<abstract xmlns="http://www.w3.org/1999/xhtml">

<sec><h3>Background</h3>
<p>Mints (<italic>Mentha</italic> L., Lamiaceae) are medicinal plants frequently used in pharmacy, cosmetics, and food industry. Among the secondary metabolites found in mint, rosmarinic acid is one of the most abundant in the whole plant. Rosmarinic acid is known as a powerful antioxidant and anti-inflammatory agent.</p>
</sec>
<sec><h3>Aim</h3>
<p>There is much information about the use and profile of secondary metabolites of mint's aerial parts. On the contrary, less is known about the secondary metabolites in the rhizomes. So, this research intended to determine the content of rosmarinic acid in the underground parts of 10 species of the genus <italic>Mentha</italic>, section <italic>Mentha</italic>.</p>
</sec>
<sec><h3>Method</h3>
<p>High-performance liquid chromatography with diode array detection (HPLC-DAD) was used to identify and determine rosmarinic acid.</p>
</sec>
<sec><h3>Results</h3>
<p>Rosmarinic acid was detected in all 10 species. The highest amount was found in <italic>Mentha × piperita</italic> L. “BULHARSKA 1” and <italic>Mentha rotundifolia</italic> (L.) Huds. The lowest content of rosmarinic acid was found in <italic>Mentha arvensis</italic> L., and a slightly higher content was measured in <italic>Mentha × piperita</italic> L.</p>
</sec>
<sec><h3>Conclusion</h3>
<p>The underground parts of mints seem to be an interesting source of natural antioxidants such as rosmarinic acid.</p>
</sec>
</abstract>

Background
Mints (Mentha L., Lamiaceae) are medicinal plants frequently used in pharmacy, cosmetics, and food industry. Among the secondary metabolites found in mint, rosmarinic acid is one of the most abundant in the whole plant. Rosmarinic acid is known as a powerful antioxidant and anti-inflammatory agent.

Aim
There is much information about the use and profile of secondary metabolites of mint's aerial parts. On the contrary, less is known about the secondary metabolites in the rhizomes. So, this research intended to determine the content of rosmarinic acid in the underground parts of 10 species of the genus Mentha, section Mentha.

Method
High-performance liquid chromatography with diode array detection (HPLC-DAD) was used to identify and determine rosmarinic acid.

Results
Rosmarinic acid was detected in all 10 species. The highest amount was found in Mentha × piperita L. “BULHARSKA 1” and Mentha rotundifolia (L.) Huds. The lowest content of rosmarinic acid was found in Mentha arvensis L., and a slightly higher content was measured in Mentha × piperita L.

Conclusion
The underground parts of mints seem to be an interesting source of natural antioxidants such as rosmarinic acid.


<div xmlns="http://www.w3.org/1999/xhtml">
<sec id="j_afpuc-2023-0006_s_001"><div>INTRODUCTION</div>
<p>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 <italic>Mentha</italic> 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 (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_001">Singh et al., 2015</a>).</p>
<p>The genus <italic>Mentha</italic> 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: <italic>Pulegium, Tubulosae, Eriodontes,</italic> and <italic>Mentha</italic> (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 (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_002">Mahendran and Rahman, 2020</a>). The object of interest, rosmarinic acid, is found not only in the genus <italic>Mentha,</italic> 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 (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_004">Hitl et al., 2020</a>). 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.</p>
</sec>
<sec id="j_afpuc-2023-0006_s_002"><div>MATERIALS AND METHODS</div>
<p>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 <a ref-type="table" href="#j_afpuc-2023-0006_tab_001">Table 1</a>. 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.</p>
<table-wrap id="j_afpuc-2023-0006_tab_001" position="float"><label>Table 1.</label>
<caption><p>The content of rosmarinic acid (RA) in the underground parts of different species of mint.</p></caption>
<table frame="box" rules="all">
<thead>
<tr>
<th align="center" valign="middle"><bold>Species of mint</bold></th>
<th align="center" valign="middle"><bold>Weight of specimen [g]</bold></th>
<th align="center" valign="middle"><bold>Amount of methanol [mL]</bold></th>
<th align="center" valign="middle"><bold>RA μg.ml<sup>−1</sup> of extract</bold></th>
<th align="center" valign="middle"><bold>RA mg.g<sup>−1</sup> of dry sample ± SD</bold></th>
<th align="center" valign="middle"><bold>% RA of dry sample ± SD</bold></th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="top"><bold><italic>M. × piperita</italic> L.</bold></td>
<td align="center" valign="top">0.4373</td>
<td align="center" valign="top">8.8</td>
<td align="center" valign="top">156,60 ± 9,23</td>
<td align="center" valign="top">3.15 ± 0.18</td>
<td align="center" valign="top">0.32 ± 0.02</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. spicata</italic> var. <italic>crispa</italic> L. “MAROCCO”</bold></td>
<td align="center" valign="top">0.5218</td>
<td align="center" valign="top">10.4</td>
<td align="center" valign="top">171,85 ± 5,99</td>
<td align="center" valign="top">3.43 ± 0.30</td>
<td align="center" valign="top">0.34 ± 0.01</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. × piperita</italic> L. “BULHARSKÁ 1”</bold></td>
<td align="center" valign="top">0.3470</td>
<td align="center" valign="top">7.0</td>
<td align="center" valign="top">301,63 ± 0,89</td>
<td align="center" valign="top">6.09 ± 0.02</td>
<td align="center" valign="top">0.34 ± 0.00</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. × piperita</italic> L. “BULHARSKÁ 2”</bold></td>
<td align="center" valign="top">0.5246</td>
<td align="center" valign="top">10.5</td>
<td align="center" valign="top">156,19 ± 0,86</td>
<td align="center" valign="top">3.13 ± 0.02</td>
<td align="center" valign="top">0.31 ± 0.00</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. spicata</italic> L.</bold></td>
<td align="center" valign="top">0.3196</td>
<td align="center" valign="top">6.4</td>
<td align="center" valign="top">181,79 ± 6,74</td>
<td align="center" valign="top">3.65 ± 0.13</td>
<td align="center" valign="top">0.36 ± 0.01</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. arvensis</italic> L.</bold></td>
<td align="center" valign="top">0.2118</td>
<td align="center" valign="top">4.3</td>
<td align="center" valign="top">138,29 ± 1,99</td>
<td align="center" valign="top">2.81 ± 0.04</td>
<td align="center" valign="top">0.28 ± 0.00</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. longifolia</italic> var. <italic>asiatica</italic> (Boriss.) Rech.f.</bold></td>
<td align="center" valign="top">0.4340</td>
<td align="center" valign="top">8.7</td>
<td align="center" valign="top">189,51 ± 0,93</td>
<td align="center" valign="top">3.80 ± 0.01</td>
<td align="center" valign="top">0.38 ± 0.00</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. × rotundifolia</italic> (L.) Huds.</bold></td>
<td align="center" valign="top">0.5203</td>
<td align="center" valign="top">10.4</td>
<td align="center" valign="top">257,90 ± 25,26</td>
<td align="center" valign="top">5.16 ± 0.50</td>
<td align="center" valign="top">0.52 ± 0.05</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. longifolia</italic> (L.) L.</bold></td>
<td align="center" valign="top">0.4688</td>
<td align="center" valign="top">9.4</td>
<td align="center" valign="top">170,67 ± 6,14</td>
<td align="center" valign="top">3.42 ± 0.13</td>
<td align="center" valign="top">0.34 ± 0.01</td>
</tr>
<tr>
<td align="left" valign="top"><bold><italic>M. spicata</italic> var <italic>crispa</italic> L.</bold></td>
<td align="center" valign="top">0.4358</td>
<td align="center" valign="top">8.7</td>
<td align="center" valign="top">167,70 ± 6,68</td>
<td align="center" valign="top">3.35 ± 0.13</td>
<td align="center" valign="top">0.33 ± 0.01</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn-group>
<fn><p><italic>Values (mg g<sup>−1</sup> of the dry sample and % RA of the dry sample) are expressed as means ± standard deviation of two measurements for each sample.</italic></p></fn>
</fn-group>
</table-wrap-foot>
</table-wrap>
<sec id="j_afpuc-2023-0006_s_002_s_001"><div>Determination of rosmarinic acid by HPLC-DAD</div>
<p>Each of the 10 samples was analyzed by HPLC-DAD using the Sykam HPLC system (Sykam, Eresing, Germany) equipped with a pump (S1125), autosampler (S5250), column oven (S4120), PDA detector (S3345), and Clarity software. HPLC separation of the extract from underground parts was performed on a TELOS LU C18 (2), 250 × 4.6 mm internal diameter (ID), 5 μm (KINESIS, Cheshire, UK), at a temperature of 30°C and a flow rate of 0.8 mL/min. Water (pH 2.45 with HOAc; Merck, Germany) and MeCN (MS grade; Honeywell, Riedel-de-Haen, Seelze, Germany) were used as mobile phases A and B, respectively. The following gradient program was used: 20% B (0 min), 35% B (10 min), 50% B (20 min), 95% B (22 min), 20% B (23 min), and 20% B (25 min), followed by column cleaning and re-equilibration (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_013">Fialová et al., 2019</a>). Quantitative determination of rosmarinic acid in the underground parts was performed using the external standard method. The samples were measured at a wavelength of 320 nm, and the standard was obtained from Sigma-Aldrich (St. Louis, MO, USA). After measuring the known concentration of the standard (5, 10, 25, 50, 100 ppm), also at 320 nm, the calibration curve was prepared. The standard showed good linearity with <italic>r</italic><sup>2</sup> = 0.9702 and the regression curve y = 50,9756*x – 498,6082. All the measured results were within the linear range of the method. The results were expressed in μg mL<sup>−1</sup> of the methanolic extract and converted to mg g<sup>−1</sup> of the dry weight of the sample. The percentage content of rosmarinic acid in the dry sample was also calculated. The measurement of each sample was performed twice, and the results were reported as mean values and standard deviation (SD).</p>
</sec>
</sec>
<sec id="j_afpuc-2023-0006_s_003"><div>RESULTS AND DISCUSSION</div>
<p>The species of the genus <italic>Mentha</italic> 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 <italic>Mentha</italic> species, but little is known about their underground parts (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_005">Cirlini et al., 2016</a>; <a ref-type="bibr" href="#j_afpuc-2023-0006_ref_006">Elansary et al., 2020</a>; <a ref-type="bibr" href="#j_afpuc-2023-0006_ref_007">Fialová et al., 2015</a>; <a ref-type="bibr" href="#j_afpuc-2023-0006_ref_008">Park et al., 2019</a>; <a ref-type="bibr" href="#j_afpuc-2023-0006_ref_009">Salehi et al., 2018</a>). 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 (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_010">Adomaco-Bonsu et al., 2017</a>) and its associated antimicrobial, cardioprotective, hepatoprotective, antidiabetic, and anti-inflammatory effects (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_004">Hitl et al., 2020</a>). This molecule of interest is the main subject of the present research. The underground parts of 10 different species of the genus <italic>Mentha</italic>, all belonging to the section <italic>Mentha</italic> 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 <italic>Mentha</italic> species.</p>
<p>The results presented in <a ref-type="table" href="#j_afpuc-2023-0006_tab_001">Table 1</a> show the highest content of rosmarinic acid in <italic>Mentha × piperita</italic> L. “BULHARSKA 1” (6.9 mg g<sup>−1</sup> of the dry sample), followed by <italic>Mentha rotundifolia</italic> (L.) Huds. (5.16 mg g<sup>−1</sup> of the dry sample). The chromatograms of these two samples are shown in <a ref-type="fig" href="#j_afpuc-2023-0006_fig_001">Figure 1</a>. The lowest yield was obtained in <italic>Mentha arvensis</italic> L. (2.81 mg g<sup>−1</sup> of the dry sample). A slightly higher content was found in the most widely used <italic>M. × piperita</italic> L. (3.15 mg g<sup>−1</sup> 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 <italic>M. rotundifolia</italic> leaves. According to them, the leaves contained 12 mg g<sup>−1</sup> rosmarinic acid in the dry sample, which is three to four times higher than in the underground parts (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_011">Alharbi et al., 2021</a>). Pavlović et al. investigated rosmarinic acid in the leaves of <italic>M. × piperita</italic> L. and recorded the content of rosmarinic acid to be 63.30 mg g<sup>−1</sup> of the dry extract (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_012">Pavlović et al., 2021</a>). In our previous studies, Bittner Fialová et al. investigated the secondary metabolites in roots and rhizomes of some <italic>Mentha</italic> 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 (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_013">Bittner Fialová et al., 2019</a>). In another study performed by Bittner Fialová et al., the rhizomes of three mint species were analyzed using HPLC-DAD: <italic>M. × piperita</italic> cv. “Perpeta,” <italic>Mentha longifolia</italic>, and <italic>Mentha × villosa</italic> cv. “Snežna.” The results showed that the infusion of <italic>M. × piperita</italic> cv. “Perpeta” contained a similar amount of rosmarinic acid as in the previous study, while the other two varieties had double the content (<a ref-type="bibr" href="#j_afpuc-2023-0006_ref_014">Bittner Fialová et al., 2020</a>). 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.</p>
<figure id="j_afpuc-2023-0006_fig_001" position="float" fig-type="figure"><h2>Figure 1.</h2>
<figCaption><p>(A) HPLC-DAD chromatogram of methanolic extract of underground parts from Mentha × piperita L. „BULHARSKÁ 1 „(λ = 320 nm). (B) HPLC-DAD chromatogram of methanolic extract of underground parts from Mentha × rotundifolia (L.) Huds. (λ = 320 nm).</p></figCaption>
<img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_afpuc-2023-0006_fig_001.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6470851571e4585e08a9f136/j_afpuc-2023-0006_fig_001.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240629T134736Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=18000&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240629%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=43f0ae5a5df5737a6f668231169f0ff05141c40e7ae9f1035269c99b175311c9" class="mw-100"/>
</figure>
</sec>
<sec id="j_afpuc-2023-0006_s_004"><div>CONCLUSION</div>
<p>Using HPLC-DAD, we analyzed the methanolic extracts from underground parts of 10 different species of the genus <italic>Mentha</italic> 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 <italic>M. × piperita</italic> L. “BULHARSKA 1” and <italic>M. rotundifolia</italic> (L.) Huds. These results suggest the possible use of <italic>Mentha</italic> rhizomes as a source of rosmarinic acid.</p>
</sec>
</div>

Rosmarinic Acid in Underground Parts of Different <italic>Mentha</italic> Species

Department of Pharmacognosy and Botany, Faculty of Pharmacy

Department of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy

European Pharmaceutical Journal

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Rosmarinic Acid in Underground Parts of Different  Species

Background
Mints (Mentha L., Lamiaceae) are medicinal plants frequently used in pharmacy, cosmetics, and food industry. Among the secondary metabolites found...

Rosmarinic Acid in Underground Parts of Different Mentha Species

Article Category: Original Paper

Published Online: Aug 31, 2023

Page range: 8 - 11

Received: Sep 28, 2022

Accepted: Mar 22, 2023

DOI: https://doi.org/10.2478/afpuc-2023-0006

Keywords
Mentha, Underground parts, HPLC-DAD, Rosmarinic acid

© 2023 M. Mergová et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Rosmarinic Acid in Underground Parts of Different Mentha Species

Article Category: Original Paper

Published Online: Aug 31, 2023

Page range: 8 - 11

Received: Sep 28, 2022

Accepted: Mar 22, 2023

DOI: https://doi.org/10.2478/afpuc-2023-0006

KeywordsMentha, Underground parts, HPLC-DAD, Rosmarinic acid

© 2023 M. Mergová et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Keywords
Mentha, Underground parts, HPLC-DAD, Rosmarinic acid