Extraction Processes Affect the Composition and Bioavailability of Flavones from Lamiaceae Plants: A Comprehensive Review

Lamiaceae plants are a widespread family of herbaceous plants with around 245 plant genera and nearly 22,576 species distributed in the world. Some of the most representative and widely studied Lamiaceae plants belong to the Ocimum, Origanum, Salvia, and Thymus genera. These plants are a rich source of bioactive molecules such as terpenes, flavonoids, and phenolic acids. In this sense, there is a subgroup of flavonoids classified as flavones. Flavones have antioxidant, anti-inflammatory, anti-cancer, and anti-diabetic potential; thus, efficient extraction techniques from their original plant matrixes have been developed. Currently, conventional extraction methods involving organic solvents are no longer recommended due to their environmental consequences, and new environmentally friendly techniques have been developed. Moreover, once extracted, the bioactivity of flavones is highly linked to their bioavailability, which is often neglected. This review aims to comprehensively gather recent information (2011–2021) regarding extraction techniques and their important relationship with the bioavailability of flavones from Lamiaceae plants including Salvia, Ocimum, Thymus, and Origanum.


Lamiaceae
The Lamiaceae family of plants, also known as the mint family, belongs to the major group Angiosperms, which is a group of herbs and shrubs that are annual or perennial, and most of which are aromatic plants [1][2][3]. The Plant List database states that around 245 plant genera are included in the Lamiaceae family, with around 7886 species [4], representing the sixth largest family of flowering plants in the world. The Lamiaceae family is distributed around the world and is widely diverse. Moreover, some of the most evaluated plant genera are Salvia (986 species), Ocimum (66 species), Origanum (56 species), and Thymus (315 species) [4]. Plants of the Lamiaceae have square stems and opposite leaves, with zygomorphic flowers that have five united petals and five united sepals. These plant species are also known for their aromatic characteristics given by their essential oils and as a source of natural compounds such as terpenes, essential oils, phenolic acids, and flavonoids [5,6]. Flavonoids are produced from the phenylpropanoid pathway [14,15]. During flavonoid biosynthesis, the main flavonoid backbone (a chalcone with a C6-C3-C6 structure), is derived from the shikimic acid pathway and is produced through the action of the enzyme 4-coumaroyl coenzyme A; this generates the synthesis of the B and C rings, and three molecules of malonyl coenzyme A, the precursor of ring A. The resulting flavonoid backbone produces the other flavonoid subclasses, which are further synthesized by the action of reductases, isomerases, hydroxylases, and other enzymes [15,16]. Subsequently, flavones are produced from the flavanone naringenin by catalysis of two oxidoreductases, flavone synthase I and II, introducing a double bond between C2 and C3 [17]. For a more detailed description of the biosynthesis of flavones, we recommend the work by Jiang, Doseff and Grotewold [17].

Flavones
Flavones are a subtype of flavonoids that are characterized by a double bond in C2-C3 in the flavonoid structure and a ketone group at C4, and a lack of oxygenation at C3 (Figure 1) [18]. Some of the most known flavones are apigenin, cirsimaritin, luteolin, scutellarein, and their derivatives ( Figure 2). Flavones can be found in nature with various substitutions, including -OH radicals, methylation, O-and C-alkylation, and glycosylation [10]. These residues may be found alone or in combination with flavones [19]. Many flavones have been reported in Lamiaceae plants, where they mostly accumulate in leaves, aerial parts, and exudates. recommend the work by Jiang, Doseff and Grotewold [17].

Flavones
Flavones are a subtype of flavonoids that are characterized b C3 in the flavonoid structure and a ketone group at C4, and a lac ( Figure 1) [18]. Some of the most known flavones are apigenin scutellarein, and their derivatives ( Figure 2). Flavones can be foun substitutions, including -OH radicals, methylation, O-a glycosylation [10]. These residues may be found alone or in com [19]. Many flavones have been reported in Lamiaceae plant accumulate in leaves, aerial parts, and exudates. In plants, flavones act as plant defenses against UV radiation have an ecological interaction with the soil microbiota [20,21]. H also attracted the attention of scientists because their consumpti with beneficial health properties including the prevention o prevention of premature aging, the decreased incidence of non such as diabetes and different types of cancer, and decreased diseases [17,22]. These noncommunicable diseases represent a g care systems and are the main cause of death worldwide [23]. The flavones depend on the number, nature, and position of the sub skeleton, which will modulate their regioselectivity and the way biological targets to exert health-promoting properties [24]. Du flavones in human health, several strategies have also been rep synthetically [24].
Moreover, some of the structural characteristics that facil flavones with biological molecules to act as antioxidants and properties are: (a) the presence of catechol on ring B; (b) the 2,3-ole group provides electron delocalization from ring B, which facilit electron to free radicals; (c) the -OH groups at C3 and C5 form hy keto group; (d) the synergy between flavones and some physiolo In plants, flavones act as plant defenses against UV radiation, favor pollination, and have an ecological interaction with the soil microbiota [20,21]. However, flavones have also attracted the attention of scientists because their consumption has been associated with beneficial health properties including the prevention of oxidative stress, the prevention of premature aging, the decreased incidence of noncommunicable diseases such as diabetes and different types of cancer, and decreased risk of cardiovascular diseases [17,22]. These noncommunicable diseases represent a global burden in health care systems and are the main cause of death worldwide [23]. The bioactive properties of flavones depend on the number, nature, and position of the substituents in the flavone skeleton, which will modulate their regioselectivity and the way flavones interact with biological targets to exert health-promoting properties [24]. Due to the importance of flavones in human health, several strategies have also been reported to produce them synthetically [24].
Moreover, some of the structural characteristics that facilitate the interaction of flavones with biological molecules to act as antioxidants and exert other bioactive properties are: (a) the presence of catechol on ring B; (b) the 2,3-olefinic bond and the keto group provides electron delocalization from ring B, which facilitates the donation of an electron to free radicals; (c) the -OH groups at C3 and C5 form hydrogen bonds with the keto group; (d) the synergy between flavones and some physiological antioxidants; and (e) the capacity of flavones to chelate metal ions attributed to the -OH at C5 and the C4 keto group [17,20].

Literature Research Strategy
To identify relevant information on Lamiaceae flavones, this review was compiled based on recent scientific literature (2011-2021) from the Scopus, Google Scholar, and Web of Science databases. The keywords used for the literature search included the terms Lamiaceae, Origanum, Ocimum, Salvia, Thymus, flavones, apigenin, luteolin, scutellarein, cirsimaritin, extraction, supercritical CO2, ultrasound-assisted extraction, and microwaveassisted extraction ( Figure 3).

Characteristics of the Salvia Genus
Salvia L. species constitute the largest genus in the family Lamiaceae with over 1000 species, organized into five subgenera (Sclarea, Audibertia, Jungia, Leonia, and Salvia); these plants grow in temperate, subtropical, arctic, and sub-arctic areas of the world and

Literature Research Strategy
To identify relevant information on Lamiaceae flavones, this review was compiled based on recent scientific literature (2011-2021) from the Scopus, Google Scholar, and Web of Science databases. The keywords used for the literature search included the terms Lamiaceae, Origanum, Ocimum, Salvia, Thymus, flavones, apigenin, luteolin, scutellarein, cirsimaritin, extraction, supercritical CO 2 , ultrasound-assisted extraction, and microwaveassisted extraction ( Figure 3).

Literature Research Strategy
To identify relevant information on Lamiaceae flavones, this review was compiled based on recent scientific literature (2011-2021) from the Scopus, Google Scholar, and Web of Science databases. The keywords used for the literature search included the terms Lamiaceae, Origanum, Ocimum, Salvia, Thymus, flavones, apigenin, luteolin, scutellarein, cirsimaritin, extraction, supercritical CO2, ultrasound-assisted extraction, and microwaveassisted extraction ( Figure 3).

Characteristics of the Salvia Genus
Salvia L. species constitute the largest genus in the family Lamiaceae with over 1000 species, organized into five subgenera (Sclarea, Audibertia, Jungia, Leonia, and Salvia); these plants grow in temperate, subtropical, arctic, and sub-arctic areas of the world and
Among Salvia bioactivities, the amoebicidal and giardicidal effects and the antidiarrheal properties of S. divinorum Epling & Jativa, S. gesneriiflora Lindl. & Paxton, S. herbacea Benth., S. microphylla Kunth, and S. shannonii Donn. Sm. have been studied, and it was found that the antiprotozoal activity of flavonoids, such as flavones, appears to be related to the phenolic and hydroxy groups at C-3, C-5, and C-7 of the flavonoid backbone. The change of a hydroxy to a methoxy group or a monosaccharide moiety at C-3 decreases the activity. Especially about flavones, a methoxy group at C-6 was favorable, and when the degree of oxygenation in the B-ring increased, the antiprotozoal activity decreased significantly; it was also observed that the 2,3-double bond was not essential for high antiprotozoal activity, but the stereochemistry could play an important role [37]. In this sense, Bautista, Calzada, Yépez-Mulia, Bedolla-García, Fragoso-Serrano, Pastor-Palacios and González-Juárez [37] determined the antiprotozoal activity of S. connivens Epling, against Entamoeba histolytica (IC 50 0.072 ± 0.006 µM) and Giardia lamblia (IC 50 0.118 ± 0.006 µM), which was comparable to the drug metronidazole; these results were related to the presence of three flavones: eupatorin, cirsiliol, and nuchensin.
There is a large variability of flavonoid structures among the Salvia genus, such as flavones; their chemical differentiation might be correlated to the geographical and ecological conditions under which they grow [31]. Researchers often find new structures in Salvia species; for example, two new flavone glycosides, with an unusual interglycosidic linkage, were isolated from the petals of S. uliginosa [29].

Characteristics of the Ocimum Genus
The genus Ocimum comprises more than 300 species of annual and perennial herbs and shrubs, and it is considered as one of the largest genera of the Lamiaceae family; this genus comprises many distinct species and varieties [38][39][40]. The typical characteristics of this genus, as with other members from the same family, are a square stem, and opposite and decussate leaves with many gland dots. The flowers (white, pink, violet) are strongly zygomorphic with two distinct lips; the stamens lie over the lower (anterior) lip of the corolla rather than ascending under the upper (posterior) lip [41]. The genus Ocimum is widely distributed in tropical and warm temperate regions over Asia, Africa, and Central and Southern America; this genus requires warmth for growth and should be protected from frost [41]. The name "Ocimum" is derived from the Greek meaning "to be fragrant"; therefore, plants of this genus are aromatic and rich in secondary metabolites, which humans have learned to use since antiquity for food preservation, flavoring, and as medicine [1,40].
Due to large variation in the morphological characteristics of species, in addition to human intervention, it has become difficult to identify some species. Therefore, it has been concluded that identification can be conducted in an auxiliary manner through molecular markers, such as the tetrahydroxyflavone luteolin 5 [38,39].
Ocimum species have been related to many different bioactivities, and most of them have been correlated to essential oils and their components. Nonetheless, a recent study showed that the polymethoxylated flavones 5-demethyl nobiletin and 5-demethyl sinensetin, together with luteolin, isolated from O. campechianum, decreased blood glucose in in vivo model; furthermore, it was proposed that these two polymethoxylated flavones can be considered as chemotaxonomical markers for the genus [18]. In addition, luteolin, and luteolin glycosides from O. sanctum leaves presented leishmanicidal properties against L. major, antituberculosis, and cytotoxic cells of prostate carcinoma in mice, and showed anti-inflammatory and antiproliferative activities [1].

Characteristics of Origanum
Origanum is an important plant genus that belongs to the Lamiaceae family. Its foremost characteristic species is Origanum vulgare L., commonly known as European oregano. The genus Origanum comprises 49 taxa and more of 42 species and 18 hybrids [43]. Origanum were divided into ten sections: (i) Amaracus Bentham, (ii) Anatolicon Bentham, (iii) Brevifilamentum Ietswaar, (iv) Longitubus Ietswaart, (v) Chilocalys Ietswaart, (vi) Majorana Bentham, (vii) Campanula ticalix Ietswaart, (viii) Elongatispica Ietswaart, (ix) Origanum Ietswaart, and (x) Prolaticorolla Ietswaart [44]. The majority of the Origanum species are located within the Mediterranean, occurring mainly in Greece and Turkey [43]. This plant grows at altitudes between the 400 and 1800 m, and in sunny areas [45]. Origanum species are annual, perennial herbs with oval to small circular leaves, with sometimes toothed margins and obtuse to pointed tips. The flowers might present white, pink or purple colors and are clustered in spikes [46].
From these studies, the most frequently identified flavones in the different polyphenolic extracts from Origanum species are luteolin and apigenin derivatives, which have shown antioxidant [53,54], anti-cancer [55], and anti-inflammatory properties [56]. Other flavones, such as didymin, isolated from O. vulgare, presented biological properties, such as anti-inflammatory activity and a reduction in the hepatic damage induced by CCl 4 , in male mice [57].

Characteristics of Thymus
The genus Thymus, belonging to the Lamiaceae family, consists of over 336 species [63]. Thymus vulgaris L., known as common thyme, is the most significant species of this genus [64]. Plants from the Thymus genera are native to the Eurasian and the Mediterranean region and are also distributed over North Africa, Australia, and South America [64,65].
Thymus species are small perennial shrubs that possess grey to green leaves that might be arranged oppositely or clustered. The flowers present light violet, purple or white coloring [64].
Polyphenolic extracts containing flavones from several Thymus species have been evaluated to determine their biological properties. For instance, it was demonstrated that decoction extracts containing glucosides of luteolin and apigenin from T. herba-barona, T. pseudolanuginosus, and T. caespititius possess antioxidant, anti-inflammatory, and antibacterial activities [71].

Conventional Methods
Flavones are usually extracted by conventional techniques, including maceration, Soxhlet extraction, hydrodistillation, and boiling, among others [80]. In almost all extraction, it is necessary to decrease particle size to help the process [81]; the plant is dried and pulverized to obtain a powder used for the extraction. Table 3 summarizes the conventional extraction techniques used to obtain flavones in Lamiaceae species; we encourage the reading of each specific research paper to obtain detailed information about the extraction process, identification technique, and compound identification. In general, apigenin, luteolin, and their glucosides are widely distributed in the genera analyzed and can be extracted by various conventional methods. Moreover, the aerial parts (leaves, stems, and flowers) are the most used in these extractions; separately, however, leaves, roots, flowers, and residues from previous processes can also be used to obtain flavones.
In Salvia species, hydrophilic solvents are widely used to extract flavones. For instance, methanolic, ethanolic, and aqueous mixtures have been used to extract various compounds, mostly luteolin and apigenin derivatives, including glucoside and glucuronides, along with hydroxylated and methylated derivatives [36,[82][83][84][85][86][87][88][89][90][91][92]. In contrast, other polar solvents such as acetone and ethyl acetate are less used to extract these compounds [32,37,[93][94][95], while dichloromethane is even less common [96,97]. Furthermore, the use of hot water to extract flavones is generally used to simulate the usual way in which these plants are consumed (infusion or decoction), with good results found when obtaining apigenin and luteolin derivatives [90], and other flavones such as cirsimaritin [98]. To improve the separation of desired compounds, it is necessary to fractionate the extracts by subsequent extractions using solvents with different polarity or by column chromatography, as seen in the hot water extract of Salvia absconditiflora, which was sequentially fractionated with ethyl acetate and n-butanol, and was found to be rich in flavones in the ethyl acetate fraction [98]. Similarly, the n-hexane extract of Salvia chloroleuca fractionated with ethyl acetate and methanol showed the first fraction as the best one to obtain these compounds. Moreover, flavones such as salpleflavone were found in the ethyl acetate fraction of the ethanolic extract of Salvia plebeian [92]. In addition, further fractionation of the ethanolic extract was useful to isolate the flavones neocafhispidulin and 6"-O-acetylhomoplantaginin, among others [86]. Similarly, the fractionation of the acetone extract of Salvia connivens was useful to isolate three bioactive flavones [37]. Meanwhile, fractionation was also used for less polar solvents such as the dichloromethane extract of Salvia circinata [97].
Polar solvents such as methanol, ethanol, and aqueous mixtures have been used to obtain extracts from species including O. basilicum, O. gratissimum, O. sanctum, and O. tenuiflorum with good results [84,[99][100][101][102][103][104]. Less used, but also effective in the extraction of flavones, are solvents such as diethyl ether, which is useful to extract such flavones as nevadensin and salvigenin in O. basilicum [105]. Fewer studies were observed with other solvents such as hot water extract that showed similar results to hydromethanolic and hydroethanolic extractions in the same plant [106]. On the other hand, some studies further fractionated the extract to isolate the compounds, as seen in the infusion of O. campechianum [107] and the methanolic extract of O. gratissimum and O. sanctum [100,101].
For Thymus species, multiple studies have been conducted using mostly ethanol, methanol, water, and their mixture as solvents, in which not much variety was observed in the flavones extraction of such species as T. alternans, T. caespititius, T. fragrantissimus, T. mastichina, T. pulegioides, T. serpyllum, and T. vulgaris, among others [68,71,[117][118][119][120][121]. However, using fractionation techniques, it is possible to obtain flavones that have not been identified in crude extracts such as 7-methoxyapigenin (genkwanin) [122] and nobiletin [123,124], while, by using dichloromethane, hydroxyluteolin and hydroxyapigenin derivatives can be extracted from T. mastichina [125]. Furthermore, the water residue from hydrodistillation from T. vulgaris process has shown to be a valuable source of flavones such as luteolin and apigenin glucuronide derivatives [126].

Alternative Methods (Environmentally Friendly)
Alternative extraction methods aim to limit the use of organic solvents, thereby reducing environmental damage and improving extraction efficiency. The most common alternative extraction techniques include ultrasound-assisted extraction (UAE), microwaveassisted extraction (MAE), pressurized liquid extraction (PLE, also known as accelerated solvent extraction), and supercritical fluid extraction (SFE), among others [142]. These technologies have been shown to be effective for extracting flavones from Lamiaceae, mostly apigenin and luteolin derivatives; the aerial parts are the most used for this. However, other parts, such as roots and even residues from previous processes, are rich in flavones. A summary of these extraction methods to obtain flavones in Lamiaceae species is shown in Table 4.
PLE has been widely used to obtain flavones from diverse Salvia species using water or ethanol as a solvent, obtaining similar compounds in them [143]. Although MAE can obtain a good variety of flavones [144], UAE was demonstrated to be more effective than MAE in some cases, because it is less time consuming [145]. On the other hand, SFE with ethanol as cosolvent was proven to be a more selective technique for the extraction of flavones such as cirsimaritin, genkwanin and salvigenin from Salvia rosmarinus leaves, compared to PLE, which obtained a greater variety of flavones [146]. Similarly, more flavone diversity was observed in the extract of roots from Salvia viridis obtained from UAE and MAE than from SFE [144]. For Origanum species, O. glandulosum (leaves and flowers) and O. majorana (leaves) have been extracted by MAE and UAE, respectively, with apigenin and luteolin derivatives in the former, but only luteolin derivatives in the latter [61,147]. However, in O. vulgare aerial parts, the same flavones profile was found using UAE, PLE, and MAE [148], while fewer flavones were found in another extract obtained by PLE [60,149]. With regard to Thymus genus, UAE is a popular extraction method to extract flavones from species such as T. marschallianus, T. seravschanicus, and T. serpyllum, and T. vulgaris [150][151][152]. Nevertheless, PLE is also effective in the extraction of flavones from Thymus species [149], including cirismaritin from T. serpyllum [60], which was also detected in T. Vulgaris after a combination of alternative extraction methods, namely pulsed electric field followed by ultrasound-assisted extraction [153]. A study by Palmieri et al. [154], with different conventional methods involving PLE and rapid solid-liquid dynamic extraction, showed that the aforementioned methods obtain better extract yield from T. vulgaris leaves and stems [154]. Subsequently, the extraction of flavones from Thymus residues using these technologies was studied, as seen in the extracts that were rich in flavones derived from steam distillation residues from T. mastichina [155], and the herbal dust and hydrodistillation residue from T. serpyllum and T. vulgaris obtained by PLE, respectively [73,156]. Finally, not many studies have been recently conducted regarding the extraction of flavones from Ocimum species using alternative methods. In this regard, the extract, obtained by UAE, of O. tenuiflorum leaves showed higher quantities of apigenin and luteolin, compared with a conventional ethanolic extract [157]; this demonstrates the high potential of these kinds of techniques in the extraction of flavones from Ocimum species.

Bioavailability and Bioactivity Relationship of Flavones in Lamiaceae
As previously mentioned, the chemical structure of flavones determines their bioactivity since it establishes the way in which they interact with biological molecules through different mechanisms of action [17,20,24]. Thus, any changes in the structures of flavones can affect (positively or negatively) their antioxidant, anti-inflammatory, anti-obesity, and anti-cancer properties. Once consumed, flavones enter the body and travel through the gastrointestinal tract. Here, flavones can be affected by physiological and biochemical conditions such as changes in pH, and interaction with other food constituents and digestive enzymes. These physiological conditions can alter the chemical structures of flavones by partial hydrolysis from the food matrix, by deprotonation of the -OH radicals of the flavone molecule, or through interaction with digestive enzymes, which will affect their bioactive properties [161,162].
Thus, it is important to evaluate the bioaccessibility and bioavailability of flavones to improve our understanding of their mechanisms of action and bioactive effects, and to develop strategies to enhance their bioavailability. Bioaccessibility is the amount of a food constituent that is released during gastrointestinal digestion and is accessible to be absorbed or pass through the enterocytes. Additionally, bioavailability is defined as the amount of the food constituent or molecule that is absorbed through the enterocytes into the bloodstream, distributed, metabolized, and excreted [161]. Bioaccessibility is assessed using simulated gastrointestinal protocols coupled with cell-based assays (Caco-2, HepG2), and bioavailability is assessed using in vivo assays that measure pharmacokinetic parameters [163][164][165][166][167].
In this sense, once consumed, flavones pass by the mouth where the saliva is present, and mastication occurs. Saliva is mainly constituted by water, electrolytes, proteins, and digestive enzymes. After that, the compounds are transported to the stomach, where pH drops to 2-4, facilitating the partial hydrolysis of these molecules from the matrix. Then, the chyme is transported from the stomach to the small intestine, passing through the duodenum, where the pH stabilizes to 7. Additionally, the small intestine is the major site of absorption for nutrients and phenolic compounds such as flavones [161,168]. The enterocytes in the small intestine are also a site of metabolism attributed to the phase I and phase II xenobiotic metabolic enzymes [169]. Generally, phenolic compounds have low bioaccessibility and bioavailability; most are degraded, metabolized, and excreted. Several factors can affect the bioavailability of flavones and phenolic compounds in general; these factors are related to the molecule and the food matrix, and others are related to the host, such as intestinal and systemic factors [170].
It was reported that aglycone flavones are more rapidly absorbed than their glycosylated derivatives due to their lower molecular weight; however, cell metabolism and transporters can mediate their active absorption [171]. In this sense, Caco-2 cell assays showed that apigenin is more permeable than apigenin-7-O-glucoside; furthermore, in vivo rat studies reported higher absorption of apigenin. Moreover, due to the xenobiotic metabolism in the body, most flavones in plasma are reported in their conjugated form by sulfation, glucuronidation, or methylation.
Few reports assess the bioaccessibility of flavones from plants of the Salvia, Thymus, Origanum, and Ocimum genus. These reports are summarized in Table 5. All reports use an in vitro gastrointestinal digestion process that simulates the digestion in the mouth, stomach, and small intestine, mimicking their physiological and enzymatic conditions; only two reports that coupled the simulated digestion to cell-based assays were found. In this sense, Chohan et al. [172] evaluated the effect of cooking and an in vitro digestion process on the total phenolic content and bioactive properties of Salvia officinalis and Thymus vulgaris. The cooking process increased the bioaccessible phenolic compounds, which might be related to the improvement of the release of phenolics from the vacuoles due to the cell wall rupture during cooking. In addition, following this, the anti-inflammatory potential of these extracts increased after the cooking and digestion process, indicating a correlation between bioaccessibility and bioactivity. Gayoso et al. [173] evaluated the bioaccessibility, through an in vitro digestion, of Origanum vulgare where an HPLC analysis showed the presence of a luteolin glycoside derivative. After digestion, luteolin glycoside had a bioaccessibility of 41%; moreover, the antioxidant activity of O. vulgare remained stable with no significant changes after the digestion process, indicating that digested phenolics are potentially bioactive. Recently, de Torre et al. [114] also evaluated the bioaccessibility of O. vulgare in an improved oral pharmaceutical form; the authors encapsulated O. vulgare and subjected it to gastrointestinal digestion. Three flavones were identified in the samples, namely two luteolin glycosides and one apigenin glycoside. The encapsulation enhanced the bioaccessibility of Origanum flavones, based on initial values with a bioaccessibility of 82.52, 85.31, and 89.28%, for the two luteolin glycosides and the apigenin glycoside, respectively. The high bioaccessibility of the flavones in the encapsulated samples is related to a higher stability as they were protected from the gastrointestinal environment.
Rubió et al. [174] showed that a mixture of olive oil and Thymus vulgaris extracts increased the bioaccessibility of luteolin as compared to thyme extract alone, with values of 16.7% and 14.6%, respectively. Moreover, a Caco-2 and Caco-2/HepG2 co-cultured assay showed that luteolin was one of the flavonoids detected after the incubation and that luteolin conjugated with sulfate and glucuronide were the main metabolites identified in the apical and basolateral sides of the cell cultures. On the other hand, incubation with HepG-2 cells only showed the presence of luteolin glucuronide.
Similarly, Villalva et al. [175] evaluated ethanol extracts of O. majorana subjected to a combination of simulated gastrointestinal digestion and a Caco-2 permeability assay. The authors identified several apigenin and luteolin derivates in the samples. The in vitro digestion process showed that the flavones with higher bioaccessibility were diosmin, luteolin-7-O-glucuronide, and luteolin-7-O-glucoside, with high stability values of nearly 99.7, 94.55, and 94.19% of the initial content. Furthermore, the contents of apigenin-7-Oglucoside and apigenin-7-glucuronide increased by 31.42 and 57.7%. The Caco-2 assay showed that luteolin and apigenin derivatives have low bioaccessibility, showing poor permeability capacity through the enterocytes. The increased levels of apigenin-7-Oglucoside and apigenin-7-glucuronide suggested cellular reflux of metabolized flavones, which are usually excreted at a physiological level. Additionally, the authors showed that rosmarinic acid enrichment of O. majorana extracts increases the content of its phenolic acids and flavonoids by 1.5-1.8 times, with luteolin and its glycosides being the main flavones detected in the enriched sample. In addition, it was suggested that the flavones luteolin and apigenin showed a synergistic effect with rosmarinic acid, protecting it from degradation during the digestion process. Furthermore, the aglycone forms of luteolin and apigenin were the main metabolites detected in the apical side of the Caco-2 monolayer culture, which might be attributed to the action of the metabolic enzyme lactase-phlorizin hydrolase. Due to the overall low bioaccessibility of phenolic compounds, it is sometimes suggested that after ingestion, their health-promoting properties could decrease. Nonetheless, in this study, the authors found that digested extracts displayed anti-inflammatory activity through inhibition of the secretion of the cytokines TNF-α, IL-1β, and IL-6 in a THP-1 cell line.
Our literature search found two recent studies that included an in vivo evaluation of the bioavailability of flavones from the Ocimum, Origanum, Salvia, and Thymus genus. Briefly, Rubió et al. [176] evaluated the effect of the combination of olive oil and Thymus vulgaris on the bioavailability and antioxidant capacity of the mixture using Wistar rats administered a dose of 1.5 mg/kg BW. The study showed that this type of extract modulated plasmatic antioxidant activity. Thyme extract and an olive oil/thyme mixture decreased the levels of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, but catalase activity was increased. In addition, the pharmacokinetic data showed that the presence of thyme extracts enhances olive oil phenolics; for instance, luteolin and apigenin were the main flavones identified in the sample, and the metabolites found in plasma were hydroxyphenylpropionic acid sulfate and dihydroxyphenylpropionic acid sulfate. The other study was reported by Zhang et al. [177], who evaluated the pharmacokinetics of danshen and huangquin (dried root of Scutellaria baicalensis Georgi), administered to Sprague-Dawley rats, which were prepared using a 1:1 ratio of weight in the mixture. Four main flavones were identified in the administered samples: baicalein, baicalin, wogonin, and wogonoside; these were found at concentrations of 13.606, 447.983 8.901, and 122.236 mg/kg, respectively. In plasma, the content of aglycone and glycoside flavones decreased significantly. Moreover, the T max values ranged from 1 to 8 h, and the C max values were 306.92, 2465.0, 373.17, and 1779.17 mg/L for baicalein, baicalin, wogonin, and wogonoside, respectively.

Thymus vulgaris
Freeze-dried olive cake and dried thyme were used for extracts by means of an accelerated solvent extractor.

Luteolin
The bioaccessibility of luteolin from thyme and in thyme and olive oil during the simulated digestion was 14.6% and 16.7%, respectively. Luteolin and its sulfate and glucuronide metabolites were detected after the incubation of Caco-2 cells. The flavone luteolin and its metabolites were the most bioaccessible. [174] Origanum majorana 100 mg of oregano dissolved in 50% ethanol were used Static simulated digestion (stomach, small intestine) coupled to a Caco-2 permeability assay Luteolin-7-O-glucoside and luteolin-7-O-glucuronide were the most stable. Luteolin and apigenin derivatives had low permeability in the Caco-2 assay.
[175] Furthermore, some chemical characteristics can aid in predicting the bioavailability of flavones and other phenolics, and these characteristics are known as the rule of five (or Lipinski's rule of five) [178]. These rules predict the drug-likeness of the passive absorption of a molecule. For instance, a molecule can permeate cells by passive absorption if the following conditions are met: The molecule has no more than 5 hydrogen bond donors; • The molecule has no more than 10 hydrogen bond acceptors; • The partition coefficient (Log p) is ≤5. Following these characteristics, conjugated flavones will often have lower cell permeability due to their higher molecular weight and numbers of H bond donors and acceptors. Nonetheless, cell transporters can metabolize these molecules via active means of absorption. The predicted passive absorption of some flavones mentioned in this study is shown in Table 6.

Conclusions
Plants of the Lamiaceae family, such as Ocimum, Origanum, Salvia, and Thymus, are rich sources of flavones. Flavones are flavonoids with antioxidant, anti-inflammatory, anticancer, and anti-diabetic potential. Some of the most abundant flavones in these species are apigenin and luteolin (and their derivates), cirsimaritin, and scutellarein. Thus, the development of methods to enhance their extraction is of interest to human health. For this, conventional techniques involving maceration, Soxhlet extraction, hydrodistillation, and boiling have been used for many years. It has been reported that the bioactive properties of flavones are highly dependent on their chemical structure, which is in turn highly dependent on the method of extraction and its further metabolism after ingestion. The factors mentioned above, plus an effort to diminish the environmental impact of conventional techniques, have led to the development of more environmentally friendly techniques such as ultrasound-assisted extraction, microwave-assisted extraction, pressurized liquid extraction, and supercritical fluid extraction. These techniques usually involve a higher initial investment expense but offer a higher yield, purity, and bioactivity of flavones. Moreover, after ingestion, flavones are highly metabolized in the gastrointestinal system by pH changes, digestive enzymes, and xenobiotic metabolic enzymes in the enterocytes and liver. In this sense, most flavones identified in plasma are conjugated derivatives rather than parental molecules. This can affect their bioactive effect and prevent their delivery at target sites in the body. Moreover, we suggest a systematic approach when evaluating the properties of flavones, including the appropriate extraction methods coupled with bioaccessibility/bioavailability studies concomitant to the evaluation of their bioactive properties.