New Phenolics from Linum mucronatum subsp. orientale

Materials

All solvents were of analytical grade and purchased from Sharlau or Caledon Company. Other reagents and materials were provided by Merck (Merck Co., Germany).

General experimental procedures

NMR spectra were recorded in Methanol-d₄ or Chloroform-d solvents on a Buker Avance 400 MHz spectrometer (400 MHz for ¹H and 100 MHz for ¹³C). Gas chromatography-mass spectrometry analyses (GC/MS) were performed on a Shimadzu GC-MS-QP 5050A gas chromatograph fitted with a DB-1 (polydimethylsiloxane, 60 m × 0.25 mm i.d.) capillary column. Carrier gas, helium with a column flow rate of 0.9 mL/min, total flow: 8.4 mL/min; injector temperature, 280 ^°C interface temperature, 310 ^°C; injection volume: 0.1 μL of extract in n-hexane (2%); split ratio, 1:5; oven temperature program: 50 ^°C (keep time: 2 min) to 305 ^°C (keep time: 10 min) with rate of 10 ^°C/min, solvent cut time: 5 min were used in the experiment. A Shimadzu LC-10A prep-HPLC coupled with a SPD-M20A detector (190-500 nm) and a prep-C18 column (CLC Shim-pack C18 column, 22 × 250 mm, 15µm) was used for separations. Column chromatography was conducted with silica gel 60 F254 (mesh; 0.063-0.200 mm) (Merck No: 1.10757.1000). Analytical and preparative thin layer chromatography (TLC) was performed on pre coated silica gel 60 F₂₅₄ (0.25 mm and 2 mm, respectively) plates.

Plant material

Aerial parts of L. mucronatum BERTOL. subsp. orientale were collected in June 2012 during the flowering period (Badlou-Miyaneh, Kaghazkonan Protected Area, Iran). A voucher specimen (Tbz-FPh-735) has been deposited in the Herbarium of the Tabriz University of Medicinal Sciences, Tabriz, Iran.

Extraction and isolation

The air-dried and grounded samples of L. mucronatum (100 g) were Soxhlet-extracted with n-hexane-DCM and methanol (1:1 L), respectively. The extracts were concentrated using a rotary evaporator under vacuum and a maximum temperature of 40 ^°C.

The DCM extract (0.9 g) was loaded on a column of silica gel (5×90 cm) and elution was done by gradient mixtures of chloroform in n-hexane (0→100). The eluted fractions (136 fraction; 10 ml each) were monitored by TLC on silica gel under the UV light (254 nm) before and after applying 70% H₂SO₄ in ethanol as spraying reagent.¹⁵ Lignans produced purple spots with H₂SO₄ reagent following heating in 120 ^°C for 5 min. The similar fractions were combined together and finally 11 sub-fractions were yielded. Lignan-containing sub-fractions were further purified using preparative TLC by double running in methanol-chloroform (10:90 v/v) and toluene-acetone (65:35 v/v), respectively. The isolated materials were extracted from silica gel and purified by crystallization/re-crystallization procedure. This procedure afforded two off-white lignans: I (1.8 mg) and II (2.3 mg). The structure of compounds I and II were elucidated using NMR spectroscopy and co-chromatography with known lignans on silica gel plate. Isolation and identification of compound III from mother liquor of crystallization was done using GC/MS technique. On the other hand, the volatile compounds of less polar sub fractions were analyzed by GC/MS technique.

A portion of the MeOH extract (2 g) was subjected to Sep-Pak fractionation using a step gradient of MeOH-water mixtures (10:90, 20:80, 40:60, 60:40, 80:20 and 100:0). Analysis of the 20% methanolic Sep-Pak fraction (234 mg) by preparative HPLC (Shim-Pak ODS column, 22×250 mm, 15 µ; mobile phase: 0-50 min, MeOH from 10% to 30% in water; 50-62 min, 30% MeOH in water; 62-64 min, MeOH from 30% to 10%; flow rate 20 mL/min; detection at 220, 280 and 350 nm) afforded compound IV (18 mg). A similar analysis of the 40% methanolic Sep-Pak fraction (312 mg) (mobile phase: 0-50 min, MeOH from 30% to 50% in water; 50-62 min, 50% MeOH in water; 62-64 min, from 50% to 30%; flow rate 20 mL/min) yielded compound V (32 mg).

Structural characterization

The DCM extract of L. mucronatum aerial parts was fractioned as described previously. The lignan-containing fraction was separated by a combination of CC and preparative TLC and a novel aryltetralin lignan (compound I) together with podophyllotoxin (compound II) were yielded. Some known alkanes were also isolated from the mother liquors of the crystallization along with the compound III (Table 1).

Compound I (6-MeO-α- peltatin), an off-white powder together with a minor impurity of 3-dodecylcyclohexanone III, was separated and purified by repeated crystallization (Fig. 1). Its structure was determined by ¹H, ¹³C-NMR spectroscopy. The ¹H- NMR of compound I was very similar to spectroscopic data of β-peltatin‏ with minor differences, especially in chemical shifts of H-3 and H-7 signals (Table 2).¹⁶ Obviously, the observed differences could be due to methoxy group which attached to C-6 position. The ¹³C-NMR data of compound I is shown in Table 3.

Fig. 1

Structure of 6-MeO-α- Peltatin and dodecylcyclohexanone isolated from L. mucronatum DCM extract

Podophyllotoxin was identified by direct comparison with authentic sample on TLC, according to Wagner et al, 1996.¹⁵ The aryltetralin lignans were isolated from various Linum species such as L. alba, L. flavum and L. capitatum.¹⁶ However, phytochemical studies are continuing on this section. This is the first report on occurrence of 6- MeO-α- peltatin, in the aerial parts of Linum species.

After fractionation of methanol extract on a SPE (C18) column, preliminary analyses of the yielded fractions with HPLC and on-line UV spectra (diode array detection: range of 220-500 nm.) revealed the presence of several flavones derivatives. The prep-HPLC of fractions afforded 2 major compounds IV and V, which were fully characterized by spectroscopic techniques (Fig. 2). The UV spectrum of IV( λmax: 270 and 336 nm) was resemble to that of apigenin and was identified as 6-C-[β-D-glucuronopyranosyl (1→6)-O-β-D-glucuronopyranoside] apigenin (Mucronatoside1) by comparison of its ¹H and ¹³C- NMR spectra (Table 4) with previously reported literature data.¹⁷

Fig. 2

Structure of two flavonoid glycosides from L. mucronatum

The UV spectra of V and IV were very similar to each other, but their NMR data was different. The ¹H- NMR spectrum of V had two doublets at δ6.50, 1.8 Hz (H6) and δ6.88, 1.8Hz (H8) typical of a 5,7 disubstituted flavonoid moiety. The ¹³ C- NMR spectrum (Table 4) was in agreement with the structure of the 7-methyl apigenin, and the connection of glycosyl moiety to position C4' and the interglycosidic (l→6) linkage was confirmed, as well. On the other hand, presence of the anomeric proton and carbon signals at δ 5.21 (d 4.3 Hz) and 103.87, respectively, confirmed the arabinose structure for the second sugar. Thus the structure of V is as 7-O-methyl[-4'-O-β-D-arabinofuranosyl (1→6)-O-β-D-glucuronopyranoside] apigenin (Mucronatoside 2). According to the published data, the 5, 4'-dihydroxy-7-methoxyflavone (genkwanin) derivatives were previously found in some genera of the families Aspleniaceae, Asteraceae, Cistaceae, Eupomataceae, Lamiaceae, Myrtaceae, Pteridaceae, Saxifragaceae and Betulaceae.^18,19 However, there have been so far no reports on the occurrence of compound V in any Linum species.