Introduction

The tropical Andes are considered one of the most diverse areas on the planet in terms of vascular plants. The flora of Perú is extremely rich, and its territory is home to some 25,000 species, almost 10% of all plants in the world. However, the percentage of them scientifically studied is quite low1. Phytochemical research on Peruvian biodiversity proved to be fundamental in the development of modern medicine, e.g. the isolation of cocaine from Erythroxylum coca was a milestone in the development of local anesthetics2, similarly the isolation of the first antimalarial agent, quinine from Cinchona ledgeriana cortex initiated “the alkaloids golden age”3. Most of those phytochemical investigations were conducted overseas, a fact that reflects the absence or the restricted access of resources and infrastructure for developing classical phytochemical research in Peru. Today, modern platforms maybe applied for the metabolic characterization of Peruvian flora, a task that can be achieved by a liquid chromatography associated with high resolution mass spectrometry (LC-HRMS) method since it is less time consuming compared to classic methods of isolation and structure identification. Some recent investigations that exemplify the use of LC-HRMS for describing the phytochemical profile of Peruvian flora include the metabolic profile on medicinal plants of the genus Chuquiraga (Asteraceae)4 and that related to Capsicum (Solanaceae) fruits5.

Perú has several traditional medicine systems, that of the northern Andes6,7, that of the southern Andes8 and that of the Amazonian forest9, each one of them with its main and minor plants and particular practices. With the passage of time, those traditional medicines are getting combined a fact that is especially noticeable in Lima city, the capital of Peru10. One aspect that is worth to highlight is that, especially in Andean medicines, but not in Amazonian ones, there is an important contribution of plants belonging to the Lamiaceae family to the traditional medicine systems.

The large family Lamiaceae has twelve subfamilies. The Nepetoideae subfamily, with 3400 species and 105 genera, has three tribes11: Elsholtzieae, Ocimeae and Mentheae, the latter with 65 genera. The Mentheae tribe is chemically characterized by having volatile terpenoids and a phenolic acid called rosmarinic acid that makes these plants aromatic and with medicinal properties12,13 Mentheae can also be classified into 3 subtribes: Menthinae (43 genera), Salviinae (10 genera) and Nepetiinae (12 genera)14,15. In Peru (Herbario Nacional Universidad de San Marcos-Perú, October 2017), the main genera of Mentheae were Clinopodium (29 species), Hedeoma (1 specie), Lepechinia (11 species), Minthostachys (7 species) and Salvia (60 species). Clinopodium, Hedeoma, and Minthostachys belong to the Menthinae subtribe, while Lepechinia and Salvia belong to the Salviinae subtribe. Investigations on the non-volatile components in Peruvian Mentheae are relatively scarce compared to the works related to essential oils16 . In a previous work17 the contents of rosmarinic acid, triterpenic acids, oleanolic and ursolic were quantified in thirteen Peruvian Mentheae. The highest content of rosmarinic acid was observed in Lepechina meyenii (Walp.) Epling and the highest content of triterpenic acids in Clinopodium revolutum (Ruiz & Pavón) Govaerts. Subsequently18, the non-volatile compounds were unambiguously or reasonably identified in two Lepechinia species: L. meyenii and L. floribunda (Benth.) Epling, by LC-HRMS, where the presence of salvianolic acids and diterpenoids were notable.

LC-HRMS methods have been used to comprehensively analyze the phenolic components of plants, this implies procedures for the systematic manually identification of mass spectra19,20 and also the use of suitable software21,22, in both cases the procedure involves recording of diagnostic ions for classification and then the identification of characteristic ionic products and neutral losses for confirmation. In the present communication, the profile of secondary metabolites by LC-HRMS is reported for ten Peruvian Mentheae: Clinopodium (4 species), Salvia (4 species), Hedeoma (1 species) and Minthostachys (1 species).

Results

Phytochemical profile

The LC-HRMS metabolite profile of the ethanolic extracts of the ten peruvian Mentheae was obtained in the negative mode (ESI (−)) and the detected compounds appear in Table 1. Assignments were made based on the literature21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37. Isomers of quinic acid (m/z 191.0556), danshensu (m/z 197.0450), protocatechuic aldehyde (m/z 137.0239), and caffeic acid (m/z 179.0350) occur in most plants. Equally abundant are the monocaffeoylquinic acids present in seven species. Minthostachys mollis contains four different monocaffeoylquinic acids. Several derivatives of ferulic acid and p-coumaric acid could also be identified. The 4 (para) substitution or the 3,4 substitution with respect to C3 cannot be determined by MS, however this is the substitution reported in Mentheae19,20,23,38,39,40,41,42,43,44,45,46,47,48,49. Caffeic acid, protocatechuic aldehyde and protocatechuic acid share the same substitution pattern. Furthermore, a diversity of flavonoids (flavonols, flavones, flavanones, flavanonols) was found in all the samples, both free and glycosylated. Minthostachys mollis, Clinopodium sericeum and Clinopodium pulchellum are the most diverse with respect to their flavonoids. The most frequent flavonoid aglycones were luteolin (m/z 285.0404), quercetin (m/z 301.0354), kaempferol (m/z 285.0404) and apigenin (m/z 269.0455). Eupatorin is present in five of the species studied50. In Clinopodium revolutum, apigenin and luteolin C- hexosides were detected. In all the samples the presence of rosmarinic acid (m/z 359.0772) was detected. In Clinopodium revolutum, salvianic acid C (m/z 377.0881), which is the result of hydrating the double bond of rosmarinic acid, has been detected, and, in Salvia sagitatta, teucrol (m/z 315.0880)51, a decarboxylated rosmarinic acid was observed. Isorinic acid (m/z 343.0827) a rosmarinic acid molecule without the 3-OH was present in Clinopodium brevicalyx, Salvia sagitatta, Salvia cuspidata and Hedeoma mandoniana. Methyl (m/z 373.0931) and ethyl (m/z 387.1088) esters of rosmarinic acid were present in Salvia cuspidata and Clinopodium brevicalyx. In Salvia cuspidata and Clinopodium revolutum, the dimer of rosmarinic acid, sagerinic acid (m/z 719.1598), which is a molecule with a stabilized cyclobutane ring, was found. Clinopodium pulchellum displayed the presence of salvianolic acid A (m/z 493.1143) and salvianolic acid F (m/z 313.0722). In Clinopodium brevicalyx, Clinopodium sericeum and Hedeoma mandoniana, the presence of salvianolic acid B (m/z 717.1443) was observed, a particularly important substance due to its effect on neurodegenerative diseases52. However, the plant with the greatest diversity of salvianolic acids was Clinopodium sericeum, "romero de jalca", in addition to salvianolic acid B, lithospermic acid (m/z 537.1038), two isomers of salvianolic acid A and two isomers of salvianolic acid F. This type of substances is very important due to its effect on cell fibrosis (scar formation) in direct relation to cancer53. Among the other substances found, it should be noted that the Rosmarinus type diterpenoids, common in Lepechinia18,54 are scarce in this work; only Salvia sagitatta and Salvia cuspidata show the presence of carnosol (m/z 329.1761) and the phenolic diterpenoid, rosmadial (m/z 343.1552) in the last one28. Salvia haenkei contains the ent-(5R,9R)-15,16-epoxy-10S-hydroxycleroda-3,7,13(16),14-tetraene-17,12S; 18,19 diolide (m/z 355.1190)26, while Salvia cuspidata had a lignan, isolariciresinol (m/z 359.1502) previously reported in Linum seeds31,55, and 5-epi-icetexone (m/z 341.1396) described as an anti- Trypanosoma cruzii molecule56. Oleanolic and ursolic triterpenic acids, quantified in a previous report by Serrano et al.17, do not appear in this analysis due to the elution program used, which does not reach 100% acetonitrile57. Figure 1 shows the typical ESI (−) chromatogram of Salvia sagitatta and Fig. 2 shows the chromatogram of Clinopodium sericeum. The chemical structures of the main metabolites detected are displayed in Fig. 3.

Table 1 Compounds detected in the ethanolic extracts of then Peruvian Mentheae by LC-HRMS.
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Figure 1
figure 1

ESI (−) chromatogram of Salvia sagitatta.

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Figure 2
figure 2

ESI (−) chromatogram of Clinopodium sericeum.

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Figure 3
figure 3

Chemical structures of main metabolites identified.

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Discussion

This is the first time that the phytochemical profile has been obtained for the ten Peruvian Mentheae (Lamiaceae) here reported. The botanical genera studied were Salvia (Salviinae), Clinopodium, Hedeoma and Minthostachys (Menthinae). While Salvia and Clinopodium are genera of worldwide distribution, Hedeoma and Minthostachys are American and South American genera, respectively. All Salvia species in this work belong to the Salvia subgenus Calosphace Benth. (Epling)63. Assignments were based on the search for diagnostic ions, characteristic product ions and neutral losses19,20,25,40,41. The fragmentation patterns shown in said references are particularly useful for this work since they are specifically directed to Lamiaceae/Mentheae. The phytochemical profiles of those Mentheae here surveyed are quite similar to their European and Asian relatives. All the species analyzed show the presence of rosmarinic acid, while, quinic acid, 3,4-dihydroxyphenyl-lactic acid “danshensu”, protocatechuic aldehyde and caffeic acid are present in most of the samples. Monocaffeoylquinic acids, also called chlorogenic acids, are also frequent but better expressed in Minthostachys. Dicaffeoylquinic acid was detected only in Clinopodium revolutum. All samples contained flavonoids with more diversity in Minthostachys and Clinopodium. Flavonoid-free aglycones predominate in several plants: In Salvia sagitatta, cirsimaritin is abundant64, while eupatorin predominates in Clinopodium revolutum50, genkwanin in Salvia haenkei36 and hesperetin in Clinopodium pulchellum27. In several plants, rosmarinic acid is the main peak: Clinopodium brevicalyx, Salvia oppositiflora, Clinopodium sericeum and Hedeoma mandoniana. Some type of salvianolic acid is present in all the samples, although in some cases, they are very small modifications of the rosmarinic acid molecule. Dimers and trimers of rosmarinic acid are present in Clinopodium brevicalyx, Salvia oppositiflora, Salvia cuspidata, Clinopodium sericeum, Hedeoma mandoniana and Clinopodium pulchellum. In Clinopodium sericeum, not only is the diversity of salvianolic acids important but also their abundance in salvianolic acid A, which would allow the preparation of the said substance from it71.

Conclusion

Peruvian Mentheae are a rich source of flavonoids, phenolic acids and terpenoids. The present study involved LC-HRMS analysis of ten species. A total of 111 compounds were detected. Most of these were identified by key ion filtering strategy and comparison with literature data. This methodology can be used to the authentication and differentiation of larger numbers of Mentheae species: The San Marcos Herbarium, Lima-Perú, in 2017 had 108 Mentheae.

Methods

Plant material

The plants used in this study are as follows: Clinopodium brevicalyx Epling (Harley & Granda) (Menthinae) (HUT 59506), Salvia oppositiflora (R. and P.) (Salviinae) (HUT 59502), Minthostachys mollis Griseb. (Menthinae) (HUT 59766), Salvia sagittata R. and P. (Salviinae) (HUT59499), Salvia cuspidata subsp. cuspidata (R. and P.) (Salviinae) (HUT 59505), Clinopodium revolutum (R. and P.) (Menthinae) (HUT 58329), Clinopodium sericeum (Briq. et Benth) Govaerts (Menthinae) (HUT 58,332), Salvia haenkei Benth. (Salviinae) (HUT 59500), Hedeoma mandoniana Wedd. (Menthinae) (HUT 59763), Clinopodium pulchellum Kunth (Govaerts) (Salviinae) (HUT 59765). All of them were collected in Peru (2014–2018) by the author (C.S.) according to the procedures of the Universidad San Antonio Abad and following the guidelines of the Herbarium Truxillense of the Universidad Nacional de Trujillo (HUT)—Perú https://facbio.unitru.edu.pe. Specimens were identified and deposited by the botanist Eric Frank Rodríguez (Herbarium Truxillense).

Sample preparation for metabolite profiling

Fifty milligrams of pulverized aerial parts were subjected to an ultrasonic bath for five minutes with 1 mL of ethanol for three times. The filtrates were evaporated in vacuo and stored at 4 °C until use.

LC-HRMS

Chromatographic separation was performed on a Thermo Scientific Dionex Ultimate 3000 UHPLC system with an Acclaim RP C18 150 × 4.6 mm × 1.8 µm chromatographic column at 25 °C and a gradient of (a) 0.1% H2CO2 in water and (b) acetonitrile: [time, % (b)]: (0.5); (5,5); (10.30); (15.30); (20,70); (25.70); (35.5) and 12 min of equilibration before each injection. The flow rate was 1 mL min−1, and the injection volume was 10 μL. The extracts were dissolved in 1.5 mL of methanol and filtered through 0.22 µm PTFE. For high resolution mass spectrometry, a Q-Exactive MS (Thermo Fisher Germany) equipped with electrospray ionization (ESI) in negative mode was used. The MS collection parameters were as follows: spray voltage 2500 V; capillary temperature, 400 °C. Sheath gas flowed at a rate of 75 units. Auxiliary gas flowed at 20 units. Scanning range of 100–1500 m/z. Resolution of 35,000. The mass tolerance threshold was 5 ppm. Data acquisition and processing were performed with XCalibur 2.3 (Thermo Fisher Scientific).

Diagnostic ions for classification

Quinic acids derivatives: 337.0929 p-coumaroylquinic acid, 367.1035 feruloylquinic acid, 353.0878 caffeoylquinic acid, 515.1195 dicaffeoylquinic acid.

Phenylpropionic acids: 163.0401 p-coumaric acid, 179.0350 caffeic acid, 359.07772 rosmarinic acid.

Flavonoids: 253.0506 chrysin, 269.0455 apigenin, 285.0404 luteonin and kaemferol, 301.0354 quercetin.