Natural Products from Leaves of the Ancient Iranian Medicinal Plant Echium amoenum Fisch. & C. A. Mey.

For several millennia, leaves of Echium amoenum Fisch. & C. A. Mey., an important Iranian medicinal plant with nutritional value as nutraceutical, have been used as tea for the treatment of several conditions, including inflammation. The nutritional value of intake of E. amoenum tea has mainly been correlated to its rich content of mainly water-soluble antioxidants. Although the entire plant is utilized, only natural products of the flowers have previously been thoroughly investigated. The rare natural products bis(3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydronaphthalene-2,3-dicarboxylate, 4-Oxy-(E)-caffeoyl-2,3-dihydroxybutanoic acid methyl ester and 4-Oxy-(Z)-caffeoyl-2,3-dihydroxybutanoic acid methyl ester, in addition to the widely distributed compounds rosmarinic acid methyl ester and (E)-caffeic acid, were purified and characterized from leaves of Echium amoenum. The structures were determined by a combination of several 2D NMR spectroscopic techniques, circular dichroism spectroscopy and high-resolution mass spectrometry. The fact that bis(3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydronaphthalene-2,3-dicarboxylate belongs to a rare group of natural products which have previously been patented for their significant anti-inflammatory activity may rationalize the traditional treatment of inflammations with E. amoenum.


Introduction
Traditional medicine is deeply rooted in Iranian culture and history. Avicenna (980-1037), the great 11th century Persian scientist and philosopher of the Middle Ages, provided comprehensive information on the preparations of various medicinal plants in his epoch-making encyclopedia Canon of Medicine (Qanun), which has been used as a textbook in medicine in the world's most renowned universities for more than 500 years. It provides extensive information on various drug preparations from a multitude of medicinal plants, which today are frequently used in Iranian traditional medicine [1]. Because of the long history of utilizing medicinal plants, in addition to the variations in climate and geographical conditions, the flora in Iran is characterized by high biodiversity and is home to a wide variety of species of the plant kingdom. At least 1000 different species of medicinal plants have been identified [1].
Echium amoenum Fisch & C.A. Mey. (Figure 1) belongs to the family Boraginaceae and is a perennial Iranian domestic plant which grows wild in the mountains along a narrow strip of land in Northern Iran and the Caucasus [2]. E. amoenum is a valuable medicinal herb which has been used in the treatment of inflammatory and infectious diseases for more than a millennium. E. amoenum is also used as a sedative in the traditional treatment of stress and anxiety. In Iran, it is a common tradition to brew portions of dried flowers of E. amoenum and drink it as tea. As far back as a millennium ago, Avicenna described the use of E. amoenum in various pharmaceutical preparations for the treatment of infectious for more than a millennium. E. amoenum is also used as a sedative in the traditional t ment of stress and anxiety. In Iran, it is a common tradition to brew portions of d flowers of E. amoenum and drink it as tea. As far back as a millennium ago, Avic described the use of E. amoenum in various pharmaceutical preparations for the treat of infectious and inflammatory diseases in his medical encyclopedia Canon of Med Among other things, he describes a pharmaceutical formulation of E. amoenum's ash effective remedy in the treatment of cold sores and oral infections. In Makhzan-al-Ad written by Khorasani, also one of the great Middle Ages Iranian scientists and physic E. amoenum was described as a remedy for coughs, sore throats, colds, pneumonia dyspnea [1].  Despite Avicenna's detailed descriptions of approaches for more research on this medicinal plant, it was not until recent years that E. amoenum was investigated in a number of scientific studies [3]. Even today, despite the great conviction and acceptance of the Iranian population of E. amoenum's versatile and unique properties in the treatment of several different diseases, only a few thorough studies have been performed on this widely used medicinal plant. There are three different species of the genus Echium (Boraginaceae) in Iran, including E. amoenum, E. russicum J.F.Gmel. (now Pontechium maculatum L.), the widespread E. italicum L. and the very rare and endemic E. khuzistanicum Mozaff. [4]. Only E. amoenum has been found to possess valuable medical applications [5]. In Iran, dried violet-red petals of E. amoenum, as well as the leaves, are used in a variety of medical contexts as anti-inflammatory, antibacterial, antioxidant, immunomodulatory, analgesic, anxiolytic, water-retardant and sedative herbal remedies. The best time to harvest the flowers is mid-May and early June, when the secondary metabolites of E. amoenum are assumed to be at their highest quantitative levels, thus accumulating the maximum amount of active substances in the flowers [6]. Various phytochemical studies conducted on E. amoenum's petals and seeds confirm the presence of various natural products such as antocyanins, flavonoids, saponins, unsaturated terpenoids, pyrrolizidine alkaloids, sterols and various fatty acids with significant amounts of γ-linolenic acid [5]. Even the presence of some essential oils in smaller amounts, with the bright yellow δ-cadinene as the major compound, is demonstrated in E. amoenum [2].
The nutritional value of intake of E. amoenum tea has mainly been correlated to the fact that the plant is a rich source of mainly water-soluble antioxidants [7]. In view of the large, frequent consumption of E. amoenum as an effective natural remedy among the population of Iran, it is evident that more research is required to solve many unanswered questions about this medicinal plant. An important aspect thereof is the isolation and characterization of new natural products from E. amoenum, with relevant biological activity in relation to those indications for which the plant is utilized, which can pave the way for the development of new drugs from this medicinal plant. Although the leaves of E. amoenum have been used for similar therapeutic applications to that of the flowers, limited knowledge is available about the chemical constituents of the leaves in the current literature. In this paper, we report the first identification of polyphenolic compounds and their structure from the leaves of E. amoenum.
The UV spectrum of compound 2 recorded on-line during HPLC analysis exhibited λ max values at 242 nm, 300 nm (sh) and 326 nm, which is in accordance with an aromatic compound with a cinnamoyl type chromophore. The aromatic region of the 1D 1 H NMR spectrum of compound 2 ( Figure S3) showed a 3H ABX system at δ 7.04 (d 2.0 Hz, H2), δ 6.99 (dd 8.2, 2.0 Hz, H6) and δ 6.75 (d 8.2 Hz, H5), a 2H AX system at δ 7.48 (d 15.9 Hz, H7) and δ 6.24 (d 15.9 Hz, H8), as well as to two phenolic hydroxyl protons at δ 9.59 (4-OH) and δ 9.13 (3-OH), in accordance with a caffeoyl moiety. This identification was confirmed by the observation of nine 13 C signals assigned to this unit by the 2D 1 H-13 C HMBC spectrum and the 2D 1 H-13 C HSQC spectrum of compound 2 ( Table 1, Figures S4 and S5). The large coupling constant of 15.9 Hz observed for H7 and H8 confirmed the (E)-configuration of the double bond of the caffeoyl moiety. The crosspeaks at δ 4.11/166.4 (H1A'/C9) and δ 4.08/166.4 (H1B'/C9) revealed that the carboxyl of compound 2 was esterified with the γ-hydroxyl of a highly hydroxylated organic acid containing four carbons, which was identified as the methyl ester of threonic acid. A comparison between the relatively weak CD spectrum of compound 2 to that of commercially available L-threonic acid provided by Sigma Aldrich ( Figure S16) revealed that both molecules exhibited a positive band around 195 nm and a negative band around 220 nm, indicating that the methyl threonyl moiety of compound 2 shares the same stereochemistry as that of L-threonic acid, which is synonymous with (2R,3S)-2,3,4-trihydroxybutanoic acid. Thus, compound 2 was identified as 4-Oxy-(E)-Caffeoyl-2 R,3 S-2,3-dihydroxybutanoic acid methyl ester ( Figure 2). Moreover, the sample of compound 2 also contained a minor component, namely compund 3. The 1 H and 13 C signals belonging to compound 3 shared many similarities with that of compound 2, indicating that compound 3 is a structural isomer of compound 2, i.e., a caffeoyl derivative esterified with threonic acid ( Table 1). The coupling constant observed between H7 and H8 (12.9 Hz) confirmed the identity of compound 3 to be 4-Oxy-(Z)-Caffeoyl-2,3-dihydroxybutanoic acid methyl ester (Figure 2). Very recently, Cao et al.      [19], the effectiveness of E. amoenum in the treatment of anxiety, depression, ischemic stroke, seizure, Alzheimer's disease and pain has been studied. Only the antidepressant and anxiolytic properties of the plant extract have been studied both clinically and experimentally [19]. Many of these effects have been attributed to the content of polyphenolic compounds such as rosmarinic acid, as well as anthocyanins and other flavonoids. Anthocyanins are exclusively present in the flowers of E. amoenum. The main anthocyanins have been identified as the relatively common compounds cyanidin 3glucoside and delphinidin 3-glucoside [19]. Even though specific polyphenolic compounds identified in extracts of E. amoenum have been considered to be responsible for the observed biological activities, the studies performed so far have only been performed on plant extracts and not on pure compounds isolated from E. amoenum. It may also be mentioned that the polyphenolic compounds previously isolated from E. amoenum are relatively common natural products, which are present in several plant species and are not specific to E. amoenum. There is a strong motivation to isolate, identify and utilize the pure active constituents of E. amoenum because of the toxicity of, particularly, extracts of flowers of E. amoenum, which is attributed to the content of toxic pyrrolizidine alkaloids [20]. In this paper, we have demonstrated that E. amoenum indeed contains natural products with very restricted occurrence in nature.

Plant Material
Seeds of E. amoenum were bought at the market in Teheran and cultivated in the Bergen Botanical Garden of the University of Bergen, Norway (coordinates 60.2499995 N 005.5191002 E). Fresh plant material was collected in August 2014 at the botanical garden. The leaves were separated and stored at −20 • C prior to extraction.

Extraction of Compounds and Partitions with Organic Solvents
Fresh leaves of E. amoenum (800 g) were extracted with 3.5 L methanol (MeOH) for 72 h at room temperature. The concentrated extract (440 mL) was purified to partition (twice) against equal volumes of petroleum ether and ethyl acetate, respectively.

Amberlite XAD-7 Column Chromatography
The residual water phase of the extract was further purified on an Amberlite XAD-7 column. The mobile phase consisted of 5 L distilled water, followed by 1 L MeOH-  Aliquots of 200 µL of each of the samples were manually injected into the HPLC column. Each peak in the chromatogram was separately collected in vials. A total of 1-1.5 mL of each of the collected fractions was transferred to HPLC vials for later identifications using analytical HPLC. Following this strategy, 1 mg of compound 1, 1.3 mg of compound 2, 1.4 mg of compound 4 and 6 mg of compound 5 were isolated.

Analytical HPLC
The HPLC instrument was equipped with a multidiode array detector, an autoinjector and a 250 × 4.6 mm, 5 µm Thermo Scientific Hypersil GOLD column. Two solvents were used for elution: A (water-TFA 99.5:0.5; v/v) and B (acetonitrile-TFA 99.5:0.5; v/v). The elution profile of the applied HPLC gradient is shown in Figure S1. The elution profile consisted of initial conditions with 90% A and 10% B, followed by gradient elution for

Spectroscopy
High resolution mass spectra were recorded using a JEOL AccuTOF TM mass spectrometer operated in positive mode at a resolving power of approximately 6000 FWHM. The atmospheric pressure interface zone was tuned for the optimization of ions below m/z 1000 and an electric potential of 2500 V (needle voltage) was applied. The TOF mass selection window was set to detect m/z values up to 2000, and the mass spectral acquisition settings applied were as follows: spectral recording interval = 0.5 s, wait time = 0.03 ns and data sampling interval = 0.5 ns. The samples were analysed as solutions in acetonitrile (~50 µg/mL) and introduced to the ESI spray chamber by weakly acidified (0.01% HCOOH) acetonitrile used as spray reagent. Internal mass drift calibration was performed using a 1 ppm solution of PEG600 (polyethylene glycol average mass 600 u) in acetonitrile.
UV-Vis absorption spectra were recorded on-line during HPLC analysis over the wavelength range 240-600 nm in steps of 2 nm. CD spectra from 180 to 260 nm (light path 1 mm) of the compounds in MeOH were recorded at 20 • C in a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control unit. The spectra obtained were the average of four scans, and MeOH buffer scans were subtracted from the obtained spectra. NMR samples were prepared by dissolving the isolated compounds in deuterated dimethylsulfoxide (DMSO-D 6 ; 99.95 atom % D, Sigma-Aldrich, Saint Louis, MO, USA). The 1D 1 H and the 2D 1 H-13 C HMBC, the 2D 1 H-13 C HSQC, the 2D 1 H-13 C HSQCTOCSY, the 2D 1 H- 13

Conclusions
For the first time, polyphenolic compounds of leaves of E. amoenum have been isolated and identified. The fact that compound 1 belongs to a rare group of natural products which have previously been patented for their significant anti-inflammatory activity by Matano et al. (1993) [16] may rationalize the traditional treatment of inflammations with E. amoenum.