Agastache Species: A Comprehensive Review on Phytochemical Composition and Therapeutic Properties

The Agastache genus is part of the Lamiaceae family and is native to North America, while one species, Agastache rugosa (A. rugosa), is native to East Asia. A review on the phytochemistry and bioactivity of Agastache genus was last performed in 2014. Since then, a lot of progress has been made on the characterization of the phytochemical and pharmacological profiles of Agastache species. Thus, the purpose of this paper is to present a summary of the findings on the phytochemistry and biological effects of several Agastache species, including both extracts and essential oil characterization. We performed a comprehensive search using PubMed and Scopus databases, following PRISMA criteria regarding the study selection process. The available data is focused mainly on the description of the chemical composition and bioactivity of A. rugosa, with fewer reports referring to Agastache mexicana (A. mexicana) and Agastache foeniculum (A. foeniculum). Agastache species are characterized by the dominance of flavonoids and phenolic acids, as well as volatile compounds, particularly phenylpropanoids and monoterpenes. Moreover, a series of pharmacological effects, including antioxidant, cytotoxic, antimicrobial, anti-atherosclerotic, and cardioprotective properties, have been reported for species from the Agastache genus.


Introduction
Agastache Clayton ex Gronov is a small genus of the Lamiaceae family that consists of 22 species known under the common name 'giant hyssop'. Agastache species (sp.) are native to North America, with one species originating from East Asia. Agastache sp. is used as a medicinal and ornamental plant and is known in traditional medicine as a natural remedy against pain, hypertension, and gastrointestinal diseases [1][2][3][4].
Overall, the Agastache genus appears to be less investigated compared with other genera, both from a phytochemical and therapeutic perspective. The chemical composition and pharmacological profile of some Agastache sp. have not been described yet, and the available data is limited compared with other medicinal plants.
Another class of major compounds in A. rugosa extracts is represented by flavonoid derivatives. Tilianin (acacetin 7-O-glucoside) and its aglycon acacetin were reported more frequently [11,23,32,33]. Some authors reported values of 21.14 mg/g tilianin and 9.94 mg/g acacetin [17], while others found a concentration of 38.29 mg/g tilianin and 7.88 mg/g acacetin [19]. Another study also confirmed that tilianin contents were noticeably higher in the leaves and flowers than acacetin concentrations [34].
The concentration of the active compounds in A. rugosa extracts depends on various factors. Some authors investigated the variation of the composition with the age of the plant and found the maximum concentration of active compounds in two-year-old A. rugosa (25.81 mg/g d.w. rosmarinic acid, 15.61 mg/g d.w. apigenin glucoside, and 7.24 mg/g d.w. chlorogenic acid) [9]. Zielińska et al. also found that two-year-old A. rugosa leaves were richer in rosmarinic and chlorogenic acids than one-year-old ones. In contrast, there was no link between the amount of these compounds in the inflorescences and the age of the plant [10].
The composition of A. rugosa extract also depends on the part of the plant used. Thus, Park et al. found that flowers had the highest content of phenolic compounds, flavonoids, and anthocyanins, followed by leaves and stems. In contrast, HPLC analysis showed that the total phenylpropanoid content was highest in the leaves [15]. Desta et al. also confirmed that the highest concentration of flavonoids was found in flowers (89.43%), followed by leaves (89.25%), stems, and roots of A. rugosa [12]. The concentration of tilianin in the leaves was 2.18 µg/g, whereas the amount in the flowers was 6.33 µg/g. The stems and roots only had trace amounts of tilianin in them, 0.49 µg/g and 0.14 µg/g, respectively [34]. Yeo et al. showed that the profile of primary and secondary metabolites is also influenced by the period of floral development. The total polyphenolic content of the methanolic extract of A. rugosa was maximum during the budding period, stage I (3.96 ± 0.06 mg/g d.w.), while the total flavonoid content was maximum at stage II, the partial flowering stage (0.68 ± 0.01 mg/g d.w.). HPLC analysis showed that 4-hydroxybenzoic acid, chlorogenic acid, trans-cinnamic acid, and quercetin levels were highest in stage I, while levels of caffeic acid, (−)-epicatechin, tilianin, and acacetin were maximum in stage II. Rutin and rosmarinic acid levels were highest in stage III, the full flowering stage [16]. Another study found that the content of rosmarinic acid is increasing during the vegetation season, from 0.63 ± 0.04 mg/g d.w. in June to 1.88 ± 0.09 mg/g d.w. in August (one-year-old plants) and from 3.7 ± 0.26 mg/g d.w. in June to 9.31 ± 0.24 mg/g d.w. in August (two-year-old plants) [9]. Other researchers showed that the highest content of tilianin and acacetin was observed in A. rugosa collected at 12-13th weeks after seeding. Thus, it is recommended to harvest the plant material 12-13 weeks after seeding due to the high content of active compounds [13].
Regarding the differences in composition depending on the origin of the plant, tilianin and acacetin were reported as the main compounds in A. rugosa from China [23,24], while A. rugosa from Korea presented a more varied composition, depending on the part of the plant used and type of extract: some reported rosmarinic acid as the major compound [11,12,18], others acacetin 7-O-(6 -O-malonyl)-β-D-glucopyranoside [13] or tilianin [16,17,19,22]. Regarding A. rugosa from Poland, researchers reported rosmarinic acid as one of the predominant compounds [9,10].

Agastache foeniculum
Regarding the composition of A. foeniculum, phenolic acids (caffeic acid and p-coumaric acid) as well as flavonoids (quercetin, genistein, hyperoside, and rutoside) were identified in the extracts. The concentrations of the bioactive compounds were higher in the methanolic extracts compared with the ethanolic extracts. Total polyphenolic content (TPC) was 485.08 mg gallic acid equivalents (GAE)/g for the methanolic extract and 403.91 mg GAE/g for the ethanolic extract [26]. These results are higher than those reported by others, who reported a TPC of 1.14 mg hydroquinone equivalents/g for the aqueous extract [36] and 2.88 mg GAE/g for the ethanolic extract [25]. However, differences regarding the extraction method, part of the plant used, and origin could lead to variations in results. A. foeniculum has more flavonoids than phenolic acids, so using gallic acid for the calibration curve is not entirely correct; the optimum choice for a calibration curve is a flavone [27]. A. foeniculum harvested at the beginning of flowering and in the afternoon had the highest amounts of polyphenols (27.19 mg GAE/g dry plant). There were no statistically significant variations in polyphenolic content after 10 days (full bloom) [27].
One study showed that the total flavonoid content was 367.32 mg quercetin equivalents (QE)/g for the methanolic extract and 355.94 mg QE/g for the ethanolic extract [26]. Lower values have been reported by others: 31.87 mg QE/g [27], 1.07 mg QE/g [36], and 2.21 mg QE/g [25]. Some authors reported the results in rutin equivalents (RE) and obtained a total flavonoid content of 6.5 mg RE/g [37].

Agastache mexicana
A. mexicana ssp. mexicana and A. mexicana ssp. xolocotziana displayed a similar chemical profile, with tilianin, acacetin [38], and (2-acetyl)-7-O-glucosyl acacetin as the most abundant compounds [39]. These findings are consistent with previous research on other Agastache species. Nevertheless, there were significant differences in the relative abundance of their constituents. For example, tilianin and acacetin were more abundant in A. mexicana ssp. xolocotziana than in ssp. mexicana [38]. Another study pointed out that acacetin 7-O-(6 -O-malonyl)-glucoside was the predominant component of subsp. mexicana (20.43 g/g extract), while subsp. xolocotziana contains only 1.26 g/g extract. Also, some authors concluded that A. mexicana ssp. mexicana has a higher total polyphenolic and flavonoid content than ssp. xolocotziana [28].

Chemical Composition of Essential Oils from Agastache Species
Agastache species are traditionally used as spices due to the characteristic aniselike aroma given by the essential oil. Different factors could determine changes in essential oil composition, such as cultivation methods, origin, age of the plant, weather conditions [40,41], and chemotype differences [42].
Yamani et al. showed that estragole was the predominant compound of the Australiangrown A. rugosa volatile oil, reaching maximum concentration in nectar (97.16%), followed by flowers (96.74%) and leaves (94.35%) [43]. In contrast, another study found that A. rugosa honey contained only 12.31% estragole, suggesting that the nectar contained only a low amount of the compound compared with flowers and leaves [44]. Najafi et al. also identified estragole as the main component of A. rugosa volatile oil and as one of the major compounds of the oil fraction, along with linolenic acid, palmitic acid, linoleic acid, and oleic acid [62]. On the contrary, pulegone was the major compound in the volatile oil from the flowers of A. rugosa from China, and p-menthan-3-one was the most abundant in the volatile oil extracted from leaves [50]. In another study, the main constituents of A. rugosa essential oil were methyl eugenol (50.51%), estragole (8.55%), and eugenol (7.54%). Most of the volatile oil (66.60%) was composed of phenylpropanoid compounds, with just 14.22% monoterpenoids and 13.34% sesquiterpenoids [53].
Seven compounds, 2,6-dimethylheptane, 4-methyl-1-pentene-3-ol, δ-limonene, transocimene, octenyl acetate, anethole, and 2-methyl-5-isopropenyl-2-cyclohexenone, were present only in the volatile oil from the flowers of A. rugosa, while β-damascenone and allyl-3-methyl-2-butanoate were present only in the volatile oil from the leaves [47]. Thus, there are differences in the composition of the volatile oil depending on the location of the plant and the part of the plant used. The concentrations of these volatile compounds fluctuated considerably throughout time, with the largest oil yield occurring at the beginning of the growth season (May) [48].
Given the diversity in the chemical composition of the volatile oil from the same species, A. rugosa was divided into several chemotypes. Chae et al. (2005) suggested that there are five major chemotypes: estragole, methyl eugenol, methyl eugenol + limonene, menthone, and menthone + pulegone [63].
Interestingly, other researchers have identified variations in the volatile oil composition in populations with the same cultivation method and location. Thus, among the 90 populations of A. rugosa, estragole, D-limonene, and isopulegone were found in the highest concentrations. Depending on the chemical composition of the volatile oil, the Plants 2023, 12, 2937 9 of 24 populations were divided into six chemotypes: estragole, pulegone, methyl eugenol, menthone, isopulegone, and nepetalactone. Thus, although the cultivated seeds had the same environmental conditions, the composition of the volatile oils varied significantly [46].
Following the same pattern, a comparative analysis performed by Dang et al. showed the differences between two chemotypes of A. rugosa: the pulegone chemotype and the estragole chemotype. The volatile oil of the pulegone chemotype was characterized by the dominance of pulegone, isomenthone, and limonene (monoterpenoids). Estragole and methyl eugenol were not found in the pulegone chemotype. In contrast, the major compounds of the volatile oil in the estragole chemotype were estragole and caryophyllene [55].
The chemical profile of the volatile constituents of the suspension culture of A. rugosa had major differences compared with the intact plant. In the suspension culture, 3-hydroxi-2-butanone and 2,4,5-trimethyl-3-oxazoline were reported as the major compounds, while estragole, the major compound of the parent plant, was not identified in the suspension culture [64].

Agastache mexicana Essential Oil
Regarding A. mexicana ssp. mexicana, one study highlighted estragole, D-limonene, and linalyl anthranilate as the major compounds of the volatile oil [65]. These results are in accordance with those previously reported by Estrada-Reyes et al., who identified estragole, limonene, and linalool as dominant compounds in A. mexicana ssp. mexicana [39]. Regarding A. mexicana ssp. xolocotziana, one study showed the dominance of phenylpropanoids (methyl eugenol and estragole) [66], and another study showed the dominance of terpenes (pulegone, menthone, and isopulegone) [39]. These results are not in concordance with the results reported by Najar et al., who showed that geranyl acetate, an oxygenated monoterpene, was the major compound in A. mexicana [4,67].

Agastache foeniculum Essential Oil
A. foeniculum is also reported to have high levels of estragole. The composition of A. foeniculum volatile oil recorded in the literature varies greatly, and these variations appear to be related to the provenance of the plant material. This suggests that A. foeniculum has several chemotypes.
As expected, the composition of the volatile oil depends on the part of the plant used. Thus, estragole was found in higher concentrations in the volatile oil of aerial parts (80-93%) and less in the essential oil extracted from leaves (18-30%) [56]. Also, estragole content was highest when the aerial parts were harvested at full bloom [60]. Irrigation regime [72], type of fertilizer used [73], and type of extraction procedure [74] were also found to influence the composition of A. foeniculum volatile oil. The volatile oil obtained using hydro distillation was approximately three times higher than that achieved from nitrogen purging. The distilled oils contained less benzaldehyde, octanone, limonene, and ocimene and higher boiling-point chemicals, particularly sesquiterpenes. The estragole concentrations of the oils and headspace were quite similar. Although this is an older study, it provides valuable information regarding the influence of the type of extraction on the composition of the essential oil [74]. Sourestani et al. showed that the relative quantities of the compounds in A. foeniculum volatile oil did not change as a result of drying procedures, storage, or distillation durations. The authors conclude that air drying (25 • C) and rapid extraction of essential oil using 2 h distillation are the optimal post-harvest treatments to optimize the amount of essential oil [57].

Agastache astromontana Essential Oil
The major compounds in both the leaves and flowers of Agastache astromontana 'Pink Pop' volatile oil were linalool, bornyl acetate, and camphene. However, essential oils extracted from flowers were richer in bornyl acetate, whereas oils extracted from leaves were richer in linalool [2].

Agastache aurantiaca Essential Oil
Studies have shown that Agastache aurantiaca volatile oil is mainly composed of oxygenated monoterpenes (88.8%), with pulegone as the major compound found in concentrations ranging from 76.7% to 77.9% [4,67].

Agastache scrophulariifolia Essential Oil
There were few reports on the composition of A. scrophulariifolia volatile oil. Isomenthone (49.7%) and pulegone (19.8%) were reported as the major compounds in A. scrophulariifolia volatile oil from leaves [61,76].
Some references regarding the composition of essential oils are older [75,76], but given the context in which the evidence is limited, they bring important information regarding the chemical composition of some less investigated Agastache species, such as Agastache 'Blue Boa' and Agastache scrophulariifolia.

The Evaluation of Therapeutic Properties
Several studies focused on the evaluation of the biological properties of Agastache species. The essential oils isolated from A. rugosa and A. foeniculum demonstrated cytotoxic properties [26,50,51], while A. rugosa extracts showed anti-inflammatory effects [6,21], anti-adipogenic and anti-atherosclerotic properties [17,18], as well as anti-osteoporotic potential [19] and antioxidant properties [12].

Cytotoxic Activity
A. rugosa and A. foeniculum volatile oils were found to display cytotoxic potential against a panel of cancerous cell lines, and their activity is often explained by the presence of certain active compounds. In this regard, A. rugosa essential oil was cytotoxic against two hormonally dependent cancer cell lines, breast cancer (MCF-7) [77] and prostate cancer (LNCaP) [51]. As a particularity of the study, the essential oil obtained using steam distillation was further extracted with diethyl ether to concentrate the active compounds and thus obtain a cytotoxic effect at a lower dose [77]. A. rugosa volatile oil also reduced the viability of lung (A549) [77] and melanoma (B16) cell lines [51]. Patchouli alcohol, one of the active compounds of A. rugosa essential oil (45.70%), also displayed cytotoxicity against LNCaP and B16 cells [51].
A. rugosa volatile oil obtained from leaves and flowers had a time-and dose-dependent cytotoxic action on the gastric cancer line SGC-7901. Also, estragole and pulegone, the main components of the essential oil, expressed antiproliferative properties in the SGC-7901 cell line [78]. Another study also confirmed the cytotoxic effect of A. rugosa volatile oil against the gastric cancer cell line SGC-7901 [50].
The volatile oil extracted from A. foeniculum "Aromat de Buzău" was cytotoxic against the MCF-7 breast cancer cell line at concentrations greater than or equal to 0.2 µg/mL [26]. In another study, estragole, the main component of A. foeniculum volatile oil, showed dose-dependent cytotoxic activity on the MCF-7 breast cancer cell line (IC 50 of 74 µg/mL), inducing apoptotic changes in the cancerous cell [79].

Anti-Inflammatory Activity
TRPA1 (transient receptor potential ankyrin1) and TRPV1 (transient receptor potential vanilloid 1) are receptors involved in processes such as pain and inflammation. TRPA1 and TRPV1 agonists can act as anti-inflammatory agents. A. rugosa stem and leaf extracts activated both receptors in vitro, indicating that A. rugosa could be associated with an anti-inflammatory effect. The authors suggested that the effect of the extract could be attributed to trans-p-methoxy cinnamaldehyde, L-carveol, methyl eugenol, p-anisaldehyde, and 4-allylanisole, which acted as TRPA1 agonists [6]. However, these volatile constituents are not usually found in extracts, but rather in the composition of essential oils. Thus, the effect of A. rugosa extract may be due to other active compounds.
Furthermore, A. rugosa essential oil exhibited in vivo anti-inflammatory activity in an adjuvant-induced arthritis rat model, reducing the level of pro-inflammatory markers such as TNF-α, IL-1, and IL-6 [51]. These findings suggest that A. rugosa contains a variety of bioactive chemicals with anti-inflammatory potential and could be beneficial for the treatment of pathologies associated with an inflammatory process.

Anti-Adipogenic and Anti-Atherosclerotic Activity
A. rugosa ethanolic extract and tilianin exhibited anti-lipogenic and anti-adipogenic effects on 3T3-L1 adipocytes. Tilianin caused a dosage-dependent decrease in lipid accumulation, whereas A. rugosa extract consistently decreased lipid accumulation regardless of dose, demonstrating that both tilianin and A. rugosa extract have anti-obesity effects in vitro. The researchers also found significant decreases in the levels of factors and proteins responsible for activating the process of adipogenesis, such as PPAR, C/EBP, SREBP1, and others [17]. Another study also confirmed that A. rugosa ethanolic extract decreased lipid accumulation by 20.4% in 3T3-L1 adipocytes [81].
Lee et al. showed that A. rugosa aqueous extract exerted an in vitro anti-atherosclerotic effect by inhibiting the proliferation of aortic vascular smooth muscle cells (VSMC). Also, by increasing the levels of the cyclin-dependent kinase inhibitors p21 WAF1/CIP1 and p27 KIP1 , A. rugosa extract produced cell cycle arrest at the G0/G1 transition phase. Although both rosmarinic acid and caffeic acid were found in high concentrations in A. rugosa extract, only rosmarinic acid showed specific action on the inhibition of DNA synthesis and cell cycle progression during VSMC proliferation [18].
The in vitro anti-atherogenic effect of A. rugosa extract was also confirmed by in vivo studies. Thus, the administration of A. rugosa methanolic extract lowered plasma cholesterol levels in mice. The study found that A. rugosa extract therapy reduced the accumulation of macrophages in atherosclerotic lesions. To better understand the mechanism of action, the study also investigated the effects of tilianin on cultured human umbilical vein endothelial cells (HUVECs). The results showed that tilianin reduces TNF-K-induced VCAM1 expression by 74% in HUVECs, an early phase in atherosclerosis pathogenesis. Also, tilianin administration significantly reduced the activity of NF-kB, another important factor in the atherosclerosis process [82].
The study performed by Jun et al. concluded that A. rugosa volatile oil administration had a hypocholesterolemic effect in vivo, significantly decreasing LDL and triglyceride levels in mice. The essential oil was also effective in reducing the oxidation of LDL and upregulating the expression of LDL receptors. Also, the administration of A. rugosa volatile oil down-regulated the expression of two transcription factors in the intrahepatic metabolism of cholesterol, SREBF-1 and SREBF-2, named sterol regulatory element binding factor [49].

Cardioprotective Activity
A. mexicana is traditionally used in herbal Mexican medicine for the treatment of hypertension [1,83]. Given the lack of scientific data that can support the empirical use of the plant, studies have investigated the cardioprotective effects of A. mexicana extracts and their main components using in vitro and in vivo models.
Tilianin isolated from A. mexicana exhibited vasorelaxant and antihypertensive effects when administered to male Wistar rats. Tilianin produced relaxation in concentrationand endothelium-dependent and independent manners in rat thoracic aortic rings. The mechanisms of action involved were NO/cGMP pathway modulation and the opening of K channels [1]. Furthermore, the same authors pointed out that extracts with a high concentration of tilianin have the best vasorelaxant effect, and the optimum method to obtain a high concentration of tilianin is by maceration with methanol, utilizing plant material dried at 50-100 • C [84]. Another study concluded that A. mexicana callus extract has vasorelaxant activity on the aortic ring. Interestingly, the callus extract with the lowest tilianin level had the best vasorelaxant effect when compared with the wild plant. This could be due to the presence of various secondary metabolites that are most likely only formed in in vitro cultures [85]. Regarding the safety profile, tilianin in doses less than 1000 mg/kg did not pose any visible toxicological effects when administered to mice: ED 50 (53.51 mg/kg) for antihypertensive effect and LD 50 (6624 mg/kg) were determined [86].
Some authors suggested that ursolic acid could also be responsible for the antihypertensive activity of A. mexicana. The dichloromethane extract produced a considerable vasorelaxant effect ex vivo in a rat aorta ring model. Moreover, the isolated ursolic acid showed antihypertensive action in hypertensive rats. Thus, the available studies support the empirical use of A. mexicana as an antihypertensive drug in Mexican traditional medicine [83].
Noteworthy, Cao et al. (2017) showed that A. rugosa extract also displayed procoagulant activity, while the isolated main components of A. rugosa extract, acacetin and tilianin, had significant anticoagulant activity in vitro [23]. Further investigation of the in vivo effects could lead to a better understanding of the possible use of Agastache sp. extracts in the management of bleeding disorders.
Another study aimed to determine the antidiabetic effect of tilianin (Acacetin 7-glucoside) in diabetic rats. Tilianin was isolated from the methanolic extract of A. mexicana. Tilianin administration (60 mg/kg body weight) for 10 days induced a significant antihyperglycemic effect at day 6. The administration of tilianin decreased IL-1b and IL-18 expression in adipose tissue, two pro-inflammatory mediators that can affect insulin sensitivity [88].
Thus, Agastache sp. extracts could be investigated in terms of antidiabetic potential, corroborating the presence of tilianin and acacetin, two active compounds with antidiabetic potential.

Hepatoprotective Activity
Cho et al. found that acacetin, isolated from aerial parts of A. rugosa, has a hepatoprotective effect in vivo against fulminant hepatic failure caused by D-galactosamine and lipopolysaccharide in mice. Acacetin pretreatment significantly increased the survival rate, reducing the level of seric aminotransferase, a biomarker for hepatocellular injury. Acacetin pretreatment also reduced the level of the pro-inflammatory tumor necrosis factorα (TNF-α) and increased the level of IL-6, a pleiotropic cytokine involved in hepatic homeostasis. The study concluded that acacetin inhibits D-galactosamine/lipopolysaccharideinduced hepatocellular damage, which could be relevant in future therapeutic applications for treating liver illnesses [89]. It is important to consider that following oral administration of A. rugosa extract in rats, acacetin was found in high concentrations in urine and feces, thus representing the main active metabolite [90]. Apart from acacetin, the unmetabolized main components of A. rugosa, tilianin, and rosmarinic acid were found in very low concentrations in urine and feces, indicating that these compounds are extensively converted to acacetin in rats. The hydrolysis of acacetin-O-glucoside (tilianin) to acacetin appears to play a significant role in the metabolism of acacetin-derivative secondary metabolites in A. rugosa [90]. Corroborating with the hepatoprotective effect of acacetin [89], Agastache sp. extracts warrant further investigation of the hepatoprotective potential in vivo.
Using the same animal model, Cho et al. demonstrated that β-caryophyllene, a sesquiterpene compound from A. rugosa essential oil, has hepatoprotective potential in acute hepatic failure. The intraperitoneal administration of β-caryophyllene prior to the induction of hepatic failure led to a reduction in the level of serum aminotransferase, tumor necrosis factor-α, and interleukin 6 [91].
Thymoquinone is an active aromatic compound found in Agastache sp. One study found that thymoquinone has a hepatoprotective effect on acetaminophen-induced acute liver injury. Administration of thymoquinone in rats reduced the levels of ALT and AST and also restored the levels of GSH, which has a significant role in the detoxification of acetaminophen. The authors also concluded that the hepatoprotective effect of thymoquinone is mediated through suppression of the MAPK signaling pathway [92].
Taken together, these studies promote the future investigation of the hepatoprotective potential of Agastache sp. extracts and volatile oils against acute liver injury.

Anti-Osteoporotic Activity
A. rugosa extract showed anti-osteoporotic potential both in vitro and in vivo. A. rugosa ethanolic extract stimulated the differentiation of MC3T3-E1 pre-osteoblasts [19]. Also, A. rugosa extract reduced the expression of NO-synthetase and inhibited NO production in ROS 17/2.8 osteoblasts. Excess NO production has been associated with osteoblast apoptosis and bone loss. Thus, A. rugosa leaf extract prevents NO-mediated apoptosis in vitro and may be beneficial in the prevention of osteoporosis and other inflammatory bone conditions [93].
In vivo, A. rugosa ethanolic extract reversed postmenopausal osteoporosis induced in an animal model by ovariectomy [19]. It has been shown that the anti-osteoporotic effect is also mediated by changes in the gut microbiome, by increasing the number of Deferribacteres, Proteobacteria, and Bacteroidetes and decreasing the number of Firmicutes bacteria [19]. Similar results were obtained by Jang et al., who discovered that A. rugosa aqueous extract reduced ovariectomy-induced bone loss in mice by suppressing RANK ligand-a marker of the osteoclast differentiation. The extract inhibited the expression of cellular oncogene fos (c-Fos) and nuclear factor NFATc1, key factors in osteoclastogenic transcription. The results support the possibility of using the aqueous extract of A. rugosa in the prevention and treatment of post-menopausal osteoporosis by suppressing osteoclastogenesis [94].

Anti-Nociceptive Activity
A. mexicana has been traditionally used in Mexican folk medicine for the treatment of pain. Decoction of A. mexicana aerial parts is empirically indicated for various pain disorders such as stomachache, colic, and intestinal pain, as well as to enhance digestion [3]. However, little scientific evidence supporting its use has been reported. Nevertheless, the latest research points towards the efficacy of A. mexicana extracts and volatile oils in the treatment of nociception.
A. mexicana ssp. mexicana and ssp. xolocotziana volatile oils showed anti-nociceptive activity in vivo, reducing abdominal pain in an animal model. The effect of the volatile oils from both subspecies could be explained by the presence of limonene and pulegone. Limonene demonstrated the strongest anti-nociceptive action in the study. It also significantly reduced intestinal inflammatory cytokines in a drug-induced ulcerative colitis model, providing gastroprotection against gastric injury. The results of the study support the traditional use of A. mexicana in the treatment of gastrointestinal conditions, showing the therapeutic potential of monoterpenes in the treatment of ulcers, colitis, and abdominal pain [95]. Another study confirmed the antinociceptive effect of pulegone in vivo. The tests that were used included the hot plate test and the formalin test. As a result, the authors suggested that pulegone could contribute to the antinociceptive effect of Agastache sp. essential oils [96].
A. mexicana extracts in hexane, ethyl acetate, and methanol inhibited the abdominal constriction generated by 1% acetic acid injection. Hexane and ethyl acetate extracts showed the highest antinociceptive efficacy. Although methanol extract was obtained in major yield, its antinociceptive effect was inferior to that of hexane and ethyl acetate extracts, suggesting less participation of the polar compounds in this type of nociception [97].
One study indicated that the antinociceptive effect of A. mexicana extracts could be partially explained by the presence of ursolic acid, a triterpene found in concentrations of 130.7 mg/g (ethyl acetate extract) and 20.3 mg/g (methanolic extract). Ursolic acid exhibited significant antinociceptive potential in different animal models, including the formalin test and the writhing test. Its efficacy was demonstrated in both neurogenic and inflammatory nociception, while its mechanism of action could involve the presence of cGMP and TRPV1 receptors [3]. These results were confirmed by another study, which showed that different types of A. mexicana extracts (hexane, ethyl acetate, and methanol), along with acacetin and ursolic acid, showed anti-nociceptive activity using the writhing test in mice [98].
Thus, the traditional use of A. mexicana as a pain reliever is sustained by in vivo studies that confirmed its antinociceptive effect in different types of nociception models.

Spasmolytic Activity
A. mexicana ssp. xolocotziana extract displayed spasmolytic properties on guinea pig ileum pre-contracted with KCl, while A. mexicana ssp. mexicana had a spasmogenic effect. Both subspecies are used in folk medicine for abdominal pain and other gastrointestinal diseases, but when administered in vivo, only ssp. xolocotziana has been shown to exhibit a spasmolytic effect. The mechanism of action could involve nicotinic receptor agonism and blockade of calcium channels. Qualitative and quantitative variances in the composition of each subspecies could be accountable for their distinct pharmacological effects [38].
Using the same animal model and inducing ileum contractions with KCl, another study found that acacetin and ursolic acid, two active components from A. mexicana extract, may be responsible for the spasmolytic effect of A. mexicana [98].

Gastroprotective Activity
A. rugosa 70% ethanolic extract (100 mg/kg/day) showed gastroprotective effects in an acute gastritis mouse model induced by administration of HCl/EtOH (clorhidric acid/ethanol). The gastroprotective effect was explained by the increased secretion of gastric mucus and the inhibition of the infiltration of inflammatory cells. At the molecular level, A. rugosa affected some signaling pathways involved in the cellular inflammatory response (PI3K/AKT and NF-kB). These results confirm the use of Korean mint as a medicinal plant in the treatment of digestive disorders such as dyspepsia, nausea, and vomiting [14].

Anxiolytic Activity
A. mexicana is traditionally used as a natural remedy for the treatment of anxiety disorders [99]. Thus, researchers evaluated the in vivo anxiolytic effect of A. mexicana extract and tilianin, one of the major bioactive compounds of the extract. The results showed that methanolic crude extract (10 to 300 mg/kg, i.p. and 300 mg/kg p.o.) and tilianin (30 to 300 mg/kg, i.p. and 300 mg/kg, p.o.) had anxiolytic effects when administered to mice. The mechanism of action could involve GABA A receptors, considering that the anxiolytic effect did not occur in the presence of flumazenil. These results demonstrated that tilianin is one of the main components of the methanolic extract responsible for the anxiolytic activity of A. mexicana and requires further research involving clinical studies [100].
Other researchers found that aqueous extracts of A. mexicana ssp. mexicana and xolocotziana exhibited anxiolytic-like activities at low doses (0.1 to 10.0 mg/kg), but at larger doses (above 100 mg/kg), they showed sedative activity that impacted general activity and motor coordination. The aqueous extract of ssp. mexicana appears to be more potent than that of ssp. xolocotziana, as it causes anxiolytic-like effects and sedative effects at lower doses [28]. Interestingly, an older study found that the effects of A. mexicana water extract are more consistent with an anxiogenic-like property than an anxiolytic-like property [101], but these results were not confirmed by other studies. These findings support the use of A. mexicana ssp. mexicana and ssp. xolocotziana in traditional medicine as anxiolytic, sedative, and tranquilizing agents.
Furthermore, a study showed that exposure to A. rugosa essential oil produced significant changes in EEG activity, stimulating brain wave activity and increasing spectral edge frequency by 50% of alpha spectra values. Also, exposure to volatile oil reduced the absolute and relative theta power spectra. These EEG changes suggest that A. rugosa essential oil can be used in aromatherapy for positive psychophysiological conditions [45].

Antioxidant Activity
Several studies evaluated the in vitro antioxidant effects of Agastache sp. extracts and volatile oils, and they demonstrated good antioxidant activity against different free radicals.
Studies showed that A. rugosa extract had DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity [12,81]. The essential oil from A. rugosa also displayed radical scavenging potential against DPPH, with an antioxidant activity of 8.79 µg/mL [51]. Regarding the mechanism of action, researchers found that A. rugosa leaf extract manifested its antioxidant activity by increasing the expression of the HO-1 (heme oxygenase 1) protein, an enzyme that protects against oxidative stress. The study showed that A. rugosa extract protects RAW264.7 macrophages against H 2 O 2 -induced oxidative stress and could be useful for the treatment of pathologies associated with cellular oxidative stress [102]. Furthermore, Gwakhyangjeonggi-san, a traditional herbal mixture that also includes A. rugosa, increased HO-1 expression and prevented the formation of reactive oxygen species in RAW 264.7 cells [80].
A. rugosa honey presents ferric-reducing antioxidant power of 3.61 ± 0.02 µmol TE/g (TE = Trolox equivalents) [103] and DPPH radical scavenging activity (IC 50 = 0.77 ± 0.02 mg/mL), with a significant positive correlation between the total antioxidant capacity and the total phenol content of the tested bee product [52].
The DPPH scavenging activity of A. aurantica flowers extract is comparable to previously published values from different Agastache flowers [104].
Plant phytochemical composition influences antioxidant activity. Typically, phenolic chemicals contribute significantly to antioxidant activity. The antioxidant activity, as evaluated by the DPPH assay, was shown to be highly correlated with the level of hydroxycinnamic acids (r = 0.77, p ≤ 0.05) as well as the total concentration of phenolic compounds (r = 0.74, p ≤ 0.05). There was also a strong association between antioxidant activity (DPPH assay) and flavonoid concentration (r = 0.58, p ≤ 0.05). A stronger link was also shown between antioxidant activity, as determined by the ABTS assay, and the total amounts of hydroxycinnamic acids and phenolic compounds [37].
Taken together, these studies show that Agastache sp. extracts and volatile oils have good antioxidant activity in different in vitro assays.

Enzyme Inhibition Activity
The inhibitory capacity of Agastache sp. extracts on the activity of some enzymes has potential applications in many fields.
A. rugosa's ethanolic extract inhibited xanthine oxidase (XO). Acacetin, one of the major compounds isolated from the extract, also exhibited xanthine oxidase inhibitory action (IC 50 = 0.58 M), which is lower compared with allopurinol, routinely used as a xanthine oxidase inhibitor. Thus, A. rugosa extract could act as a functional component and natural medicine for the treatment of hyperuricemia and gout [32]. Lee et al. showed that acacetin and its derivative, acacetin 7-O(6-O-malonylglucoside), two active compounds isolated from the methanolic extract of A. rugosa leaves, had inhibitory activity on MAO-A and MAO-B, two enzymes implicated in the degradation of amines, including dopamine. Thus, acacetin and its derivative could be further tested for their potential as MAO inhibitors, with application in the treatment of some neurological disorders, including Parkinson's disease [32].
A. foeniculum extract inhibited α-amylase and α-glucosidase, enzymes related to carbohydrate digestion. Amylase inhibitory activity was highly correlated with hydroxycinnamic acid concentration and moderately correlated with flavonoid concentration [37]. The inhibition of α-amylase and α-glucosidase could be beneficial in the treatment of type 2 diabetes; thus, further investigation of the antidiabetic potential of A. foeniculum extract is needed.

Antibacterial Activity
The essential oil obtained from A. rugosa flowers and leaves was found to have moderate to high antimicrobial activity. A strong in vitro inhibitory effect against Staphylococcus aureus (S. aureus-MIC 21 µg/mL) and Escherichia coli (E. coli-MIC 21 µg/mL) was noticed in the case of the essential oil from flowers, while the essential oil from leaves strongly inhibited E. coli (MIC 9.4 µg/mL). Both products were able to inhibit biofilm formation [50]. The antibacterial activity of the essential oil against S. aureus and E. coli was also confirmed by another study, which in addition reported antimicrobial activity against two other Gram-negative bacteria (Salmonella enteritidis and Pseudomonas aeruginosa). The antibacterial capacity could be attributed to estragole, the major compound of essential oil, but also to other trace volatile compounds such as terpenoids, including dl-limonene, linalool, β-caryophyllene, and germacrene-D [48]. E. coli was also susceptible to the action of A. rugosa methanolic extract, together with Aeromonas hydrophila, Aeromonas salmonicida, Staphylococcus haemolyticus, and Cronobacter sakazakii [15].
Antimicrobial activity evaluation showed that A. foeniculum essential oil had a moderate effect on some Gram-positive (Curtobacterium flaccumfaciens, Listeria monocytogenes, Bacillus subtilis) and Gram-negative bacteria (Salmonella sp., Proteus vulgaris, Klebsiella pneumoniae), with MICs ranging from 0.157 to 2.50 µL/mL. [68]. Regarding the antibacterial capacity of the extracts, silver nanoparticles obtained from A. foeniculum aqueous extract showed in vitro properties against several bacterial strains (Staphylococcus aureus, Staphylococcus haemolyticus, Klebsiella pneumoniae, and Streptococcus pneumoniae) [36].

Antifungal Activity
Shin et al. found that A. rugosa essential oil fraction and estragole exhibited antifungal activity against Aspergillus and Candida species (MICs ranging from 2.5 and 10 mg/mL). Also, essential oil and estragole displayed synergism with ketoconazole against Blastoschizomyces capitatus, a rare fungus that causes serious infections in immunocompromised patients [105]. These results were also confirmed by other studies, which pointed out the synergism of action between the estragole isolated from A. rugosa and ketoconazole against Candida species (C. albicans, C. utilis). However, this synergism was not confirmed for the combination of estragole and amphotericin B [106]. C. albicans was also susceptible in vitro to the action of A. foeniculum essential oil [68].
Another study evaluated the antifungal capacity of essential oil from A. rugosa against Trichophyton species. Synergistic effects were noticed between A. rugosa essential oil and estragole with ketoconazole against the studied Trichophyton species, with fractional inhibitory concentration indices that ranged from 0.05 to 0.27 [107].
Juárez et al. showed that A. mexicana ssp. xolocotziana volatile oil had antifungal activity against a panel of fungal strains, including Aspergillus sp., Penicillium sp., and Eupenicillum sp., with MICs ranging from 0.3 to 30 g/mL [66].
Other researchers investigated the antifungal properties of Agastache honey in comparison to other commercial honeys. Agastache honey showed superior antifungal activity against dermatophytes (T. mentagrophytes and T. rubrum) and C. albicans. Agastache honey contained several volatile compounds with antifungal activity, whereas other honeys contained few antifungal compounds. Phenol-2,4-bis(1,1-dimethylethyl), estragole, nonanoic acid, and ethyl ester were found as chemical markers in Agastache honey. Agastache honey products could be developed for topical use against fungal skin infections [44].

Insecticidal Activity
A. foeniculum volatile oil displayed strong insecticidal activity against Tribolium castaneum and Rhyzopertha dominica [70]. Moreover, the essential oil of A. foeniculum was found to be effective as a natural pesticide against Oryzaephilus surinamensis and Lasioderma serricorne [58]. These findings suggest that A. foeniculum volatile oil can be used to manage and reduce the negative impacts of modern insecticides.

Anti-Photoaging Activity
Some in vitro studies investigated the protective potential of A. rugosa extract against photoaging induced by UV-B radiation in HaCaT keratinocytes. The results of the studies showed that A. rugosa extract inhibited the activity of elastase and hyaluronidase, enzymes associated with the skin aging process [29]. Also, the extract presented an effective radical scavenging activity against DPPH, superoxide, nitrite [29], and ABTS + [30] free radicals. Moreover, A. rugosa extract reduced the level of NO, an important intracellular pro-inflammatory mediator [29,108], and the level of reactive oxygen species [29,30,108,109], thus demonstrating anti-inflammatory and antioxidant potential.
A. rugosa extract's anti-UV-B protective properties are augmented when using probiotic fermentation [108,109]. Both non-fermented and fermented extracts significantly reduced the level of ROS induced by UVB in keratinocytes and enhanced the activity of glutathione and superoxide dismutase, two antioxidant components involved in protection against oxidative stress [30,109].
Later, other researchers investigated the anti-photoaging effect of A. rugosa extract in an animal model. The extract was given orally to hairless mice at doses of 100 or 250 mg/kg/day for 12 weeks, along with UVB exposure. UVB-induced wrinkle formation, epidermal thickening, erythema, and hyperpigmentation have been histologically improved. The extract also restored skin moisture by improving skin hydration and transepidermal water loss, increasing hyaluronic acid levels, and significantly increasing collagen density [110]. Thus, these findings suggest that A. rugosa extract could be used as a preventive and therapeutic agent against photoaging and support the extract's cosmetic value.

Effects on the Respiratory System
A herbal mixture containing Glycyrrhiza glabra, Agastache rugosa, and tilianin significantly reduced neutrophil levels in the lungs and bronchoalveolar lavage fluid in an animal model of chronic obstructive pulmonary disease, thus showing potential in the management of this severe pathology [111].
A. mexicana ssp. mexicana volatile oil produced relaxation of the trachea smooth muscle, attenuating carbachol-and histamine-induced contractions. The study also suggested that the mechanism of action involved calcium channel blockade and that estragole and D-limonene could be responsible for the relaxant effect of the essential oil [65]. Also, A. mexicana extract had a relaxant effect on rat-isolated trachea contracted with carbachol [112]. Thus, A. mexicana extract and volatile oil could be further investigated in terms of their anti-asthmatic potential.

Conclusions and Future Perspectives
In this paper, we have performed a comprehensive review of the literature on the phytochemical profile and pharmacological properties of several species from the Agastache genus, a small genus of aromatic plants from the Lamiaceae family. Studies on Agastache sp. have seen significant progress in the last decade. Studies evaluating the chemical composition and therapeutic potential pointed out that Agastache sp. represent valuable natural sources of bioactive compounds. Agastache sp. extracts contain mainly flavonoids (tilianin, acacetin, apigenin, quercetin) and phenolic acids (rosmarinic acid, chlorogenic acid, caffeic acid). Tilianin (acacetin-7-O-glucoside) and its aglycone, acacetin, were identified as major compounds in the hydroalcoholic extracts of Agastache sp. Many of the therapeutic effects of the extracts have been attributed to the presence of these secondary metabolites. Some of the most important pharmacological effects of the extracts include anti-adipogenic, anti-atherosclerotic, cardioprotective, anti-diabetic, anti-osteoporotic, and hepatoprotective properties. The anti-atherosclerotic and cardioprotective effects of the extracts have been explained by the presence of tilianin, a glycosidic flavonoid with therapeutic potential in the cardiovascular field. Tilianin exhibited anti-lipogenic, anti-atherosclerotic, antihypertensive, and anticoagulant activity. Although these are promising results, most of the studies used in vitro models. To better understand the therapeutic potential of Agastache sp. extracts and their main components, in vivo studies are needed. Subsequently, standardization of the extracts of tilianin and providing a specific concentration of the active compound could enhance therapeutic efficacy and guarantee the quality of the extracts. Another option would be the structural modeling of tilianin with the development of structural analogues and the investigation of structure-activity relationships.
The volatile oils obtained from Agastache sp. contain mainly phenylpropanoids (estragole and methyl eugenol) and terpenoids (limonene, germacrene, and pulegone). Estragole is the major volatile compound reported in most of the studies; however, there are many chemotypes within the same species. Some chemotypes are characterized by the dominance of phenylpropanoids, while others are characterized by the dominance of terpenes. Thus, depending on the chemical composition, Agastache sp. volatile oils have demonstrated different pharmacological effects: cytotoxic, antimicrobial, anti-nociceptive, anti-inflammatory, and anti-atherosclerotic. Estragole also exhibited cytotoxic, antibacterial, and antifungal effects. Future research directions include testing the cytotoxic effect of volatile oils and estragole on other cancerous cell lines (colon, triple-negative breast, lung, and others) and investigating the cytotoxic potential of other Agastache species. The cytotoxic effect has been determined so far only for A. rugosa and A. foeniculum.
In the few studies conducted on animals, the authors do not report the adverse effects or toxicity of Agastache sp. volatile oils. Also, the dosage and mode of administration of the volatile oils are not always clearly mentioned. The extraction of the active volatile compounds from the essential oils and the investigation of their pharmacological effects may considerably enhance the therapeutic potential of the volatile oils.
Although research on Agastache species has seen a lot of progress in recent years, there are still many scientific gaps that require investigation. Of the 22 species of the genus, only a few of them have been characterized phytochemically and pharmacologically. Despite the promising therapeutic potential of some Agastache sp. extracts, volatile oils, or their active components, clinical studies are lacking. Moreover, some pharmacological properties have been tested only in vitro, and most of them were attributed to crude extracts and not to active compounds. Thus, the investigation of the pharmacological effects using animal models, the testing of the active compounds, and subsequently the standardization of extracts from these compounds represent important research directions.
In conclusion, Agastache species are aromatic plants considered natural sources of important bioactive compounds, representing potentially effective clinical drug candidates. Compared with other genera, Agastache genus is less investigated; thus, our present work gives a scientific foundation for future research and medicinal applications on the plants of this genus.