition. In addition, the complexity of plant matrices, often containing hundreds of constituents with potential synergistic or antagonistic interactions, complicates the identification of appropriate marker compounds and limits the effectiveness of single-compound standardization approaches. Another critical issue is the lack of harmonized regulatory requirements and standardized methodologies across different regions, which hampers global consistency and comparability of products. Furthermore, extraction processes themselves play a decisive role, as variations in solvent type, temperature, pressure and processing time can significantly affect both the qualitative and quantitative composition of the final extract. The importance of standardization has increased considerably with the growing global market for botanical supplements and plant-derived pharmaceuticals. By controlling the concentration of specific active constituents, standardized extracts enable more accurate dosing, improved reproducibility in clinical studies and better regulatory acceptance [17,18]. Furthermore, standardization increasingly relies on advanced analytical approaches, including chromatographic fingerprinting, spectroscopic techniques and multi-component quantification, which together provide a more comprehensive characterization of extract composition. Nevertheless, the integration of such analytical strategies with robust and reproducible extraction technologies remains a key challenge and a critical area for further development [19,20].

Plant extracts standardized in bioactive constituents are widely utilized across multiple industrial sectors. In the food industry, standardized extracts rich in polyphenols and natural antioxidants are incorporated into functional foods, beverages and natural preservatives to improve nutritional value and shelf life [21,22]. Similarly, in the nutraceutical sector, botanical extracts are commonly formulated as capsules, tablets or liquid supplements designed to support health and prevent disease [23]. In pharmaceutical applications, standardized plant extracts serve as active pharmaceutical ingredients or as sources of lead compounds for drug development. Well-known examples include standardized extracts of Ginkgo biloba, Hypericum perforatum and Panax ginseng, which have been extensively studied for their therapeutic properties and are used in clinically validated formulations [24,25]. The increasing consumer demand for natural and plant-based products has further stimulated research into new extraction strategies capable of producing highly reproducible and bioactive plant extracts suitable for these applications.

The recovery of phytochemicals from plant materials typically relies on conventional extraction techniques such as maceration, percolation, Soxhlet extraction and hydrodistillation. These methods often require large volumes of organic solvents, long extraction times and elevated temperatures, which may lead to degradation of thermolabile compounds and reduced extraction efficiency [26,27]. Furthermore, the use of organic solvents raises environmental and safety concerns, particularly when extracts are intended for food or pharmaceutical applications. In addition, variations in solvent composition, extraction conditions and plant-to-solvent ratios may significantly influence the chemical profile of the obtained extracts, thereby complicating efforts to produce standardized preparations [28]. These drawbacks have prompted increasing interest in the development of alternative extraction technologies that are more efficient, environmentally friendly and capable of producing extracts with consistent phytochemical composition.

SWE has emerged as a promising green technology for the recovery of bioactive compounds from plant materials. This technique utilizes water at temperatures between 100 and 374 °C under sufficient pressure to maintain the liquid state. Under these conditions, the physicochemical properties of water change significantly, enabling selective extraction of compounds with different polarities. As a result, subcritical water can efficiently extract a wide range of phytochemicals, including polyphenols, alkaloids, terpenoids and glycosides with shorter extraction times, improved yields and minimal environmental impact [29,30,31,32].

Given the growing demand for high-quality botanical preparations and environmentally sustainable processing technologies, the integration of green extraction methods with strategies for extract standardization has been recognized as an important area of research [33,34]. In this context, SWE offers significant potential for the production of standardized plant extracts with consistent chemical composition and biological activity. Although numerous review papers have addressed the application of SWE for the extraction of plant materials [28,35,36], to the best of our knowledge no review has specifically focused on its applicability for the development of standardized extracts with defined active constituents. Therefore, the objective of this review is to provide a comprehensive overview of subcritical water extraction as a green technology for the development of standardized plant extracts. Particular emphasis is placed on the relationship between extraction conditions, phytochemical composition and extract standardization. The review also discusses recent advances in SWE applications for the recovery of bioactive compounds from plant materials and highlights its potential for producing reproducible extracts suitable for food, nutraceutical and pharmaceutical applications. Together with the particular active constituents and extraction conditions (temperature, time, pressure, particle size, etc.), mechanistic plant cell disruption, compound stability and hydrothermal transformations under SWE conditions are also recognized as important factors crucial for extract standardization. To maintain a focused scope, this review primarily addresses extracts containing low-molecular secondary metabolites, including phenolics, flavonoids, alkaloids and terpenoids. Extracts dominated by primary metabolites such as proteins, polysaccharides, sugars, amino acids or lipids are beyond the scope of the present review. The same is valid for extracts obtained with surfactants- and organic modifiers-assisted SWE.

2. Fundamentals of Subcritical Water Extraction

This section summarizes the fundamental principles of SWE, with particular emphasis on the thermophysical properties of subcritical water, solvent polarity tuning and the dominant extraction mechanisms involved.

2.1. Definition and Thermophysical Properties of Subcritical Water

Extraction is a mass transfer process in which target compounds are recovered from a solid or liquid matrix through contact with a suitable liquid phase, commonly referred to as the extractant or extragent [37]. Depending on whether the operating parameters remain constant or vary with time, extraction processes may proceed under steady-state or non-steady-state conditions [38]. In SWE, the extractant is pressurized liquid water, and its transport properties and solvent strength are primarily governed by temperature and pressure.

In the case of solid–liquid extraction, the overall mechanism can be described as a sequence of five consecutive stages [39,40]. Initially, the solvent is transported from the bulk liquid phase to the surface of the solid matrix. This step is followed by internal diffusion, during which the solvent penetrates the porous structure of the solid material. The third stage involves the dissolution of the target compounds, which is thermodynamically controlled and strongly dependent on temperature and solubility. Subsequently, the dissolved compounds undergo internal mass transfer back toward the surface of the solid matrix. In the final stage, the solutes are transferred from the solid surface into the bulk liquid phase, where they accumulate in the solvent [41].

Together, these stages define the general mass transfer framework that governs solid–liquid extraction processes and provide the basis for understanding more specific extraction mechanisms. The overall efficiency of extraction can be quantitatively characterized using the mass transfer coefficient (k), which is widely applied as a key parameter for evaluating extraction performance [42].

A thermodynamic approach is commonly employed to further analyze and control the extraction process. By examining the temperature dependence of the equilibrium constant (K*) (Equation (1)), fundamental thermodynamic parameters, including enthalpy (ΔH⁰), entropy (ΔS⁰), and Gibbs free energy (ΔG⁰), can be determined. These parameters provide valuable insight into the spontaneity of the extraction process and its sensitivity to temperature variations [43,44,45].

$$\ln {\mathrm{K}}^{*}=-{\displaystyle \frac{{\Delta \mathrm{H}}^0}{\mathrm{R}}}·{\displaystyle \frac{1}{\mathrm{T}}}+{\displaystyle \frac{{∆\mathrm{S}}^0}{\mathrm{R}}}$$

(1)

where K* is the apparent equilibrium constant, ΔH⁰ is the differential enthalpy (J·mol⁻¹), ΔS⁰ is the differential entropy (J·mol⁻¹·K⁻¹), T is the absolute extraction temperature (K), and R is the universal gas constant (8.314 J·mol⁻¹·K⁻¹).

In this context, K* represents the distribution of phytochemicals between the plant matrix and the solvent. This thermodynamic model has been successfully applied to analyze ultrasound-assisted extraction in the temperature range 303–343 K [43], microwave-assisted extraction in the range 313–373 K [44], and SWE in the range 413–493 K [45], indicating the predominantly endothermic nature of these processes and their thermodynamic favorability, particularly at elevated temperatures.

The Gibbs free energy change can be expressed according to Equation (2).

$${∆\mathrm{G}}^0=∆{\mathrm{H}}^0-\mathrm{T}·{∆\mathrm{S}}^0$$

(2)

From a kinetic perspective, the effect of temperature on the extraction rate (k) is frequently described using the linearized Arrhenius equation (Equation (3)), which enables estimation of the activation energy (E_a) and the pre-exponential factor (A).

$$\ln \mathrm{k}=\ln \mathrm{A}-{\displaystyle \frac{{\mathrm{E}}_{\mathrm{a}}}{\mathrm{R}}}·{\displaystyle \frac{1}{\mathrm{T}}}$$

(3)

where A is the pre-exponential factor (min⁻¹), and E_a is the activation energy of the extraction process (J·mol⁻¹).

The combined application of thermodynamic and kinetic analyses therefore provides a robust scientific framework for understanding, modeling and optimizing extraction processes [46].

Within this general mass transfer framework, extraction from plant raw materials is of particular interest due to its relevance for the recovery of bioactive compounds [47,48,49]. During such processes, bioactive constituents are released from the inert plant matrix and transferred into the solvent, enabling their subsequent utilization in various industries, including pharmaceuticals, cosmetics and food processing [37]. The primary objectives of any extraction process are to achieve high yield, selectivity toward the target compounds and high purity of the final extract. Overall extraction efficiency is governed by multiple factors related to the characteristics of the plant material, the properties of the solvent and the applied technological parameters [50], as summarized in Figure 1.

2.2. Dielectric Constant, Solvent Polarity Setting, and Surface Tension

SWE employs water as a solvent at temperatures ranging from 100 °C to 374 °C and at pressures sufficiently high to maintain it in the liquid state (critical pressure of water, 22.1 MPa) [51,52]. Under these conditions, the physicochemical properties of water undergo significant changes, including decreases in dielectric constant, viscosity and surface tension [53]. As a result, water becomes progressively less polar and acquires solvent properties comparable to those of organic solvents, enabling the dissolution of both polar and moderately nonpolar compounds that are otherwise insoluble under ambient conditions [54]. Consequently, the spectrum of bioactive compounds accessible via SWE is substantially expanded.

The phase behavior of water as a function of temperature and pressure is illustrated in Figure 2. Under ambient conditions, water is a highly polar solvent with a high dielectric constant (ε ≈ 78.5 at 20 °C), which favors the dissolution of ionic and polar compounds [35]. This behavior arises from several intrinsic features of water, including its strong molecular polarity, extensive hydrogen-bonding capability, high polarizability and dynamic three-dimensional structure, which together stabilize charged species and facilitate solvent–solute interactions [26].

Water’s high polarity originates from the uneven distribution of electron density between oxygen and hydrogen atoms, resulting in a permanent dipole moment [55]. This polarity enables strong electrostatic interactions among water molecules and between water and other polar or charged species, thereby enhancing its capacity to modulate electric fields within the medium. Hydrogen bonding between adjacent water molecules further contributes to the structural organization and internal cohesion of liquid water. These interactions play a key role in stabilizing solvated ions and polar molecules, leading to a high dielectric constant and excellent solvent properties for polar substances [56,57]. In addition, water forms a dynamic three-dimensional hydrogen-bonded network that continuously rearranges as hydrogen bonds form and break. This structural flexibility enhances water’s adaptability as a solvent and allows it to respond effectively to diverse chemical environments [58]. Owing to this high dielectric constant, water has long been regarded as unsuitable for the extraction of nonpolar or hydrophobic compounds, as its strong polarity and extensive hydrogen bonding limit the solubility of such species [59,60]. However, as temperature and pressure increase under subcritical conditions, hydrogen bonds progressively weaken and reorganize. This results in a marked reduction in dielectric constant, viscosity and surface tension. The decrease in polarity enables subcritical water to dissolve not only polar but also moderately nonpolar compounds, thereby expanding its extraction capacity [61]. In parallel, surface tension decreases from approximately 72 mN/m at 25 °C to below 30 mN/m at 250 °C, significantly improving capillary penetration into plant tissues. Enhanced wetting facilitates solvent access to small pores and intracellular structures, while the reduced viscosity increases solvent mobility. Together, these effects accelerate internal diffusion and overall extraction kinetics [62].

The selectivity of a solvent toward specific metabolites is largely governed by its dielectric constant (ε), which reflects solvent polarity. Based on ε values, organic compounds can be broadly categorized as polar (water-soluble, ε ≈ 50–100), moderately polar (alcohol-soluble, ε ≈ 10–50) and nonpolar (fat-soluble, ε ≈ 2–10) [63]. During extraction, the solvent preferentially dissolves compounds whose polarity and dielectric properties are comparable to its own. In this context, the dielectric constant of subcritical water is of particular importance, as it decreases continuously with increasing temperature [64]. This temperature-dependent behavior enables fine-tuning of solvent selectivity in SWE, allowing the extraction of compounds with a wide range of polarities by simple adjustment of operating temperature (Figure 3). As it is evident from the figure, the continuous decrease in ε with increasing temperature allows subcritical water to mimic solvents of different polarities, thereby enabling selective extraction of polar, moderately polar and low-polarity compounds through simple adjustment of the operating temperature in SWE. Thus, temperature-controlled modulation of ε represents a central physicochemical principle underlying SWE selectivity.

2.3. Extraction Mechanisms (Solubilization, Hydrolysis, Diffusion)

Based on the general mass transfer framework and the temperature-dependent physicochemical properties of subcritical water discussed above, the efficiency of SWE can be attributed to the combined action of several interrelated mechanisms, namely solubilization, hydrolysis and diffusion. These mechanisms operate simultaneously and are strongly influenced by extraction temperature, pressure and residence time [65,66].

Solubilization represents the primary mechanism responsible for transferring target compounds into the extracting medium. Under subcritical conditions, the reduced dielectric constant of water lowers its polarity, thereby facilitating the dissolution of not only highly polar compounds but also moderately polar and certain nonpolar constituents [66]. As a result, a wide range of bioactive compounds, including phenolic acids, flavonoids, alkaloids and selected terpenoids, exhibit enhanced solubility with increasing temperature. The enhanced solubilization capacity of subcritical water is closely associated with the weakening of hydrogen-bonding networks and the reduced polarity mismatch between solvent and solute. Consequently, compounds that are poorly soluble in water at ambient conditions can be efficiently extracted without the use of organic solvents [26]. Hydrolysis constitutes an additional mechanism that differentiates SWE from conventional extraction techniques. At elevated subcritical temperatures, the ionic product of water increases, resulting in higher concentrations of hydronium (H₃O⁺) and hydroxide (OH⁻) ions. This enables water to act simultaneously as a weak acid and a weak base, thereby promoting hydrolytic reactions within the plant matrix [32,67]. As a consequence, glycosidic bonds, ester linkages and other hydrolysable moieties may be cleaved, facilitating the release of bioactive compounds present in bound, conjugated or polymerized forms [68]. While hydrolysis can significantly enhance extraction efficiency, excessive temperatures may lead to the degradation of thermolabile compounds, highlighting the importance of careful optimization of extraction conditions [69].

Diffusion plays a crucial role in the overall mass transfer process during SWE. The pronounced reduction in water viscosity and surface tension under subcritical conditions improves solvent penetration into the plant matrix and enhances internal diffusion within cellular structures [70]. Moreover, elevated temperatures increase molecular mobility and diffusion coefficients, further accelerating mass transfer. The combined effects of improved diffusion, reduced boundary layer resistance and enhanced solvent accessibility result in significantly shorter extraction times and higher extraction efficiencies compared to conventional solvent-based methods [71].

Importantly, solubilization, hydrolysis and diffusion do not act independently during SWE but instead exhibit strong synergistic effects. Enhanced solubilization increases concentration gradients, thereby promoting diffusion, while hydrolysis liberates bound compounds and renders them more accessible for dissolution and transport [26,72,73]. The relative contribution of each mechanism depends on the extraction conditions, particularly temperature, pressure and residence time, as well as on the chemical nature of the target compounds and the structural characteristics of the plant matrix [26]. Overall, the interplay of these mechanisms under subcritical conditions provides a fundamental explanation for the high efficiency, selectivity and versatility of SWE in the extraction of plant-derived bioactive compounds.

3. Degradation of Plant Polymers Under SWE Conditions and Its Influence on Extract Quality

Despite the active interest in applying SWE, a significant amount of research is still required to better understand its influence on target compounds and plant cell wall constituents. In particular, more insight is needed into the formation of hydrolysis products from the complex plant matrix, as well as their subsequent degradation and interactions. Plant materials are highly reactive due to their heterogeneous composition. The cell walls of terrestrial plants represent a highly organized network consisting mainly of polysaccharides such as cellulose, hemicelluloses and pectins [28,74], along with structural proteins, phenolic compounds, lignins, minerals, cutin and suberin [75]. Together with extractives and cytoplasmic contents, these components form a reactive system where SWE can promote not only extraction but also the formation of intermediate and secondary products, thereby affecting extract composition.

3.1. Degradation of Carbohydrates Under SWE Conditions

Carbohydrates (polysaccharides) constitute more than 90% of primary cell wall structural polymers and undergo hydrothermal degradation under SWE, leading to depolymerization into oligomers and simple sugars. The extent of degradation depends on process conditions, polysaccharide type, and catalytic effects. Cellulose, composed of β-(1→4)-linked D-glucose units, degrades at elevated temperatures into oligosaccharides, glucose and secondary products such as 5-hydroxymethylfurfural (5-HMF), furfural and organic acids [76,77,78,79]. Its hydrolysis rate increases significantly with temperature, with limited decomposition below 200 °C and rapid breakdown above ~240 °C [77,78,79]. Similar degradation products are observed under both acidic and hydrothermal conditions, indicating that near-critical water can catalyze hydrolysis reactions [80]. Starch, although not structural, is a major reserve polysaccharide and degrades more readily than cellulose due to its α-linkages. SWE induces gelatinization followed by depolymerization into maltose, oligosaccharides, and glucose, which further convert into compounds such as pyruvaldehyde and 5-HMF [81,82,83]. High liquefaction can be achieved at relatively low temperatures (~180 °C), while higher temperatures promote further degradation and gas formation [82,84]. Inulin, another storage polysaccharide composed of fructose units, shows even higher susceptibility to degradation. Under SWE, it yields fructose and intermediates such as pyruvaldehyde, along with acids and furan derivatives [85,86,87]. Milder conditions produce oligosaccharides and difructose anhydrides, while higher temperatures favor conversion to 5-HMF, especially under acidic conditions [88,89,90]. Pectins, composed mainly of galacturonan-based structures, exhibit increased solubility at moderate temperatures (100–160 °C), followed by depolymerization at higher temperatures [91]. Polygalacturonan is particularly susceptible, degrading readily into oligomers and monomers [83]. Hydrothermal treatments generate mixtures of oligogalacturonides and other fragments, while more severe conditions lead to further decomposition into furan derivatives [92,93,94]. Significant conversion into soluble products can occur even at relatively short treatment times [95].

In addition to chemical alterations in extract composition, co-extracted “by-products/impurities” remain an integral part of the final extracts, resulting in complex multicomponent systems. In such systems, interactions between constituents play a crucial role in determining functional properties, drying behavior and the overall physical profile of the extracts [96]. For example, depending on their molecular weight and concentration, solubilized polysaccharides, particularly pectin and starch, can significantly increase extract viscosity during cooling [96,97]. Moreover, the presence of polysaccharides with amorphous and porous structures may lead to poor flow properties while simultaneously enhancing the solubility of the dried extract. Conversely, severe thermal treatment can induce aggregation and denaturation of co-extracted proteins, reducing their water solubility and causing phase separation and turbidity in liquid extracts [98]. Similarly, solid humins formed during SWE are largely insoluble in water and common solvents, further affecting the physical characteristics of the extracts. Regarding drying behavior, the gel-forming capacity and amorphous nature of certain polysaccharides (e.g., maltodextrin, pectin) can improve extract stability and protect target compounds during drying by increasing the glass transition temperature, thereby extending shelf life [99]. In contrast, higher levels of free sugars and low-molecular-weight polymers promote crystallinity and moisture retention, increasing the likelihood of stickiness and caking during drying [100,101].

3.2. Degradation of Proteins and Lignin Under SWE Conditions; Protein-Polyphenol Interactions

Proteins present in plant cell walls are generally more thermally stable than polysaccharides but undergo hydrolysis above ~190 °C [102,103,104]. Under SWE, they decompose into peptides and amino acids, which can further degrade into smaller molecules such as organic acids, ammonia, and gases [105,106,107,108]. Secondary reactions, including cyclization and oxidation, lead to compounds such as diketopiperazines and other nitrogen-containing products [109,110]. Lignin, another major structural component, is a complex cross-linked phenolic polymer associated with secondary cell walls [111]. It consists of p-hydroxyphenyl (H), syringyl (S), and guaiacyl (G) units linked by C–C and ether bonds, the latter contributing to its resistance to degradation [112,113]. Thermal degradation begins near the glass transition temperature (~90–180 °C) and intensifies with increasing temperature. Significant cleavage of ether linkages occurs around 260 °C and continues rapidly up to 350–450 °C [114,115]. Under SWE, lignin removal begins at ~170 °C and reaches a maximum around 190 °C, although higher temperatures promote repolymerization of degradation products [116]. Depolymerization proceeds through cleavage of ether bonds and formation of phenolic monomers such as guaiacol, vanillin, and homovanillic acid [117,118,119].

It should be noted that the coexistence of proteins and phenolic constituents can promote both covalent and non-covalent interactions, leading to the formation of protein–phenolic complexes [120,121,122]. Reactive phenolic intermediates commonly interact with amino acid side chains such as lysine, cysteine, tyrosine, methionine, tryptophan, and histidine within polypeptides [123]. These interactions can significantly modify protein structure and functionality, affecting parameters such as secondary structure, ζ-potential, surface hydrophobicity, and thermal stability, which in turn influence solubility, foaming, and emulsifying properties [124]. Furthermore, protein–phenolic interactions may affect protein digestibility, absorption, and overall bioavailability due to modifications of essential amino acids [124]. Conversely, such interactions can enhance the stability of phenolic compounds against enzymatic degradation and pH variations, thereby improving their bioavailability and antioxidant capacity [125,126]. The extent and direction of these effects depend on multiple factors, including the type of (poly)phenols, the phenolic-to-protein ratio [124], and SWE conditions, particularly pH and temperature [127].

Overall, a deeper understanding of hydrolysis products and their interactions is still required, as these transformations may alter biological activity (sometimes adversely) and highlight the need to combine SWE with appropriate purification strategies. At the same time, the enrichment of extracts with peptides and oligosaccharides may enhance biological activity or even confer new functional properties. Furthermore, hydrolysis-driven generation of new bioactive forms deserves particular attention. During SWE, the combined effect of elevated temperature and the altered dielectric properties of water promotes the hydrolysis of glycosidic bonds, leading to the conversion of glycosides into their corresponding aglycones. These aglycones often exhibit enhanced biological activity due to improved membrane permeability and higher affinity for biological targets. For example, quercetin glycosides such as rutin or quercetin-3-O-glucoside can be partially hydrolyzed under SWE conditions to yield quercetin aglycone, which is widely reported to possess stronger antioxidant, anti-inflammatory and enzyme-inhibitory activities compared to its glycosylated counterparts.

3.3. Formation of Potentially Toxic Metabolites Under SWE Conditions

In real plant matrices, the reviewed degradation pathways are highly interconnected. Interactions among carbohydrates, proteins, lignin, and extractives give rise to complex reaction networks. Carbohydrate-derived intermediates play a central role, participating in reactions such as caramelization and the Maillard reaction [103,105]. The Maillard reaction between reducing sugars and amino compounds generates intermediates that can further degrade into compounds such as 5-HMF and furfural, ultimately leading to the formation of melanoidins [103,128]. While some of these products may exhibit beneficial bioactivities, others (e.g., acrylamide and N-nitrosamines) raise safety concerns [129]. Caramelization, for example, contributes to the sensory properties of extracts but also leads to the formation of potentially toxic furan derivatives and polymeric products [96,128,130]. Consequently, the safety assessment of subcritical water extracts and the evaluation of potential risks to animal and human health require careful consideration, particularly from a regulatory perspective with regard to compounds such as 5-HMF.

The formation of 5-HMF during SWE is strongly influenced by raw material composition, temperature, and residence time. Significant accumulation typically begins at temperatures of 120–140 °C [131], while formation remains relatively low at shorter extraction times (e.g., <60 min) under moderate conditions. This suggests that such parameters may be considered relatively safe for processing carbohydrate-rich, thermolabile plant materials. However, 5-HMF formation increases markedly at higher temperatures, reaching a maximum in the range of 180–200 °C [132,133]. Beyond this range, although 5-HMF is rapidly formed, it also undergoes further degradation into levulinic and formic acids or polymerizes into humins and pseudo-lignin. Accordingly, the formation of 5-HMF and related furan derivatives becomes a practical concern for food- and pharmaceutical-grade extracts when processing temperatures exceed approximately 160 °C and approach the critical range of 180–200 °C [133]. In terms of residence time, prolonged SWE treatment (≥30–45 min) at elevated temperatures (>200–220 °C) promotes the degradation of 5-HMF, thereby reducing its final concentration in the extract [132,133].

At present, neither the European Food Safety Authority nor the U.S. Food and Drug Administration (FDA) has established a formal limit for human dietary exposure to 5-HMF. European Food Safety Authority has identified a No Observed Adverse Effect Level of 80–100 mg/kg body weight per day based on animal studies [134]. In contrast, the Codex Alimentarius Commission sets a quality limit of 40 mg/kg for honey, primarily as an indicator of excessive heat treatment or improper storage. Similarly, the FDA considers 5-HMF mainly as a marker of processing and storage conditions rather than a direct carcinogenic compound [135].

It is well established that 5-HMF is metabolized into several derivatives, among which 5-sulfoxymethylfurfural is regarded as the most reactive metabolite with potential carcinogenic effects [136,137]. Despite these concerns, the levels of 5-HMF typically detected in SWE extracts are generally lower than those found in highly heat-processed foods such as coffee or caramel, and are therefore not considered to pose a significant safety risk under normal conditions [132,137,138].

Therefore, controlling such transformations under SWE conditions remains challenging and requires detailed knowledge of biomass composition and degradation pathways to optimize extraction and ensure product quality [139,140,141].

4. Applications of Subcritical Water Extraction for Recovery of Plant Bioactives and Development of Standardized Botanical Extracts

4.1. Process Parameters Affecting SWE Efficiency; Optimization of the Extraction

Numerous studies have reported the application of SWE for the recovery of various classes of low-molecular-weight bioactive compounds, including phenolic compounds (e.g., flavonoids), alkaloids, terpenoids, and saponins, from a wide range of plant sources and tissues. However, in the context of developing standardized botanical extracts, it is important to distinguish between two key aspects of SWE efficiency. The first relates to the efficiency of recovering target bioactive metabolites into the extraction medium, which is governed by multiple process parameters, including temperature, pressure, extraction time, particle size, solvent-to-solid ratio, and the physicochemical properties of the compounds of interest [26,31,32,142], as schematically illustrated in Figure 4.

4.1.1. Temperature

Temperature is the most influential parameter in SWE, as it directly governs the physicochemical properties of water, including its dielectric constant, viscosity, surface tension, and diffusion coefficient [143]. Increasing temperature under subcritical conditions reduces water polarity, thereby enhancing the solubility of moderately polar and nonpolar compounds. At the same time, elevated temperatures accelerate mass transfer and diffusion processes, leading to shorter extraction times and improved extraction efficiency [54]. However, excessively high temperatures may induce hydrolytic or thermal degradation of sensitive bioactive compounds [144]. Therefore, an optimal temperature range must be established for each class of target compounds to achieve an appropriate balance between extraction efficiency and compound stability.

4.1.2. Pressure

In SWE, pressure is primarily applied to maintain water in the liquid state at elevated temperatures. Although its effect on solvent polarity is less pronounced than that of temperature, pressure influences solvent density and penetration into the plant matrix [71]. Increased pressure can enhance solvent–matrix contact, thereby facilitating improved mass transfer. In most SWE applications, pressure is selected to ensure phase stability rather than to directly control extraction selectivity. Nevertheless, precise pressure control remains essential for process safety, reproducibility, and scalability.

4.1.3. Extraction Time

Extraction time determines the extent to which mass transfer and equilibrium between the solid matrix and the solvent are achieved. Insufficient extraction times may result in incomplete recovery of target compounds, whereas excessively long extraction durations increase the risk of compound degradation, particularly at elevated temperatures [145]. Accordingly, extraction time is routinely considered a key parameter in SWE process optimization, with studies demonstrating its significant influence on target yields (e.g., an optimized extraction time of 36.21 min reported for SWE systems) [146].

Due to intensified mass transfer under subcritical conditions, SWE generally requires significantly shorter extraction times compared to conventional solvent extraction methods [147]. Therefore, careful optimization of extraction time is essential to achieve high extraction efficiency while preserving the integrity of bioactive compounds.

4.1.4. Solid-to-Liquid Ratio

The solid-to-liquid ratio, often referred to as the hydromodule, affects both extraction yield and extract concentration. Higher solvent-to-solid ratios typically enhance extraction efficiency by preventing premature solvent saturation and maintaining favorable concentration gradients [148]. Optimization studies frequently identify solid–liquid ratio as a statistically significant variable influencing yields in SWE, with various works reporting optimum effects in the range studied experimentally [149,150]. However, excessively high ratios may dilute the final extract and increase energy consumption during downstream processing steps, such as solvent removal or concentration. Therefore, an optimal solid–liquid ratio must be selected to balance extraction efficiency with process economy.

4.1.5. Particle Size and Matrix Characteristics

Particle size and the structural characteristics of the plant matrix play a crucial role in SWE performance, as they directly affect solvent accessibility, internal diffusion resistance, and mass transfer efficiency. Smaller particle sizes increase the specific surface area and shorten diffusion pathways, thereby facilitating solvent penetration into plant tissues and enhancing the release of target compounds [35,65]. For example, studies on the subcritical water extraction of caffeine from tea waste have shown that extraction yield varies significantly with particle size, with intermediate particle sizes often providing optimal mass transfer and higher yields under otherwise constant conditions [151]. However, excessive particle size reduction may lead to matrix compaction, channeling effects, or increased pressure drop in dynamic SWE systems. In addition, the chemical composition and rigidity of plant cell walls influence solvent accessibility and compound release, making matrix characteristics a critical factor in process optimization [152].

4.1.6. pH and Water Modifiers

Under subcritical conditions, water exhibits an increased ionic product and altered solvent properties, enabling it to function simultaneously as a weak acid and a weak base. This dual behavior promotes hydrolytic reactions without the need for added acids or bases. As a result, processes such as the hydrolysis of polysaccharides, glycosides, and ester linkages are facilitated during SWE, enhancing the extractability of structurally bound compounds [35,67,153]. In some cases, the addition of small amounts of modifiers, such as organic co-solvents, has been shown to influence extraction efficiency and selectivity by altering solvent polarity and solubilization capacity. For example, the incorporation of ethanol (10% v/v) during SWE of pectic polysaccharides from passion fruit peel led to increased total phenolic content and antioxidant activity, highlighting the potential of co-solvent-assisted approaches [154]. More broadly, several studies suggest that controlling the pH environment, either intrinsically through temperature-dependent water chemistry or through carefully selected modifiers, can enhance SWE yields and antioxidant properties [155]. Nevertheless, the use of modifiers may compromise the environmental advantages of SWE by increasing process complexity and downstream separation requirements and, as noted, is beyond the scope of the present review.

Collectively, these parameters govern mass transfer, phytochemical solubility, and the disruption of plant cell structures, thereby influencing extraction yield and selectivity. Together, they define the operational window within which SWE can be effectively applied while minimizing degradation of thermolabile compounds. Optimal SWE conditions, therefore, depend on both the specific plant material or organ (e.g., roots, leaves, flowers) and the chemical nature and stability of the target metabolites. As discussed in Section 3, bioactive compounds may undergo transformation or degradation under subcritical conditions, potentially reducing their concentration in the final extract. Consequently, SWE parameters must be carefully tailored to both the plant matrix and the target compounds.

In practice, such optimization is commonly achieved through systematic experimental design approaches, including response surface methodology and related statistical tools. Design of experiments enables the identification of key factors influencing extraction performance and the evaluation of their effects on response variables such as the recovery of target bioactive compounds [8,10]. Compared with traditional one-variable-at-a-time approaches, multivariate experimental designs allow the simultaneous evaluation of factor interactions, thereby improving process optimization while reducing the number of experiments, associated costs, and environmental impact. Properly designed experimental strategies are therefore essential for obtaining reliable and reproducible results and for identifying optimal conditions that maximize both selectivity and extraction efficiency in complex plant matrices [8,10,11].

When discussing standardized extracts, it is essential to emphasize that they must be standardized to a defined quantity of one or more marker compounds, or groups of bioactive constituents, within a specified range. These marker compounds are typically well-characterized bioactive substances that serve as indicators of extract quality, consistency, and efficacy. Such marker compounds are described in numerous authoritative monographs on medicinal plants, including those published in the United States Pharmacopeia [156], Chinese Pharmacopeia [157], WHO Monographs on Selected Medicinal Plants [158,159], Japanese Pharmacopeia [160], and expanded Commission E monographs on herbal medicines [161]. These references play a critical role in defining both the identity and acceptable concentration ranges of marker compounds for specific herbal drugs. In addition to setting compositional standards, these documents also provide validated analytical methodologies for the identification and quantification of marker compounds. This establishes a robust analytical framework that enables reliable comparison of subcritical water extraction (SWE) with pharmacopoeial and conventional extraction techniques in terms of efficiency, selectivity, and reproducibility.

Table 1. Application of subcritical water extraction for recovery of biologically active components from different plant sources.

Plant Source	Subcritical Water Extraction Conditions	Bioactive Compounds	Yield, mg/g		Pharmacopoeial Requirements	Reference
			Subcritical Water Extraction	Conventional Extraction
Betula pendula (birch) bark	160–200 °C, 10–30 min, static, 1 g/40–60 mL	Betulinic acid	1.60–2.80	-	No	[162]
Brassica napus pressings (canola meal)	110–160 °C, 30 min, dynamic, 1 g/30 mL	Total phenolics	7.03–8.29;	5.60–10.00	No	[53]
			15.42–16.30
Carum carvi (caraway) seeds	110–190 °C, 5–15 min, static, 1 g/22 mL	Carvone	28.48	≈15.00–20.00	Yes	[163]
		Limonene	0.28	≈0.20–1.60
		Carveol	0.11	≈0.10–0.15
Castanea sativa (sweet chestnut) bark	150–250 °C, 10–60 min, static, 1 g/10–30 mL	Total phenolics	38.30–85.20	-	No	[164]
		Total tannins	42.80–98.30
Centella asiatica (Gotu kola) whole plants	150–250 °C, 300 min, static, 50 g/232 mL	Asiatic acid:	0–7.00	0–10.00	Yes	[165]
		Asiaticoside	1.10–8.40	1.3–17.7
Chamomilla matricaria (chamomile) flowers	65–210 °C, 5–60 min, static, 1 g/30–100 mL	Total phenolics	74.84–215.15	-	Yes	[166]
		Total flavonoids	15.99–38.89
		Apigenin	1.30–11.50
Chlorella sp. microalgae	100–250 °C, 5–20 min, static, 1 g/5 mL	Ferulic acid	0.015–0.032	0.004–0.021	No	[167]
		p-Coumaric acid	0.012–0.030	0.003–0.029
		Caffeic acid	0.018–0.033	0.002–0.023
Citrus unshiu (Satsuma orange) pomaces	25–250 °C, 10–60 min, static, 0.1 g/10 mL	Total phenolics	0.03–0.50	-	No	[168]
Coffea arabica (spent coffee grounds)	110–190 °C, 15–75 min, static, 1 g/30–70 mL	Total phenolics	21.09–56.59	-	No	[169]
Coriandrum sativum (coriander) seeds	100–200 °C, 10–30 min, static, 10 g/100 mL	Total phenolics	5.11–26.29	-	Yes	[170]
		Total flavonoids	2.31–6.18
Fagopyrum esculentum (common buckwheat) aerial parts	80–120 °C, 20 min, dynamic, 2 g/14 mL	Rutin	62.00–75.00	-	No	[171]
Garcinia mangostana (mangosteen) pericarps	120–160 °C, 30–180 min, dynamic, 2.5 g/50 mL	Total phenolics	75.87–372.69	-	No	[172]
		Xanthone	3.10–27.15
Glycyrrhiza uralensis (licorice) root	50–300 °C, 10–60 min, static, 0.1 g/10 mL	Total phenolics	0.20–1.52	-	Yes	[173]
		Glycyrrhizin	0.03–1.44
		Glycyrrhetic acid	0.01–0.24
		Liquiritin	0.03–0.60
Linum usitatissimum (flax) seeds	160–180 °C, 5–60 min, static, 5 g/33 mL	Total phenolics	1.91–10.81	-	No	[174]
		Total flavonoids	0.04–0.96
Matricaria chamomilla (German chamomile) flowers	100 °C, 30 min, static, 10 g/300 mL	Apigenin	0.06–1.34	-	Yes	[175]
Melissa officinalis (lemon balm) leaves	100–150 °C, 10–20 min, dynamic, 100 g/2000 mL	Rosmarinic acid	12.20–16.20	13.80–15.3	Yes	[176]
		Total phenolics	80.00–92.60	78.90–79.10
Morinda citrifolia (noni) fruits	100–140 °C, 20 min, dynamic, 2 g/14 mL	Scopoletin	0.36–0.53	-	No	[177]
		Alizarin	0.01–0.08
		Rutin	0.36–0.45
Onosma mutabilis aerial parts, roots	100–200 °C, 30–180 min, static, 1–25 g/10–100 mL	Total phenolics	14.49–184.58	-	No	[178]
		Pyrocatechol	0.54–1.06	0.01–0.02
		Epicatechin	0.51–1.10	0.02–0.07
Rheum tanguticum (Chinese rhubarb) root	100–200 °C, 33–67 min, dynamic, 10 g/50 mL	Total anthraquinones	15.50–37.90	-	Yes	[179]
Salvia miltiorrhiza (red sage) aerial parts	75–150 °C, 30 min, static, 2 g/10 mL	Protocatechualdehyde	0.01–1.76	0.01	No	[180]
		Caffeic acid	0.01–0.05	0.05
		Ferulic acid	0.001–0.04	-
		Tanshinone I:	0.004–0.07	0.0002
		Tanshinone IIA	0.003–0.01	0.0008
Teucrium montanum (mountain germander) aerial parts	60–200 °C, 30 min, static, 1 g/10 mL	Total phenolics	143.89–174.61	-	No	[181]
Theobroma cacao (cocoa) shells	120–220 °C, 15–75 min, static, 1 g/10–30 mL	Total phenolics	33.41–130.33	-	No	[182]
		Theobromine	13.10–47.70
Thymbra spicata (spiked savory) leaves	100–175 °C, 30 min, dynamic, 1.5 g/10.4 mL	p-Cymene	6.40–29.30	-	No	[183]
		γ-Terpinene	3.70–19.00
		E-3-caren-2-ol	30.80–77.10
		Thymol	9.70–36.70
		Carvacrol	795.00–862.00
Vitis vinifera (grapes, var. Arkansas-1575) skins	100–160 °C, 5–7 min, static, 0.4 g/22 mL	Total phenolics	32.90–52.30	22.90–53.70	No	[51]
		Total anthocyanins	29.60–49.40	21.50–50.20
Vitis vinifera (grapes, var. Pinot Nero) seeds	80–120 °C, 120 min, dynamic, 2 g/100 mL	Total phenolics	44.30–77.00 44.00–124.00	-	No	[184]

summarizes representative examples of SWE applications for the recovery of various plant metabolites from different plant species and organs, along with the corresponding extraction conditions employed. The reviewed literature demonstrates that SWE has been widely applied for the extraction of various plant species and their respective organs. However, many of these investigations primarily evaluate the effectiveness of the technique based on the recovery of broad groups of compounds (e.g., total polyphenols, total flavonoids), which is insufficient for achieving precise extract standardization. While such global assays are useful for preliminary screening, they lack the specificity, selectivity, and reproducibility required to ensure batch-to-batch consistency and compliance with pharmacopoeial standards. In particular, they do not provide adequate information on the presence, concentration, and stability of defined marker compounds or active constituents, which are essential for quality control and regulatory acceptance. This trend further suggests that SWE has often been underestimated or, in some cases, inadequately applied in the context of developing standardized extracts. The technique has predominantly been used to maximize overall extraction yield rather than being systematically optimized for the selective recovery of target compounds with well-defined chemical profiles. Such an approach overlooks the significant potential of SWE to fine-tune extraction parameters, such as temperature, pressure, and solvent polarity, in order to enable targeted extraction of specific marker compounds and alignment with pharmacopoeial requirements.

Other studies reveal that SWE is particularly effective when targeting specific bioactive compounds. For instance, high selectivity and yield were achieved for triterpenes such as betulinic acid from birch bark (1.60–2.80 mg/g) [162]. Similarly, SWE showed strong performance in extracting oxygenated terpenes from caraway seeds, with carvone reaching 28.5 mg/g, indicating its selectivity toward volatile and semi-volatile compounds [163]. Several studies highlight SWE efficiency for glycosides and specialized metabolites. In stevia leaves, high recoveries of rebaudioside A and stevioside (up to ~35–38 mg/g) were obtained, demonstrating the suitability of SWE for extracting thermally stable sweet diterpene glycosides [183]. Likewise, SWE proved effective for saponins and triterpenoids from Centella asiatica, yielding up to 8.40 mg/g asiaticoside, although longer extraction times were required [165]. Notably, SWE has shown strong potential for flavonoids and phenolic acids when targeting individual compounds as demonstrated for rutin (62–75 mg/g) from buckwheat aerial under relatively mild temperatures [172]. Similarly, selective recovery of apigenin from chamomile (up to 11.50 mg/g) demonstrates that SWE conditions can be tuned for specific flavones rather than total phenolic fractions [166]. The method is also effective for alkaloids and quinone-type compounds. For example, SWE enabled efficient extraction of anthraquinones (15.5–37.9 mg/g) from Rheum tanguticum, highlighting its applicability for pharmaceutically relevant compounds [179]. In cocoa shells, significant yields of theobromine (up to 47.7 mg/g) were obtained, further confirming SWE suitability for methylxanthines [182]. In addition, SWE shows promising selectivity for essential oil components and phenolic terpenes. Extremely high recoveries of carvacrol (up to 862 mg/g) and other monoterpenes from Thymbra spicata emphasize its effectiveness for hydrophobic bioactives typically extracted by conventional distillation [183]. Similarly, xanthones from mangosteen pericarps (up to 27.15 mg/g) demonstrate that SWE can efficiently recover complex polyphenolic structures when optimized [172].

4.2. Development of Standardized Dry Botanical Extracts

The second very important aspect of SWE efficiency relates to the content of the target metabolites in the final dried extract, which is typically obtained after solvent removal and drying of the liquid extract. As discussed in detail in Section 3 of the current review, under SWE conditions, elevated temperatures may cause partial degradation of plant cell walls and promote the solubilization of structural polysaccharides, proteins and other macromolecules from plant tissues [150,185,186]. As a result, the total extraction yield may increase due to the co-extraction of these additional components. Although this phenomenon may appear beneficial from the perspective of overall extraction efficiency, it can lead to dilution of the target bioactive metabolites in the dry extract, thereby decreasing their relative concentration and complicating the development of standardized preparations. This has already been demonstrated in the SWE extraction of rosmarinic acid from lemon balm, where up to a 41% increase in overall extract yield was achieved depending on the conditions, but with a concomitant decrease in the relative content of rosmarinic acid in the extracts from 5.55% to 2.36% [176]. The higher yield was attributed to enhanced degradation of plant matrix components by subcritical water, as evidenced by increased extraction and breakdown of proteins, pectin and cellulose. Therefore, the optimization of SWE processes should consider not only the recovery of target metabolites in the extraction solution, but also their final concentration in the dried extract, which is ultimately the parameter most relevant for extract standardization and product quality.

Table 2. Application of SWE for development of dry botanical extracts.

Plant Source	Subcritical Water Extraction Conditions	Bioactive Compounds	Content in Dry Extract, mg/g		Pharmacopoeial Requirements	Reference
			Subcritical Water Extraction	Conventional Extraction
Achillea asplenifolia flower	130 °C, 30 min, static, 1 g/20 mL	Total phenolics	75.02	23.51–34.99	No	[187]
		Total flavonoids	4.13	4.64–6.03
Achillea clypeolata flower	130 °C, 30 min, static, 1 g/20 mL	Total phenolics	77.61	25.75–34.70	No	[187]
		Total flavonoids	4.78	3.99–9.76
Achillea crithmifolia flower	130 °C, 30 min, static, 1 g/20 mL	Total phenolics	90.12	24.59–34.59	No	[187]
		Total flavonoids	7.38	4.55–7.55
Achillea millefolium flower	130 °C, 30 min, static, 1 g/20 mL	Total phenolics	93.63	25.71–30.01	Yes	[187]
		Total flavonoids	6.24	4.28–5.44
Achillea nobilis subsp. Nelreichii flower	130 °C, 30 min, static, 1 g/20 mL	Total phenolics	67.38	31.83–37.76	No	[187]
		Total flavonoids	7.75	6.79–11.11
Allium hookeri (garlic chives) root	160 °C, 30 min, static, 1 g/20 mL	Gallic acid	2.70	0.20–0.50	No	[188]
		Total phenolics	29.40	2.40–3.10
		Total flavonoids	30.60	4.10–6.20
Allium sativum (garlic) root	120–180 °C, 10 min, dynamic, 5 g/10 mL	Alliin	100.52–136.82	31.54–65.18	Yes	[189]
Camellia oleifera seed	120–160 °C, 20–60 min, static, 1 g/18–22 mL	Saponins	17.80–316.90	-	No	[190]
Castanea mollissima (Chinese chestnut) exocarp	100–220 °C, 20 min, static, 5 g/600 mL	Total phenolics	159.37–746.71	87.64–295.23	No	[191]
Castanea sativa (chestnut) shells	51–249 °C, 6–30 min, static, 10 g/100 mL	Total phenolics	315.21–496.80	-	No	[192]
Castanea sativa (chestnut) peels	100–220 °C, 0–45 min, static, 1 g/20–30 mL	Total phenolics	60.00–160.70	-	No	[193]
		Catechin	112.85
		Total tannins	828.40
		Hydrolyzable	63.54
		Nonhydrolyzable	764.86
Citrus unshiu (Satsuma orange) peel	150 °C, 15 min, static, 190 °C, 5 min, static, 3 g/22 mL	Hesperidin	38.45	2.10–22.00	No	[194]
		Narirutin	6.56	3.10–7.50
Coffea genus (coffee) silverskin	120–240 °C, 10–60 min, static, 1 g/10–40 mL	Total phenolics	19.01–93.83	-	No	[144]
		Total chlorogenic acid	0.48–2.33
Curculigo latifolia root	100–200 °C, 30–120 min, dynamic, 1 g/10 mL	Total phenolics	34.43–92.55	-	No	[195]
		Total flavonoids	5.72–13.26
Hibiscus sabdariffa (calyces of roselle)	≈100–140 °C, 5 min, dynamic, 0.2 g/25 mL	Total phenolics	0.07–0.39	-	Yes	[196]
		Total flavonoids	1.82–7.62
		Total anthocyanins	6.56–9.74
Hippophae rhamnoides (sea buckthorn) leaves	80–260 °C, 15 min, static, 2 g/33 mL	Total phenolics	76.07–93.72	28.35–77.85	No	[197]
		Total flavonoids	47.06–66.40	14.14–42.93
		Quercetin-3-galactoside	0.16–0.26	0.04–0.17
		Kaempferol	0.01–0.04	0.01–0.02
		Isorhamnetin	0.02–0.11	0.02–0.06
Malpighia emarginata (acerola) seeds, pomace	70–130 °C, 15 min, dynamic, 5 g/90 mL	Total phenolics	119.10–362.00	292.60	No	[198]
Melissa officinalis (lemon balm) leaves	100–150 °C, 10–20 min, dynamic, 100 g/2000 mL	Rosmarinic acid	23.60–53.10	55.50	Yes	[176]
		Total phenolics	0.26–0.30	0.27
Origanum vulgare (oregano) leaves	25–200 °C, 15–30 min, static, 0.75 g/11 mL	Total phenolics	84.00–182.00	-	Yes	[199]
Orostachys japonicus stems, leaves	110–240 °C, 15 min, static, 1 g/22 mL, 30 g/900 mL	Total phenolics	9.40–39.90	3.50–10.70	No	[200]
		Total flavonoids	2.50–11.40	3.6–6.1
Oryza sativa rice bran	125–200 °C, 30 min, static, 4 g/10 mL	Total phenolics	2.01–19.48	-	No	[201]
Pseuderanthemum palatiferum (Hoan-Ngoc) leaves	110–270 °C, 15 min, static, 2 g/140 mL	Total phenolics	9.48–33.68	6.94–15.27	No	[202]
		Total flavonoids	8.16–20.71	6.22–14.71
		Total saponins	5.12–33.82	1.38–4.09
Pueraria lobata (kudzuvine) root	120–160 °C, 20–60 min, static, 1 g/15–20 mL	Total flavonoids	130.00	48.00–150.00	Yes	[203]
		Puerarin	48.00	22.00–69.00
		Daidzin	9.00	4.00–10.00
		Daidzein	0.60	0.30–0.50
Punica granatum (pomegranate) seed	80–280 °C, 15–120 min, dynamic, 1 g/10–50 mL	Total phenolics	9.92–27.52	3.12–21.94	No	[204]
Stevia rebaudiana (stevia) leaves	100–150 °C, 30–60 min, dynamic, 1 g/10 mL	Total phenolics	28.96–48.63	-	No	[205]
		Total flavonoids	24.27–29.81
		Rebaudioside A	6.20–35.68
		Stevioside	3.27–38.67
Tagetes erecta flower	80–260 °C, 15–90 min, dynamic, 1 g/20–60 mL	Total phenolics	116.00	10.94–96.66	No	[206]
		Total flavonoids	135.00	12.78–127.54
Thymus vulgaris (thyme) leaves	50–200 °C, 5–30 min, static, 5 g/100 mL	Total phenolics	131.00–262.00	-	Yes	[207]

summarizes available studies on the development and characterization of dry botanical extracts.

The studies summarized in the table further confirm that SWE can be highly effective for the targeted recovery of specific bioactive compounds, although its selectivity is strongly dependent on process conditions. For instance, efficient recovery of citrus flavanones has been demonstrated by high yields of hesperidin (38.45 mg/g) and narirutin (6.56 mg/g) from Citrus unshiu peel, highlighting the potential of SWE for valorizing agro-industrial by-products rich in glycosylated flavonoids [194]. Similarly, SWE enabled the selective extraction of chlorogenic acids from coffee silverskin, although the yields remained relatively moderate (0.48–2.33 mg/g), suggesting partial degradation or limited accessibility within the matrix [144]. Notably, SWE shows high efficiency for phenolic acids when appropriately optimized. For example, the extraction of rosmarinic acid from lemon balm reached 23.6–55.5 mg/g, confirming the strong recovery potential for this compound. However, as discussed above, increasing extraction severity may reduce its relative content due to concurrent matrix degradation [176]. In another case, gallic acid was successfully recovered from Allium hookeri roots (2.70 mg/g), indicating that SWE can facilitate both the release and possible formation of simple phenolic acids from more complex precursors [188]. Isoflavones and related compounds also exhibit favorable recoveries under SWE conditions. High yields of puerarin (48.0 mg/g) and daidzin (9.0 mg/g) from Pueraria lobata root further demonstrate the suitability of SWE for extracting relatively polar bioactive glycosides [203].

Overall, these studies indicate that SWE is highly effective for recovering defined compounds such as flavanones, phenolic acids, and isoflavones. In this context, pharmacopoeial standards could play an important role in defining minimum content requirements for marker compounds in dried extracts (i.e., 2.0% rosmarinic acid in dry extract produced from Melissa leaf (Melissa officinalis L.) [208] not less than 2.5% of total withanolides in dry ashwagandha root extract (Withania somnifera) [156], etc. However, studies adopting this systematic approach are relatively scarce, and the compliance of available studies on subcritical water extracts from medicinal plants with pharmacopoeial requirements is summarized in

Table 3. Compliance of subcritical extracts with pharmacopoeial requirements.

Plant Source	Pharmacopoeial Requirements for Standardization	Bioactive Compounds in SWE	Compliance with Pharmacopoeial Requirements	Comment	Reference
Achillea millefolium (yarrow) flower	Essential oil: minimum 2 mL/kg in dried drug; Proazulenes, expressed as chamazulene: minimum 0.02 per cent in dried drug.	Total phenolics Total flavonoids	No	The study does not analyze the content of essential oil.	[187]
Carum carvi (caraway) seeds	Minimum 30 mL/kg of essential oil	Carvone Limonene Carveol	No	The study does not analyze the content of essential oil.	[163]
Centella asiatica (Gotu kola) whole plants	Minimum 6.0 per cent of total triterpenoid derivatives, expressed as asiaticoside in dried drug	Asiatic acid 0–0.70% Asiaticoside 0.11–0.84%	No	The study does not use pharmacopoeial assay.	[165]
Coptis chinensis (Chinese goldthread) rhizome	Minimum 5.0 per cent of berberine in dried drug	Extracted berberine from dried drug 6.07–7.19%	Yes	All tested SWE temperatures extracted more than 5% berberine.	[209]
Coriandrum sativum (coriander) seeds	Minimum 3 mL/kg of essential oil in dried drug	Total phenolics Total flavonoids	No	The study does not analyze the content of essential oil.	[170]
Hibiscus sabdariffa (calyces of roselle)	Minimum 13.5 per cent of acids, expressed as citric acid in dried drug	Total phenolics Total flavonoids Total anthocyanins	No	The study does not analyze the content of acids.	[196]
Hydrastis canadensis (Goldenseal) rhizoma	Minimum 2.5 per cent hydrastine in dried drug Minimum 3.0 per cent berberine in dried drug	Extracted hydrastin and berberine from dried drug 4.30%	No	The study does not use pharmacopoeial assay and does not present the individual contents of the extracted hydrastin and berberine.	[210]
Matricaria chamomilla (German chamomile) flowers	Minimum 4 mL/kg blue essential oil in dried drug; Minimum 0.25 per cent apigenin 7-glucoside in dried drug	Extracted total flavonoids (1.75–2.04%) and apigenin (0.20–3.20%) from dried drug	No	The study does not analyze the content of essential oil, does not use pharmacopoeial assay and analyze apigenin instead of apigenin 7-glucoside.	[166]
Melissa officinalis (lemon balm) dry extract	Minimum 2.0 per cent of rosmarinic acid in dried extract.	Rosmarinic acid content in dry extract 2.36–5.28%	Yes	Extracts obtained at different extraction conditions (temperature 100 °C and 150 °C) and duration (10 min and 20 min) colmply with pharmacopoeial requirements.	[176]
Melissa officinalis (lemon balm) leaves	Minimum 1.0 per cent of rosmarinic acid in dried drug	Extracted rosmarinic acid from dried drug 1.22–1.51%	Yes	All tested extraction parameters (temperature 100 °C and 150 °C) and duration (10 min and 20 min) extracted more than 1% rosmarinic acid.	[176]
Origanum vulgare (oregano) leaves	Essential oil: minimum 25 mL/kg in anhydrous drug; Sum of the contents of carvacrol and thymol in the essential oil.	Total phenolics	No	The study does not analyze the content of essential oil and the sum of the contents of thymol and carvacrol in the essential oil.	[199]
Pueraria lobata (kudzuvine) root	Minimum 6.5 per cent of total isoflavonoids, expressed as puerarin in dried drug, of which minimum 45 per cent consists of puerarin	Total flavonoids, Puerarin, Daidzin and Daidzein in dry extract	No	The methodology used to analyze total flavonoids does not comply with the pharmacopoeial procedure that analazyes the conent of 3-hydroxypuerarin, puerarin, 3-methoxypuerarin, 6-O″-d-xylosylpuerarin and daidzin.	[203]
Rheum tanguticum (Chinese rhubarb) root	Minimum 2.0 per cent of the sum of hydroxyanthracene glycosides, expressed as rhein-8-glucoside in dried drug	Total anthraquinones 1.55–3.79%	Yes	The study did not employ pharmacopoeial assay.	[179]
Scutellaria baicalensis (Baical skullcap) root	Not less than 9.0 per cent of baicalin in dried drug	Extracted baicalin from dried drug 2.28–2.43%	No	Extracted baicalin content lower than pharmacopoeial requirements.	[209]
Thymus vulgaris (Thyme) leaves	Essential oil: minimum 12 mL/kg (anhydrous drug); Sum of the contents of thymol and carvacrol: minimum 40 per cent in the essential oil	Total phenolics Individual phenolic compounds in dry extract	No	The study does not analyze the content of essential oil and the sum of the contents of thymol and carvacrol in the essential oil.	[207]
Glycyrrhiza glabra (Liquorice) root	Minimum 4.0 per cent of 18β-glycyrrhizic acid in dried drug	Extracted glycyrrhizin from dried drug 1.72–1.84%	No	The study does not use pharmacopoeial assay.	[209]

. As evident from the table, among the limited number of studies using pharmacopoeial plant materials, only three report SWE extracts that meet the specified requirements for marker compound content, either in the dried herbal material or in the resulting dry extracts.

The enrichment of plant extracts with components derived from the degradation of plant cell wall polymers (carbohydrates and proteins) may necessitate additional purification steps. Several approaches can be employed for this purpose. Liquid–liquid extraction can be effective for the isolation of marker compounds; however, it requires the use of organic solvents [31,211]. To reduce solvent consumption, solid-phase extraction is frequently applied, offering improved reproducibility and selectivity through the use of various sorbents. SPE is particularly effective when combined with modified extraction systems or tailored solvent mixtures [212,213]. Nevertheless, these techniques may still rely on organic solvents for elution of target compounds, thereby limiting the environmental advantages of SWE. In contrast, membrane-based technologies such as ultrafiltration and nanofiltration provide efficient, non-thermal alternatives for the separation, purification, and concentration of high-value plant bioactives without the use of organic solvents. Ultrafiltration (typically 0.01–0.1 µm) enables the removal of large macromolecules such as proteins and polysaccharides, whereas nanofiltration (approximately 0.001 µm) facilitates the concentration of smaller molecules based on size and charge. These characteristics make membrane processes particularly suitable for the separation and purification of marker compounds from SWE-derived plant extracts [214]. An integrated process flow diagram for the production of standardized botanical extracts is presented in Figure 5.

5. Advantages, Limitations and Technical Challenges of SWE

In conventional solvent-based extraction, the efficiency of bioactive compound recovery is strongly influenced by the intrinsic characteristics of the plant material [48]. Each plant species exhibits a distinct matrix architecture, chemical composition, and phytochemical profile, resulting in considerable variability in the extractability of target compounds. Even within the same species, different plant parts, such as leaves, stems, roots, and flowers, show varying extraction efficiencies due to differences in tissue structure and compound localization [215]. Furthermore, the physical state of the raw material (e.g., fresh, dried, or ground) and particle size distribution can significantly affect extraction performance under otherwise identical operating conditions [216].

5.1. Solvent Use

Conventional extraction techniques typically employ organic solvents such as ethanol, methanol, acetone, or their mixtures, selected according to the polarity of the target compounds. Although effective, the extensive use of these solvents increases process costs and raises environmental and safety concerns related to toxicity, flammability, and the need for subsequent solvent removal and recovery steps [217]. In contrast, SWE utilizes water as the extraction medium, which is inexpensive, non-flammable, and non-explosive, thereby eliminating the use of organic solvents and significantly reducing the environmental footprint of the extraction process.

5.2. Energy Consumption

Conventional solvent extraction is typically associated with prolonged extraction times, ranging from several hours to days, and often requires multiple extraction cycles. These factors contribute to high overall energy consumption, particularly when solvent heating and evaporation are involved [217,218]. Subcritical water extraction (SWE) is inherently energy-intensive, as substantial thermal input is required to heat water to 110–250 °C and maintain elevated pressures. Nevertheless, enhanced mass transfer and accelerated extraction kinetics enable significantly shorter processing times, which may lead to a more favorable overall energy balance. Furthermore, energy efficiency can be improved through the implementation of heat recovery systems and continuous processing designs [67].

5.3. Selectivity

The selectivity of conventional solvent extraction is primarily dictated by the inherent polarity of the chosen solvent. Consequently, the selective recovery of different classes of compounds often requires the use of multiple solvents or sequential extraction steps [219]. In contrast, SWE allows solvent selectivity to be continuously tuned by adjusting temperature and pressure, enabling the extraction of compounds with varying polarity using water alone. However, due to the extensive disruption and degradation of plant cell wall components under subcritical conditions, SWE extracts may become enriched with degradation products of polysaccharides and proteins. This co-extraction can reduce the relative concentration of marker bioactive compounds in the final dried extract, thereby complicating standardization.

5.4. Suitability for Standardization

Conventional solvent extraction processes are often sensitive to variations in raw material properties, solvent composition, and operating conditions, which can hinder process standardization and industrial scale-up [220]. In contrast, SWE offers improved reproducibility and process control due to the precise regulation of temperature, pressure, and extraction time, making it more suitable for standardized and scalable industrial applications.

To facilitate a concise comparison between conventional solvent-based extraction and SWE, key differences can be highlighted in terms of solvent use, process efficiency, selectivity, environmental impact, and potential for standardization [35,65,221]. Numerous studies demonstrate that SWE frequently outperforms conventional techniques by achieving higher extraction efficiencies and significantly shorter processing times, while eliminating the need for toxic organic solvents, in line with the principles of green chemistry [187]. Furthermore, the tunable physicochemical properties of water under subcritical conditions enable the effective extraction of both polar and moderately nonpolar compounds, with a reduced environmental burden and simplified downstream processing compared to traditional solvent-based methods, which typically involve prolonged extraction cycles and solvent removal steps [222]. These advantages, including enhanced mass transfer, adjustable selectivity, and reduced reliance on hazardous solvents, highlight the technological maturity and industrial relevance of SWE. However, the main limitations of the technology are associated with the requirement for specialized high-pressure and high-temperature equipment, which increases operational costs and process complexity, thereby limiting its widespread industrial adoption. In addition, elevated temperatures may lead to the degradation of thermolabile bioactive compounds, potentially affecting product quality. Therefore, precise control of process conditions is essential to minimize unwanted side reactions and maintain the desired extract composition.

5.5. Regulatory Framework

SWE-derived extracts are not currently covered by a specific regulatory framework as a distinct extraction technology; instead, they are assessed within existing regulatory systems based on their intended use (e.g., food, food supplement, or medicinal product), composition, and safety profile. In the European Union, botanical extracts intended for use in foods or food supplements fall under the general provisions of food law, including Regulation (EC) No 178/2002 [223]. For herbal medicinal products, compliance with guidelines issued by the European Medicines Agency and relevant monographs of the European Pharmacopoeia is required, particularly with respect to marker compounds, purity, and manufacturing consistency [224].

In the United States, SWE-derived products are likewise not regulated according to the extraction technology itself. Instead, botanical ingredients are assessed within the regulatory framework of the U.S. Food and Drug Administration. For food applications, ingredients may be marketed if they are Generally Recognized as Safe or approved as food additives, in accordance with the Federal Food, Drug, and Cosmetic Act [225]. For dietary supplements, compliance with the Dietary Supplement Health and Education Act is required, including notification procedures for new dietary ingredients [226].

A key regulatory consideration for SWE is whether the use of elevated temperature and pressure conditions leads to the formation of new compounds or alters the phytochemical profile in a manner that may affect safety or efficacy. In such cases, additional toxicological and compositional data may be required by regulatory authorities [225,227]. Furthermore, irrespective of the extraction method, the standardization of botanical extracts for pharmaceutical use is governed by established pharmacopoeial requirements and quality guidelines, including Good Manufacturing Practice and specifications for marker compounds and contaminants [224,228].

5.6. Scaling Up

Mass transfer coefficients derived from laboratory-scale batch or static systems cannot be directly extrapolated to industrial dynamic or continuous configurations, due to fundamentally different flow regimes and heat transfer characteristics. Nevertheless, pilot-scale SWE systems have been successfully applied to a variety of plant matrices, including citrus peels and brewer’s spent grain, achieving high recovery of target compounds [229,230]. Furthermore, SWE shares technological similarities with hydrothermal processing platforms that are already implemented at demonstration and industrial scales for biomass valorization, processing up to several tons per day and enabling the recovery of functional ingredients such as antioxidants, oligosaccharides and protein hydrolysates [67]. Despite these advances, the broader industrial adoption of SWE for the production of standardized plant extracts remains constrained by economic and engineering considerations. Capital expenditure is relatively high due to the need for corrosion-resistant, high-pressure equipment, as well as specialized pumping and heat-exchange systems. Consequently, although SWE represents a technically validated and environmentally sustainable approach with strong potential for the development of standardized plant extracts, its industrial implementation remains at an emerging stage, with economic and energy considerations playing a critical role in determining its feasibility at scale.

6. Conclusions and Future Perspectives

The present review demonstrates that SWE has been widely applied for the extraction of a broad range of plant species and tissues. However, the majority of the studies evaluated focus primarily on the recovery of general compound groups (e.g., total polyphenols, total flavonoids), which is insufficient for achieving precise extract standardization. Only a limited number of studies adopt a systematic approach aligned with pharmacopoeial requirements. These studies indicate that SWE can be successfully applied to the development of standardized extracts containing defined levels of marker compounds, such as rosmarinic acid from lemon balm leaves or berberine from Chinese goldthread rhizomes. Nevertheless, the overall trend suggests that SWE has often been underestimated or, in some cases, inadequately applied in the context of standardized extract development. This limited approach overlooks the potential of SWE to fine-tune extraction parameters (e.g., temperature, pressure, and solvent polarity), enabling targeted recovery of specific marker compounds and alignment with pharmacopoeial standards. Given the diversity of medicinal plants and their bioactive constituents, this gap presents significant opportunities for future research and the development of standardized botanical extracts. However, following key aspects must be carefully considered:

Extraction efficacy: it depends on the efficient recovery of target metabolites and is governed by parameters such as temperature, extraction time, particle size, and solvent-to-solid ratio. While higher temperatures can enhance mass transfer and solubility, they may also promote degradation, necessitating careful optimization to balance yield, selectivity and compound stability.

Extract purity: it is related to the concentration of target metabolites in the final dried extract. Elevated SWE temperatures may increase the co-extraction of polysaccharides, proteins and other macromolecules, thereby increasing overall yield but diluting target compounds and complicating standardization. This highlights the need for a better understanding of hydrolysis products and their interactions, as well as for integrating SWE with green downstream purification techniques (e.g., membrane filtration).

Safety and toxicity: these considerations are also critical, as the complexity of plant matrices may lead to the formation of potentially harmful compounds during processing. For instance, Maillard reactions can generate compounds such as 5-HMF and furfural, which may further transform into other undesirable products. Although the reviewed studies indicate that their levels under commonly applied SWE conditions are generally low and not considered hazardous compared to other food processing practices, their formation should not be neglected.

Despite its demonstrated potential, SWE remains at a relatively early stage of maturity for pharmacopeial standardization, as current applications are often limited by insufficient compound-specific optimization and a lack of validated, reproducible protocols. Key challenges include controlling hydrothermal transformations, ensuring consistent marker compound recovery, and integrating efficient downstream purification strategies. Overall, future research should focus on a deeper understanding of hydrothermal transformations, their interactions, and their impact on extract composition, as well as on the development of SWE protocols tailored for standardized extracts with controlled degradation and reproducible phytochemical profiles. Controlling these processes remains challenging and requires detailed knowledge of biomass composition and the underlying degradation pathways.