New isoquinoline alkaloid from Carduus crispus L

A new isoquinoline alkaloid, along with other known 4 bioactive compounds (1 isoquinoline alkaloid, 2 flavonoids, 1 coumarin), were isolated from the ethanol extract of aerial parts of Carduus crispus L. Crispine А N-oxide was newly isolated compound, two of them (quercetin and rutin) were compounds isolated for the first time from indicated plant. The structures of these compounds were elucidated and confirmed with spectroscopic methods e.g. 1 H-NMR, 13 C-NMR, EI-MS, COSY, HSQC, HMBC, and by comparison with literature data.


Introduction
he genus Carduus (Compositae) comprises 95 species all over the world [1]. Two species (C.crispus L. & C.nutans L.) are widely distributed in Mongolia [2]. Investigations of chemical constitutes showed that flavonoids, alkaloids and coumarins are the main components of this genus [1,3,4,5]. C.crispus is used in Mongolian traditional medicine for treatment of gland's cancer and as anaelgestics [6]. It has been shown that this plant has antioxidant and cell wall strengthening activity. It has been established the cytotoxic activity of the alcoholic extract of C.crispus. [3]. Isoquinoline alkaloids (crispine A-E, carcrisine A, B) and flavone glycoside were isolated from this plant collected in Inner Mongolia have been reported [1,3,5].
Herewith, we report about the isolation and structural elucidation of a new isoquinoline alkaloid; crispine A N-oxide. The 1 H and 13 C NMR spectral data of this compound are given for the first time.

Apparatus
Melting point was determined on a Stuart SMP 10 apparatus. FABMS were measured on Bruker APEX II spectrometer. 1 H NMR and 13 C NMR (400 MHz) spectra (all in CDCl 3 ) were recorded with a Bruker AM 400, using TMS as internal standard. Silica gel 60 (Merck 0.063-0.200 mesh) was used for column chromatography. Al 2 O 3 plates were used for TLC. Plates were visualized by spraying with Dragendorff's reagent.
samples were dried at room temperature and grinded.

Extraction and isolation
The plant material (4.5kg) was weighed and extracted with 95% ethanol three times. The ethanol extract was concentrated by reducing pressure to dryness. After was added 5% sulfuric acid to adjust pH 1 and thoroughly shaked. The yielding alkaloidal acidic solution was filtered and treated with NaHCO 3 to adjust pH 5. Basic solution was extracted with chloroform. Distillation of chloroform solution gave extract-A 25g. Adding to acidic solution Na 2 CO 3 to adjust pH 8 and following extraction with chloroform after distillation yield extract-B, 23g. The extract-B was chromatographed over a silica gel with gradient CHCl 3 -MeOH (100→99:1→70:30) to give 85 fractions. Fraction 35-39 were chromatographied on Al 2 O 3 column eluted with CHCl 3 -MeOH (98:2) to yield compound CC-1 (64 mg).

Results and Discussion
The structural identification Isoquinoline alkaloids were isolated from the ethanol extract of the aerial parts of C.crispus. C NMR spectral data with those of crispine A [3]. As shown in Table 1   Researchers have established that, chemical shifts of carbon atoms adjacent to N atom undergo a downfield shift due to oxidation of nitrogen compounds to N-oxides [7]. This proves convertion of nitrogen atom in crispine A into N-oxide form. Because of absence of any information in literature about crispine A N-oxide we assign that crispine A N-oxide is new naturally occuring compound (Fig.1). Chemical method have used for provement of chemical structure of crispine A N-oxide. We have carried out reduction (deoxygenation) of crispine A N-oxide and convert it to crispine A. The spectral data of newly synthesized product was similar to spectral data of crispine A and proves that crispine A is a precursor of crispine A N-oxide.

Method of deoxygenation of crispine A
Crispine A N-oxide was dissolved in 5ml 5% sulfuric acid, added Zn powder, shaked permamently during 24 hours for providing chemical reaction. After end of reaction, the solution was filtered. Filtrate was alkalified by 5% sodium hydroxide to adjust pH 8-9 and was extracted by chloroform. Chloroform extract was concentrated in vacuo until dryness. Residue was chromatographed by using TLC with pure crispine A. Only one spot of crispine A have been detected in chromatogram, that prove that crispine A Noxide have been completely converted to crispine A by deoxygenation reaction (Fig.2). It is known to be that this method widely used for deoxygenation of N-oxides [7].

Conclusion
New isoquinoline alkaloid has been isolated from the aerial parts of C.crispus L. It's structure was confirmed by chemical and spectral analysis and named as crispine A Noxide.