Rubiothiazepine a Novel Unusual Cytotoxic Alkaloid from Ixora undulata Roxb. Leaves

Leaves of Ixora undulata Roxb. were investigated phytochemically for the first time for its alkaloidal content, which resulted in the isolation of a novel unusual thiazepine alkaloid glycoside identified as 7-[(β-D-glucopyranosyl)oxy]-6hydroxy-2-methoxy-4,5-dihydro-1,3-thiazepine and trivially named as Rubiothiazepine. Its structure was determined on the basis of 1D and 2D NMR (1H-, 13C-NMR, 1H-1H COSY, 1H-13C HETCOR, 1H-13C HMBC, 1H-15N HMBC and DIFNOE), high resolution ESI-CID-MS/MS, UV and IR spectroscopy. The isolated compound showed cytotoxic activity against EL4 (Murine Leukemia) with IC50 >100 μg/mL, and also showed cytotoxic and HIV-1 activity against MT-4 and HIV-1IIIB with CC50 >100 μg/mL and EC50 >100 μg/mL, respectively. *Corresponding author: Magdy Mostafa Desoky Mohammed, Danish Institute of Agricultural Sciences, Department of Food Science, Research Center Aarslev, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark, Tel: 00201022610332; E-mail: melhenawy111@gmail.com Received November 26, 2013; Accepted February 05, 2014; Published February 07, 2014 Citation: Mohammed MMD, Ibrahim NA, Chen M, Zhai L (2014) Rubiothiazepine a Novel Unusual Cytotoxic Alkaloid from Ixora undulata Roxb. Leaves. Nat Prod Chem Res 2: 128 doi:10.4172/ 2329-6836.1000128 Copyright: © 2014 Mohammed MMD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Ixora undulata Roxb. is native to North Africa, Southern Europe and Asia, and it can be cultivated in moderate climates. I. undulata is an evergreen shrub 6 to 8 ft., it belongs to family Rubiaceae which comprises of 400 genera, from which the genus Ixora consists of 300 species; three of which were cultivated in Egypt; Ixora coccinea, Ixora finlaysoniana and Ixora undulata [1]. The genus Ixora has been used in the Ayurvedic system of medicine for a variety of ailments e.g., leaves in diarrhea, antimicrobial and anti-inflammatory; roots in hiccough, fever, scores, chonic ulcers and skin diseases; flowers in catarrhal bronchitis, dysentery, cytotoxic and antitumor principles. The aerial parts were used as central nerves system (CNS) depressant, hypothermic and semen coagulant activity [2]. Literature survey revealed little information concerning the chemical constituents, Saleh et al. [3] reported the possible presence of tannins in the plant leaves. Mohammed [4] reported the isolation of D-mannitol from I. undulata leaves, which was reported to ameliorate some metabolic disorders in schistosomal mansoni infected mice [5]. Thus, our group started the phytochemical investigation searching for the alkaloidal content of I. undulata leaves, and then evaluating the cytotoxicity of the isolates against EL4 (Murine Leukemia), and its anti-HIV-1 activity against MT-4 and HIV-1 IIIB .

Plants materials
The leaves of Ixora undulata were collected in May 2006, from El-Orman Botanical Garden -Giza Governorate -Egypt. The plant samples were kindly identified by Miss. Tressa Labib-Head of Taxonomist Specialists at the garden, a voucher specimen (No.23) of the plant was kept at the Herbarium of El-Orman Botanical Garden.

Apparatus
Melting point: (uncorrected) was determined on a BÜCHI melting point apparatus. 1 H-, 13 C-NMR, 1 H− 1 H COSY, 1 H− 13 C HETCOR, DEPT and NOE spectra were obtained using a pulse sequence supplied from Varian VXR-Unite-300 MHz spectrometer (in DMSO-d 6 ). Chemical shifts were given in values (ppm) relative to trimethylsilane (TMS) as an internal reference. Nitrogen ( 15 N) chemical shifts are reported relative to liquid ammonia using a nitromethane chemical shift of δ 380.2. Gradient 1 H-13 C HMBC (J, 10 Hz), and gradient 1 H-15 N HMBC experiments (J NH , 5 Hz) were performed with standard pulse programs on a Bruker Advance DPX 500. ESI/MS: was obtained using Nano-electrospray tandem (MS/MS) mass spectrometry on a hybrid quadrupole time-of-flight (Q-TOF) MS instrument equipped with Protana's Nano-ESI source for HRESI/MS and Nano-spray needles from Proxeon (Applied Biosystems/MDS Sciex) (QSTAR, prototype, PE-Sciex, Canada). Tandem (MS/MS) spectra were interpreted using the programs BioMultiView (PE Sciex, Canada) and GPMAW (Lighthouse Data, Denmark).
For accurate mass measurements the instrument was calibrated using a 10 mM solution of NaI in isopropanol/water. The instrument's mass scale was calibrated for each determined ion mass using the cluster ions Na n+1 I n + closest to the sought mass. Collision induced dissociation (CID) spectra were obtained using N 2 in the collision cell and collision energies between 30 -40 eV (E lab ). Analyses were first conducted using ESI/MS in positive mode to obtain ionized molecular species. Then tandem MS/MS spectra were obtained by Collision Induced Dissociation (CID) of the [M+Na] + ion. The product ion spectra were obtained in the continuous mode of acquisition of the quadruple analyzer. Reversed Phase-High Performance Liquid Chromatography (RP-HPLC): Consists of L-6200 Intelligent Pump (Merck-HITACHI) equipped with UV-VIS Detector SPD-10AV (SHIMADZU), the column used in HPLC separation is (20ϕ x 250 mm, Develosil ODS-HG-5, Nomura Chemicals). Samples of 8 mL volume were injected into 10 mL loop (after prefiltration with Nylon Filter 0.45 µm). HPLC solvents used for all analyses were of grade M (Sigma-Aldrich chemie, UK) with ultra-pure water. All solvents used were of AR grade. Kiesel gel 60 F 254 (Merck) was used for analytical TLC.

Assays for cytotoxicity
The cell line EL 4 (Murine Lymphoma) was purchased from National Cancer Institute (NCI). A standard high-flux anticancer-drug screening method was employed in this study [6]. Briefly, cancer cells were plated in 96-multiwell plate (10 4 cells/well) for 24 h before treatment with the samples to allow attachment of the cell to the wall of the plate. Different concentrations of the samples under test (0, 1, 5, 10, 25, 50, 100, 250, 500, and 1000 μg/mL) were added to the cell monolayer, triplicate wells were prepared for each individual dose. The monolayer cells were incubated with the test samples at 37 °C for 48h in atmosphere of 5% CO 2 . Cultures were fixed with trichloroacetic acid, then stained with sulforhodamine B and the colour intensity was measured at 490 nm by ELISA reader. All experiments were performed at least two times in triplicates. Thapsigargin (T-9033, >96%, Sigma) was used as positive potent cytotoxin with IC 50 1.9 ± 0.5 μM/mL. Data were given as IC 50 (μM/mL) mean ± SEM from 4 different experiments.

Assay for antiviral
It was performed at department of science and biomedical technology. Cittadella University, Monserrato, Italy Samples were solubilized in DMSO at 100.000 γ and then diluted in culture medium.
HIV titration: Titration of HIV was performed in C8166 cells by the standard limiting dilution method (dilution 1:2, four replica wells per dilution) in 96-well plates. The infectious virus titer was determined by light microscope scoring of syncytia after 4 days of incubation. Virus titers were expressed as CCID50/mL.

Anti-HIV assays:
The activity of tested compound against multiplication of HIV-1 wild type IIIB, N119, A17, and EFV R in acutely infected cells was based on inhibition of virus-induced cytopathicity in MT-4 cells. Briefly, an amount of 50 μL of culture medium containing 1.0 x 10 4 cells was added to each well of flat-bottom microtiter trays containing 50 μL of culture medium with or without various concentrations of test compounds. Then an amount of 20 μL of HIV-1 suspensions (containing the appropriate amount of CCID 50 to cause complete cytopathicity at day 4) was added. After incubation at 37°C, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) method [7]. The cytotoxicity of test compounds was evaluated in parallel with their antiviral activity and was based on the viability of mock-infected cells, as monitored by the MTT method.

Results and Discussion
Compound (1)   In accord with the molecular formula C 12 H 19 NO 8 S, 12 carbon signals were resolved in the 13 C-NMR spectrum (Table 1), and categorized by DEPT 135° and HETCOR experiments as; one methoxyl, one methylene, nine methines and one quaternary carbon, which can be divided into one methylene and five methines corresponding to glucosyl moiety, and suggested that the other signals (four methines, one methoxyl and one quaternary carbons) belongs to the aglycone part, this suggestion was confirmed from 1 H− 1 H COSY (Figure 1), which showed three cross-peaks represented the correlation of the broad doublet signal at δ H 4.39 with the signals at δ H 5.04, 6.12 and 6.21, another cross-peak corresponding to the correlation of the signal at δ H 6.12 with that at δ H 6.21 with J value (10.9 Hz) corresponding to AM system in the cisconfiguration. The CID-MS/MS spectra of the sodium adduct [M+Na] + of compound (1) showed a molecular ion peak at m/z 247 corresponding to molecular formula C 7 H 12 O 6 SNa of m/z (224 + 23) confirmed that, the attachment of the glucosyl moiety to be at C-7 adjacent to the sulphur atom. The EIMS of compound (1) showed a fragment ion peak at m/z 91 corresponding to 1,3-thiaza group (HS−C(OCH 3 )=NH). The above data suggested that compound (1) has 1,3-thiazepine glucoside skeleton, and its backbone was established as shown in the HMBC (Figure 2), which showed the correlations of the methoxyl and four methene protons with each other and their correlations toward the quaternary carbon of the thiazepine ring. The glucosyl moiety was proved to be attached to C-7 of the thiazepine ring as indicated from the cross-peak between H-1` at H 4.44 and C-7 at δ C 82.8, another two cross-peaks revealed the correlations of the quaternary carbon (C-2) at δ C 147.9 with the methoxyl signal at δ H 3.86 and H-4 at δ H 6.21.