Camptothecinoids from the seeds of Taiwanese Nothapodytes foetida

Two new alkaloids, 9-methoxy-18,19-dehydrocamptothecin (1) and 5-hydroxymappicine-20-O-β-glucopyranoside (2a/2b as a racemic mixture), together with nine known compounds: camptothecin (3), 9-methoxy-camptothecin (4), 5-hydroxy-camptothecin (5a/5b racemic mixture), 5-hydroxy-9-methoxycamptothecin (6a/6b racemic mixture), diosmetin (7), apigenin (8), apigenin-7-O-glucopyranoside (9), rosin (cinnamyl-O-β-D-glucopyranoside) (10) and amarantholidoside IV (11) were isolated from the immature seeds of Nothapodytes foetida (Wight) Sleumer. The structures were elucidated by spectroscopic analyses. In the present research, compounds 1, 3, 4, 5a/5b and 6a/6b, also showed in vitro cytotoxicity against six cancer cell lines (HepG2, Hep3B, MDA-MB-231, MCF-7, A549, and Ca9-22). Among them, compound 1 exhibited significant cytotoxicity against these cancer cell lines, with IC50 of 0.24-6.57 μM. Furthermore, HPLC profiles were developed for qualitative and quantitative analysis of these active constituents in different parts of this plant, including mature and immature seeds, leaves, stems and roots. The results revealed that compounds 3 and 4 have the highest concentrations, which are found in the roots part of the plant.

The development of camptothecin-containing plants as a cash crop is becoming an important issue in southeastern Asia.Camptotheca acuminata and Nothapodytes foetida were both cultured in Taiwan successfully.N. foetida is the only species native to Orchid Island, where it is used for hedges or firewood and is cultured in Taiton Hsien, Taiwan [13].In 1995, we published a paper that revealed a new camptothecinoid from N. foetida [14].Meanwhile, the anti-cancer agent, "Campto Injection," [Irinotecan Hydrochloride] was approved as a medicine for treating several cancers in Japan, France, and United States with camptothecin originating from Taiwanese N. foetida.Since Taiwanese N. foetida is an important resource for anticancer drugs, this plant has been reinvestigated.A number of camptothecinoids, other alkaloids and phytochemicals have been reported from this plant [13,14,15].In the current investigation, camptothecinoids, including two new ones, have been identified and quantified by HPLC, in different parts of Taiwanese N. foetida, including mature and immature seeds, leaves, stems, and roots.
HRESIMS of the new compound 2 showed an [M+H] + ion at m/z 485.1925, corresponding to the molecular formula C 25 H 29 N 2 O 8 .The IR spectrum indicated the presence of hydroxyl (3397 cm -1 ), lactam carbonyl (1670 cm -1 ) and aromatic functional (1596 cm -1 ) groups.In the 1 H-NMR spectrum of compound 2, the resonances and multiplicities largely matched those of the A, B, and D rings in camptothecin (3), however, in 1 H-and 13 C-NMR spectra, the overlapping signals and two sets of signals for part of molecules could be distinguished, revealing 2 to be a racemic mixture.The absence of geminal protons (H-7) at δ 5.43, the absence of lactone carbonyl (C-21) at δ 172.5, and the presence of a singlet at δ 2.25 (3H, H-17) and a triplet at d 5.27 (1H, J=6.9 Hz, H-20) suggested that 2 lacked the E ring of camptothecin.As another difference in the 1 H-NMR spectra, compound 2 showed a pair of downfield-shifted singlets at δ 6.97/7.00(1H for 2a and 2b in a ratio of 1:1) in place of the proton singlet at δ 5.28 (H-5, 2H) found in 3.In addition, in the 13 C-NMR spectrum, C-5 was found at δ 84.3/84.4 in 2, rather than 50.6 as in 3. Based on this data, a hydroxyl group was attached on C-5.The NMR data for 5-hydroxyl substitution of camptothecinoids could be assigned on the basis of data of the known synthetic product, 5-hydroxycamptothecin [17].The proton signals between δ 3.18 to 4.10 were assigned to a sugar moiety.The coupling constant of the anomeric proton (δ 4.10, d, J=7.8, H-1') indicated the β-configuration, and H-1' showed a HMBC correlation with C-20.This established that the glucose residue was attached at C-20.For the C-20, it could not be found clear positive or negative peak between 300-400 nm.We defined C-20 in an S configuration according to the past literature [18].Because of the limited amount of 2, we could not determine the stereochemistry of C-5 in means of chemical reactions, such as the Mosher ester method.Therefore, the stereochemistry of C-5 remains undefined and the structure of compound 2, was determined as 5-hydroxy-mappicine-20-O-βglucopyranoside.

Qualitative and quantitative analysis
Compounds 1 (1.69 mg), 3 (11.51mg), 4 (3.79 mg), 5a, 5b (8.19 mg), and 6a, 6b (8.12 mg) were obtained from the organic layer.Separation on a reversed phase C-18 column (250×4.6 mm) with acetonitrile-H 2 O (25:75, v/v) as a solvent system provided good separation of these camptothecinoids.The HPLC profile of a mixture of compounds 1, 3, 4, 5a/5b, and 6a/6b carried out by the above condition is shown in Figure 2A.The crude extracts from different parts of N. foetida were prepared and analyzed, and the HPLC profiles were shown in Figure 2B.immature seeds; and 6. mature seeds.
Calibration curves were established with six concentrations (0.015-0.5 mg/mL) of compounds 3 and 4 (see Experimental section).The linearity of the plot of concentration (x, mg/mL) for each compound versus peak area (y) was investigated.Under these analytical conditions, good linearities for all of the calibration curves were obtained (Table 2).As indicated in Table 3, camptothecin (3) was more abundant in the roots (9.73%) than other parts of N. foetida.The other important component, 9-methoxy-camptothecin (4), also showed the highest concentration in the roots.

General
Optical rotations were recorded on a JASCO P-1020 polarimeter.IR spectra were measured on a Perkin Elmer system 2000 FT-IR spectrophotometer in CHCl 3 .UV spectra was obtained on a JASCO V-530 UV/VIS spectrophotometer.NMR spectra were run on Varian Unity-plus 400 MHz FT-NMR, Varian Mercury-plus 400 MHz FT-NMR and Varian Unity-plus 600 MHz FT-NMR.The chemical shift (δ) values are in ppm (part per million) with DMSO and CD 3 OD as internal standard, and coupling constants (J) are in Hz.Low resolution ESI-MS spectra were obtained on an API 3000TM (Applied Biosystems) in positive or negative mode (solvent: CH 3 OH), high resolution ESI-MS spectra were obtained on a Bruker Daltonics APEX II 30e spectrometer in positive or negative mode (solvent: CH 3 OH).Circular dichroism was measured on a Jasco J-810 spectrophotometer.Shimadzu LC-10AT pump, Shimadzu SPO-M10A diode array detector, Shimadzu SIL-10A autoinjector and Varian Polaris 5 C18-A (250×4.6mm)were employed for the HPLC qualitative and quantitative analysis.Jasco PU-980 pump, Jasco UV-970 detector and Discovery HS C18 column (250×10 mm) were employed for separations.Silica gel 60 (70-230, 230-400 mesh, Merck), Sephadex LH-20 and Diaion HP-20 were used for column chromatography (CC).Silica gel plates (Kieselgel 60, F254, 0.20nm, Merck) were used for TLC.

Plant Materials
The immature seeds (2.28 Kg), mature seeds (2.3 Kg), stems (1.21 Kg), leaves (0.2 Kg) and roots (0.15 Kg) of N. foetida, were collected in 2004 from the farm of Taiwan Sugar Corporation, Tainan, Taiwan.Only immature seeds were used to isolate pure constituents.

Crude samples prepared from different parts of N. foetida for qualitative and quantitative analysis
Dried leaves, stems, roots, mature seeds, and immature seeds were ground, 10 g were weighed and extracted with methanol at 24-25°C for 5 days, to mimic the large scale extraction conditions.All extracts were evaporated under reduced pressure to give five residues (leaves: 3.05 g, stems: 0.93 g, roots: 1.60 g, mature seeds: 0.44 g and immature seeds: 0.42 g).Each dry extract (5.0 mg) was dissolved in DMSO/MeCN (0.6 mL), filtered on a pre-column and injected to HPLC (each injection was 30 µL).

Preparation of reference samples
Camptothecinoids were isolated from immature seeds of N. foetida and their structures were determined by spectroscopic methods.These reference compounds were used for qualitative analysis.Standard stock solutions containing 1 mg/mL of compounds 3, 4 were prepared with 1 mg compounds 3, 4 in 1.0 mL DMSO for quantitative analysis.Standard sample solutions were injected (injection volume: 10 µL) directly into the HPLC system.

Analytical HPLC
HPLC analyses were performed on a Shimadzu model LC-10AT HPLC (Japan) equipped with a two solvent delivery system, a SIL-10A automatic sample injector and a model SPD-M10A diode array detector.The detector was at 365 nm.Data acquisition and quantification were performed using Shimadzu Class-VP software (version: 6.12SP5).Chromatography was carried out on a Varian Polaris 5 C18-A (250×4.6mmi.d.) column.Isocratic elution was performed with water and HPLC-grade acetonitrile/H 2 O (25:75, v/v) at a flow rate of 1 mL/min.The solvents were filtered through a 0.45 μm filter prior to being used.Total HPLC running time for the assay was 22 minutes.

Calibration
In the standard HPLC chromatogram, six different concentrations of compounds 3 and 4, in the linear range of 0.015 to 0.5 mg/mL, were prepared in DMSO, respectively.Six replicates (n=6) of each concentration were subjected to HPLC.

Cytotoxicity assay
Fractions and isolates were tested against lung (A549), breast (MEA-MB-231 and MCF7), and liver (HepG2 and Hep3B) cancer cell lines using established colorimetric MTT assay protocols [21].Doxorubicin was used as a positive control.Freshly trypsinized cell suspensions were seeded in 96well microtiter plates at densities of 5000-10000 cells per well with tested compounds added from DMSO stock solution.After 3 days in culture, attached cells were incubated with MTT (0.5 mg/mL, 2 h) and subsequently solubilized in DMSO.The absorbance was measured at 550 nm using a microplate reader.The IC 50 is the concentration of agent that reduced cell growth by 50% under the experimental conditions.

Table 2 .
Regression equations and retention times of compounds 3, 4 determined for the HPLC assay.

Table 3 .
The contents of compounds 3, 4 from different parts of N. foetida (g/Kg).