A Systematic Approach to Agastache mexicana Research: Biology, Agronomy, Phytochemistry, and Bioactivity

Mexico is the center of origin of the species popularly known as toronjil or lemon balm (Agastache mexicana Linton & Epling). Two subspecies have been identified and are commonly called purple or red (Agastache mexicana Linton & Epling subspecies. mexicana) and white (Agastache mexicana subspecies xolocotziana Bye, E.L. Linares & Ramamoorthy). Plants from these subspecies differ in the size and form of inflorescence and leaves. They also possess differences in their chemical compositions, including volatile compounds. Traditional Mexican medicine employs both subspecies. A. mexicana exhibits a broad range of pharmacological properties, such as anti-inflammatory, anxiolytic, and antioxidant. A systematic vision of these plant’s properties is discussed in this review, exposing its significant potential as a source of valuable bioactive compounds. Furthermore, this review provides an understanding of the elements that make up the species’ holistic system to benefit from lemon balm sustainably.


Introduction
Lamiaceae is the eighth most diverse plant family in Mexico and 5.5% of the species worldwide are found in this country. Thus, Mexico may be one of the most important diversification centers [1]. This family contains a wide range of aromatic plants possessing agronomical, pharmacological, and commercial potential. Mexican Agastache belongs to this family and its use and commercialization for traditional Mexican medicine make it the most important member of the Agastache genus in Mexico [2,3]. The species Agastache mexicana divides into two subspecies, based on anatomical characteristics [2] and chemical composition [4]: red lemon balm, Agastache mexicana Linton & Epling subspecies mexicana, and white toronjil, Agastache mexicana subspecies xolocotziana Bye, E.L. Linares & Ramamoorthy [5].
The species is distributed in the states of Guanajuato, Mexico, Michoacán, Puebla, Querétaro, Hidalgo, Veracruz, Chihuahua, Morelos, and Tlaxcala, as well as in Mexico City ( Figure 1). The species concentrates in the volcanic axis of Central Mexico [2,6].
This article presents a review of the research on Agastache mexicana from the perspective of a holistic system to understand its components and interactions. A literature search was sourced from meta-analyses of available Agastache data. Data were sourced from bibliographic engines like Web of Science ® , Scopus ® , ScienceDirect ® , and Google Scholar ® using "Agastache mexicana" as a keyword. This article presents a review of the research on Agastache mexicana from the perspec tive of a holistic system to understand its components and interactions. A literature searc was sourced from meta-analyses of available Agastache data. Data were sourced from bib liographic engines like Web of Science ® , Scopus ® , ScienceDirect ® , and Google Scholar ® us ing "Agastache mexicana" as a keyword.

A Holistic Approach to Agastache mexicana Usage
Systems biology analysis allows the understanding of different biological element and their interactions with non-biological elements, such as the environment or huma impacts (for example, the analysis of various traditional medicine systems like traditiona Chinese medicine) [8]. Systems biology, in tandem with reverse pharmacology, may allow discovering new active biological compounds [9].
Life science studies relying on systems biology and holistic approaches shy awa from reductionist views and incorporate biological effects and their interaction with th environment [8,9]. A biological system contains numerous components interacting in vast variety of combinations. Once the components and interactions of a system ar known, a system's behavior may be understood [10].
We used systems biology principles for a holistic analysis of different component within the lemon balm plant system and its environment ( Figure 2). This system's insight are derived from a general vision that includes the system components' relationships an interactions. This approach may provide new collaborative information, fresh insight and research prospects for the species.

A Holistic Approach to Agastache mexicana Usage
Systems biology analysis allows the understanding of different biological elements and their interactions with non-biological elements, such as the environment or human impacts (for example, the analysis of various traditional medicine systems like traditional Chinese medicine) [8]. Systems biology, in tandem with reverse pharmacology, may allow discovering new active biological compounds [9].
Life science studies relying on systems biology and holistic approaches shy away from reductionist views and incorporate biological effects and their interaction with the environment [8,9]. A biological system contains numerous components interacting in a vast variety of combinations. Once the components and interactions of a system are known, a system's behavior may be understood [10].
We used systems biology principles for a holistic analysis of different components within the lemon balm plant system and its environment ( Figure 2). This system's insights are derived from a general vision that includes the system components' relationships and interactions. This approach may provide new collaborative information, fresh insights, and research prospects for the species.

Biology
A. mexicana is a native vascular plant of Mexico [11]. It is a perennial herb. Plants of both subspecies have a typical Lamiaceae morphology: opposite, petiolate leaves, a fourangled stem, and numerous trichomes [12]. In the two subspecies, the stem is quadrangular in cross section; in A. mexicana ssp. mexicana, the basal and middle part of the stem is purple. The cuticle is smooth, but in the angles, it is observed to be crenulated, with a thickness of 4-8 µm. The three types of epidermal appendages described for the leaf are observed, but uniseriate non-glandular trichomes are abundant in A. mexicana ssp. xolocotziana, while they are more scattered and not visible to the naked eye in A. mexicana ssp. mexicana [2]. The plant height reaches between 50 and 150 cm [2] and the base chromosome number is 9. The plants have slender and spreading rhizomes [5].
Various plants from the Agastache genus are used for bee forage and honey production [13,14]. Toronjil is a honey plant; its flowers produce nectar for bee collection [15].

Biology
A. mexicana is a native vascular plant of Mexico [11]. It is a perennial herb. Plants of both subspecies have a typical Lamiaceae morphology: opposite, petiolate leaves, a fourangled stem, and numerous trichomes [12]. In the two subspecies, the stem is quadrangular in cross section; in A. mexicana ssp. mexicana, the basal and middle part of the stem is purple. The cuticle is smooth, but in the angles, it is observed to be crenulated, with a thickness of 4-8 μm. The three types of epidermal appendages described for the leaf are observed, but uniseriate non-glandular trichomes are abundant in A. mexicana ssp. xolocotziana, while they are more scattered and not visible to the naked eye in A. mexicana ssp. mexicana [2]. The plant height reaches between 50 and 150 cm [2] and the base chromosome number is 9. The plants have slender and spreading rhizomes [5].
Various plants from the Agastache genus are used for bee forage and honey production [13,14]. Toronjil is a honey plant; its flowers produce nectar for bee collection [15].
The stem of A. mexicana ssp. mexicana is erect, branched, and four-angled. The basal and middle part of the stem is purple. The form of the leaves is ovate-lanceolate, measuring 4.4 to 6.3 cm long and 2.1 to 2.5 cm wide [2]. The petiole is 1 cm long [2], and the corolla is purplish red to red [2,5]. The seeds measure approximately 4 to 5 mm ( Figure 3).  The stem of A. mexicana ssp. mexicana is erect, branched, and four-angled. The basal and middle part of the stem is purple. The form of the leaves is ovate-lanceolate, measuring 4.4 to 6.3 cm long and 2.1 to 2.5 cm wide [2]. The petiole is 1 cm long [2], and the corolla is purplish red to red [2,5]. The seeds measure approximately 4 to 5 mm ( Figure 3).

A. mexicana ssp. xolocotziana
The stem of A. mexicana ssp. xolocotziana is erect, branched, and four-angled. The form of the leaves is ovate-lanceolate, measuring 4.6 to 6.2 cm long and 1.7 to 3.0 cm wide [2]. The petiole is 1 cm long [2]. Inflorescences end in ramifications of interrupted whorls of cymes with numerous flowers. The calyx is 1.0 to 1.3 cm long and the corolla is white and approximately 2.4 cm long. Stamens are didynamous and exserted. Its anther is approximately 1 mm long. The style is 2.8 cm long, and its tip is bifid with the upper arm slightly longer. Ovules are only 0.5 mm tall [5]. The seeds measure approximately 3 to 4 mm ( Figure 4).  The stem of A. mexicana ssp. xolocotziana is erect, branched, and four-angled. The form of the leaves is ovate-lanceolate, measuring 4.6 to 6.2 cm long and 1.7 to 3.0 cm wide [2]. The petiole is 1 cm long [2]. Inflorescences end in ramifications of interrupted whorls of cymes with numerous flowers. The calyx is 1.0 to 1.3 cm long and the corolla is white and approximately 2.4 cm long. Stamens are didynamous and exserted. Its anther is approximately 1 mm long. The style is 2.8 cm long, and its tip is bifid with the upper arm slightly longer. Ovules are only 0.5 mm tall [5]. The seeds measure approximately 3 to 4 mm ( Figure 4).

Ethnobotanical Uses
The genus Agastache includes ornamental plants and aromatic plants that c sential oils [12]. For example, both A. mexicana subspecies have therapeutic and tal uses [2]. Knowledge about lemon balm healing properties is cited in sourc back to pre-Hispanic culture, such as in the De la Cruz Badiano Codex [16]. In th

Ethnobotanical Uses
The genus Agastache includes ornamental plants and aromatic plants that contain essential oils [12]. For example, both A. mexicana subspecies have therapeutic and ornamental uses [2]. Knowledge about lemon balm healing properties is cited in sources dating back to pre-Hispanic culture, such as in the De la Cruz Badiano Codex [16]. In the Nahuatl tongue, A. mexicana is known as tlalahuehuetl [6]. Traditional Mexican medicine labels the plant as "hot". It is prescribed to cure fright, stomach pain, excessive bile, cough, vomit, chills, and anxiety [2].
The holistic method to study plants with medicinal properties examines the interactions and relationships among the environment's biological and cultural components. Rural and urban populations use this plant for in-home treatments in the form of herbal teas (infusions and decoctions) [17]. In Mexico, A. mexicana is identified for its medicinal properties against anxiety and as a sleep-promoting plant [3,16]. The subspecies have specific uses: A. mexicana ssp. mexicana is preferred for wound healing, as an antispasmodic agent, and against stomach pain, while A. mexicana ssp. xolocotziana is employed to treat heart disease.
Many modern drugs originated from ethnopharmacology and knowledge of traditional medicine [18]. Results from research on the medicinal effects of A. mexicana ssp. mexicana and ssp. xolocotziana support their use in traditional medicine as an anxiolytic, tranquilizer, and sedative, as well as a remedy to alleviate "nervousness" [3,19].

Agronomy
Many medicinal and aromatic plants are industrially sown, but most are still obtained by harvesting wild populations. The need for renewable sources and protection of plant biodiversity creates an opportunity for farmers to grow these crops [20]. In Mexico, overharvesting of medicinal plants is counteracted by collecting seeds, cuttings, or roots to propagate the plant. Most of these collected samples are planted in small home gardens to be later sown on cultivated fields [21].
A. mexicana is a candidate species for structured cultivation as a source for active principles, extracts, essential oils, and pharmaceutical products [22]. Propagation is mainly asexual [23], through vegetative propagation, and depends on its rhizomes' division, as seed viability is low; A. mexicana ssp. xolocotziana seems to have even lower viability. A further complication arises as seed formation is hindered since harvesting occurs during flowering [5]; inflorescences are the main commercialized product. However, red lemon balm exists in wild populations, unlike white lemon balm. A hybridization process between the Mexican subspecies and Agastache palmeri possibly originated A. mexicana ssp. xolocotziana [23].
A. mexicana blooms from June to November [22]. Subspecies show phenotypic differences in leaf shape, flower color, and flavor [2]. Farmers from Santiago Mamalhuazuca (State of Mexico) have empirically gathered knowledge that the xolocotziana subspecies is more susceptible to extreme temperature and humidity. The cultivation of both subspecies begins in April, and the stems and inflorescences are harvested in November. The rhizome promotes stem sprouting, which allows a new harvest in the following February [23]. No technological packages based on crop physiology, detailing handling on its phenological stages, leading to higher biomass yields or providing information on bioactive production per cultivation area, exist for A. mexicana cultivation.

Commercialization
Ethnobotanical studies have impacted toronjil research. Empirical observations have detected that mexican markets sold a different subspecies from the typical A. mexicana subspecies mexicana. Identification of A. mexicana subspecies xolocotziana occurred through differential characterization of morphological, chemical, and pharmacological features [2,5]. The commercialization of botanical products promotes the cultural exchange of traditional knowledge and the exploitation of natural resources. Studies illustrate the influence popu-lar markets have on the demand for plants with novel applications. Attention should also focus on the dangers of overcollection of wild species in response to increasing demand and supporting natural habitats' conservation [21].
White and red lemon balm are commercially sown and traded in various Mexican regions, including Hidalgo, Mexico, Morelos, Puebla, and Veracruz. Inflorescence bundles or dried plants are distributed through different regional sales channels in the State of Mexico, Southeast Puebla, Morelos, and Mexico City [24].
The chemical composition of essential oils is influenced by the subspecies, environmental conditions of the crop, harvest time and type of extraction [4,12]. Table 1 shows the chemical compositions of different essential oils obtained from A. mexicana subspecies. However, some chemical studies do not specify the studied subspecies, which makes it difficult to establish a defined chemical profile for each subspecies. Plants introduced to other countries have essential oils with different chemical compositions. For example, the essential oil of A. mexicana grown in Scotland (subspecies not specified) is characterized by pulegone as the main compound, followed by menthone and limonene [25]; in contrast, A. mexicana ssp. mexicana plants introduced in Belarus produced methyl eugenol and estragole as the main compounds [26]. Extraction methods also influence the variability of the physical and chemical characteristics of the essential oils, but different distillation apparatus does not affect the quality of A. mexicana essential oil [27].

Biological Activity
The biological activity attributed to A. mexicana differs between subspecies because each one has a different chemical profile [4]. It also differs with respect to essential oils or extracts, as well as secondary metabolites present in them. Terpenes and flavonoids, such as ursolic acid, oleanolic acid, acacetin, apigenin, and tilianin, are the most active [33]. In some studies, where the biological activity of A. mexicana is determined, the studied subspecies is not specified. Tables 1 and 3 show the biological activities of the essential oils, extracts, and compounds for each subspecies.
Antihypertensive, Vaso-Relaxant, Spasmolytic, and Spasmogenic Properties Pharmacological studies correlate with the ethnomedicinal uses of A. mexicana. In 1982, infusions from both subspecies underwent pharmacological studies. Results showed contrasting effects for each subspecies. It was found that A. mexicana ssp. xolocotziana contracted the aorta, bladder, intestinal and uterine muscles, and the heart in experiments with frogs [5]. Studies performed in 2009 and 2010 showed antioxidant and vasoactive activities for A. mexicana ssp. mexicana extracts (Table 3) [19]. Additionally, the flavonoid tilianin, extracted from the plant, had antihypertensive and vasorelaxant effects on in vitro experiments, as observed on rat aortic rings and in vivo experiments in spontaneously hypertensive rats (SHR) [35]. A later study validated a liquid chromatographic method to detect tilianin in aqueous and organic (methanolic and hydroalcoholic) extracts of A. mexicana ssp. mexicana and correlated the biological activity with tilianin content and extraction conditions. The methanolic extracts had higher concentrations of tilianin and were the more vasorelaxant on thoracic aorta rat rings compared to carbachol, while the methanol extracts from dried biomass at 100, 90, and 50 • C were potent vasorelaxants [40]. Tilianin did not have toxic effects in sub-acute and acute oral administration in mice [36]. The vasorelaxant activity of the dichloromethane soluble extract from A. mexicana ssp. mexicana and its components (ursolic acid, oleanolic acid, and acacetin) also showed therapeutic effects: ursolic acid and acacetin had antihypertensive activity [38] (Table 3).
Furthermore, spasmogenic and spasmolytic activities differ between the two subspecies [28]. A. mexicana ssp. mexicana extracts were spasmogenic in guinea pig ileum, while A. mexicana ssp. xolocotziana extracts had a spasmolytic effect. Additionally, subs. xolocotziana contains a higher amount of acacetin and tilianin. Thus, only A. mexicana ssp. xolocotziana should be used to treat gastrointestinal afflictions [28] (Table 3). These results disagree with those reported in 1982 [5], but confirm each toronjil subspecies' contrasting pharmacological effects.
Studies of the effect of A. mexicana ssp. xolocotziana hexanic, dichloromethanic and methanolic extracts on tracheal rat rings found a relaxant-like activity [41], while the essential oil of A. mexicana ssp. mexicana caused relaxation of guinea pig tracheal tissue. The essential oil contains primarily estragole and D-limonene, which act as relaxants and anti-asthmatic compounds [31] (Tables 1 and 3). These studies support the potential therapeutic use of A. mexicana for asthma treatment.
Plant-tissue cultures of A. mexicana ssp. mexicana further confirmed the observed in vivo antihypertensive and vasorelaxant effects in SHR [33,35]. Tilianin isolated from methanolic extracts obtained from in vitro plantlets and calli confirmed the conservation of its vasorelaxant effects. The in vitro methanolic extracts contained a higher concentration of tilianin and produced a stronger vasorelaxant effect on aorta rat rings than extracts from wild plants [33] (Table 3).

Analgesic and Anti-inflammatory Properties and Effects in the Central Nervous System
The first pharmacological study on the effects of water-soluble A. mexicana extract on the central nervous system showed an anxiogenic-like effect in behavioral experiments at the doses tested in male rats [42]. Chemical and pharmacological studies performed in 2014 to identify the effects aqueous extracts from both subspecies have on the central nervous system found similar chemical profiles but different compound abundances.
Low doses of the extracts produced an anxiolytic effect, but higher doses sedated mice. Flavonoid derivatives may be responsible for the observed pharmacological effect [3]. Additionally, organic extracts of A. mexicana ssp. xolocotziana contain acacetin and ursolic acid and produce anxiolytic, spasmolytic, and antinociceptive effects in in vitro and in vivo experiments in mice [39]. In vivo experiments in different pain models in rodents confirmed the antinociceptive effect of organic A. mexicana ssp. xolocotziana extracts and ursolic acid [24,39]. Further behavioral experiments in mice determined the anxiolytic effects of A. mexicana ssp. mexicana methanolic extract and tilianin; they confirmed lemon balm contains tilianin, an anxiolytic compound, plus acacetin and ursolic acid [37] (Table 3). Taken together, results from pharmacological studies validate the traditional use of toronjil (lemon balm) to relieve gastrointestinal disorders, stomach pain, asthma, anxiety, insomnia, and hypertension.

Antioxidant and Nutraceutical Properties
Traditional Mexican medicine promotes lemon balm as an herbal product. However, herbal products lack strict quality control to guarantee their chemical composition or authenticity for manufacture. Thus, the consumer may not experience the expected therapeutic effect. However, various herbal products containing A. mexicana found significant antioxidant activity [43]. Additional reports detailed similar antioxidant activity of A. mexicana [22,34] (Table 3).
A. mexicana var. "Sangria" (ssp. mexicana) inflorescences are edible and have nutraceutical potential as they contain sugars and secondary metabolites. Compared to other Agastache species and the Lamiaceae family members, lemon balm inflorescences have higher polyphenols and flavonoid content and higher antioxidant properties [22].

Antifungal and Phytotoxic Properties
Aside from its use as a medicinal plant, A. mexicana produces bioactive compounds with antifungal activity ( Table 1). The essential oil contains monoterpenes and phenylpropanoids, such as estragole and methyl eugenol, which act as antifungal compounds. Notably, the essential oil did not show toxicity against human macrophages and brine shrimp. Research has shown the potential for its use as a non-toxic botanical fungicidal and as an alternative to synthetic fungicides [32]. A recent study tested the effect of adding A. mexicana essential oil to wheat grains as a food preservative for flour and dough. The quality of the dough and cookies prepared with the treated flour did not decrease and the growth of fungal pathogens of Aspergillus, Eurotium, Eupenicillum and Penicillium species were reduced (see Table 1). Additionally, by day 49, 79.2% of the added amount persisted. These properties indicate the essential oil as a candidate non-toxic food preservative [44].
In addition, the phytotoxic potential of organic extracts obtained with hexane, acetone and ethanol was explored ( Table 3). The acetone extract of A. mexicana (subspecies not specified) leaves was the most active, with an IC 50 of 71 µg/mL on the radical growth of Amaranthus hypochondriacus L. [45].  [35,40]. LD 50 of 6624 mg kg −1 in mice and antihypertensive effect (ED 50 53.51 mg kg −1 ) in SHR [36].

Potential and Perspectives
The holistic approach to studying the A. mexicana species focuses on biology, ethnobotany, chemical composition, and biological activity. The species has potential pharmacological uses as a source of bioactive compounds, such as tilianin, acacetin, apigenin, ursolic acid, and oleanolic acid [33] in areas such as drug development, disease modeling, and other biological explorations [47].
Agro-industrial applications and the production of essential oils require greater knowledge and understanding of endemic and native species of cultivated aromatic plants, such as Agastache mexicana. It is also essential to develop appropriate technologies for industrial applications and products. The information provided in this review supports the cultivation of lemon balm to take advantage of the plant, extracts, and essential oils. Red lemon balm has high essential oil yields, averaging 2.26%, regardless of the type of distillation device [27], while white lemon balm yields about 1.2%. This essential oil has proven antifungal activity against eleven strains isolated from wheat grains during storage [32]. Extracts from the leaves of A. mexicana contain reducing compounds like phenols and flavonoids and have been successfully used to provide a reducing medium for the synthesis of nanoparticles as well as their stability [46].
Progress has been made in the botanical and anatomical differentiation of the two identified subspecies. Evidence from biotechnological studies show that A. mexicana plant tissue cultures have great potential as a source of tilianin and other bioactive compounds [33], but information is scarce in terms of a technological package of cultivation and standardization of its components. There is phenotypic variability between subspecies and populations concerning wild or cultivated plants [23]. These results suggest that there may be genetic variability and the potential for genetic improvement of A. mexicana to increase plant biomass, improve resistance to climatic factors, resistance to pests and diseases. Furthermore, this variability could allow for the development of populations with specific chemotypes. For this reason, a holistic approach to the study of the species could help visualize a broader panorama that allows the sustainable use of lemon balm. Data Availability Statement: Publicly available datasets were analyzed in this study ( Figure 1). This data can be found here: https://datosabiertos.unam.mx/biodiversidad/ (accessed on 20 April 2021).