Terpenes From the Root of Salvia hypoleuca Benth

Background The genus Salvia, with nearly 900 species, is one of the largest members of Lamiaceae family. In the Flora of Iran, the genus Salvia is represented by 58 species of which 17 species are endemic. Salvia hypoleuca Benth., is one of these species growing wildly in northern and central parts of Iran. Salvia species are well known in folk medicine and widely used for therapeutic purposes. Literature review shows that there is no report on phytochemical investigation of the roots of S. hypoleuca. Results The separation and purification process were carried out using various chromatographic methods. Structural elucidation was on the basis of NMR and MS data, in comparison with those reported in the literature. The isolated compounds were identified as sitosteryl oleate (1), β-sitosterol (2), stigmasterol (3), manool (4), 7α-acetoxy royleanone (5), ursolic acid (6), oleanolic acid (7), 3-epicorosolic acid (8), 3-epimaslinic acid (9) and coleonolic acid (10). Conclusions In the present study, three sterols, two diterpenes and five triterpenes were isolated from the ethyl acetate extract of the roots of S. hypoleuca. As the chemotaxonomic significance, some of the isolated compounds (1–7, 9) have not been previously reported from the species S. hypoleuca, while the triterpenes 8 and 10 are now documented from Salvia genus for the first time.


Background
The genus Salvia L. (Lamiaceae), with more than 900 species throughout the world, is represented 58 species in Iran, 17 of which are endemic. Most of the species are used as herbal tea and flavoring agent by people and also used in traditional medicine as tonic, antirheumatoid, antimicrobial and carminative [1][2][3]. Flora Orientalis includes as many as 107 species of Salvia [4]. Salvia hypoleuca Benth., is one of these species which growing wildly in northern and central parts of Iran [1].
Several sesterterpene lactones, isomeric epoxides, monolactone and hypoleuenoic acid have been reported from varies fractions of S. hypoleuca [12][13][14]. The main aromatic components of the essential oil of S. hypoleuca roots have been identified as hexadecanoic acid (27.4%) and viridiflorol (14.9%) [15], while germacrene D (15.1%) and β-caryophyllene (22.0%) identified as the major constituents during flowering stages [16]. A great number of diterpenes exhibited interesting biological activities e.g. anti-tuberculous, antitumour, antibacterial, antileishmanial and antispasmolytic, and Salvia species are the excellent source of diterpenoids [17]. In this study, we aim to report the isolation and identification of some sterols, diterpenoids and triterpenoids from the root extract of S. hypoleuca which have not been previously reported from this species.

Methods
Instruments and materials 1 H-NMR and 13 C-NMR spectra were recorded on a Brucker Avance 500 DRX spectrometer W with tetramethylsilane as an internal standard and chemical shifts are given in δ (ppm). Multiple-pulse experiments (HSQC, HMBC and H-H COSY) were performed using the standard Bruker W programs. Silicagel 60 F 254 and Silicagel 60 RP-18 F 254 S pre-coated plates (Merck W ) were used for TLC. The spots were detected by spraying with anisaldehyde-H 2 SO 4 reagent followed by heating.

Plant materials
The roots of Salvia hypoleuca Benth., were collected from Tehran province (near to Damavand city), Iran, at flowering stage in August 2008 and dried at room temperature. Voucher specimen was deposited at the Herbarium of Complex of Academic Center for Educational and Cultural Research under number ACECR-266.

Discussion
Literature reviews show that Salvia species are important medicinal and food plants. About 200 triterpenoids have been isolated and identified from about 100 Salvia species and presented in a review article by Topcu [29]. The oleanane, and ursane triterpenes display various pharmacological activities. These triterpenes can be considered as the lead compounds for the development of new multi-targeting bioactive agents [30]. Both oleanolic and ursolic acid have been documented to protect liver against chemically induced injuries in laboratory animals via inhibition of toxicant activation and enhancement of immune systems. These two triterpenes have also been long-recognized as anti-inflammatory and antihyperlipidemic agents. Furthermore, anti-tumor activity has been noted from both non-toxic compounds [31].
Corosolic acid, a triterpenoid compound has been proved to have anti-diabetic effects on animal and human via enhancing glucose uptake in L6 myotubes and facilitating glucose transporters isoform 4 translocation in CHO/hIR cells. In addition, corosolic acid has been reported to inhibit the enzymatic activity of several non-receptor protein tyrosine phosphatases (PTPs) [32]. The abietane diterpene 7 α-acetoxy-royleanone, containing quinone moiety in its structure, was demonstrated to possess cytotoxic activity on cancer cell lines and also alkylating properties using the nucleophile 4-(4-nitrobenzyl) pyridine [33]. Among the reported antimicrobial labdane-type diterpenes, manool is the most active, since it furnished very promising MIC values for several tested bacteria that are closely associated with periodontitis [34]. According to chemotaxonomic significance, the isolated terpenes (manool (4), 7α-acetoxy-royleanone (5), ursolic acid (6), oleanolic acid (7), 3-epimaslinic acid (9)) were previously reported from other Salvia species such as S. sclarea [21], S. pubescens [35], S. lavandulifolia [36] and S. officinalis [37]. To the best of our knowledge, there is no report about the presence of the above mentioned compounds from S. hypoleuca. The triterpene 3epicorosolic acid (8) and coleonolic acid (10) has not been reported from Salvia species, while some other genus of Lamiaceae such as Perilla frutescens [38] and Coleus forskohlii [39] contains these triterpenes.