Phytochemical analysis on the aerial parts of Teucrium capitatum L. with aspects of chemosystematics and ethnobotany

Abstract The phytochemical analysis on the aerial parts of Teucrium capitatum L. collected from a new population in Central Italy, led to the identification of eight compounds, i.e. pheophytin a (1), poliumoside (2), apigenin (3), luteolin (4), cirsimaritin (5), cirsiliol (6), 8-O-acetyl-harpagide (7) and teucardoside (8) belonging to four different classes of secondary metabolites. Pheophytin a (1) represents a newly identified compound in the genus whereas compounds (7–8) are newly identified compound in the species. The chemotaxonomic and ethnobotanical aspects relative to the presence of these compounds were widely discussed suggesting important conclusions for both. Graphical Abstract


Introduction
Teucrium capitatum L. is a perennial sub-shrub belonging to the Lamiaceae family. The etymology of its name derives from the Greek word se tjqiom (te ucrion) and the Latin word c aput (head) and they refer to Teῦjqo1 (Teucer), the first king of Troy to use species of this genus in the medicinal field, and to the typical disposition of its inflorescence , a glomerule, respectively. Linnaeus was the first one to scientifically describe this species (Pignatti 1982).
From the morphological point of view, this species is characterized by an erected stem which is woody only at the base and is spread with slight and small glandular hairs. Leaves are sessile with an entire margin and hairy on both surfaces. The inflorescence is composed to form small and branched terminal and axillary glomerules. The corolla is extremely hairy and is formed by five small white or pink petals. The fruit is a schizocarp constituted by four oval reticulated light brown mericarps. The pollination is through insects (Pignatti 1982).
The species is widely distributed in the entire Mediterranean basin and along the Black Sea (Navarro 2020). In Italy, it can be found everywhere except in Valle d'Aosta, Trentino Alto Adige and Lombardia in arid and rocky areas and in garriga (sub-shrub open Mediterranean vegetation type), up to 1800 m. a.s.l. (Conti et al. 2005). The species is morphologically similar and taxonomically close to Teucrium polium L.. Actually, it was considered in the past as a subspecies of this (Teucrium polium L. subsp. capitatum) but it recently raised to the rank of nominal species after genetic and morphological considerations. In fact, T. capitatum presents some morphological differences from T. polium like its reduced dimensions and its reduced inflorescences (www.worldfloraonline.org 2022).
In general, some works are present in literature on the phytochemistry of the species with the old and new name. Despite, most of them focused on the essential oil composition (Cozzani et al. 2005;Kerbouche et al. 2015;Khani and Heydarian 2014;Chabane et al. 2021;Maccioni et al. 2021). Indeed, only a few phytochemical studies treated about some non-volatile compounds like phenyl-ethanoid glycosides (Pedersen 2000), flavonoids (Harborne et al. 1986;Stefkov et al. 2011) and diterpenoids (Marquez et al. 1981;Camps et al. 1987;Fernandez et al. 1985).
The species has been used in the folklore medicine of several countries. In particular, in Jordan, the decoction or the infusion of its leaves is administered to treat gastro-intestinal diseases and diabetes (Oraib et al. 2013) and to treat diarrhea, wounds, scab and colic in sheep, goats and cows (Mohammed et al. 2016). In addition, in Palestine, the decoction of the leaves is used against psoriasis (Shawahna and Jaradat 2017). Notwithstanding, recent studies demonstrated the acute toxicity of this species (Dourakis et al. 2002) as well as many other Teucrium (Stickel et al. 2000;Savvidou et al. 2007;Grafakou et al. 2020) and Ajuga species (El Hilaly et al. 2004;Luan et al. 2019). These toxic effects are only due to the presence of specific secondary metabolites, i.e. neo-clerodane diterpenes having a furan ring, which are quite common in Teucrium and Ajuga species (Frezza et al. 2019a). In the liver, this furan ring is oxidized by the cytochrome P450 3A4, an enzyme with the task to oxidize small molecules to favor their removal from the body, producing reactive epoxide molecules with a strong alkylating nature which makes them very toxic for the liver even at micromolar concentration (Zhou et al. 2004(Zhou et al. , 2007. For this reason, the use of Ajuga and Teucrium species in the folklore medicine is being discouraged. Yet, further considerations and evaluations are needed.
The aims of this work were multiple: to perform a phytochemical analysis on the species; to study a new population; to draw deeper chemotaxonomic conclusions on the species at the genus and family levels; to verify the possibility to also use this exemplar in the ethnobotanical field.
To the best of our knowledge, during this study, pheophytin a (1) was identified in the genus for the first time whereas 8-O-acetyl-harpagide (7) and teucardoside (8) were identified in the species for the first time. In fact, given its nature, pheophytin a (1) , is potentially ubiquitous in the plant kingdom. It has been found in various plant species belonging to different families, e.g. in some Lamiaceae species such as Ocimum labiatum (N.E.Br) A. J. Paton (Kapewangolo et al. 2017) and Origanum onites L. (Azcan et al. 2000) but also in other families like Sapindaceae (Semaan et al. 2018), Theaceae (Kusmita et al. 2015), Moraceae (Bafor and Kupittayanant 2020) and Plantaginaceae (Frezza et al. 2019b). For this reason, this compound cannot be considered of any chemotaxonomic value. Poliumoside (2) has been already evidenced in the species (Andary et al. 1985(Andary et al. , 1988 but also in other species of the Teucrium genus like T. polium L. (Chabane et al. 2021;Venditti et al. 2017a), T. yemense Deflers (Essam 1995) and T. chamaedrys L. (Mitreski et al. 2014) as well as in the genus Callicarpa L. of the Lamiaceae family (Liu et al. 2013) and in other families like Oleaceae (Andary et al. 1992), Paulowniaceae (He et al. 2000), Plantaginaceae (Zhou et al. 1998), Orobanchaceae (Tuncay Agar and Cankaya 2020) and Scrophulariaceae (Grice et al. 2003). The co-occurrence of phenyl-ethanoid glycosides such as poliumoside (2) and iridoids such as 8-O-acetyl-harpagide (7) and teucardoside (8) has a taxonomical relevance in Asteridae (Jensen 1992), the subclass that comprises the Lamiales order where the family Lamiaceae is included. All the flavonoids identified in this accession (apigenin (3), luteolin (4), cirsimaritin (5) and cirsiliol (6)) have been already found in the species (Harborne et al. 1986;Stefkov et al. 2011) but also in many other species of the genus (Venditti et al. 2017a(Venditti et al. , 2017bMitreski et al. 2014;Harborne et al. 1986;Jari c et al. 2020), in the Lamiaceae family (Frezza et al. 2019a) and in many other families like Compositae, Plantaginaceae, Scrophulariaceae, Leguminosae, Apiaceae, Rosaceae and Cactaceae (Miean and Mohamed 2001; L opez-L azaro 2009; Tom as- Barberan et al. 1988;Porter and Harborne 1994;Di Bella et al. 2022). According to these data, none of these flavonoids can be used as chemotaxonomic marker. 8-O-acetyl-harpagide (7) has been already identified in the genus only in Teucrium chamaedrys (Frezza et al. 2018) and T. orientale L. (Oganesyan et al. 1986) even if it is quite known in the Lamiaceae family especially in Ajuga L. (Venditti et al. 2016a), Melittis L. (Venditti et al. 2016b), Sideritis L. (Venditti et al. 2016c), Lamium L. (Alipieva et al. 2003) and Stachys L. species (Venditti et al. 2017c). As a matter of fact, this compound is considered as one of the main chemotaxonomic markers of the Lamiaceae family (Frezza et al. 2019a). Nevertheless, it has been also found in other families like Scrophulariaceae (Venditti et al. 2016d) and Plantaginaceae (K€ upeli et al. 2005) even if less frequently. Lastly, teucardoside (8) has been evidenced, in general, only in Teucrium marum L., T. subspinosus Pourr. ex Willd. (Bianco et al. 2004), T. polium (Jaradat 2015) and T. yemense (Abdel-Sattar 1998). As a matter of fact, this compound is considered as one of the main chemotaxonomic markers of the Teucrium genus (Frezza et al. 2019a). The presence of both compounds (7) and (8) is perfectly in accordance with the biosynthetic pathway of iridoids which is peculiar of Lamiales, involving the biogenetic Route II with 8-epi-loganic acid and 8-epi-loganin as precursors, that leads to the production of iridoid derivatives showing the 8a-stereochemistry such as 8-O-acetyl-harpagide, which are further transformed into aucubin derivatives with the insertion of a double bond at the C7-C8 positions (Jensen 1991(Jensen , 1992. Given the identified compounds, from the phytochemical point of view, the studied accession is surely a member of the Teucrium genus within the Lamiaceae family since some of their chemotaxonomic markers were identified. Anyway, even if there are macroscopic differences between T. polium and T. capitatum that have justified their distinction from a morphological point of view, no chemotaxonomic markers have been evidenced till now that may justify this from this point of view . In this sense, further phytochemical analyses are absolutely necessary studying other known and new populations collected in different areas of the world since it is well known that the secondary metabolite pattern is deeply affected by several factors like environment, genetics and growth area (Toniolo et al. 2014).
It is noteworthy to underline that no neo-clerodane diterpene has been evidenced during this study. The absence of neo-clerodane diterpenes is also very important under the chemotaxonomic standpoint since, as already explained, neo-clerodane diterpenes having a furan ring are considered as other fundamental chemotaxonomic markers of Teucrium and Ajuga species (Frezza et al. 2019a). Actually, this same situation has been already observed in T. chamaedrys (Frezza et al. 2018) as well as in Ajuga chamaepitys (L.) Schreb. (Venditti et al. 2016a), Ajuga genevensis L. (Venditti et al. 2016e), Ajuga tenorei C. Presl. (Frezza et al. 2017) and Ajuga reptans L. (Frezza et al. 2019c), all collected in Central and Northern Italy. This accession was also collected in Central Italy, and this almost suggests the possibility that the absence of neo-clerodane diterpenes may even be a peculiarity of Ajuga and Teucrium species growing in these places despite further phytochemical studies on different populations and species are needed to confirm this hypothesis. In addition, the absence of neo-clerodane diterpenes having a furan ring is very important under the ethnobotanical standpoint since these compounds are extremely toxic as already explained (Zhou et al. 2004(Zhou et al. , 2007. This absence and the presence of the identified compounds in the studied accession suggest the possibility to use it in the ethnobotanical filed because of the really beneficial biological properties associated to them. In fact, pheophytin a (1) exerts strong antioxidant, antimutagenic, chemopreventive and anti-inflammatory activities (Hsu et al. 2013;Queiroz Zepka et al. 2019). Poliumoside (2) possesses strong antioxidant and antiproliferative properties (He et al. 2000(He et al. , 2001. Apigenin (3) is a potent anti-inflammatory, antioxidant, antitumoral, antiproliferative, antidiabetic, antidepressive, neuroprotective, anxiolytic and sedative compound (Salehi et al. 2019). Luteolin (4) exhibits strong anti-inflammatory, antioxidant, antimicrobial, antitumoral and chemopreventive effects (L opez-L azaro 2009). Cirsimaritin (5) has good antispasmodic, antimicrobial, antioxidant, anti-inflammatory, antitumoral and antidiabetic properties (Pathak et al. 2021). Cirsiliol (6) exerts strong antitumoral, sedative, hypnotic and muscle-relaxing effects (Al-Shalabi et al. 2020;Viola et al. 1997;Mustafa et al. 1995). 8-O-acetyl-harpagide (7) possesses potent antifungal, antibacterial, antipyretic and antitumoral effects (Shafi et al. 2004;Singh et al. 2006). Lastly, teucardoside (8) shows strong antioxidant and antitumoral activities (Elmasri et al. 2016). Yet, further pharmacological and toxicological studies are necessary before the use of this accession in the ethnobotanical filed can really take place.

Experimental part
See the supplementary material.

Conclusions
The phytochemical analysis on the aerial parts of T. capitatum collected from a new population in Italy evidenced the presence of eight compounds belonging to four different classes of non-volatile secondary metabolites. One of these was identified in the genus for the first time while two of these were identified in the species for the first time, during this work. The presence of all these compounds confirms the belonging of the studied accession to the Teucrium genus within the Lamiaceae family since some of their chemotaxonomic markers were identified. Yet, this was not enough to confirm the correct promotion of T. capitatum to the rank of a nominal species from the chemotaxonomical point of view.
No neo-clerodane diterpene was identified in the studied accession and this is important under the chemotaxonomic and ethnobotanical standpoints since this may reinforce a phytochemical peculiarity of Teucrium and Ajuga species collected in certain areas of Italy as well as suggest the possibility to use this accession in the folklore medicine even if further studies in different fields are necessary in both cases.