Pseudolycorine N-oxide, a new N-oxide from Narcissus tazetta

Abstract A new N-oxide, Pseudolycorine N-oxide (1) was characterised along with eleven known alkaloids homolycorine (2), O-methylmaritidine (3), 8-O-demethylhomolycorine (4), homolycorine N-oxide (5), lycorine (6), narciclasine (7), pseudolycorine (8), ungeremine (9), 8-O-demethylmaritidine (10), zefbetaine (11) and lycorine N-oxide (12), from Narcissus tazetta. Their structures were established on the basis of spectroscopic data analysis. The extract, fractions and isolated compounds were screened for in vitro cytotoxicity against two human cancer cell lines, human cervical cancer (SiHa) and human epidermoid carcinoma (KB) cells. The study demonstrated the cytotoxic potential of extract and its chloroform and n-butanol fractions. Further, the results revealed the bioactive potential of narciclasine, pseudolycorine and homolycorine alkaloids. However, new N-oxide (1) was not active against these cell lines. Graphical Abstract


Introduction
Narcissus tazetta L. commonly known as nargis, daffodils belong to family Amaryllidaceae. Different species of Narcissus are used traditionally in different countries for the treatment of variety of diseases from the time of Hippocrates (Dweck 2002). In China, bulbs were applied topically to cure tumors (Duke 1983). In Turkey, bulbs were used for treatment of wounds, painful joints, abscesses and skin infections (Dweck 2002;Bastida et al. 1998;Kornienko and Evidente 2008). Different parts of the plants are used for the treatment of tumors, boils, skin diseases, wounds, painful joints, whooping cough, uterine tumors and abscesses (Dweck 2002). Various alkaloids along with flavonoids, lectins and glycosides have been reported from this genus (Bastida et al.1998;Bastida and Viladomat 2002;Rezanka et al. 2010;Fu et al. 2013;Ooi et al. 2000;Jin 2013). Alkaloids of this genus are generally classified as Amaryllidaceae alkaloids (Bastida et al. 1998). Pharmacological activities associated with these plants include acetylcholinesterase inhibitory, antitumor, antiviral and emetic (Bastida et al. 1998;Kornienko and Evidente 2008;Nair et al. 2016;Jin 2016). Galanthamine, which has been approved as a drug for dementia had attracted the attention of researchers to explore more potential molecules from the plants of this family (Heinrich and Teoh 2004).
In continuation to our research interest on characterisation of alkaloids from different plants, (Katoch et al. 2012(Katoch et al. , 2013(Katoch et al. , 2018, present study focussed on isolation of bioactive molecules from the active fractions of N. tazetta. Herein, we report the isolation and characterisation of new N-oxide, pseudolycorine N-oxide (1) along with eleven known molecules. Cytotoxicity of extract, fractions and the isolated alkaloids were evaluated against human cervical cancer (SiHa) and human epidermoid carcinoma (KB) cells. Active chloroform fraction led to the isolation of five known compounds (2-6) and chemical investigation of n-butanol fraction resulted in one new compound (1) along with six known compounds (7-12).

Results and discussion
The extract and four fractions of whole plants of N. tazetta were screened for cytotoxicity. The result revealed the cytotoxic potential of extract and its chloroform and n-butanol fractions against KB and SiHa cell lines. The active n-butanol fraction on further investigation has led to the isolation of one new compound (1) along with six known alkaloids (7-12). The active chloroform fraction led to isolation of five known alkaloids (2-6).
The trans relationship between H-10b (d H 3.21) and H-4a (d H 3.93) was established by their coupling constant (J ¼ 11 Hz) which was typical for lycorine type alkaloids (Bastida et al. 1998). The present data analysis was consistent with previous spectral data of pseudolycorine, a lycorine type alkaloid that has been isolated from N. requienii bulbs (Llabres et al. 1986). However, the signals of C-4a (d c 72.9), C-6 (d c 68.6) and C-12 (d c 69.1) attached to nitrogen atom were deshielded as compared to values in pseudolycorine 8 carbon spectra (Llabres et al. 1986). Further the mass fragmentation shows the loss of oxygen which suggested that the compound contains N-oxide.
The NOESY experiment of compound 1, showed the correlation between H-1 and H-10b suggesting that these protons are on the same side. No correlation was observed between H-2 and H-10b suggesting that these protons are on opposite side (Kaya et al. 2011). Because in contrast when H-2 and 10 b are on the same side as in case of pancratinine D (a lycorine type compound) the correlation between H-2 and H-10b was observed in NOESY experiment (Cedron et al. 2009). Thus, the above data indicated that the compound 1 is the N-oxide of pseudolycorine. However the orientation of N-oxide is tentatively defined by comparing the NMR data of compound 1 with the reported NMR of galanthine N-b-oxide and carinatine N-a-oxide (Zhan et al. 2017) for resonances at H-6a (d H 4.44, d, J ¼ 14.6), H-6b (d H 4.65, d, J ¼ 14.5), and at C-4a (d C 72.9), C-6 (d C 68.6), C-10b (d C 35.3), C-11(d C 27.2), C-12 (d C 69.1) these values closely resembles with galanthine N-b-oxide (Zhan et al. 2017) values at these positions which helped us to deduce the orientation of N-oxide in compound 1 to be in the b-orientation and not in a-orientation. Finally, above data confirmed the proposed structure of compound 1 as pseudolycorine N-b-oxide (Figure 1). Different N-oxides have been reported from plants of this family (Dembitsky et al. 2015;Kobayashi et al. 1991;Kaya et al.2011;Sarikaya et al. 2012).

Cytotoxicity assay
Results for extract and fractions are shown in Figure S3 and their IC 50 values in Table  S2. The extract showed good to moderate activity. Further the result revealed the cytotoxic potential of n-butanol fraction against SiHa cells and chloroform fraction against both SiHa and KB cells. Therefore, chloroform and n-butanol fractions are further taken up for isolation of pure molecules and the isolated molecules were further tested for their cytotoxicity against these cells, however two other molecules could not be tested due to their less quantity. Results for ten pure alkaloids are shown in Figure S4 and their IC 50 values in Table S2. The IC 50 value for these alkaloids lies in the range <10 to >100 lM where dose dependent effect was exhibited by these alkaloids. The study showed that narciclasine is the most potent cytotoxic alkaloid against both the cells and pseudolycorine as second promising alkaloid. Homolycorine act as other potent molecules against both the cells whereas 8-O-demethylmaritidine as subsequent potent molecule against SiHa cells. The new N-oxide was found to be inactive (IC 50 > 100 lM) as compared to pseudolycorine indicating the N-oxide group as a deactivating group against these cells. However, all other alkaloids have IC 50 value more than 100 lM when tested against both the cells.

General experimental procedures
IR spectra were recorded in Shimadzu IR Prestige-21 with ZnSe Single reflection ATR accessory. Optical rotation was recorded in MCP 100 modular circular polarimeter, Anton Paar. Mass spectra were recorded on a Q-TOF triple quadrupole mass spectrometer equipped with an ESI source (Micromass, Manchester, UK). NMR experiments were performed on a Bruker Avance-600 spectrometer with TMS as internal standard. UVvisible absorption spectra were recorded on Cary series UV-VIS spectrophotometer, Agilent technologies. Silica gel of 230-400 mesh size and precoated TLC sheets of silica gel 60 F 254 were used for column chromatography (CC) and thin layer chromatography (TLC), respectively. Spots were visualised under UV light (254 and 366 nm) and further detected on TLC plate after spraying with Dragendorff's reagent for alkaloids, the chemicals used were purchased from Merck India Ltd.

Plant material
The whole plants of N. tazetta was collected in January 2015 from Chauntra (1300 m), Himachal Pradesh, India and was authenticated by Dr. Brij Lal, CSIR-IHBT, Palampur, India. The voucher specimen has been deposited in the herbarium of the institute (PLP 16584).

Extraction and isolation
The whole plants of N. tazetta were washed, chopped and air dried. The dried plant material (2 kg) was grinded to coarse powder and extracted with ethanol at room temperature (3 Â 2 L, 24 h each) in a percolator. The filtrate was dried in vacuum using rotary evaporator and lyophilized in lyophilizer to yield 270 g of dried extract. Dried extract was fractionated after dissolving in water using n-hexane, chloroform and nbutanol (3 Â 200 ml, each). Further the extract and fractions were screened for cytotoxicity. The active chloroform fraction (20 g) was taken up for isolation of pure molecules by subjecting this fraction to CC over silica gel 230-400 mesh size using gradient of chloroform: methanol (10:0; 10:0.5; 10:0.75; 10:1.0; 10:1.4; 10:2.0; 10:2.4; 10:3.0; 10:4.0; 10:5.0, v/v) as eluting solvent to afford 7 fractions (Fr.1-Fr.7) according to their TLC analysis. Further purification of fr.3 led to afford 2 (100 mg) by CC over silica gel using 20% ethyl acetate in methanol as eluting solvent. Fr.5 was subjected to CC over silica gel using 30% ethyl acetate in methanol as eluting solvent to yield 3 (20 mg). Fr.6 yielded 4 (25 mg) by CC over silica gel using 30% ethyl acetate in methanol as eluting solvent. Further fr.7 was subjected to CC over silica gel using 25% ethyl acetate in methanol as eluting solvent to provide 5 (20 mg) and 6 (2 mg).

Cell lines and cell culture
SiHa and KB cells were obtained from National Centre for Cell Sciences, Pune, India. All the cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (Ref. No. 15240-062, Invitrogen, India). Cells were incubated in a CO 2 incubator at 37 C.

Cytotoxicity assay
The cytotoxicity was measured using the sulforhodamine B (SRB) dye method (Kumar et al. 2015). The cells were incubated at a density of 20000 cells/well in 100 lL of complete medium. Various concentrations (25, 50, 100, 200 mg/mL for extract, fractions and 10, 25, 50, 100 mM for pure compounds) were tested against SiHa and KB cells in 100 lL of complete medium. Cells alone were used as negative control, however Vinblastine (1 lM) used as standard control. The plates were then incubated for 48 h at 37 C. After incubation, 50 mL of 50% trichloroacetic acid was added and incubation was followed for 1 h at 4 C. After this, the plates were washed with tap water (five times) and air-dried. 100 mL of SRB was added to the wells and plates were placed in the dark for 30 min at room temperature. Subsequently, plates were washed with 1% glacial acetic acid (five times) and air dried. After that 100 lL of 10 mM Tris base (Sigma) was added. The absorbance was measured using microplate reader (BioTeK Synergy H1 Hybrid Reader) at wavelength of 540 nm (Katoch et al. 2013). The growth inhibition rate was calculated as percentage of parallel negative controls and further IC 50 values were calculated.

Conclusion
In conclusion, the present study reports isolation of a new N-oxide, along with eleven known amaryllidaceae alkaloids from N. tazetta. The N-oxide alkaloid is the new one from this family. Cytotoxic studies of the extract and fractions against SiHa and KB cells showed chloroform and n-butanol fraction as the active fractions of the extract. Further the cytotoxic studies of the isolated alkaloids from the respective active fraction indicates narciclasine as the most active and pseudolycorine, homolycorine as the potent alkaloids and new N-oxide alkaloid was found inactive against these cells.