Phytochemical profile and rosmarinic acid purification from two Peruvian Lepechinia Willd. species (Salviinae, Mentheae, Lamiaceae)

The phytochemical profile of Lepechinia meyenii (Walp.) Epling and Lepechina floribunda (Benth.) Epling obtained by liquid chromatography associated with high-resolution mass spectrometry is presented. Forty eight compounds were detected exhibiting a variety of salvianolic acids and abietane phenolic diterpenoids. A simple procedure by cold evaporative crystallization to purify rosmarinic acid from these botanical species was also shown.

Traditional medicines in Peru like others in different parts of the world are characterized by the use of plants of the big family Lamiaceae 1-4 . One of these approaches reports 25 Lamiaceae species out of a total of 510 medicinal plants (4.9%) 4 . Lamiaceae is a cosmopolitan family with more than 230 genera and approximately 7000 species 5,6 . The Lamiaceae family in Peru has about 21 genera and 190 species, mostly herbs and shrubs, 57 species are endemic in 9 genera 7 . Lamiaceae has twelve subfamilies, one of them being Nepetoideae (3400 species, 105 genera) 6,8 . Mentheae is the largest and economically important tribe of the Nepetoideae subfamily (2000 species, 60 genera, 3 tribes) 6,9 . The Mentheae tribe in turn is divided into five sub-tribes: Menthinae, Salviinae, Nepetinae, Prunellinae and Lycopinae 6,9,10 . In Peru Salviinae tribe is represented fundamentally by two genera: Lepechinia and Salvia. The pan american genus Lepechinia Willd. is constituted by ca 45 species, 30 of which occur in South America at elevations from 1500 to 4000 m within a broad range of habitats 11,12 . Lepechinia in Peru has 4 endemic species: Lepechinia marica Epling & Mathias, Lepechinia mollis Epling, Lepechinia scobina Epling Lepechinia tomentosa (Benth.) Epling 6 . In the Mentheae tribe, the presence of volatile and non-volatile terpenoids, the absence of iridoids (monoterpenglycosides) and the abundance of rosmarinic acid and their higher derivatives, salvianolic acids, is characteristic 13 . The presence of rosmarinic acid is not exclusive to Mentheae, but for all Nepetoideae 6,13 . In contrast, rosmarinic acid is not present in the Lamoideae subfamily, but iridoids are 13 . In addition, it is known that rosmarinic acid is also present in very diverse taxa in dicots, monocots, ferns and hornworts and that for this reason it is not a good chemotaxonomic indicator but very useful to distinguish intrafamilial taxonomic subgroups in Lamiaceae 14 . This varied taxonomic distribution suggests a complicated evolutionary history in rosmarinic acid biosynthesis, for example Lamiaceae and Boraginaceae (order Lamiales) have different mechanisms for producing rosmarinic acid 15 so it is likely that each case has different biosynthetic mechanisms. Rosmarinic acid and salvianolic acids, particularly salvianolic acid A and B has interesting effects on fibrosis, cancer and neurodegenerative processes [16][17][18] . Other important effects of rosmarinic acid are as an anti-inflammatory, UV protector, antioxidant, cytoprotector 18 and as an antihepatotoxic 19 . Lepechinia meyenii (Walp.) Epling is a medicinal herb that grows at 3800-4000 m of altitude where is employed for respiratory diseases 2 . In the southern andes of Perú this plant "Puna Salvia" 20 was the third most used plant in communities whose health then depended almost exclusively on the use of medicinal plants. Lepechinia floribunda (Benth.) Epling is a sub-shrub that grows in the foothills of the eastern Andes of southern Peru and according to the inhabitants of the collection area is used as a tonic and comforting for the body. In a recent work on Argentinean Lepechinia meyenii (Walp.) Epling 21,22 was reported the presence of three hydroxycinnamic acids: caffeic, p-coumaric and rosmarinic acids and seven abietane diterpenoids: carnosol, rosmanol, carnosic acid, carnosic acid ɤ-lactone, 20 25 . The rosmarinic acid content in these two Lepechinias is high when compared with the content in species of the genus Salvia from other parts of the world 26,27 . Lepechinia also has a higher rosmarinic acid content than Peruvian Salvia, and than Peruvian Minthostachys, Clinopodium and Hedeoma (Menthinae) 25 . Considering all the studies mentioned above, we see that Lepechinia meyenii (Walp.) Epling and Lepechinia floribunda (Benth.) Epling have not been fully investigated for their non-volatile composition. Ultra-performance liquid chromatography associated with tandem mass spectrometry (UHPLC/MSMS) is an important structural tool for the study of complex plant extracts, for this we will use the UHPLC-Q-OT-MS technology, the Q-Exactive mass spectrometer hybridizes the high mass resolving power of orbitrap mass analyzer with the selectivity of a quadrupole, multiple precursor ions are fragmented in a high energy collision cell and the product ions could be detected with a mass error of less than 5 ppm for a wide range of analyte concentrations 28,29 . Data processing includes spectral similarity prospection and characteristic product ions-neutral loss searching 29,30 .
In the present work, the first objective is the phytochemical profile of the ethanolic extract of the aerial parts of both plants by UHPL-Q-OT-MS. And, our second objective is the purification of rosmarinic acid from these two species.
The final stage of purification involves precipitating solid rosmarinic acid from a concentrated aqueous solution. This was done by placing said solution in a vacuum desiccator with a strong desiccant such as sulfuric acid, which concentrates the cold solution. The solid obtained can be recrystallized by the same procedure.

Discussion
This is the first UHPLC/MSMS phytochemical profile of L. meyenii and L. floribunda showing salvianolic acids and diterpenoids like principal components. There are the typical monomers, caffeic acid, protocatechuic aldehyde , protocatechuic acid and "danshensu", which are considered to be the building blocks of dimeric salvianolic acids, rosmarinic acid, salvianic acid C , (caffeoyl-4-hydroxy-phenyl)lactic acid "isorinic acid" and Salvianolic acid F. The trimeric Yunnaneic acid E, salvianolic acid F and the isomeric tetramers sagerinic and clerodendranoic acid H are the more structured salvianolic acids. In addition to the salvianolic acids, we found the diterpenoid phenolics rosmanol, carnosol, carnosic acid and rosmaridiphenol, beside the phenantrenequinone horminone, the same terpenoids as Rosmarinus officinalis, an old world medicinal Mentheae-Salviinae. These substances support the presence of Lepechinia within Salviinae beside Salvia, Melissa, Rosmarinus, among other genera 27,31,32,34,36,39,54,56,57,59 . However, a recent work 53 , based on DNA, chloroplastic, nuclear ribosomal and lowcopy nuclear gene regions, lumps the small genera Dorystaechas, Meriandra, Perovskia, Rosmarinus and Zhumeria within Salvia genus while Melissa and Lepechinia do not. It is also observed that in the analyzed Lepechinias, flavonoids and chlorogenic acids are not significantly present as in the case of Rosmarinus 54 . In this work, a luteolin hexoside has been detected in Lepechinia meyenii as the only flavonoid in the same way as, luteolin-3-Oglucuronide, is shown in Melissa officinalis 34,58 .
The rosmarinic acid of both species were easily purified from the hydroethanolic extract without any preparative chromatographic method by a classical procedure 47 with an initial extraction which adapts the concept that extractability is not the same as solubility-rosmarinic acid is much more soluble in ethanol than in water, but hydroalcoholic mixtures access vacuoles more effectively than pure ethanol because it makes non-permeable to vacuolar membrane 49,55 . Then, partitions with low toxicity solvent and precipitation of rosmarinic acid by cold evaporation of the aqueous solution with the help of a desiccant in vacuo and final recrystallization from hot aqueous solution. The yields of rosmarinic acid are approximately half of those of the analytical report 25 . If the solution is simply left to the environment, it takes too long or nucleation conditions are never reached. This process of evaporating aqueous solutions without the application of heat could be applied to purify other types of phenolic acids that are usually difficult to precipitate and tend to remain glassy. Thus, high purity rosmarinic

Disclosure statement
The authors declare no conflict of interest. C.S. declares that the botanical material collected was made with permission of Universidad Nacional de San Antonio Abad del Cusco-PERU in quantities less than 300 g of dried material in accord to institutional rules. www.nature.com/scientificreports/