Rosmarinic Acid in Underground Parts of Different Mentha Species

Medicinal plants are a significant part of medicine and pharmacy worldwide. They are invaluable sources of various medications. Almost all therapy depends on the plant kingdom in some parts of the world. Plants of the genus Mentha are considered one of the oldest and best-known medicinal plants. They have been known and used since ancient times for their healing and soothing effects on digestive and pulmonary diseases (Singh et al., 2015).

The genus Mentha belongs to the family Lamiaceae, subfamily Nepetoidae, and according to Tucker et al., it includes about 18 species and 11 hybrids, which are divided into four sections: Pulegium, Tubulosae, Eriodontes, and Mentha (Tucker et al., 2007). All plants of the genus contain some groups of secondary metabolites, such as essential oil consisting of monoterpenes and sesquiterpenes (menthol, menthone, carvone, β-caryophyllene, linalool, etc.), phenolic compounds including flavonoids (eriodictyol, apigenin, etc.), and phenolic acids such as rosmarinic acid (Mahendran and Rahman, 2020). The object of interest, rosmarinic acid, is found not only in the genus Mentha, but also in many others in the families Lamiaceae and Boraginaceae. In recent years, rosmarinic acid has received much attention, mainly due to its pharmacological activities. It shows a promise in the therapy and prevention of some diseases. Thanks to its structure with four hydroxyl groups, it acts as a powerful antioxidant and anti-inflammatory agent. A study on the antioxidant effects of rosmarinic acid has revealed other effects – cardioprotective, neuroprotective, antimicrobial, hepatoprotective, and anti-inflammatory (Hitl et al., 2020). Based on numerous experiments and studies, there is a strong demand for the natural sources of rosmarinic acid. The aerial parts of mint are known for their high content of rosmarinic acid, but mint also produces large, thin rhizomes that are not known to be used in pharmacy. The study aimed to determine the content of rosmarinic acid in the underground parts of 10 mint species using high-performance liquid chromatography (HPLC). The results may help to consider mint as a source of rosmarinic acid.

The underground parts of 10 species of mint were obtained from the Medicinal Plant Garden of the Faculty of Pharmacy, Comenius University in Bratislava. The underground parts were harvested in May before flowering and dried in the laboratory at room temperature (25°C) for 7 days. The dried samples were then used for the preparation of the methanolic extract. All chemicals used for the extraction were purchased from Merck (Germany). About 0.5000 g of each sample was weighed for the extraction. The amount of methanol corresponding to a ratio of 1:20 was added to each specimen. The exact specimen weights and the amounts of methanol used are listed in Table 1. Extraction was performed by sonication for 2 × 20 min. After filtration, 1 mL of the extract was used for HPLC with diode array detection (HPLC-DAD) analysis, and 1 mL of the extract was placed in the freezer for possible repeat measurement. Part of the extract (5 mL) was evaporated and weighed.

The species of the genus Mentha are distributed almost all over the world, except Antarctica. Different species are typical for each continent or country, and each species differs a little in the content of secondary metabolites. Much has been written about numerous groups of secondary metabolites in the aerial parts of many Mentha species, but little is known about their underground parts (Cirlini et al., 2016; Elansary et al., 2020; Fialová et al., 2015; Park et al., 2019; Salehi et al., 2018). Mints produce a large number of long rhizomes in a relatively short time. Mint is mainly valued and used for its essential oil content, but many efforts have also been made to study the phenolic compounds in it. Rosmarinic acid, which is a phenolic acid, is nowadays extensively studied, mainly for its antioxidant properties (Adomaco-Bonsu et al., 2017) and its associated antimicrobial, cardioprotective, hepatoprotective, antidiabetic, and anti-inflammatory effects (Hitl et al., 2020). This molecule of interest is the main subject of the present research. The underground parts of 10 different species of the genus Mentha, all belonging to the section Mentha and growing in Slovakia, were studied. HPLC-DAD analysis of methanolic extracts was performed, and quantitative determination of rosmarinic acid was performed by an external standard method. Methanol was used as an extracting agent due to its polarity to obtain a better yield. To our knowledge, there are only less studies on the content of rosmarinic acid in the underground parts of different Mentha species.

The results presented in Table 1 show the highest content of rosmarinic acid in Mentha × piperita L. “BULHARSKA 1” (6.9 mg g⁻¹ of the dry sample), followed by Mentha rotundifolia (L.) Huds. (5.16 mg g⁻¹ of the dry sample). The chromatograms of these two samples are shown in Figure 1. The lowest yield was obtained in Mentha arvensis L. (2.81 mg g⁻¹ of the dry sample). A slightly higher content was found in the most widely used M. × piperita L. (3.15 mg g⁻¹ of the dry sample). In recent years, some published papers deal with the evaluation of rosmarinic acid content in methanolic extracts of the aerial parts of mints. Alharbi et al. studied the secondary metabolites in methanolic extracts of M. rotundifolia leaves. According to them, the leaves contained 12 mg g⁻¹ rosmarinic acid in the dry sample, which is three to four times higher than in the underground parts (Alharbi et al., 2021). Pavlović et al. investigated rosmarinic acid in the leaves of M. × piperita L. and recorded the content of rosmarinic acid to be 63.30 mg g⁻¹ of the dry extract (Pavlović et al., 2021). In our previous studies, Bittner Fialová et al. investigated the secondary metabolites in roots and rhizomes of some Mentha species, and one of the investigated compounds was rosmarinic acid. HPLC-DAD analyses of the infusion of peppermint rhizomes were performed and the amount of rosmarinic acid was three times lower than in leaves (Bittner Fialová et al., 2019). In another study performed by Bittner Fialová et al., the rhizomes of three mint species were analyzed using HPLC-DAD: M. × piperita cv. “Perpeta,” Mentha longifolia, and Mentha × villosa cv. “Snežna.” The results showed that the infusion of M. × piperita cv. “Perpeta” contained a similar amount of rosmarinic acid as in the previous study, while the other two varieties had double the content (Bittner Fialová et al., 2020). Despite mint rhizomes content less amount of rosmarinic acid than leaves, they still might be interesting for rosmarinic acid extraction. Mint rhizomes might be an additional source of phenolic acids, chiefly rosmarinic acid.

Figure 1.

Using HPLC-DAD, we analyzed the methanolic extracts from underground parts of 10 different species of the genus Mentha found in Slovakia. We focused on phenolic acids, especially rosmarinic acid, known for different biological activities. We focused on the underground parts as too little is known about their constituent compounds. Rosmarinic acid was detected as a major compound in every sample. The highest content was found in M. × piperita L. “BULHARSKA 1” and M. rotundifolia (L.) Huds. These results suggest the possible use of Mentha rhizomes as a source of rosmarinic acid.