Journal of Ethnopharmacology 222 (2018) 34–51

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Journal of Ethnopharmacology

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Review Aloysia citrodora Paláu (Lemon verbena): A review of phytochemistry and T pharmacology

Roodabeh Bahramsoltania,b, Pourouchista Rostamiasrabadic, Zahra Shahpiria,b, ⁎ André M. Marquesd,e, Roja Rahimia,b, Mohammad Hosein Farzaeif,g, a Department of Pharmacy in Persian Medicine, School of Persian Medicine, Tehran University of Medical Sciences, Tehran, Iran b PhytoPharmacology Interest Group (PPIG), Universal Scientific Education and Research Network (USERN), Tehran, Iran c Department of Chemistry, Faculty of Science, University of Tehran, Tehran, Iran d Oswaldo Cruz Foundation (FIOCRUZ), Institute of Technology in Pharmaceuticals (Farmanguinhos), Rio de Janeiro, RJ, Brazil e PhytoPharmacology Interest Group (PPIG), Universal Scientific Education and Research Network (USERN), Rio de Janeiro, Brazil f Pharmaceutical Sciences Research Center, Kermanshah University of Medical Sciences, Kermanshah 6734667149, Iran g Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

ARTICLE INFO ABSTRACT

Keywords: Ethnopharmacological relevance: Aloysia citrodora Paláu (Lippia citriodora Kunth), commonly known as "lemon Lippia citrodora verbena" is a medicinal plant native to South America, North Africa, and South of Europe which is used by native Aloysia triphylla people for several indications such as diarrhea, flatulence, insomnia, and rheumatism. Lemon beebrush Aim of the review: Despite the wide biological activities of lemon verbena, there is no current review summar- Anxiety izing medicinal properties of the plant; thus, this paper aims to discuss current state of the art regarding the Sedation phytochemistry, pharmacology, and therapeutic applications of A. citrodora considering in vitro, in vivo, and clinical studies. Materials and methods: Electronic databases including PubMed, Scifinder, Cochrane library, Scopus, and Science direct were searched with the scientific name of the plant and its synonyms, as well as the common name. All studies on the ethnobotany, phytochemistry, pharmacology, and clinical application of the plant until October 2017 were included in this review. Results: Despite the few number of studies on the ethnopharmacology of the plant, A. citrodora is widely assessed regarding its phytochemistry and biological activities. Neral and geranial are the main ingredients of the es- sential oil; whereas verbascoside is the most significant component of the extract. Biological activities such as antioxidant, anxiolytic, neuroprotective, anticancer, anesthetic, antimicrobial, and sedative effects are proved in cell cultures, as well as animal studies. Conclusions: Several pharmacological activities have been reported for A. citrodora; however, the plant is not fully assessed regarding its safety and efficacy in human. Future well-designed human studies are essential to confirm the therapeutic benefits of this plant in clinical settings.

1. Introduction therapies (Farzaei et al., 2015). Lemon verbena, with the scientific name of Aloysia citrodora Paláu, is one of the well-known medicinal Medicinal plants have long been used by people all over the world. plants with several therapeutic activities (PDR, 2007). Traditional medicine of different nations have various treatment ap- A. citrodora belongs to the family Verbenaceae and has several other proaches amongst which the most important ones are plant-based synonyms for the scientific name including A. triphylla (L'Hér.) Britton,

Abbreviations: AC, Aloysia citrodora; Min, minimum; EO, Essential oil; MIC, minimum inhibitory concentration; AEC, aqueous extract of cedrón; Ach, acetylcholine; TBARS, thio- barbituric acid-reactive substances; CL, chemiluminescence; MDA, malondialdehyde; FRAP, ferric reducing ability of plasma; SOD, superoxide dismutase; CAT, catalase; Gpx, glutathione peroxidase; MPO, myeloperoxidase; Gred, glutathione reductase; TAG, triacylglycerol; HDL-C, high-density lipoprotein cholesterol; ROS, reactive oxygen species; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; AST, aspartate transaminase; ALT, alanine transaminase; ALP, Alkaline Phosphatase; Ig, immunoglobulin; PTZ, pentylenetetrazole; MES, maximal electroshock; CCI, chronic constriction injury of the sciatic nerve; MIA, monoiodoacetate; EPM, elevated plus maze; DSS, dextran sodium sulfate; MS, multiple sclerosis; IL, interleukin; IFN, interferon; DRC, dose-response curve; TEA, tetra ethyl ammonium; MMP, mitochondrial membrane potential; PBMCs, normal human peripheral blood mononuclear cells; IC, inhibitory concentration; EtOH, ethanol; MeOH, methanol ⁎ Corresponding author at: Pharmaceutical Sciences Research Center, Kermanshah University of Medical Sciences, Kermanshah 6734667149, Iran. E-mail address: [email protected] (M.H. Farzaei). https://doi.org/10.1016/j.jep.2018.04.021 Received 15 November 2017; Received in revised form 16 April 2018; Accepted 17 April 2018 Available online 23 April 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved. R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

A. citridora Paláu, A. citriodora Paláu, Lippia citriodora Kunth, L. ci- usually prepared as aqueous extracts, i.e. decoction or infusion trodora Kunth, and L. triphylla (L'Hér.) Kuntze (www.itis.gov). The plant (Portmann et al., 2012), and is used as an antispasmodic, diuretic, di- is a perennial shrub with up to 3 m height, striate and scabrous bran- gestive, cardiotonic, and sedative remedy (Lira et al., 2013; Daniel ches, and lanceolate 7–10 cm margined leaves with short petioles. The et al., 2014; Parodi et al., 2013a). The species has a history of use in tiny flowers have white or light blue color which appear on a hairy circulatory affections by both traditional as well as scientific in- calyx with four tips in the panicle-like spikes. The petals form a 4–5mm vestigations (Pochettino et al., 1997). In an ethnobotanical study in San funnel at the base which covers 2 long and 2 short stamens (PDR, Clemente (approximately 70 km from Cordoba) and Chancani (ap- 2007). proximately 200 km from Cordoba), the species is popularly consumed The plant has a wide geographical distribution from South America for the treatment of heart conditions (Toledo et al., 2007). Also, the to North Africa and South of Europe. It should be mentioned that due to processed leaves are used in tea manufacturing and as an ingredient of the pleasant lemony fragrance and its application in food industries and alcoholic or non-alcoholic herbal drinks (Gattuso et al., 2008). The cosmetics, as well as its use as a home remedy for several health pro- plant “cedrón” is also included in the Código Alimentario Argentino blems, the plant is currently available in other parts of the world, as (CAA) as a corrective and coadjutant, in the section referred to vegetal well (Carnate et al., 1999). Current paper aims to provide an overview condiments (Código Alimentario Argentino). The species is recognized of the phytochemistry, pharmacology, and therapeutic activities of A. and described in the Farmacopea Nacional Argentina, VI Edición (FNA) citrodora. as “the dried leaves with young stems, flowers and fruits of Aloysia triphylla (L´Hérit) Britt”. The increasing interest in this species has 2. Traditional uses largely contributed to expanding the plant crops in Argentina, Chile, and Paraguay (Oliva et al., 2010). The species Aloysia citrodora is popularly known in Latin-America as In many countries of South America, the infusions are typically “cedrón”, “cidron”, “citró”, “hierba Luisa”, “Maria Luisa” (Spanish made from dried or fresh leaves. It is consumed as a tincture, tea-like speaking countries) and “limonete”, “erva-luísa”“falsa-erva-cidreira”, infusion, or added to yerba mate (known as chimarrão or cimarrón, a “salva-limão”, “Lúcia-lima” or “cidrão” in Brazil (Argueta and Rodarte, traditional South American caffeine-rich infused drink made of the 1994; Freddo et al., 2016; Jimenez-Ferrer et al., 2017). The plant is leaves of Ilex paraguariensis)(Sartoratto et al., 2004; Pereira and widely used for medicinal and aromatic purposes in South America. Meireles, 2007; Bilia et al., 2008). According to literature data, reports about the traditional use of this In Equador, A. citrodora is used to alleviate fever, gastrointestinal species dates back to the 17th century, showing its ethnopharmacolo- problems, spasms and nervous attacks. For rheumatism, the cooked and gical importance as a medicinal plant used by the Inca culture (Elechosa warm leaves in a cloth, placed on the affected area, calms and relieves et al., 2017). The “kallawayas”, an itinerant group of traditional healers the pain. The infusion, prepared by boiling 15 g of the fresh plant in Andes, used to call the plant as “quechua” or “wari pankara” which material in two cups of water, to be taken after each meal is also used was used as a digestive, antispasmodic, and a remedy for bronchitis and for heart problems. In Ecuadorian gastronomy, “cedrón” occupies an heart problems (Girault, 1987). The most common use of this aromatic important place as a condiment once it brings its fragrant citric aroma plant was preparation of infusions. Many bioactivities were latterly and flavor to all traditional drinks such as the purple wash, the “chi- confirmed in the volatile fraction (Gil et al., 2007). A. citrodora is chas” (Sarmiento, 2012). worldwide considered as a citral-bearing plant species and hence is In Colonia del Sacramento (Uruguay), the researchers collected data cultivated in several countries due to its high economic value (Parodi about twenty-four urban and periurban gardens, small in size but quite et al., 2013a, 2013b). productive, and the second cultivated medicinal plant was the species In Americas, Brazil, Cuba, Peru, Bolivia, Argentina and Mexico have of A. citrodora. The plant was reported by 33% as a good digestive and a long history in the traditional use of medicinal plants for the treat- tranquilizer (Madaleno, 2012). In Paraguay (the Rural Paraguay re- ment of many conditions (Duarte et al., 2005). Traditionally, leaf in- gion), an ethnobotanical study investigated the seasonal differences and fusions of A. citrodora are used as a refreshing drink (Fonnegra and difference between urban and rural residents regarding the type of Jiménez, 2007; Santos-Gomes et al., 2005), as a liquor flavoring, and medications used to treat thirteen different common ailments and the also as an ingredient of a soft drink called “Inka kola”, which is po- source of medicinal plants used to treat those diseases. Interviews pularly consumed in Peru (Bandoni et al., 2003; Jimenez-Ferrer et al., performed in 2015 revealed that the species “Cedron Paraguay”, A. 2017). The plant is also used for the management of insomnia and citrodora, is cited as one of the traditionally used herbs in the region anxiety in Latin-American countries (Pascual et al., 2001; Santos-Gomes (Goyke, 2017). et al., 2005; Parodi et al., 2013a, 2013b). In Mexican traditional med- In Portugal, the plant is called "Lúcia-lima" and the leaves are used icine, the plant is used for stomach disorders, sadness and nervousness for diarrhea, insomnia, and rheumatism (Gião et al., 2007). (Argueta and Rodarte, 1994). However, in Brazil, a study in 40 healthy volunteers, subjected to a temporary state of anxiety, showed no benefit 3. Phytochemistry from the infusions of green branches of the plant (2, 6 or 18 g/200 ml) (Wannmacher et al., 1990). Several categories of phytochemicals have been identified in dif- Many plants from Brazilian biomes have been used as natural ferent parts of A. citrodora. Various flavonoids are isolated from me- medicines by local populations in the treatment of tropical diseases thanolic, ethanolic, or aqueous extracts of the plant. In addition, the (Alves et al., 2000). In Brazil, A. citrodora is grown in gardens and essential oil is mostly composed of monoterpenes and monoterpenoids, kitchen gardens, mainly for medicinal purposes and occasionally as sesquiterpenes and sesquiterpenoids, as well as some fatty alcohols. seasoning in cooking and salads (Lorenzi and Matos, 2008). The plant is Table 1 shows the isolated phytochemicals from different parts of the popularly used as a tincture or essential oil as a bactericide and for plant in detail. Also, Fig. 1 represents the structure of the most im- dermal disorders (Duarte et al., 2005). Also, the tea is consumed for portant phytochemicals of the plant. many purposes such as cold and fever, influenza, nerve problem, acne, and as an insecticide, bactericide, tonic, antispasmodic, carminative, 3.1. Terpenes and stimulant in South Region of Brazil (Maia and Fransisco, 2001; Ritter et al., 2002; Negrelle et al., 2007; Lorenzi and Matos, 2008; Terpenes are one of the most diverse classes of phytochemicals. Merétika et al., 2010; Santos et al., 2015). Terpenes, especially those found in the essential oils, are considered as In Argentina, the plant is commonly known as “cedrón” and grows suitable penetration enhancers in pharmaceutical industries since they in all Central and North regions of the country (Elechosa, 2009). It is can improve the permeability coefficient of poorly-absorbed drugs

35 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

Table 1 Phytochemicals isolated from different parts of Aloysia citrodora.

Phytochemical category Phytochemical name Part/extract Reference

Aldehyde Photocitral A Leaves/essential oil (Lira et al., 2008) Benzoid Benzyl alcohol O-β-D-glucopyranoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Fatty acid glycoside Tuberonic Acid Glucoside Aerial part/ MeOH, aqueous extract (Quirantes-Pine et al., 2010) Fatty alcohol 1-octen-3-ol Leaves/essential oil (Lira et al., 2008) Fatty alcohol Heptacosanol Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Fatty alcohol n-Octanol Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Fatty aldehyde 2,6-dimethyl-5-heptena Leaves/essential oil (Lira et al., 2008) Fatty esters Geranyl propanoate Leaves/essential oil (Argyropoulou et al., 2007) Fatty esters Geranyl propionate Leaves/essential oil (Lira et al., 2008) Flavonoid 6-hydroxyluteolin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid 6-Hydroxylated flavones Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Acacetin-7-diglucuronide Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Flavonoid Apigenin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Apigenin-7-diglucuronide Aerial part/Aqueous extract (Quirantes-Pine et al., 2009) Flavonoid Chrysoeriol Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Chrysoeriol-7-diglucuronide Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Flavonoid Cirsiliol Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Cirsimaritin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Diosmetin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Eupafolin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Eupatorin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Hispidulin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Jaceosidin Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Flavonoid Luteolin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Nepetin Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Flavonoid Nepitrin Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Flavonoid Pectolinarigenin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Flavonoid Salvigenin Leaves/MeOH extract (Skaltsa and Shammas, 1988) Glycoside Gardoside Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Hydroxycinnamic acid derivative β-hydroxyacteoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Iridoid glycoside Asperuloside Aerial part/ MeOH, aqueous extract (Quirantes-Pine et al., 2010) Iridoid glycoside Hastatoside Leaves/Aqueous extract (Benelli et al., 2017) Iridoid glycoside Ixoside Aerial part/MeOH, Aqueous extract (Quirantes-Pine et al., 2010) Iridoid glycoside Theveside Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Iridoid glycoside Verbenalin Leaves/Aqueous extract (Benelli et al., 2017) Isoamyl butyrate Methyl butyl 2-methyl butanoate Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Ketoalcohol Cinerolone Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Ketone 6-Methyl-5-hepten-2-one Leaves/essential oil (Argyropoulou et al., 2007) Ketone 6-methyl-5-hepten-2-one Leaves/essential oil (Lira et al., 2008) Lignan (+)-Lariciresinol-9-O-β-d-glucopyranoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Lignan (+)-Pinoresinol 4-O-β-D-glucoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Lignin sub-unites Dehydrodiconiferyl glucoside D Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Lignin sub-unites Dehydrodiconiferyl glucoside E Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Lipid Linoleic acid, ethyl ester Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Methyl Esters (9,12) – Hexadecadienoic acid methyl ester Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monocyclic sesquiterpene α-Humulene Leaves/essential oil (Ebadi et al., 2016) Monoterpene trans -Limonene oxide and cis Leaves/essential oil (Meshkatalsadat et al., 2011) Monoterpene (Z)-β-Ocimene Leaves/essential oil (Argyropoulou et al., 2007) Monoterpene Camphene Leaves/essential oil (Ebadi et al., 2016) Monoterpene Limonene Leaves/essential oil (Ebadi et al., 2016) Monoterpene Myrcene Leaves/essential oil (Lira et al., 2008) Monoterpene Neral Aerial part/volatile oil (Hudaib et al., 2013) Monoterpene Nerol Leaves/essential oil (Ebadi et al., 2016) Monoterpene Neryl acetate 1. Leaves/essential oil (Ebadi et al., 2016) 2. Supercritical fluid extracts from leaves Monoterpene p-cymene Leaves/essential oil (Lira et al., 2008) Monoterpene Sabinene Leaves/essential oil (Ebadi et al., 2016) Monoterpene Terpinolene Leaves/essential oil (Ebadi et al., 2016) Monoterpene Thujone Aerial part/essential oil (Dambolena et al., 2010) Monoterpene trans- and cis-Sabinene hydrate Leaves/essential oil (Meshkatalsadat et al., 2011) Monoterpene α-pinene Leaves/essential oil (Ebadi et al., 2016) Monoterpene γ-terpinene Leaves/essential oil (Ebadi et al., 2016) Monoterpene alcohol α-terpineol Leaves/essential oil (Ebadi et al., 2016) Monoterpene alcohol γ-terpineol Aerial part/volatile oil (Hudaib et al., 2013) Monoterpene ketone Camphor Whole plant (Kim and Lee, 2004) Monoterpene ketone Myrcenone Leaves/essential oil (De Figueiredo et al., 2004) Monoterpenoid (−)-loliolide Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Monoterpenoid (6S)-3,7-Dimethyl-7-hydroxy-2(Z)-octen-6- Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) olide Monoterpenoid 1,8-cineole Leaves/essential oil (Ebadi et al., 2016) Monoterpenoid Carvone Leaves/essential oil (Lira et al., 2008) Monoterpenoid Carvotanacetone Leaves/essential oil (Lira et al., 2008) Monoterpenoid Chrysanthemal Leaves/essential oil (Argyropoulou et al., 2007) (continued on next page)

36 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

Table 1 (continued)

Phytochemical category Phytochemical name Part/extract Reference

Monoterpenoid Chrysanthenol Leaves/essential oil (Argyropoulou et al., 2007) Monoterpenoid cis-sabinol Leaves/essential oil (Ebadi et al., 2016) Monoterpenoid Citronellal Leaves/essential oil (Ebadi et al., 2016) Monoterpenoid Citronellyl acetate Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monoterpenoid Cubebene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monoterpenoid Geranial 1. Leaves/essential oil (Ebadi et al., 2016) 2. Supercritical fluid extracts from leaves Monoterpenoid Geraniol Leaves/essential oil (Lira et al., 2008) Monoterpenoid Geranyl acetate Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monoterpenoid Linalool Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monoterpenoid Neoisothujyl alcohol Leaves/essential oil (Lira et al., 2008) Monoterpenoid Nerol Leaves/essential oil (Lira et al., 2008) Monoterpenoid Pinocarvone Leaves/essential oil (Lira et al., 2008) Monoterpenoid Piperitone Leaves/essential oil (Lira et al., 2008) Monoterpenoid p-Mentha-triene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monoterpenoid Shanzhiside Aerial part/ MeOH, aqueous extract (Quirantes-Pine et al., 2010) Monoterpenoid Tagetone Supercritical fluid extract/leaves (Parodi et al., 2013a, 2013b) Monoterpenoid Terpinen-4-ol Leaves/essential oil (Lira et al., 2008) Monoterpenoid trans-carveol Leaves/essential oil (Lira et al., 2008) Monoterpenoid Transpinocarveol Leaves/essential oil (Ebadi et al., 2016) Monoterpenoid trans-verbenol Leaves/essential oil (Lira et al., 2008) Monoterpenoid Z-Citral dimethoxy Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Monoterpenoid trans-p-menth-2,8-dienol Leaves/essential oil (Lira et al., 2008) Naphtoquinone Avicennone A Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Norisioprenoid 9-Hydroxymegastigm-5-en-4-one Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Oligosaccharide Jionoside C Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Phenolic glycoside Forsythoside A Aerial part/Aqueous extract (Quirantes-Pine et al., 2009) Phenylpropanoid glycoside Lippianosides A-E Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Phenylethanoid Isoverbascoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Phenylethanoid glycoside cis-acteoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Phenylethanoid glycoside trans-acteoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Phenylethanoid glycoside Verbascoside (acteoside) Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Phenylethanoid glycoside β-hydroxyverbascoside Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Phenylethanoid glycoside β-hydroxyisoverbascoside Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Phenylpropanoid Campneoside I Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Phenylpropanoid glycoside cis-tanoside F Aerial parts/MeOH and EtOH extract (Quirantes-Pine et al., 2009; Zhang et al., 2015a, 2015b) Phenylpropanoid glycoside Eukovoside Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Phenylpropanoid glycoside Martynoside Aerial parts/MeOH and EtOH extract (Quirantes-Pine et al., 2009’ Zhang et al., 2015a, 2015b) Sesquiterpene Aromadendrene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpene Cubenol Leaves/essential oil (Ebadi et al., 2016) Sesquiterpene E-Caryophyllene Leaves/essential oil (Ebadi et al., 2016) Sesquiterpene Germacrene D Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpene Globulol Leaves/essential oil (Ebadi et al., 2016) Sesquiterpene Nerolidol Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpene Spathulenol Aerial part/volatile oil (Hudaib et al., 2013) Sesquiterpene Turpinionoside D Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Sesquiterpene α-Cedrene Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpene α-Copaene Leaves/essential oil (Ebadi et al., 2016) Sesquiterpene α-Gurjunene/ γ- Gurjunene Leaves/essential oil (Ebadi et al., 2016) Sesquiterpene α-Muurolene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpene α-Zingiberene Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpene β-Bourbonene Leaves/essential oil (Meshkatalsadat et al., 2011) Sesquiterpene β-Himachalene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpene γ-Elemene Leaves/essential oil (Ebadi et al., 2016) Sesquiterpene alcohol Spathulenol Leaves/essential oil (Ebadi et al., 2016) Sesquiterpenoid (Z)-Nerolidol Leaves/essential oil (Lira et al., 2008) Sesquiterpenoid 2E, 6E-Farnesyl acetate Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid 8S-13-Cedranediol Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid 9-epi-E-Caryophyllene-14-ol Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid Acorenone B Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid ar-Turmerol Leaves/essential oil (Lira et al., 2008) Sesquiterpenoid Bicyclogermacrene Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpenoid Cadinene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid Caryophyllene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid Caryophyllene acetate Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid Caryophyllene oxide Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid cis-α -Bergamotene Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpenoid Curcumene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid Dihydrovomifoliol-O-β-D-glucopyranoside Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Sesquiterpenoid Eudesm-4(15)-ene-1β,6α-diol Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Sesquiterpenoid Hexahydro farnesyl acetone Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid Humulene epoxide II Leaves/essential oil (Lira et al., 2008) (continued on next page)

37 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

Table 1 (continued)

Phytochemical category Phytochemical name Part/extract Reference

Sesquiterpenoid Isoaromadendrene epoxide Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid trans-Cadina-1(2)-4-diene Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpenoid Z-Muurol-5-en-4β - Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Sesquiterpenoid α-Muurolol Leaves/essential oil (Lira et al., 2008) Sesquiterpenoid β-Acoradiene Leaves/essential oil (Argyropoulou et al., 2007) Sesquiterpenoid alcohol epi-α-cadinol Leaves/essential oil (Ebadi et al., 2016) Terpene Calamenene Leave/essential oil (Santos-Gomes et al., 2005) Terpenoid Citral Leaves/essential oil (Carnate et al., 1999) Flavonoid Luteolin-7-diglucuronide Aerial parts/MeOH extract (Quirantes-Pine et al., 2009) Triterpene Squalene Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Triterpene Ursolic acid Arial part/ EtOH extract (Zhang et al., 2015a, 2015b) Triterpenoid Oleanolic acid MeOH extract/ whole plant (Rao et al., 2013) Unsaturated hydrocarbon 2-Methyl-7-octadecyne Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b) Unsaturated hydrocarbon Octadecyne Leaves/supercritical fluid extract (Parodi et al., 2013a, 2013b)

Abbreviations: EtOH: ethanol, MeOH: methanol.

(Williams and Barry, 2012). They are also used as flavor and fragrance can be used as an indicator of storage duration (Ebadi et al., 2016). in food industries due to their pleasant organoleptic properties Meshkatalsadat et al. (2011) introduced 1,8-cineole (23.66%) and α- (Merfort, 2002). A wide variety of terpenes and terpenoids have been curcumene (14.83%) as main ingredients of A. citrodora from Ker- detected in A. citrodora which are discussed as follow: manshah, Iran. Although the essential oil had a high amount of geranial (13.74%) and limonene (13.46%), neral, one of the major components of the essential oil in the two latter studies, was not detected. Also, a 3.1.1. Monoterpenes and monoterpenoids study on lemon verbena in Morocco reported 1,8-cineole (12.4%) as the Monoterpenes are mostly detected in the essential oil obtained from principle essential oil component, followed by geranial (9.9%), 6-me- aerial parts of A. citrodora. According to Argyropoulou et al. (2007), thyl-5-hepten-2-one (7.4%), and neral (6.9%) (Bellakhdar et al., 1994). geranial (26.8–38.7%), neral (21.8–24.5%), and limonene (5.8–17.7%) Lira et al. (2008) evaluated several samples of the plant from Argentina, are the major monoterpenes detected in the essential oil of the plant Chile, and Paraguay. Paraguay samples had a similar composition to leaves which is grown in Greece. The amount of geranial and neral has those from Argentina. In samples from Argentina, three different types been decreased from May to September; while there was an increase in of the plant were identified based on the essential oil composition. In the percentage of limonene, which shows the impact of harvesting time the most common type, geranial, neral, and limonene were the major on the chemical composition of the essential oil (Argyropoulou et al., components. Two other types of lemon verbena with high content of 2007). Santos-Gomes and Fernandes-Derreira (2005) reported the same sabinene, limonene, and citronellal, as well as 1,8-cineole and β-thu- monoterpenes as the major components of the plant essential oil from jone were identified as Type B and Type C, respectively (Lira et al., Portugal. In a plant sample from Jordan, limonene (17.7%), along with 2008). In the Chilean samples, 1,8-cineole and sabinene had higher geranial (10.1%) and neral (9.8%), was the major components of the amounts in comparison to the other samples (Lira et al., 2008). Some essential oil (Hudaib et al., 2013). Ebadi et al. (2016) also assessed the monoterpene alcohols like α- and γ-terpineol and monoterpene ketone impact of different packaging methods on the quality and quantity of A. such as myrcenone have also been identified in the essential oil citrodora essential oil. In the plant samples cultivated in Tehran (Iran), (Table 1). Monoterpenes seem to have an important role in the anti- neral, geranial, and limonene were the major essential oil components. bacterial activity of A. citrodora since low amounts of these compounds All evaluated packaging methods including packaged under vacuum, showed less potent antibacterial activity (Parodi et al., 2013a, 2013b). with air, or nitrogen, resulted in decreased essential oil content; how- Iridoides, a subgroup of monoterpenoids, were also identified in their ever, the percentage of limonene and 1,8-cineole was increased during glycosylated form in the aqueous and methanolic extract of A. citrodora an eight-month period. In addition, the study suggests camphene as a aerial parts (Quirantes-Pine et al., 2009). suitable marker for the quality control of A. citrodora essential oil as it

Fig. 1. Chemical structure of the most popular components of Aloysia citrodora. A: neral, B: geranial, C: citronellal, D: 1,8-cineole, E: verbascoside, F: isoverbascoside, G: nepitrin.

38 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

3.1.2. Sesquiterpenes aureus, and Pseudomonas aeruginosa with the MICs range from 2.84 to Sesquiterpenes, with 15-carbon skeleton, are also present in the 8.37 mg/ml (Oukerrou et al., 2017). The essential oil also enhanced the essential oil of A. citrodora; however, they are found to a lesser extent. survival of silver catfish against Aeromonas hydrophila infection, which Argyropoulou et al. (2007) reported the presence of 79.5–83.3% of is mediated by strong antibacterial function (Dos Santos et al., 2017a, monoterpenoids in the essential oil of A. citrodora; whereas sesqui- 2017b). The ethanolic extract from the aerial parts of A. citrodora terpenes constitute only 14.1–15.1% of the essential oil. showed the maximum inhibitory effect on growth of the gram-negative Meshkatalsadat et al. (2011) also identified 59.54% monoterpene de- bacterium ‘P. aeroginosa’ and the least effect on the gram positive rivatives (including 15.70% monoterpenes and 43.84% oxygenated bacterium ‘B. subtilis’, which indicates the presence of strong anti- monoterpenes) and 26.93% sesquiterpenoids from the essential oil of bacterial compounds in this plant (Mirzaie et al., 2016). In contrast, the plant cultivated in Kermanshah, Iran. It should be mentioned that, aqueous and ethanolic extract of the leaf in the concentration range of unlike monoterpenes and their derivatives, the presence of sesqui- 625–20,000 μg/ml showed no significant inhibitory effect on the terpene compounds is not limited to the essential oil, i.e. they can be growth of clinically-isolated Streptococcus mutans and S. sobrinus isolated from different extracts of the plant. In a pressurized leaf extract ( Shafiee et al., 2016). obtained by supercritical CO2, total of 26% sesquiterpenes were iden- Larvicidal effects of the essential oil against Culex quinquefasciatus, tified and the highest percentage was related to β-caryophyllene (6.5%) southern house mosquito, showed that it is a natural larvicide, espe- and germacrene D (2.4%) (Parodi et al., 2013a, 2013b). Ebadi et al. cially in combination with Satureja montana (ratio 1:1), the larvicidal (2016) reported a series of sesquiterpene structures in the essential oil properties can be increased. The effect is suggested to be related to the of A. citrodora leaves. Ethanolic extract of the plant also contained some high neral and geranial content of the essential oil which have also sesquiterpenes such as turpinionoside D, dihydrovomifoliol-O-β-D-glu- demonstrated adulticidal effects in the previous studies (Benelli et al., copyranoside, and Eudesm-4(15)-ene-1β,6α-diol (Zhang et al., 2015a, 2017). 2015b). The effects of the pure oxygenated monoterpenoids, pulegone and citral were assessed on the head lice (Pediculus humanus capitis). 3.2. Phenylethanoids and phenylpropanoids Knockdown and mortality activities of the different hydroalcoholic solutions were evaluated which resulted in 47–53% and 42–55% of Phenylpropanoids are a class of plant secondary metabolites which mortality and knockdown percentages, respectively (Gonzalez-Audino are the products of shikimic acid pathway (Korkina, 2007). Probably, et al., 2011). the most important phenylethanoid isolated from A. citrodora plant is verbascoside (acetoside) and its derivatives which are isolated from the 4.2. Neuropsychological effects methanolic extract of the aerial parts. However, Quirantes-Pine et al. (2009) reported that β-hydroxyverbascoside and β-hydro- A. citrodora essential oil possesses significant neuroprotective ac- xyisoverbascoside were not distinguishable due to their similar frag- tivities in the hydrogen peroxide-induced (100 µm H2O2) and β-amy- mentation pattern in MS-MS spectrometry. The commercial extract of loid-induced (2.5–25 µm) neurotoxicity in the CAD (Cath.-a-differ- A. citrodora aerial parts also contained other phenylethanoids and entiated) neuroblastoma cell line. Neuroprotective effect was observed phenylpropanoids, which are represented in Table 1. in the concentrations above 0.1 mg/ml A. citrodora in both models of the in vitro experiments. 3.3. Flavonoids Radioligand binding profile was used to detect the molecular targets of A. citrodora oil as a neuroprotective agent. For this purpose, different Flavonoids are a sub-group of polyphenolic compounds with anti- concentrations of the oil (0.0001–0.1 mg/ml) were used in competition oxidant, anti-inflammatory, antimicrobial, cytoprotective, and anti- binding assays by means of [3 H] flunitrazepam, [35 S] TBPS, [3 H] neoplastic activities. These plant secondary metabolites are divided into MK801 as well as [3 H] nicotine in neuroblastoma cells. These radi- six main sub-categories: flavonols, flavanones, flavones, flavanols, iso- oligand binding evaluations were performed to understand the role of flavones, and anthocyanins (Pandey and Rizvi, 2009), and are usually the essential oil in the main neuropharmacological neurotransmitter found as colorful pigments in several plant species (Havsteen, 2002). pathways, including acetylcholine, glutamate, ligand-gated ion channel In 1988, Skaltsa could isolate a series of fl avonoids from the leaf receptors (NMDA), neuronal nicotinic receptor and the both benzo- extract of A. citrodora using column chromatography with silica gel. All diazepine site and ion channel site of Gamma-aminobutyric acid of the purified compounds had flavone structures (Skaltsa and (GABA) receptors. The results of this study showed that the oil only Shammas, 1988). Later, in 2009, Quirantes-Pine et al. could detect the performed a concentration-dependent modulatory effect on the [3 H] glycosides of the previously isolated flavones like apigenin-7-diglucur- nicotine binding with the IC50 of 0.0018 ± 0.0008 mg/ml onide and chrysoeriol-7-diglucuronide from the aqueous extract of A. (Abuhamdah et al., 2015). citrodora aerial parts. Recent studies also identified some new flavo- The anesthetic effect of the essential oil of A. citrodora in silver noids (jaceosidin, nepetin, and nepitrin), all with flavone structures catfish has been confirmed. The essential oil induced anesthesia at the − (Zhang et al., 2015a, 2015b). concentrations of 150 and 300 μLL 1, and the addition of diazepam (150 µM) had a synergistic effect on faster induction of anesthesia with 3.4. Miscellaneous compounds no significant change in the recovery times. Flumazenil reversed the diazepam-induced anesthesia; however, it did not change the an- In addition to the above-mentioned phytochemicals, some other esthesia induced by A. citrodora essential oil, indicating that the ben- chemical structures such as fatty alcohols, ketones, lignans, and tri- zodiazepine site of the GABA receptors has no significant role in its terpenes are also isolated from the essential oil, aqueous extract, and neuropharmacological mechanism (Dos Santos et al., 2016). Another alcoholic extracts of A. citrodora aerial parts (Table 1). study evaluated the effect of the essential oil on the recovery times of sub-adult and post-larvae white shrimp from anesthesia. In sub-adult 4. Pharmacological activities of A. citrodora shrimp, the minimum concentration of the essential oil for the induc- − tion of stage II anesthesia was 100 μLL 1 (16 min); the shortest stage II 4.1. Antimicrobial and insecticidal effects anesthesia induction time (5 min) was observed at the concentration of − 500 μLL 1, and the shortest recovery time (10 min) was found at − The essential oil obtained from the leaves of A. citrodora demon- 300 μLL 1. In post-larvae shrimp, the shortest induction and recovery − strated antibacterial function against Escherichia coli, Staphylococcus times (less than 10 min) were both seen at 300 μLL 1 (Parodi et al.,

39 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

2012). Induction of anesthesia in the gray and albino strains of silver assessment of malondialdehyde (MDA) generation, ferric-reducing catfish showed that the essential oil is an effective anesthetic for both ability of plasma (FRAP) and superoxide dismutase activity (SOD) in strains and can induce deep anesthesia without mortality (Parodi et al., the plasma samples proved the strong antioxidant activity of the plant 2013a). Also, the exposure of silver catfish to 40 μL/L of the essential oil (Funes et al., 2009). The effect of A. citrodora administration in rats on as a sedative during transportation improved the chemical and sensory the oxidative-associated biochemical parameters demonstrated an in- qualities of the fish during refrigerated storage, and diminished sensory crease in the antioxidant enzymes activities, namely glutathione per- demerit scores (Daniel et al., 2014). oxidase (Gpx), catalase and glutathione reductase (Gred), reduced The aqueous extract of A. citrodora as a coordinator for anxiety myeloperoxidase (MPO) activity and protection of blood cells against conducted important sedative effect mechanistically similar to benzo- oxidative damage (Quirantes-Pine et al., 2013). Aqueous extract (in- diazepines (Ragone et al., 2010). Veisi et al. (2015) investigated the fusion and decoction) of A. citrodora protected against lipid peroxida- effects of the intraperitoneal injection of the aqueous extract of A. ci- tion and protein carbonylation. Also, the decoction showed higher an- trodora on male wistar rats which, in doses higher than 10 mg/kg, in- tioxidant capacity compared to the infusion (Portmann et al., 2012). A. creased anxiety in rats in elevated plus maze (EPM) (Veisi et al., 2015). citriodora-leaf infusion in mouse bone marrow cells assessed by the By contrast, a fatty acids and terpenes-enriched fraction of A. citrodora comet assay technique has been reported to inhibit the capacity of extract showed significant improvements in a dose of 500 mg/kg in cisplatin to induce genetic damage and increase cell viability. There- EPM test (Jimenez-Ferrer et al., 2017). This effect seems to be involved fore, infusion as a free radical scavenger has an antigenotoxic activity with GABAergic and serotonergic neurotransmission. The controversial by enhancing the antioxidant status (Zamorano-Ponce et al., 2004). results obtained in the two studies might be due to the difference in the Furthermore, in another study, the essential oil of Aloysia species ex- nature of the assessed extracts. While the former used an aqueous ex- hibited a high antigenotoxic effect against ultraviolet radiation-induced tract (Veisi et al., 2015), the latter evaluated a lipophilic fraction DNA damage (Quintero Ruiz et al., 2017). (Jimenez-Ferrer et al., 2017) which might have a higher ability to pass Verbascoside, a caffeoyl phenylethanoid glycoside (CPG), was used the blood brain barrier. Thus, future investigations are recommended in piglets in order to decrease the response to stress induced by high for better clarification of the effect of lemon verbena extract and its n − 6 polyunsaturated fatty acids. Oxidative damage of the liver was constituents on anxiety-like behavior. modified without changing the systemic responses to the oxidative Ethanolic extract from the leaves of A. citrodora showed antic- stress (Di Giancamillo et al., 2015). Despite that, evaluation of the es- onvulsant activity against electro- and chemoconvulsant-induced sei- sential oil on the antioxidant status showed an elevation in the oxida- zures in mice. The extract caused a reduction in the duration and an tive protection and an alleviation in the oxidative stress (Gressler et al., increase in the latency of the seizures in the pentylenetetrazole (PTZ) 2014). model, as well as a decrease in the hind limb tonic extension duration in The effect of an oral administration of verbascoside (maximum dose the maximal electroshock (MES) test (Rashidian et al., 2016). Since the of 3 mg/day) as a food supplement to hares indicated a protective effect anticonvulsant effect of the extract was reversed by flumazenil, this on the ocular tissue and fluids from oxidative stress by thiobarbituric effect, at least in part, is related to the interaction of the extract with acid-reactive substances (TBARS) and trolox equivalent antioxidant benzodiazepine site of the GABA receptors. capacity (TEAC) assays (Mosca et al., 2014). The effects of verbascoside treatment during the in vitro maturation 4.3. Gastrointestinal effects of juvenile sheep oocytes and embryo development were analyzed. Bioenergetic/oxidative status of the matured oocytes was tested by the Ragone et al. (2007) reported that the aqueous extracts of A. ci- confocal analysis of mitochondria and ROS, quantitative polymerase trodora decreased the contraction, and inhibited the dose–response chain reaction (PCR) of bioenergy/redox-related genes, and lipid per- curves DRC of acetylcholine (ACH) in a non-competitive manner. The oxidation assays. Verbascoside reduced oocyte ROS and lipid perox- aqueous extract of A. citrodora can directly relax the intestinal smooth idation. Also, verbascoside increased the blastocyst quality and mi- muscle. Maximal relaxation of A. citrodora was 81.0 ± 3.2% of the tochondria/ROS colocalization (Martino et al., 2016). maximal effect of papaverine, a non-selective phosphodiesterase (PDE) The effect of verbascoside-based dietary supplement on the pro- inhibitor, and 78.1 ± 5.0% of quercetin, a selective PDE inhibitor. ductive performance, plasma oxidative status, and some blood meta- Moreover, methylene blue (10–30 µmol/L) non-competitively inhibited bolites was assessed in suckling lambs. A remarkable decrease in TBARS A. citrodora relaxation and tetraethylamonium (40 mmol/L) competi- (p < 0.05) and reactive oxygen metabolites (P < 0.01), as well as a tively antagonized it (Ragone et al., 2007). Vitexin and isovitexin, significant increase in the serum levels of vitamin A (p < 0.05) and identified as the major flavonoids of the extract by HPLC, were in- vitamin E (P < 0.01) was observed (Casamassima et al., 2013). The dividually tested, as well. However, only vitexin demonstrated a spas- effect of A. citrodora extract on reproductive/productive performance molytic activity (Ragone et al., 2007). and plasma biochemical parameters in white rabbit was tested. There Methanolic crude extract of the plant was assessed on the char- was an increase in the plasma level of vitamin A and E (P < 0.01), coal–gum acacia-induced hyperperistalsis in rats, and the charcoal meal along with a reduction of plasma reactive oxygen metabolites and test was applied. Moderate inhibitory effect (32% inhibition) on the TBARS values (P < 0.01) (Casamassima et al., 2017). hyperpropulsive movement of the small intestine was obtained with A. Antioxidant properties of A. citrodora as a sport supplement has citrodora extract which is almost equal to loperamide and support the been investigated in several trials. Antioxidant effect of a beverage traditional use of the plant as an antidiarrheal agent (Calzada et al., containing lemon verbena extract was demonstrated in 10 swimmers 2010). after 26 days of supplementation in regard to the increased activity of A. citrodora infusion was evaluated regarding its efficacy in the Gred and Gpx (Mestre-Alfaro et al., 2011). A randomized, double-blind, treatment of dextran sulfate sodium (DSS)-induced colitis in rats. placebo-controlled clinical trial in 15 male athletes for 21 days was Administration of 40 g/L of DSS for 7 days significantly reduced body conducted to evaluate the capacity of the extract (10% verbascoside w/ weight gain and colon length. Likewise, the results showed a significant w, 600 mg/capsule) to improve the oxidative stress and antioxidant improvement in the colitis, histological colonic alterations and the co- enzyme activities. The results showed a significant improvement in the lonic damage in rats (Lenoir et al., 2012). erythrocytes and lymphocytes (p < 0.05) and the reduction of oxida- tive stress markers such as protein carbonyls and MDA in plasma 4.4. Antioxidant and cytoprotective effects (p < 0.05) (Carrera-Quintanar et al., 2012). Another study evaluated the effect of the same supplement, alone or in combination with an After the oral administration of A. citrodora extract in rats, almond beverage, in subjects performed eccentric exercise. The results,

40 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51 in line with the previous study, supported the positive effects of the 4.7. Estrogenic effects plant on the endogenous antioxidant defense mechanisms (Carrera- Quintanar et al., 2015). Also, lemon verbena supplement showed a A clinical trial in female swimmers for 26 days evaluated the effects synergistic effect with cellular adaptive response to intense exercise, i.e. of A. citrodora extract containing 20 mg/100 ml of verbascoside on the it did not inhibit the exercise-induced reductions of the pro-in- sex hormone levels, antioxidant responses and oxidative damage. flammatory cytokines IL-6 and IL-1β, but further decreased neutrophils Hypothalamic modification of estradiol synthesis showed a reduction in oxidative damage (Funes et al., 2010). In another clinical trial in 43 17-β-estradiol and the testosterone levels, and an enhancement of the healthy subjects, A. citrodora leaves improved oxidant/ antioxidant sex hormone binding globulin levels (Mestre-Alfaro et al., 2011). balance by a decrease in the lipid peroxidation (p < 0.05) and increase in the total antioxidant ability of the serum (p < 0.05) (Malekirad 4.8. Cardiovascular effects et al., 2011). Cardiovascular effects of the aqueous extracts of A. citrodora were 4.5. Antinociceptive and anti-inflammatory effects investigated in vivo and in vitro. Measurement of blood pressure in normotensive rats showed no significant effect on the heart rate, but Several pharmacological experiments showed anti-inflammatory regarding the left ventricular pressure in isolated rat hearts, a dose- and anti-nociceptive effects of the plant. Ponce-Monter et al. (2010) dependent negative cardiac inotropism and a transitory hypotension showed that the administration of the hexane extract and citral, the were observed (Ragone et al., 2010). Use of specific receptor antago- main chemical component of the plant, possessed anti-inflammatory nists revealed that this cardiac effect is not mediated through mus- activity. In different systems of traditional medicine, A. citrodora has carinic, NO, or α1 receptors, but is the result of a direct effect on the been used as an antispasmodic agent. In an in vitro study, the relaxant smooth muscles (Ragone et al., 2010). In a randomized, double-blind effect of the hexane extract from the dried leaves of A. citrodora and clinical trial, 100 patients with high cardiovascular risk (having one or citral was evaluated. Both extract and citral showed an inhibitory ac- more cardiovascular risk factors) were treated with 50 or 100 mg ver- tivity on the painful feeling and contraction of uterus muscle in a bascoside. After two weeks of supplementation, the higher dose of concentration dependent manner (Ponce-Monter et al., 2010). verbascoside could significantly reduce the platelet aggregation in- The effects of verbascoside on the neuropathic pain induced by the duced by arachidonic acid or adenosine diphosphate, in comparison to chronic constriction injury of sciatic nerve were studied in rats. This an- placebo. Authors suggested cyclooxygenase and thromboxane A2, as tinociceptive effects might be through the inhibition of the microglia ac- well as adenosine diphosphate (ADP) and P2Y12 receptor (a main re- tivation, apoptotic pathways, and antioxidant properties. Modification of ceptor in the ADP-dependent platelet aggregation) to be involved in the the behavioral changes associated with neuropathy were due to decreased antiplatelet aggregating effects of verbascoside (Campo et al., 2015). Bax (a proapoptotic factor), Iba protein (a marker of microglia activation), and increased Bcl-2 (Amin et al., 2016). 4.9. Anticancer effects The antihyperalgesic activity of pure verbascoside of the hydro- alcoholic extract of A. citrodora was studied in two models of neuro- Microculture tetrazolium test (MTT) assay, flow-cytometry and real- pathic pain. Oral and intraperitoneal administration of the agents de- time PCR methods on the human colon cancer (HT29) cells were used creased hyperalgesia in both animal models of neuropathic pain. Also, a to investigate the anticancer effect of A. citrodora extract. The ethanolic dose of 100 mg/kg for the intraperitoneal injection and 300 mg/kg for extract enhanced BAX (pro-apoptotic gene) and reduced Bcl-2 (anti- the oral administration were the most effective doses to decrease hy- apoptotic gene) expression level. Likewise, the extract induced apop- peralgesia (Isacchi et al., 2011). tosis in the colon cancer cell line (38.66%), with a significant inhibition A randomized, double-blind, placebo-controlled study assessed the of the cell proliferation in a dose-dependent manner (maximum in- efficacy of a combination of A. citrodora extract and fish oil omega-3 hibition in 1000 mg/ml) (Mirzaie et al., 2016). fatty acids or placebo as an alternative treatment for joint management in subjects with joint discomfort. The Western Ontario MacMaster 4.10. Growth improvement in animals (WOMAC) and Lequesne's questionnaires were used for both groups to record pain and stiffness of the joints. Administration of the supplement The effects of verbascoside on the growth of suckling lambs, ame- for 9 weeks alleviated stiffness and symptoms of the pain, and ame- liorated the average daily gain, milk consumption and high density li- liorated the physical function, compared to the placebo (Caturla et al., poprotein cholesterol (HDL-C) levels, along with a reduction in TAG 2011). level (p < 0.05) (Casamassima et al., 2013). Progressive daily weight A randomized, double-blind, placebo-controlled clinical trial as- gaining from birth to weaning, enhancement of kit body weight at sessed the effect of A. citrodora in 30 participants with multiple sclerosis weaning, increase in HDL-C, and decrease in bilirubin, activities of ALP, (MS). Compared with the control group, C reactive protein (CRP) levels and AST/ ALT were observed in white rabbits fed with two doses of A. in the secondary progressive MS patients, cytokines/inflammatory citrodora supplement (Casamassima et al., 2017). A study on the piglets markers, such as IL-12 levels in the relapsing-remitting type, and IFN-γ supplemented with a dietary extract from A. citrodora showed positive levels in all types of MS patients were reduced which further confirms effect on the growth, antioxidant status, and serum immunoglobulin the anti-inflammatory effect of the plant. Besides, the concentrations of (IgA) concentration (Pastorelli et al., 2012). Also, in a study of the the anti-inflammatory cytokine, IL-4 and IL-10, were increased in the broilers fed with dietary A. citrodora, elevated average weight gain, feed secondary progressive MS patients (Mauriz et al., 2015). intake and Gpx level (an antioxidant enzyme which reduces the nega- tive effects of heat stress), along with the reduction of heterophyl/ 4.6. Metabolic effects lymphocyte ratio and LDL-C were observed (Rafiee et al., 2016).

The effects A. citrodora polyphenols were tested by an insulin-re- 5. Safety and toxicity sistant hypertrophic 3T3-L1-adipocyte model on the obesity-induced metabolic disturbances in mice. The results demonstrated a remarkable There are few studies in which the toxicological aspects of lemon improvement in the fat metabolism. In addition, the extract decreased verbena have been assessed. A study by Oskouei Shirvan et al. (2016) the production of ROS and triacylglycerol (TAG) accumulation, and demonstrated a dose of 0.5 mg/kg of the plant extract to be safe in enhanced the mitochondrial membrane potential (MMP) (Herranz- pregnant animals without any teratogenic properties (Oskouei Shirvan Lopez et al., 2015). et al., 2016). Etemad et al. (2016a) also assessed the teratogenic effect

41 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51 of verbascoside at a dose of 1 g/kg/day in mice. The results showed that joint disease, one trial on its cardiovascular effects, as well as one trial verbascoside has no negative impact on the embryo development. In regarding its estrogenic effects (Table 2). However, not all of the ethnic two other studies by Etemad et al., acute and sub-acute toxicity of uses of the plant have yet been assessed in clinic. lemon verbena aqueous extract, as well as its main constituent, ver- The current human studies on the efficacy of lemon verbena have bascoside, was evaluated. Both studies reported the median lethal dose mostly focused on its antioxidant activity in different ways such as the

(LD50) of the aqueous extract and verbascoside to be higher than 5 g/kg improvement of antioxidant ability of blood cells, induction of en- in the acute toxicity evaluations. In sub-acute analysis, no significant dogenous antioxidant defense mechanisms, and decrease in oxidative pathological, hematological, or biochemical toxic effect was observed damage (Table 2). Although antioxidant effect is an important biolo- after 21 days of administration (Etemad et al., 2015, 2016b). gical activity, a lot of plants have demonstrated such properties in In addition to the above-mentioned studies which directly assessed clinical studies (Pandey and Rizvi, 2009; Firuzi et al., 2011; Zhang the toxicity of the plant, toxicological data can also be inferred from the et al., 2015a, 2015b); thus, it is somehow a general therapeutic activity clinical trials on the therapeutic activity of the plant. Most clinical trials which might be achievable using several medicinal plants. In other have evaluated lemon verbena administration for a period of words, this is not an effect specific for lemon verbena. Although most 21–28 days in which significant improvements in the endogenous an- clinical trials have focused on the antioxidant activity in athletes, how tioxidant defense mechanisms were observed; however, no adverse ef- much this effect can influence the pathogenesis of chronic diseases is fect is reported. Thus, we can conclude that 3–4 weeks of lemon ver- still questionable unless future clinical trials demonstrate its efficacy. bena administration does not induce significant complications. There On the other hand, traditional uses of lemon verbena suggest this are information suggesting that high doses of lemon verbena can cause plant to be a good anxiolytic and hypnotic, as well as a gastrointestinal gastric irritations. The high content of citral in the essential oil might antispasmodic agent; however, none of these effects are clinically as- also cause photosensitivity (https://articles.mercola.com/herbal-oils/ sessed. Pre-clinical studies discussed in this review support the bene- lemon-verbena-oil.aspx). In a clinical trial by Campo et al. (2015), ficial activity of this plant for such indications. The essential oil of verbascoside was administered for 2 weeks and the only adverse effect lemon verbena mostly contains neral and geranial. The anxiolytic effect was dyspepsia; though, the prevalence was similar to the group con- of other plant species with the same composition of the essential oil, sumed placebo which suggest this side effect to be unrelated to the such as lemon balm and lemon grass, is demonstrated in clinical studies active ingredient. (Alijaniha et al., 2015; Goes et al., 2015). Relaxing effect on intestinal smooth muscles, anticolitis activity, and antihyperpropulsive effects, 6. Conclusions which are proved in animal models, suggest lemon verbena as a suitable supplement to be investigated in clinical settings for gastrointestinal A. citrodora is a valuable medicinal plant with several demonstrated spasms, diarrhea-predominant inflammatory bowel disease, and irri- biological activities in the literature. table bowel syndrome. The most important medically relevant product obtained from A. Another gap in the clinical application of lemon verbena is the lack citrodora aerial parts is the essential oil, containing neral and geranial as of enough toxicological studies. Although a history of traditional use the main components, which is responsible for a series of biological supports the safety of this plant (PDR, 2007), the few number of activities of the plant such as antimicrobial, anesthetic, neuroprotec- documents regarding the traditional use of the plant makes the decision tive, and sedative effects (Table 2). Due to the impact of the environ- difficult about the administration of A. citrodora, especially in patients mental factors on the quality and quantity of the essential oil, optimum with underlying diseases or in especial populations such as elderlies, cultivation conditions should be obtained for A. citrodora. Also, geo- children, pregnant women and nursing mothers. Thus, safety of the graphical site of cultivation showed an important impact on the quality long-term use of the plant, especially in chronic conditions, needs to be and quantity of essential oil components, so that neral and geranial scientifically investigated. It should be mentioned that lemon verbena, reached to a very low level in some samples. Thus, if looking for a similar to several other medicinal plants (Bahramsoltani et al., 2017; pharmacological activity related to a specific volatile component, in- Soleymani et al., 2017), has phytochemicals which may be substrates vestigators should consider the geographical source from which the for drug metabolizing enzymes; thus, its concomitant use with con- plant is provided. ventional drugs in patients with underlying disorders might cause the In the plant extract, verbascoside seems to be one of the most im- risk of herb-drug interaction. Despite all of the above-mentioned con- portant components, which could show pharmacological activity in the siderations, lemon verbena is a generally regarded as safe (GRAS) plant purified form; thus, in studies assessing the effect of the plant extract, in US (Jimenez-Ferrer et al., 2017); thus, in patients without underlying verbascoside would be a suitable chemical for standardization. chronic conditions can be safely used. Additionally, some studies assessed the biological activity of vitexin, as In conclusion, A. citrodora is a valuable medicinal plant with several one of the spasmolytic components of the extract. So, based on the demonstrated pharmacological activities in experimental studies; required biological activity, lemon verbena products can be standar- however, future clinical trials are necessary to clinically prove the dized according to the suitable compound. It should be mentioned that safety and efficacy of this medicinal plant in human. several other phytochemicals have also been detected in A. citrodora extract which are possibly involved in the pharmacological activities of the plant and could be subjected to the investigation in the future Conflict of interest studies. One of the important gaps regarding the investigations on lemon None declare. verbena is the few number of studies discussing the traditional medic- inal uses of this plant. As Lemon verbena is native to some specific parts of the world, not all people are familiar with the medicinal properties of Authors contribution this plant; thus, investigations regarding the traditional uses of this plant by native people through ethnopharmacological surveys would be MH F & RR designed the study, wrote the main text and approved of a great value to introduce new therapeutic applications for A. ci- the final edition of the manuscript. RB performed the search, edited the trodora. Lack of such ethnomedicinal evidences might be one of the tables, wrote the main text, and approved the final edition of the reasons for the few number of clinical trials on this plant. There are four manuscript. AM screened the papers and wrote the main text. PR and ZS clinical studies on the antioxidant activity of A. citrodora, one trial on its screened the papers, prepared the tables, and approved the final edition anti-inflammatory activity, one study regarding beneficial effects on of the manuscript.

42 .Bhaslaie al. et Bahramsoltani R. Table 2 Biological activities of Aloysia citrodora.

Pharmacological activity Plant part Plant extract Method Results Evaluated dose/ concentration Active constituents References

Food preservatives Leaves EO In vivo- silver catfish infected Promoting fish survival, 2.0 ml/kg diet β-citral and α-citral (Dos Santos et al., 2017a, by Aeromonas hydrophila maintaining blood/ biochemical 2017b) parameters similar to basal values and antibacterial effect against Aeromonas hydrophila Antimicrobial Aerial parts Ethanolic extract In vitro – disc diffusion Maximum antibacterial effect on 10–40 mg/ml – (Mirzaie et al., 2016) method gram-negative bacteria Leaves Aqueous and ethanolic In vitro- disc diffusion, agar No antibacterial activity against 625–20,000 μg/ml – (Shafiee et al., 2016) extract well diffusion, and macrotube Streptococcus mutans and dilution Streptococcus sobrinus isolated from clinical samples Leaves EO In vitro- agar disc diffusion MICs ranged between 2.84 and Serial dilutions (1: 2) from EO terpenes and (Oukerrou et al., 2017) 8.37 mg/ml against Escherichia 2.5% (v/v) of EO in DMSO terpenoids coli, Staphylococcus aureus, and Pseudomonas aeruginosa Anesthetic Leaves EO In vivo – the time Induction of stage II anesthesia at 25, 150 or 300 μLL_1 β-citral and α-citral (Dos Santos et al., 2016) measurement of anesthesia 25 μLL_1 and stage III at 150 and induction and recovery of the 300 μLL_1, in 30 min; synergistic silver catfish after exposure effect with diazepam in the to EO of AC alone or with induction of anesthesia, without diazepam 150 µM and significant change in recovery evaluation of flumazenil times. Flumazenil did not change effect (5 or 10 µM) on anesthesia induced by EO and recovery from EO recovery time – μ − 1 43 Aerial parts EO In vivo anesthesia induction - In sub-adult shrimp: Minimum 50, 100, 300 or 500 LL for Z-citral and E-citral (Parodi et al., 2012) in sub-adults and post-larvae concentration of EO for inducing sub-adults and 100, 300, 400 or − shrimp by different stage II anesthesia was 500 μLL 1 for post-larvae − concentrations of EO 100 μLL 1(16 min); the shortest stage II anesthesia induction time (5 min) was observed at − concentration 500 μLL 1 and the shortest recovery time (10 min) − was found at 300 μLL 1 - In post- larvae shrimp: The shortest induction and recovery times (less than 10 min) was seen at − 300 μLL 1 − Aerial parts EO In vivo – induction of The most prolonged anes- 20–800 μLL 1 – (Parodi et al., 2013a, anesthesia in both strains thetic induction and recovery 2013b)

(gray and albino) of silver time was noticed in the Journal ofEthnopharmacology222(2018)34– catfish (Rhamdia quelen) albinos. Effective concentration range to induce anesthesia was 100–800 lL − L 1 (11.1–1.24 min) without mortality. The best response considering time to anesthesia was at 200 − lL L 1 (5.35 min) Neuroprotective Leaves EO In vitro – β-amyloid-induced Significant neuroprotective 0.01 & 0.001 mg/ml Limonene, β-citral, (Abuhamdah et al., 2015) and hydrogen peroxide- activity α-citral, 1, 8-cineole, induced neurotoxicity in curcumene, Cath.-a-differentiated (CAD) spathulenol and neuroblastoma cells caryophyllene oxide (continued on next page) 51 .Bhaslaie al. et Bahramsoltani R. Table 2 (continued)

Pharmacological activity Plant part Plant extract Method Results Evaluated dose/ concentration Active constituents References

Leaves and aerial Infusion and decoction In vitro- measurement of Strong inhibition of brain lipid- -Spontaneous brain CL: infusion Polyphenolic (Lasagni Vitar et al., 2014) parts TBARS and Spontaneous CL peroxidation, free radical 1/20 (0, 5, 20 and 40 ml) or compounds of brain homogenates trapping ability and antioxidant decoction 1/20 (0, 2.5, 10 and obtained from female Swiss activity 40 ml) - TBARS: infusion 1/10 mice and decoction 1/10 (0, 5, 10 and 25 ml) Antioxidant Leaves Extract In vivo – (a) MDA generation Strong antioxidant activity in vivo 2180 mg/kg Phenylpropanoid (Funes et al., 2009) assay (b) FRAP assay (c) SOD (verbascoside) activity assay in plasma samples of rat Leaves Extract In vivo – determination of Accelerating antioxidant enzymes 1440 mg/kg Phenylpropanoids (Quirantes-Pine et al., GPx, CAT, MPO and GRed activities and descending MPO (verbascoside) 2013) activities in blood cells activity, protection of blood-cells isolated from rats after acute against oxidative damage administration of AC extract Aerial parts Aqueous extracts (infusion In vitro - (a) Spontaneous CL Decoction showed higher 1 mg/ml EO and phenolic (Portmann et al., 2012) and decoction) of rat brain homogenates antioxidant capacity. compounds method (b) TBARS and Aqueous extracts protected protein carbonylation against lipid peroxidation techniques performed on and protein carbonylation brain homogenates Leaves Extract In vivo – high n − 6 Modifying oxidative damage of 5 mg verbascoside/kg feed Verbascoside (Di Giancamillo et al., polyunsaturated fatty acids- the liver without changing the 2015) induced oxidative stress in systemic responses to oxidative piglets stress

44 Leaves EO In vivo – EO- anaesthetized Elevation in oxidative protection 135 & 180 mg/L Citral (neral and (Gressler et al., 2014) silver catfish and alleviation in stress geranial) and limonene Antinociceptive – Verbascoside In vivo – neuropathic pain Modification of behavioral 50, 100, 200 mg/kg Phenylpropanoid (Amin et al., 2016) induced by chronic changes associated with (verbascoside) constriction injury of sciatic neuropathy, decrease Bax on day nerve in rats 3 and Iba protein (a marker of microglia activation) on days 3 and 7 and increase Bcl-2 Anti hyperpropulsive Aerial parts Methanol extracts In vivo – charcoal –gum Moderated inhibitory effect 300 mg/kg – (Calzada et al., 2010) acacia-induced (32%) hyperperistalsis in rats and charcoal meal test Homoeostatic stability Leaves Extract In vivo – Lacaune suckling Remarkable decrease in TBARS 2.5 & 5.0 mg Phenylpropanoid (Casamassima et al., 2013) effect lambs fed orally a and ROS, significant increase in glycoside

verbascoside-based dietary serum levels of vitamin A and (verbascoside) Journal ofEthnopharmacology222(2018)34– supplement vitamin E Leaves Extract In vivo – white rabbit fed with Enlargement in the amount of 1 g (5 mg verbascoside/kg feed) & Phenylpropanoid (Casamassima et al., 2017) two doses of AC supplement plasma vitamin A and E, 2 g (10 mg verbascoside/kg feed) glycoside for three consecutive diminution in plasma TBARS and (verbascoside) reproductive cycles ROS Growth improvement Leaves Extract In vivo – Lacaune suckling Ameliorate average daily gain 2.5 & 5.0 mg Phenylpropanoid (Casamassima et al., 2013) lambs fed orally a and milk consumption glycoside verbascoside-based dietary (verbascoside) supplement Leaves Extract In vivo – white rabbit fed with Progressive daily weight gaining 1 g (5 mg verbascoside/kg feed) & Phenylpropanoid (Casamassima et al., 2017) two doses of AC supplement from birth to weaning, 2 g (10 mg verbascoside/kg feed) glycoside for three consecutive enhancement kit body weight at (verbascoside) reproductive cycles weaning (continued on next page) 51 .Bhaslaie al. et Bahramsoltani R. Table 2 (continued)

Pharmacological activity Plant part Plant extract Method Results Evaluated dose/ concentration Active constituents References

Leaves Extract In vivo – piglets supplemented Positive effect on growth 5 & 10 mg verbascoside/kg of diet Verbascoside (Pastorelli et al., 2012) with a dietary AC performance Aerial parts Powder In vivo – chronic heat- improvement in performance 0.5% & 1% AC powder Verbascoside (Rafiee et al., 2016) stressed broilers fed with parameters: elevating average dietary AC weight gain and feed intake Improvement blood Leaves Extract In vivo – Lacaune suckling Reducing TAG, enhancement in 2.5 & 5.0 mg Phenylpropanoid (Casamassima et al., 2013) parameters lambs fed orally a HDL-C levels (p < 0.05) glycoside verbascoside-based dietary (verbascoside) supplement Leaves Extract In vivo – white rabbit fed with Decreasing in bilirubin, activities 1 g (5 mg verbascoside/kg feed) Phenylpropanoid (Casamassima et al., 2017) two doses of AC supplement of ALP, AST, ALT, TC, LDL-C and and 2 g (10 mg verbascoside/kg glycoside for three consecutive TAG, increase HDL-C feed) (verbascoside) reproductive cycles Leaves Extract In vivo – piglets supplemented Improvement antioxidant status 5 & 10 mg verbascoside/kg of diet Verbascoside (Pastorelli et al., 2012) with AC and serum IgA concentration Aerial parts Powder In vivo – chronic heat- Improvement blood metabolites: 0.5% & 1% AC powder Verbascoside (Rafiee et al., 2016) stressed broilers fed with increasing Gpx level and dietary AC reduction in heterophyl/ lymphocyte ratio and LDL-C Sedative Fresh leaves ES In vivo – exposure of silver Delay in degradation of IMP into 27 & 36 mg/L Monoterpenoids and (Daniel et al., 2014) catfish to 40 μL/L of the EO HxR, onset and resolution of rigor sesquiterpenoids (α- during transport mortis; lower sensory demerit citral, β-citral and scores; longer sensory shelf life limonene) Leaves Aqueous extract In vivo – evaluation Important sedative effect 0.15, 1 & 10 mg/kg – (Ragone et al., 2010) exploratory activity and mechanistically similar to

45 spontaneous locomotion of benzodiazepines mice in the open field test Anticonvulsant Leaves Ethanolic extract In vivo – PTZ and Reduction in duration and 200, 400, 800 mg/kg Flavonoids and (Rashidian et al., 2016) MES‑induced seizures in mice increase in latency of the seizures alkaloids in the PTZ model- decrease in hind limb tonic extension duration none dose dependently in the MES test Antihyperalgesic Leaves Purified verbascoside (12%) In vivo- paw-pressure test in Reversion hyperalgesia in both 100, 300, 600 mg/kg Verbascoside (Isacchi et al., 2011) from hydroalcoholic extract two animal models of CCI and MIA treated rats after neuropathic pain: CCI or oral and intraperitoneal intra-articular injection of administration sodium MIA Anxiolytic Leaves Aqueous extract In vivo – Intraperitoneal Acute administration of AC with 10, 100, 500, 1000 mg/kg Flavonoids (Veisi et al., 2015) injection of plant extract in doses ≥10 mg/kg increased

male Wistar rats and anxiety in rats in EPM, possibly Journal ofEthnopharmacology222(2018)34– evaluation of anxiety level in low doses of extract (< 10 mg/ EPM kg) induce anxiolytic reactions in rats Leaves and stems Fatty acids and terpenes In vivo- Oral administration of Improvement of time spent in 125, 250, 500, 750 mg/kg Fatty acids and (Jimenez-Ferrer et al., fraction different fractions to ICR open arms and number of open terpenes 2017) mice and evaluation of arm entries with a dose of anxiety level in EPM 500 mg/kg Anticolitis Leaves Infusion In vivo – DSS-induced colitis Partially protection against DSS- 31.6 ml Phenolic acids and (Lenoir et al., 2012) in rats induced inflammation flavone derivatives (continued on next page) 51 .Bhaslaie al. et Bahramsoltani R. Table 2 (continued)

Pharmacological activity Plant part Plant extract Method Results Evaluated dose/ concentration Active constituents References

Anti-inflammatory Dried leaves Hexane extract In vivo – carrageenan- Administration of extract and Extract concentration: Citral (trans citral (Ponce-Monter et al., 2010) induced rat hind paw edema citral exhibited anti-inflammatory 100–800 mg/kg Citral and cis citral) model activity concentration: 100–800 mg/kg Spasmolytic effect Dried leaves Hexane extract In vitro and in vivo– KCl Treatment with the extract and Extract concentration: 3–56 µg/ Citral (trans citral (Ponce-Monter et al., 2010) 60 mM, oxytocine 10 mIU/ citral inhibited contraction of ml Citral concentration: and cis citral) ml, charbacol 10 µM and uterus muscle in a concentration 3–230 µg/ml PGF2α 5 µM induced dependent manner contractions in isolated uterus strips from estrogen primed rats Dried leaves Aqueous extract In vitro –DRC to acetylcholine Non-competitively inhibition DRC 0.1, 0.2, 0.6, 1, 2 or 6 mg/ml Flavonoid vitexin (Ragone et al., 2007) and to CaCl2 in rat of Ach (IC50:1.34 ± 0.49 mg duodenums lyophilized/ml) and DRC of CaCl2 (IC50: 2.64 ± 0.23 mg/ml), potentiating non-competitive inhibition of 30 µmol/LW-7 and 5–15 µmol/L papaverine on the Ca2+ -DRC Muscle relaxant Dried leaves Aqueous extract In vitro – muscles contraction AC relaxed muscles contraction 0.6, 1, 2 & 3 mg/ml Flavonoid vitexin (Ragone et al., 2007) of rats duodenums induced (IC50 of 2.6 ± 0.2 mg/ml), by (a) ClK 10% and Maximal relaxation of AC was dose–relaxation curve to AEC 81.0 ± 3.2% of the maximal and addition papaverine and effect of papaverine and quercetin (b) ClK and 78.1 ± 5.0% of the quercetin. dose–relaxation curve of Methylene blue (10–30 µmol/L)

46 quercetin or dose–relaxation non-competitively inhibited and curve of AEC in the absence TEA (40 mmol/L) competitively and in the presence of antagonized AC relaxation methylene blue (c) acetylcholine and dose–relaxation curve of AEC in the absence and in the presence of TEA Antigenotoxic Leaves Infusion In vivo- cisplatin-induced Inhibition capacity of cisplatin to Infusion (5%) Polyphenolic (Zamorano-Ponce et al., genetic damage in mouse induce genetic damage and compounds and EO 2004) bone marrow cells using increasing cell viability comet assay technique – EO in vitro- UV-induced DNA Significant inhibition of UV Different EO dilutions between Neral/ Geranial (Quintero Ruiz et al., 2017) damage in PQ37 Escherichia genotoxicity (minimal 0.05 & 1.66% coli strain using SOS concentration producing

Chromotest significant genotoxicity inhibition Journal ofEthnopharmacology222(2018)34– 0.8%) − Larvicidal Leaves EO Bioassay – Mosquito Larvicidal effect against C. 5–100 μLL 1 Geranial, neral, (Benelli et al., 2017) larvicidal assessment on quinquefasciatus larvae with a limonene, 1,8- −1 Culex quinquefasciatus larvae LC50 value (65.6 μLL ) cineole, ar- 24 h after treatment with the curcumene, EO spathulenol Anti-obesity-related Leaves Extract In vitro – insulin-resistant Extract diminished production of In vitro:50–400 μg/ml In vivo: Polyphenolic (Herranz-Lopez et al., metabolic disturbances hypertrophic 3T3-L1- ROS and TAG accumulation and 750 mg/kg compounds 2015) adipocyte model In vivo- diet- enhanced MMP, remarkable induced obesity in LDL improvement in fat metabolism receptor-deficient male mice (continued on next page) 51 .Bhaslaie al. et Bahramsoltani R. Table 2 (continued)

Pharmacological activity Plant part Plant extract Method Results Evaluated dose/ concentration Active constituents References

Ocular- protective Leaves Extract In vivo- TBARS and trolox Protection against dose Verbascoside dose: 2, 3, 4 mg/day Phenylpropanoid (Mosca et al., 2014) equivalent antioxidant dependently ocular tissue and glycosides capacity assays conducted on fluids from naturally oxidative (verbascoside and eye fluids and tissues of male stress (maximum up to 3 mg/day) isoverbascosid) hares fed with AC Insecticidal Leaves Monoterpenoids pulegone Immersion bioassay- toxicity Development of lice control: Concentration 5% Pulegone and citral (Gonzalez-Audino et al., and citral isolated from EO assessment on Pediculus mortality percentages between 2011) humanus capitis 10 min and 47% and 53% and knockdown 18 h after treatment with the percentages between 42% and pulegone and citral 55% Cytoprotective – Verbascoside In vitro – adding verbascoside Elevation of embryo 1 nM &10 nM Verbascoside (Martino et al., 2016) supplementation during in development: reduction oocyte vitro maturation of juvenile ROS and lipid peroxidation as sheep oocytes and evaluation increasing blastocyst quality and developmental rates, formation and mitochondria/ROS bioenergetic/oxidative status colocalization of oocytes matured by confocal analysis of mitochondria and reactive oxygen species, quantitative PCR of bioenergy/redox- related genes and lipid peroxidation assay Anti-cancer Aerial parts Ethanolic extract In vitro – MTT assay, flow- Significant inhibition ofHT29) ell 7.8, 15.6, 31.25, 62.5, 125, 250, – (Mirzaie et al., 2016) cytometry and real-time proliferation in a dose-dependent 500 & 1000 mg/ml

47 polymerase chain reaction manner (maximum inhibition in methods on human colon 1000 mg/ml),,increase in BAX cancer HT29 cells and decrease in Bcl− 2 expression level, apoptosis induction in colon cancer cell line (38.66%) Leaves EO In vitro- MTT assay in murine Dose-dependent cytotoxic activity 0–160 μg/ml EO terpenes and (Oukerrou et al., 2017) mastocytoma cells (P815), with IC50 ranging from 6.60 to terpenoids human breast 79.63 μg/ml adenocarcinoma cells (MCF7), monkey kidney carcinoma cells (VERO), and PBMCs Cardiovascular effect Leaves Aqueous extract In vivo – measurement Induction a transitory In vivo: 1, 3, 10 & 30 mg/kg In – (Ragone et al., 2010) blood pressure in hypotension in rats and a dose- vitro: 0.2, 0.6, 2 & 6 mg/ml

normotensive rats dependent negative cardiac Journal ofEthnopharmacology222(2018)34– In vitro – measuring the inotropism in vitro left ventricular pressure LVP in isolated rat hearts Toxicological evaluations Leaves Ethanolic extract In vivo- acute toxicity study No death/ behavioral 2000 mg/kg – Rashidian et al. (2016)) was performed in NMRI mice abnormality was observed, no with a single dose of the LD50 was determined. extract and the signs of toxicity/ abnormal behavioral reactions were observed for 48 h. (continued on next page) 51 .Bhaslaie al. et Bahramsoltani R. Table 2 (continued)

Pharmacological activity Plant part Plant extract Method Results Evaluated dose/ concentration Active constituents References

– Verbascoside In vivo – Single LD50 value of − 0, 1, 2 and 5 g/kg (acute Verbascoside (Etemad et al., 2015) intraperitoneal injection Verbascoside > 5 g/kg; no model) (acute model) and 21 statistically significant differences − 0, 10, 30 and 60 mg/kg days administration in the values of hematological, (subacute model) (subacute model) of biochemical and pathological verbascoside at the parameters in comparison with different dose range in control group; viability in all mice groups > IC50 value In vitro – MTT assay in HepG2 and NIH cells (cellular toxicity) Leaves – In vivo – Intraperitoneally No statistically significant 1 g/kg/day Verbascoside (Etemad et al., 2016a) administration of difference in mean number of verbascoside or vehicle implantation sites, live and control to pregnant mice and resorbed fetuses between two measurement of maternal groups, no difference in mean of body weights, external maternal weight gain malformations and skeletal abnormalities Leaves Aqueous extract In vivo – Single LD50 value = 5 g/kg of body − 0, 1, 2, and 5 g/kg of body Verbascoside (Etemad et al., 2016b) intraperitoneal injection weight; no significant change in weight (acute model) (acute model) in mice any of the hematological, − 0, 50, 100, and 200 mg/kg and 21 days biochemical, or pathological of body weight (subacute administration in rats parameters in comparison with model) (subacute model) of control group, except for a − 0, 50, 100, and 200 µg/ml

48 aqueous extract at the reduction in triglyceride levels; (cellular toxicity) different dose range viability in all groups > IC50 In vitro – MTT assay in value HepG2 cells (cellular toxicity)

AC: Aloysia citrodora; Min: minimum; EO: Essential oil; AEC: aqueous extract of cedr´on; Ach: acetylcholine; TBARS: thiobarbituric acid-reactive substances; CL: chemiluminescence; MDA: malondialdehyde; FRAP: ferric reducing ability of plasma; SOD: superoxide dismutase; CAT: catalase; Gpx: glutathione peroxidase; MPO: myeloperoxidase; Gred: glutathione reductase; TAG: triacylglycerol; HDL-C: high-density lipoprotein cholesterol; ROS: reactive oxygen species; TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; AST: aspartate transaminase; ALT: alanine transaminase; ALP: alkaline transferase; Ig: immunoglobulin; PTZ: pentyle- netetrazole; MES: maximal electroshock; CCI: chronic constriction injury of the sciatic nerve; MIA: monoiodoacetate; EPM: elevated plus maze; DSS: dextran sodium sulfate; MS: multiple sclerosis, IL: interleukin; IFN: interferon; DRC: dose-response curve; TEA: tetra ethyl ammonium; MMP: mitochondrial membrane potential; PBMCs: normal human peripheral blood mononuclear cells, IC: inhibitory concentration. Journal ofEthnopharmacology222(2018)34– 51 R. Bahramsoltani et al. Journal of Ethnopharmacology 222 (2018) 34–51

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