Genus Stachys: A Review of Traditional Uses, Phytochemistry and Bioactivity

Background: The genus Stachys L. (Lamiaceae) includes about 300 species as annual or perennial herbs or small shrubs, spread in temperate regions of Mediterranean, Asia, America and southern Africa. Several species of this genus are extensively used in various traditional medicines. They are consumed as herbal preparations for the treatment of stress, skin inflammations, gastrointestinal disorders, asthma and genital tumors. Previous studies have investigated the chemical constituents and the biological activities of these species. Thus, the present review compiles literature data on ethnomedicine, phytochemistry, pharmacological activities, clinical studies and the toxicity of genus Stachys. Methods: Comprehensive research of previously published literature was performed for studies on the traditional uses, bioactive compounds and pharmacological properties of the genus Stachys, using databases with different key search words. Results: This survey documented 60 Stachys species and 10 subspecies for their phytochemical profiles, including 254 chemical compounds and reported 19 species and 4 subspecies for their pharmacological properties. Furthermore, 25 species and 6 subspecies were found for their traditional uses. Conclusions: The present review highlights that Stachys spp. consist an important source of bioactive phytochemicals and exemplifies the uncharted territory of this genus for new research studies.


Introduction
The genus Stachys L., a large member of the Lamiaceae family, comprises more than 300 species, dispersing in temperate and tropical regions of Mediterranean, Asia, America and southern Africa [1][2][3]. Up to now, the most established and comprehensive classification of the genus is introduced by Bhattacharjee (1980), categorizing into two subgenera Betonica L. and Stachys L. [2,3]. The subgenus Stachys includes 19 sections, while the subgenus Betonica comprises 2 sections [1]. However, the two subgenera present important botanical and phytochemical differences which differentiate them [1,4,5].
Stachys species grow as annual or perennial herbs or small shrubs with simple petiolate or sessile leaves. The number of verticillate ranges from four to many-flowered, usually forming a terminal spike-like inflorescence. Calyx tubes are tubular-campanulate, 5 or 10 veined, regular or weakly bilabiate with five subequal teeth. Corolla has a narrow tube, 2-lipped; upper lip flat or hooded and generally hairy, while the lower lip is 3-lopped and glabrous to hairy. The nutlets are oblong to ovoid, rounded at apex [6].
The genus name derived from the Greek word «stachys (=στάχυς) », referring to the type of the inflorescence which is characterized as "spike of corn" and resembles to the inflorescences of the species of genus Triticum L. (Gramineae). In ancient times, the name "stachys" referred mainly to the S. acerosa Boiss. Iran Common cold Decoction [31] S. affinis Bunge (=S. sieboldii Miq.)

Iran
Infections, asthmatic, rheumatic, inflammatory disorders Extracts of aerial parts (non flowering stems) [36,37] Iran Common cold, Analgesic, high blood pressure Decoction of aerial parts [31] S. iva Griseb. Greece Common cold and gastrointestinal disorders Decoction, infusion [56] S. kurdica Boiss & Hohen var. kurdica Turkey Cold, stomach-ache Decoction of branches/flowers Drink one glass of the plant on an empty stomach in the morning [50] S. lavandulifolia Vahl.

Serbia, Egypt, Montenegro
Skin disorders, antibacterial purposes, against headache, nervous tension, anxiety, menopausal problems, as a tobacco snuff Tea of dried leaves [22] Italy Dye wool yellow Plant [51] Italy Wounds, in the sores of pack animals Oily extract of flowers [54] S. palustris L.
Anatolia Antibacterial and healing effects Tea of the whole part [21] Anatolia Sedative, antispasmodic, diuretic and emmenagogic properties Tea of the leaves [21] -Bronchitis, asthma, stomach pain and gall and liver disorders - [65]  S. recta L.

Europe
Anxiolytic properties Herbal tea, Oral administration [11] Italy Headache Infusion of leaves to wash face [51] Italy Bad influence/spirit Decoction [53] Italy Depurative Decoction of the aerial parts [54] S. recta L. subsp. recta Italy Tootache and other pain Aerial parts applied in body parts [53] against anxiety, pain and toothache Decoction of flowering tops for bath or to wash face, hands and wrists for 3 days S. schtschegleevii Sosn. ex Grossh.
A few studies mentioned the existence of flavonols in Stachys spp. (Tables 4 and 18), mainly in species occurred in Greece. Afouxenidi and colleagues (2018) isolated kaempferol (91) from the n-butanol residue of the aerial parts of S. tetragona [100], which was also identified in the aerial parts of S. cretica subsp. smyrnaea [81]. Moreover, isorhamnetin (92) was isolated from the methanol extract of the aerial parts of S. swainsonii subsp. swainsonii and S. swainsonii subsp. argolica [102]. A study conducted by Marin et al. (2004) identified the presence of quercetin 3-O-rutinoside (93) and isorhamnetin 3-O-glucoside (94) from the aerial parts of S. palustris [5].
In addition, three flavanones were isolated from three species of the genus Stachys (Tables 5 and 19). Eriodictyol (95) was mentioned in S. cretica [108] and in one subspecies of S. swainsonii [102], while naringenin (96) was isolated from the aerial parts of the species S. aegyptiaca [104]. A flavanone rutinoside, known as hesperidin (97), was identified as one of the major compounds of the aerial parts of S. cretica subsp. smyrnaea [81].
Of great interest is the isolation of a rare diflavone ester of µ-truxinic acid, namely stachysetin (98). It is well-known that diglycoside flavone esters of dicarboxylic acids are rare compounds in plant kingdom. Stachysetin was firstly isolated from the ethanol extract (70% v/v) of the aerial parts of S. aegyptiaca [69]. Then, Murata and co-workers (2008) reported it in the methanol residue (80% v/v) of the aerial parts of S. lanata [82]. In a current study carried out by Pritsas et al. (2020), stachysetin was isolated from the methanol: aqueous (5:1) extract from the flowering aerial parts of the cultivated S. iva (Tables 6 and 20) [56]. Up to now, there is no report of this secondary metabolite in the species of the subgenus Betonica. The presence of this rare natural compound in the sections Ambleia, Eriostomum and Candida of the subgenus Stachys might be considered as a chemotaxonomic marker among the two subgenera and of the genus Stachys. of S. aegyptiaca [69]. Then, Murata and co-workers (2008) reported it in the methanol residue (80% v/v) of the aerial parts of S. lanata [82]. In a current study carried out by Pritsas et al. (2020), stachysetin was isolated from the methanol: aqueous (5:1) extract from the flowering aerial parts of the cultivated S. iva (Tables 6 and 20) [56]. Up to now, there is no report of this secondary metabolite in the species of the subgenus Betonica. The presence of this rare natural compound in the sections Ambleia, Eriostomum and Candida of the subgenus Stachys might be considered as a chemotaxonomic marker among the two subgenera and of the genus Stachys.   Table 16. Cont.

Lignans
Lignans are types of polyphenols with diverse structures. Although these bioactive compounds were presented in Lamiaceae family [149], a few studies reported their existence in plants of genus Stachys. Specifically, three lignans categorizing into two furanofuran-type derivatives (sesamin and paulownin) and one benzofuran-type lignan (urolignoside) were reported in two species of the subgenus Stachys (Tables 9 and 23). Laggoune et al. (2016) isolated sesamin (112) and paulownin (113) from the aerial parts of S. mialhesii [103], while urolignoside (114)

Lignans
Lignans are types of polyphenols with diverse structures. Although these bioactive compounds were presented in Lamiaceae family [149], a few studies reported their existence in plants of genus Stachys. Specifically, three lignans categorizing into two furanofuran-type derivatives (sesamin and paulownin) and one benzofuran-type lignan (urolignoside) were reported in two species of the subgenus Stachys (Tables 9 and 23). Laggoune et al. (2016) isolated sesamin (112) and paulownin (113) from the aerial parts of S. mialhesii [103], while urolignoside (114) was isolated from the aerial parts of S. tetragona [100]. Given that up to now there is no study reported the presence of lignans in the subgenus Betonica, the identification of lignans might be considered as a chemotaxonomic difference between the two subgenera Stachys and Betonica. of S. tetragona [100]. Given that up to now there is no study reported the presence of lignans in the subgenus Betonica, the identification of lignans might be considered as a chemotaxonomic difference between the two subgenera Stachys and Betonica.

Phenylethanoid Glucosides; Phenylpropanoid Glucosides
The present review unveiled 29 phenylethanoid glucosides in 17 Stachys species (Tables 10 and  24). Acteoside or verbascoside (118) was the most abundant found in 16 Stachys spp. of all sections through this survey. Additional phenylethanoid glucosides isolated and identified from this genus includes martynoside, leucosceptoside A and lavandulifoliosides. Lavandulifolioside A (or stachysoside A) (129) was firstly isolated from the methanol extract of the aerial parts of S. lavandulifolia in 1988 [115], while in 2011 Delazar et al. (2011) isolated lavandulifolioside B (130) from the same plant, for the first time [12]. Moreover, three phenylethanoid glucosides were reported from the aerial parts of S. byzantina (section Eriostomum), including verbascoside (118), 2′-O-arabinosyl verbascoside (122) and aeschynanthoside C (133) [35]. Among them, the first and the last compound has been isolated only from the specific species. A survey conducted by Murata and co-workers (2008) reported ten phenylethanoid glucosides from different plant parts [82]. In the aforementioned study, leonoside B (or stachysoside D) (134) and martynoside (135) were mentioned from the aerial parts of S. lanata, while from the roots of the specific species were reported eight phenylethanoid glucosides, namely rhodioloside (115), verbasoside (116), 2-phenylethyl-D-xylopyranosyl-(1→6)-Dglucopyranoside (117), verbascoside (118), isoacteoside (119), darendoside B (120), campneoside II (121) and campneoside I (136). It is remarkable to point out that compounds 115, 117 and 120 haven′t been reported in other Stachys species. This might be attributed to the fact that the plant material was roots. Another study carried out by Karioti et al. (2010) focused on the phenolic compounds from the aerial parts of S. recta, and reported many phenylethanoid glucosides from its aerial parts, including acteoside (118), isoacteoside (119), β-OH-acteoside (121), betunyoside E (127), campneoside I (136), forsythoside B (137), β-OH-forsythoside B methyl ether (138) [14]. Furthermore, lamiophloside A (141) was isolated with some other phenylethanoid glucosides from the aerial parts of S. tetragona [100]. Of great interest is that our survey revealed that this constituent is mentioned only in the specific species. Two rare phenylethanoid glucosides, parviflorosides A-B (142-143) were isolated from the whole plant of S. parviflora [120]. These two compounds are characterised by the presence of a third saccharide (rhamnose) linked to the proton H-2′ of glucose, comparing to others common phenylethanoid glucosides where the connection of the third saccharide is in proton H-3′ of glucose. Of great interest is that S. parviflora is now considered as the monotypic genus Phlomidoschema (only P. parviflorum (Benth.) Vved.) [2]. Furthermore, leonoside A (or stachysoside B) (139) was isolated with other three phenylethanoid glucosides from the whole plant of S. riederi [114]. To be mentioned that phenylethanoid glucosides were reported in both subgenera of genus Stachys.
Apart from phenylethanoid glucosides, Murata et al. (2008) mentioned two phenylpropanoid glucosides in the roots of S. lanata (subg. Stachys; sect. Eriostomum), coniferin (144) and syringin (145) Medicines 2020, 7, x FOR PEER REVIEW 44 of 78 of S. tetragona [100]. Given that up to now there is no study reported the presence of lignans in the subgenus Betonica, the identification of lignans might be considered as a chemotaxonomic difference between the two subgenera Stachys and Betonica.

Phenylethanoid Glucosides; Phenylpropanoid Glucosides
The present review unveiled 29 phenylethanoid glucosides in 17 Stachys species (Tables 10 and  24). Acteoside or verbascoside (118) was the most abundant found in 16 Stachys spp. of all sections through this survey. Additional phenylethanoid glucosides isolated and identified from this genus includes martynoside, leucosceptoside A and lavandulifoliosides. Lavandulifolioside A (or stachysoside A) (129) was firstly isolated from the methanol extract of the aerial parts of S. lavandulifolia in 1988 [115], while in 2011 Delazar et al. (2011) isolated lavandulifolioside B (130) from the same plant, for the first time [12]. Moreover, three phenylethanoid glucosides were reported from the aerial parts of S. byzantina (section Eriostomum), including verbascoside (118), 2′-O-arabinosyl verbascoside (122) and aeschynanthoside C (133) [35]. Among them, the first and the last compound has been isolated only from the specific species. A survey conducted by Murata and co-workers (2008) reported ten phenylethanoid glucosides from different plant parts [82]. In the aforementioned study, leonoside B (or stachysoside D) (134) and martynoside (135) were mentioned from the aerial parts of S. lanata, while from the roots of the specific species were reported eight phenylethanoid glucosides, namely rhodioloside (115), verbasoside (116), 2-phenylethyl-D-xylopyranosyl-(1→6)-Dglucopyranoside (117), verbascoside (118), isoacteoside (119), darendoside B (120), campneoside II (121) and campneoside I (136). It is remarkable to point out that compounds 115, 117 and 120 haven′t been reported in other Stachys species. This might be attributed to the fact that the plant material was roots. Another study carried out by Karioti et al. (2010) focused on the phenolic compounds from the aerial parts of S. recta, and reported many phenylethanoid glucosides from its aerial parts, including acteoside (118), isoacteoside (119), β-OH-acteoside (121), betunyoside E (127), campneoside I (136), forsythoside B (137), β-OH-forsythoside B methyl ether (138) [14]. Furthermore, lamiophloside A (141) was isolated with some other phenylethanoid glucosides from the aerial parts of S. tetragona [100]. Of great interest is that our survey revealed that this constituent is mentioned only in the specific species. Two rare phenylethanoid glucosides, parviflorosides A-B (142-143) were isolated from the whole plant of S. parviflora [120]. These two compounds are characterised by the presence of a third saccharide (rhamnose) linked to the proton H-2′ of glucose, comparing to others common phenylethanoid glucosides where the connection of the third saccharide is in proton H-3′ of glucose. Of great interest is that S. parviflora is now considered as the monotypic genus Phlomidoschema (only P. parviflorum (Benth.) Vved.) [2]. Furthermore, leonoside A (or stachysoside B) (139) was isolated with other three phenylethanoid glucosides from the whole plant of S. riederi [114]. To be mentioned that phenylethanoid glucosides were reported in both subgenera of genus Stachys.

Phenylethanoid Glycosides; Phenylpropanoid Glucosides
The present review unveiled 29 phenylethanoid glycosides in 17 Stachys species (Tables 10 and 24). Acteoside or verbascoside (118) was the most abundant found in 16 Stachys spp. of all sections through this survey. Additional phenylethanoid glycosides isolated and identified from this genus includes martynoside, leucosceptoside A and lavandulifoliosides. Lavandulifolioside A (or stachysoside A) (129) was firstly isolated from the methanol extract of the aerial parts of S. lavandulifolia in 1988 [115], while in 2011 Delazar et al. (2011) isolated lavandulifolioside B (130) from the same plant, for the first time [12]. Moreover, three phenylethanoid glycosides were reported from the aerial parts of S. byzantina (section Eriostomum), including verbascoside (118), 2 -O-arabinosyl verbascoside (122) and aeschynanthoside C (133) [35]. Among them, the first and the last compound has been isolated only from the specific species. A survey conducted by Murata and co-workers (2008) reported ten phenylethanoid glycosides from different plant parts [82]. In the aforementioned study, leonoside B (or stachysoside D) (134) and martynoside (135) were mentioned from the aerial parts of S. lanata, while from the roots of the specific species were reported eight phenylethanoid glycosides, namely rhodioloside (115), verbasoside (116), 2-phenylethyl-D-xylopyranosyl-(1→6)-D-glucopyranoside (117), verbascoside (118), isoacteoside (119), darendoside B (120), campneoside II (121) and campneoside I (136). It is remarkable to point out that compounds 115, 117 and 120 haven t been reported in other Stachys species. This might be attributed to the fact that the plant material was roots. Another study carried out by Karioti et al. (2010) focused on the phenolic compounds from the aerial parts of S. recta, and reported many phenylethanoid glycosides from its aerial parts, including acteoside (118), isoacteoside (119), β-OH-acteoside (121), betunyoside E (127), campneoside I (136), forsythoside B (137), β-OH-forsythoside B methyl ether (138) [14]. Furthermore, lamiophloside A (141) was isolated with some other phenylethanoid glycosides from the aerial parts of S. tetragona [100]. Of great interest is that our survey revealed that this constituent is mentioned only in the specific species. Two rare phenylethanoid glycosides, parviflorosides A-B (142-143) were isolated from the whole plant of S. parviflora [120]. These two compounds are characterised by the presence of a third saccharide (rhamnose) linked to the proton H-2 of glucose, comparing to others common phenylethanoid glycosides where the connection of the third saccharide is in proton H-3 of glucose. Of great interest is that S. parviflora is now considered as the monotypic genus Phlomidoschema (only P. parviflorum (Benth.) Vved.) [2]. Furthermore, leonoside A (or stachysoside B) (139) was isolated with other three phenylethanoid glucosides from the whole plant of S. riederi [114]. To be mentioned that phenylethanoid glycosides were reported in both subgenera of genus Stachys.
Apart from phenylethanoid glucosides, Murata et al. (2008) mentioned two phenylpropanoid glucosides in the roots of S. lanata (subg. Stachys; sect. Eriostomum), coniferin (144) and syringin (145) (Tables 11 and 25) [82]. It is worth to mention that the isolation of phenylpropanoid glucosides only from the specific plant, might be assigned to the different studied plant material (roots).  (Tables 11 and 25) [82]. It is worth to mention that the isolation of phenylpropanoid glucosides only from the specific plant, might be assigned to the different studied plant material (roots).  (Tables 11 and 25) [82]. It is worth to mention that the isolation of phenylpropanoid glucosides only from the specific plant, might be assigned to the different studied plant material (roots).  (Tables 11 and 25) [82]. It is worth to mention that the isolation of phenylpropanoid glucosides only from the specific plant, might be assigned to the different studied plant material (roots).

Iridoids
Iridoids are among the major chemical compounds found in genus Stachys. According to Tundis et al. (2014), iridoids are considered as good chemotaxonomic markers of this genus [3]. Accumulating phytochemical studies have reported diverse types of iridoids [3]. The present review summarises all these studies, exemplifying 38 Stachys species which their iridoid cargo has been investigated (Tables 12 and 26). Harpagide (148; 31 species) and its acetyl derivative; 8 acetyl-harpagide (150; 28 species) are of common occurrence in genus Stachys and might be considered as characteristic iridoids of these plants. Furthermore, ajugol (146; 18 species), ajugoside (147; 18 species), melittoside (166; 17 species), monomelittoside (165; 4 species) and 5-allosyloxy-aucubin or 5-O-allopyranosyl-monomelittoside (167; 4 species/1 subsp.) were also mentioned in various species. Allobetonicoside (161) was firstly isolated from the aerial parts of S. officinalis [127] and then from the aerial parts of S. glutinosa [122] and of S. macrantha [117]. The latter study also mentioned the isolation of cinnamoyl-harpagide derivative, macranthoside (156) [123]. A study conducted by Háznagy-Radnai (2006) examined the phytochemical profiles of Stachys spp. growing in Hungary, reporting the iridoid content of ten taxa [124]. Murata and co-workers (2008) isolated five new esters of monomelittoside from the aerial parts and roots of S. lanata [82]. In particular, stachysosides E (168), G-H (170-171) were found in roots, while stachysosides E (168) and F (169) were discovered from the aerial parts of the specific species. It is important to be mentioned the detection of a new iridoid diglycoside, 4 -O-β-D-galactopyranosyl-teuhircoside (162), which was isolated from the flowering aerial parts of S. alopecuros subsp. divulsa [119]. Muñoz et al. (2001) reported the presence of 5-desoxy-harpagide (151) and 5-desoxy-8-acetyl-harpagide (152) from the aerial parts of S. grandidentata [129]. Notably, this review unveiled some differences in iridoids among subgenera Stachys and Betonica. Firstly, it was observed that there is no report for the presence of monomelittoside or melittoside derivatives in the subgenus Betonica. Secondly, reptoside (153) was found in two species of subgenus Betonica (S. macrantha and S. officinalis) and not in the plants of subgenus Stachys. harpagide (152) from the aerial parts of S. grandidentata [129]. Notably, this review unveiled some differences in iridoids among subgenera Stachys and Betonica. Firstly, it was observed that there is no report for the presence of monomelittoside or melittoside derivatives in the subgenus Betonica. Secondly, reptoside (153) was found in two species of subgenus Betonica (S. macrantha and S. officinalis) and not in the plants of subgenus Stachys. Secondly, reptoside (153) was found in two species of subgenus Betonica (S. macrantha and S. officinalis) and not in the plants of subgenus Stachys.
In the context of chemotaxonomic significance, it could be observed that species of subgenus Stachys product mainly neo-clerodane and labdane type derivatives, while the plants of subgenus Betonica biosynthesized diterpene lactone derivatives. Thus, the latter derivatives might be recognised as characteristic chemotaxonomic markers of subgenus Betonica. Another important chemotaxonomic point is reported by Piozzi et al. (2002), mentioning that (+)-6-deoxyandalusol has been determined only in three Stachys species of eastern part of the Mediterranean region [139].

Other Chemical Categories
Notable among the above-mentioned classes of compounds are the megastigmane derivatives from Stachys spp. (Tables 15 and 29). Takeda and colleagues (1997) isolated from the aerial parts of S. byzantina five bioactive compounds from this group, including byzantionosides A-B (244,245), icariside B2 (246), (6R, 9R)-and (6R, 9S)-3-oxo-α-ionol glucosides (247) and blumeol C glucoside (248) [148]. Furthermore, vomifoliol (249) and dehydrovomifoliol (250) were reported from the aerial parts of S. lanata, while citroside A (251) was isolated from the roots of this species [82]. This study also mentioned the presence of sugar ester (cistanoside F) from the roots of S. lanata [82]. At this point, we should note that few studies reported some oligosaccharides from Stachys spp. [3]. For instance, stachyose is a tetrasaccharide which consists one of the most common oligosaccharides in genus Stachys and shows beneficial effects for the gastrointestinal system as it can be directly consumed [3,23,119,150]. Precisely, the species S. sieboldii is a major source of this constituent [27,151,152]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151].

Other Chemical Categories
Notable among the above-mentioned classes of compounds are the megastigmane derivatives from Stachys spp. (Tables 15 and 29). Takeda and colleagues (1997) isolated from the aerial parts of S. byzantina five bioactive compounds from this group, including byzantionosides A-B (244,245), icariside B2 (246), (6R, 9R)-and (6R, 9S)-3-oxo-α-ionol glucosides (247) and blumeol C glucoside (248) [148]. Furthermore, vomifoliol (249) and dehydrovomifoliol (250) were reported from the aerial parts of S. lanata, while citroside A (251) was isolated from the roots of this species [82]. This study also mentioned the presence of sugar ester (cistanoside F) from the roots of S. lanata [82]. At this point, we should note that few studies reported some oligosaccharides from Stachys spp. [3]. For instance, stachyose is a tetrasaccharide which consists one of the most common oligosaccharides in genus Stachys and shows beneficial effects for the gastrointestinal system as it can be directly consumed [3,23,119,150]. Precisely, the species S. sieboldii is a major source of this constituent [27,151,152]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. stachyose is a tetrasaccharide which consists one of the most common oligosaccharides in genus Stachys and shows beneficial effects for the gastrointestinal system as it can be directly consumed [3,23,119,150]. Precisely, the species S. sieboldii is a major source of this constituent [27,151,152]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151].  (248) Vomifoliol (249) stachyose is a tetrasaccharide which consists one of the most common oligosaccharides in genus Stachys and shows beneficial effects for the gastrointestinal system as it can be directly consumed [3,23,119,150]. Precisely, the species S. sieboldii is a major source of this constituent [27,151,152]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. Stachys and shows beneficial effects for the gastrointestinal system as it can be directly consumed [3,23,119,150]. Precisely, the species S. sieboldii is a major source of this constituent [27,151,152]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. Stachys and shows beneficial effects for the gastrointestinal system as it can be directly consumed [3,23,119,150]. Precisely, the species S. sieboldii is a major source of this constituent [27,151,152]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151]. Stachyose is an oligosaccharide, which can be directly consumed for the benefit of gastrointestinal system [150]. Furthermore, Yin and colleagues (2006) mentioned that the bitter taste of some Stachys species, such as S. annua and S. balansae, might be attributed to their bitter diterpene derivatives, like stachylone [22,151].

Pharmacological Activities
This section includes the most interesting pharmacological data of the last five years (from 2015 to 2020). Many studies exemplified the great antimicrobial, antioxidant and cytotoxic effects of the essential oils of these plants [3,15]. Tundis et al. (2014) described in detail the biological studies (in vitro and in vivo) of the essential oils, extracts and compounds [3]. Thus, in the present review, we focused on the current available pharmacological researches of the extracts and isolated compounds from Stachys spp. as they are presented in Table 30. Anti-diabetic α-Amylase inhibition (mmol ACEs/g extract): 0.31 ± 0.01 α-Glucosidase inhibition (mmol ACEs/g extract): 1.95 ± 0.20  Anti-diabetic α-Amylase inhibition (mmol ACEs/g extract): 0.34 ± 0.02 α-Glucosidase inhibition (mmol ACEs/g extract): 6.17 ± 0.51 S. iva Griseb.

Cytotoxicity and Antiproliferative Activity
Venditti et al., (2017) investigated the cytotoxic activity and the anti-reactive oxygen species activity of the ethanol extract from tubers of the Chinese artichock (S. affinis) [27]. Regarding the cytotoxicity, the specific extract didn t demonstrate any activity in K562, SH-SY5Y and Caco-2 cell lines, even at the highest concentrations (1.0 mg/mL). The cytotoxic activity of extracts and isolated flavonoids from the aerial parts of S. lavandulifolia were studied by Delnavazi et al. (2018) through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [13]. The dichloromethane extract showed the highest cytotoxic activity in brine shrimp lethality test (BSLT) (LD 50 = 121.8 ± 5.6 µg/mL), while as a positive control was used podophyllotoxin (LD 50 = 3.1 ± 0.6 µg/mL). Afterwards, they explored the cytotoxic activity of isolated flavonoids in three cancer cell lines (MDA-MB-231, HT-29 and MRC-5), using as reference compound tamoxifen. All the nine isolated flavonoids moderated the cytotoxicity activated on the studied cell lines. However, chrysosplenetin (84) was reported as the most active compound in the first two cell lines. In MRC-5 cell line, apigenin (1) exhibited the greatest activity. It is remarkable to point out that the specific study also mentioned the selective activity against cancer cells, reporting that chrysosplenetin (84), kumatakenin (79) and viscosine (78) exhibited higher selective toxicity against MDA-MB-231 cell line than tamoxifen. At this point, we should underlie that the great cytotoxic activity of these compounds is attributed to their substitutions with (poly)-methylated groups which increase this effect. Another study evaluated the methanol extract, the alkaloid and the terpenoid fractions of S. pilifera for their cytotoxic and antiproliferative activity in vitro (HT-29 cell line), indicating great results [45]. The terpenoid fraction was found to have the best cytotoxic activity compared to the other fractions and as reference compound was used cisplatin. Moreover, they investigated the antiproliferative activity, studying the effects on the activity of caspase-8 and caspase-9, Nuclear factor-κB (NF-κB) and Nitric Oxide (NO), reporting that the extract/fractions increased the activity of caspase-8/-9 and decreased NF-κB and subsequently NO level. Of note, they compared their results with previous data of cytotoxic activity in vitro of other Stachys species such as S. acerosa, S. benthamiana, S. floridana, S. lavandulifolia, S. obtusicrena, S. persica, S. pubescens and S. spectabilis. Three isolated compounds from the extract (CH 2 Cl 2 :MeOH 1:1) of the aerial parts of S. aegyptiaca were investigated for the cytotoxic activity in HepG2 cell line, using MTT assay [132]. Precisely, the IC 50 values of stachaegyptin D (193), stachysolon monoacetate (178) and stachysolon diacetate (180) were 94.7, 63.4 and 59.5 µM, respectively, with stachysolone diacetate being the most active. In another study, the cytotoxic effect of the ethanol extract of S. riederi var. japonica on UVA-irradiated HDFs was evaluated at different concentrations for 48 h by MTT assay, showing no or little cytotoxicity [160]. Shakeri et al. (2019) mentioned that the methanol extract of S. parviflora demonstrated no cytotoxic activity toward the cancer cell lines, namely A2780, HCT, and B16F10 in all tested concentrations (>100 µg/mL) [64]. Moreover, the genotoxic activity of the extracts from four different plants were investigated by Slapšytė and colleagues (2019) [157]. They reported that all the plant extracts induced DNA damage, using the comet assay, whereas the extract of S. officinalis induced the increase of sister chromatid exchange value. The methanol extract of the Lebanese species S. ehrenbergii was investigated for its antioxidant and cytotoxic activity [154]. The cytotoxicity was examined by MTT assay where the methanol extract showed the highest cytotoxicity (IC 50 = 420 ± 104 µg/mL) at a concentration of 3000 mg/mL.

Polycystic Ovary Syndrome (PCOS)
In Iran, S. sylvatica is used for the treatment of women with polycystic ovary syndrome (PCOS). A current study carried out by Alizadeh et al. (2020) evaluated the hydroalcoholic extract of this plant in a rat model of PCOS [47]. It was observed that the extract at the dose of 500 mg/kg increased gonadotropins FSH and LH (5.95 ± 0.02 mIU/mL; 6.48 ± 0.09 mIU/mL) and reduced the level of estrogen (0.9 ± 0.07 mIU/mL) compared to the PCOS group (FSH level: 1.69 ± 0.08 mIU/mL; LH level: 6.29 ± 0.04 mIU/mL; estrogen level: 1.42 ± 0.05 mIU/mL), causing the ratio of LH/FSH to be close to 1:1 (6.48/5.59). According to the literature, this ratio LH/FSH is almost 1:1 in normal cases, while in PCOS women is higher e.g., 2:1 or 3:1. They also mentioned that these great results of the extract of S. sylvatica could be correlated to the flavonoid content of the plant. Previous studies showed that flavonoids could decrease the level of estrogen and could also act as GABA receptor agonists, regulating gonadotropins. Given that women with PCOS showed high concentrations of inflammation factors, they assumed that the extract could act as anti-inflammatory and antioxidant agent as flavonoids and iridoids demonstrated antioxidant and anti-inflammatory properties.

Anticholinesterase and Anti-Alzheimer's Activity/Neuroprotective Activity
The aqueous extract from the tubers of S. sieboldii ("chorogi") was studied in vivo in mice model for its neuroprotective potential [152]. Specifically, the study examined the effects of chorogi's extract on celebral ischemia and scopolamine-induced memory impairment, using as positive control the extract of Gingko biloba, proving that S. sieboldii improves the learning and memory dysfunction correlated with ischemic brain injury. Another work examined the cholinesterase inhibitory activity of S. lavandulifolia extracts and isolated compounds [116]. Specifically, the most active extract against anticholinesterase (AChE) was the n-hexane extract with an IC 50 value of 13.7 µg/mL. However, the dichloromethane extract was the most effective against butyrylcholinesterase (BChE) (IC 50 = 143.9 µg/mL) where its major constituent, stachysolone (177), inhibited the activity of this enzyme with a percentage of inhibition of 50% at 0.06 mg/mL. Among the studied polar extracts, the methanol extract exhibited a selective inhibitory activity against AChE with an IC 50 value of 211.4 µg/mL and the isolated compounds, arbutin (107) and 5-allosyloxy-aucubin (167), showed a percentage of inhibition of 50 and 23.1% at 0.06 mg/mL, respectively, against AChE. Notably, the other constituents of this species were inactive at the maximum concentration tested of 0.25 mg/mL. Ferhat et al. (2016) examined the AChE activity of n-butanol, the ethyl acetate and the chloroform extracts of the aerial parts of S. guyoniana, demonstrating that the n-butanol extract (IC 50 = 5.78 ± 0.01 µg/mL) was a little less active than the used standard drug against Altzheimer's disease; galantamine (IC 50 = 5.01 ± 0.10 µg/mL). Furthermore, they exhibited that this extract inhibited the BChE, having an IC 50 value of 39.1 ± 1.41 µg/mL which was better than the standard (IC 50 = 39.10 ± 1.41 µg/mL) [155]. Moreover, the anti-Alzheimer's activity of two subspecies of S. cretica (S. cretica subsp. smyrnaea; S. cretica subsp. mersinaea) were evaluated in different works [81,108]. In addition, the potential effects of 20% ethanol extract of S. sieboldii was evaluated against oxidative stress induced by H 2 O 2 in SK-N-SH cells and memory enhancement in ICR mice [162]. This study showed that the daily intake of the extract (dose: 500 mg/kg) through dietary supplementation produced memory enhancing effects in animals. Recently, Ertas and Yener (2020) reported that the acetone extract of S. thirkei demonstrated good activity against AChE and BChE with a percentage of inhibition of 52.46 ± 1.26% and 75.04 ± 1.91%, respectively [84].

Anti-diabetic Activity
Bahadori et al. (2018) evaluated the anti-diabetic activity of the extracts of S. cretica subsp. smyrnaea [81]. Specifically, the methanol extract demonstrated strong anti-diabetic activity against α-amylase (61.4 mg ACEs/g dry plant) and α-glucosidase (47.8 mg ACEs/g dry plant), following by ethyl acetate extract. They assumed that the above good properties were attributed to the phenolic constituents of the methanol extract since the anti-glucosidase activity is associated with caffeic acid, trans-cinnamic acid, and vanillin, whereas the amylase inhibitory activity is related to kaempferol and p-hydroxybenzoic acid. A year later, the anti-diabetic activity of the extracts of S. cretica subsp. mersinaea was studied, reporting that the ethyl acetate extract had best activity against α-amylase (396.50 mgACEs/g), while the methanol extract exerted strong activity against α-glucosidase (734 mg ACEs/g) [108]. Furthermore, the α-amylase inhibition of the methanol and water extract of S. cretica subsp. vacillans was evaluated, with the methanol extract exhibited stronger activity (433.99 ± 5.10 mg ACE/g extract) [112]. Currently, Pritsas et al. (2020) studied the anti-diabetic activity in silico of 17 isolated compounds from the cultivated S. iva, mentioning that stachysetin (98) interacted with five out of ten proteins implicated in diabetes [56]. This is the only study reported a pharmacological activity of this rare compound.

Antimicrobial Activity
Regarding the antibacterial activity, the n-butanol extract of S. guyoniana showed strong activity against Staphylococcus aureus (MIC = 32 ± 0.90 µg/mL) and Enterobacter aerogenes (MIC = 32 ± 0.70 µg/mL), while it was not active against Pseudomonas aeruginosa and Morganella morganii [155]. The ethyl acetate extract demonstrated the best inhibition against Escherichia coli (MIC = 64 ± 0.60 µg/mL), whereas it didn t show any activity against P. aeruginosa and M. morganii. Shakeri et al. (2019) reported the antimicrobial activity of the methanol extract of the aerial parts of S. parviflora which exerted the highest activity against the Gram-positive bacterium, Bacillus cereus, with a MIC of 0.12 mg/mL [64]. Furthermore, the antimicrobial activity of extracts of S. thirkei against different microorganisms were studied according to inhibition zone diameter and MIC value [84]. The acetone and methanol extract demonstrated good activity against S. aureus, Streptococcus pyogenes and E. coli. Intriguingly, S. thirkeis extracts were not active against P. aeruginosa (Gram-negative bacterium) and Candida albicans (yeast).

Hepatoprotective
The hepatoprotective property of the ethanol extract of S. pilifera was studied in carbon tetrachloride (CCl 4 )-induced hepatotoxicity in rats and indicated that this extract could act as hepatoprotective agent [158]. They assumed that this property might be also related to the strong antioxidant activity of the species. Later, Mansourian et al. (2019) exhibited the hepatoprotective and antioxidant activity of hydroalcoholic extract of S. pilifera on hepatotoxicity induced by acetaminophen (APAP) in male rats [159]. Precisely, the extract reduced hepatotoxicity by decreasing liver function markers/enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and protein carbonyl (PCO) compared to the APAP group. It also diminished the oxidative stress through inhibiting protein oxidation and inducing the activity of glutathione peroxidase (GPX) enzyme. So, they assumed that this great activity was attributed to the antioxidant activity of this plant.  (70) reduced the binding affinity for MOR (Ki = 18.5 µM), whereas the replacement of this group with a methoxy moiety, as in 8-methoxycirsilineol (71), eliminated the affinity for MOR (Ki > 50 µM). Furthermore, they evaluated the antinociceptive activity of xanthomicrol in an animal model (in mice) of acute pain (tail-flick test). In another study, the n-butanol extract of S. mialhesii exhibited significant anti-inflammatory activity in vivo, reducing the weight of edema: 52.03% induced by carrageenan in the rat's paw, whereas indomethacin (dose: 5 mg/kg; decrease 83.36%) was used as a reference drug [103]. In the same study, the n-butanol extract exerted antinociceptive effect at dose-dependent manner. Ramazanov et al. (2016) evaluated the wound healing activity of the extract of S. hissarica on rats, showing that the extract improved the healing process of linear skin wounds at an oral dose of 10 mg/kg [67]. Of note, the wound healing activity of the extract was more effective than the known drug methyluracil (2,4-dioxo-6-methyl-1,2,3,4tetrahydropyrimidine), especially in case of alloxan induced diabetic animals. A study carried out by Iannuzzi et al. (2019) studied the antiangiogenic activity in two in vivo models (zebrafish embryos and chick chorioallantoic membrane assays) of the isolated compounds of the leaf extract of S. ocymastrum. The isolated compounds with the best antiangiogenic activity in both assays were β-hydroxyipolamiide (173) and ipolamiide (174) [123]. Recently, Lee et al. (2020) studied the anti-obesity and anti-dyslipidemic property of the roots powder of S. sieboldii in rats, following a high-fat and high-cholesterol diet (HFC) [161]. This powder demonstrated the anti-adipogenic and lipid-lowering effects through enhancing lipid metabolism.

Others
Taken together all the above pharmacological studies, we could observe that these findings confirmed most of the traditional medicinal uses of Stachys spp. However, the present review unveiled that there are still species pharmacologically uncharted.

Clinical Studies
Through our literature survey, four clinical studies for the species S. lavandulifolia were revealed. The first clinical study carried out by Rahzani et al. (2013) reported the effects of the aqueous extract of the specific plant (dose; infusion from 3 g aerial parts of plant, twice daily) on the oxidative stress in 26 healthy humans, underlying that the participants demonstrated a significant reduction in oxidative stress [163]. In parallel, another randomized clinical trial (33 women) examined the effects of S. lavandulifolia and medroxyprogesterone acetate (MPA) in abnormal uterine bleeding (AUB) in PCOS [164]. This study exemplified that the infusion of the aerial parts of wood betony (dose; 5 g of plant in 100 mL boiling water; duration 3 months) showed a reduction of AUB, recommending its consumption for the treatment of AUB related to PCOS. They also mentioned that this result might be attributed to the flavonoid content of the plant and mainly to apigenin. In addition, Monji et al. (2018) evaluated on a clinical trial the therapeutic effects of standardized formulation of S. lavandulifolia on primary dysmenorrhea, indicating that the standardized capsules of plant's extract could diminuish the menstrual pain, and might be recommended as an auxiliary therapy or an alternative remedy to nonsteroidal antiinflammatory drugs (NSAIDs) with fewer side effects in primary dysmenorrhea [165]. Recently, a double-blind randomized clinical study mentioned the analgesic activity of the herbal tea of S. lavandulifolia (10 g in 200 cc of boiling water) in 50 patients with migraine [166], showing the capability of this herbal tisane to decrease and also improve the pain intensity in these patients. In addition, Ashtiani et al. (2019) considered that the therapeutic properties of this plant associated with its rich phytochemical profile which include iridoids, flavonoids and phenylethanoid glucosides [166].
To sum up, the above clinical studies confirm the ethnomedicinal uses of S. lavandulifolia as a traditional medicine. Although these promising results, more clinical studies should be performed for obtaining data for diverse Stachys spp. As a future prospective, further studies should strengthen the research of bioavailability, dosage, toxicity and potential drug interactions in order to endorse the observed pharmacological activities of these plants.

Toxicity
S. lavandulifolia is popularly claimed as an abortifacient agent by Iranian women. The effect of its hydroalcoholic extract on fertility was investigated, revealing that the extract had a dose dependent abortifacient activity. Thus, its use during pregnancy may cause abortion and consequently, the plant should be considered as contraindicated or be used with caution [167]. In addition, the nephrotoxicity of the same extract was studied on male Wistar rats and a mild degeneration of renal tubular epithelial cell after one month was observed, while in the second month the histologic lesions were significantly more. However, further studies need to evaluate renal complications of this plant in human [168]. Moreover, the acute and subchronic toxicological evaluation of S. lavandulifolia aqueous extract in rats indicated that the high dose (2 g/kg) did not produce any symptoms of toxicity and there was no significant difference in body weights between the control and treatment groups of the animals [169].

Conclusions
In the present review, we attempted to describe in detail all the current knowledge and research advances of genus Stachys, focusing on pointing the significance of this genus as herbal supplement and medicine.
Taken together with all the analyzed studies in the current review, we categorized the used literature data into four categories according to their general characteristics; ethnobotanical (no of used studies: 48), phytochemical (no of used studies: 91), pharmacological (no of in vitro studies: 22, no of in vivo studies: 8 and 2 in silico study), clinical studies (no of used studies: 4) and reviews (no of used studies: 4). The general characteristics of the analyzed studies in the current review are showed in Table 31. Table 31. General characteristics of the analyzed studies in the current review. Several Stachys spp. have been used as traditional herbal medicines for thousands of years. Therefore, accumulating studies have been performed in order to explore the chemical compounds and the pharmacological properties of these species to validate their claimed ethnomedicinal properties. However, the present review data shows that there are still species phytochemically and pharmacologically unexplored. This comprehensive survey could serve as useful tool for scientists searching uncharted and interesting species to study, as well as it could be an informative guide for researchers aimed to identify leads for developing novel drugs. Although many pharmacological studies have demonstrated the great properties of these plants, only the clinical effects of one species have been investigated. As a result, further studies should be performed to validate the clinical efficiency of several Stachys spp. and if there is any potential toxicity. To be mentioned that there are still yet much to be done on the detailed documentation (safety and efficacy data) of genus Stachys in order to be developed an official monograph as a traditional use or well-established use plants.