Virginicin , a New Naphthalene from Kosteletzkya virginica ( Malvaceae )

A new naphthalene, 8-methoxy-2,7-dihydroxyl-4-(1'-hydroxyl-isopropyl)-6-methyl-1-naphthaldehyde, together with twelve known compounds were isolated from the tubers of Kosteletzkya virginica. Their structures were elucidated by spectroscopic methods, including 1D-, 2D-nuclear magnetic resonance (NMR) and high-resolution time of flight electrospray ionization mass spectrometry (HRTOFESIMS). Some compounds were evaluated for their potential in scavenging diphenyl-picryl hydrazyl radical (DPPH•), inhibition of nitric oxide (NO) induced by lipopolysaccharide (LPS), and cytotoxic activity. The new compound showed activities against DPPH•, NO, human acute promyelocytic leukemia (HL-60) and human colorectal adenocarcinoma (LOVO), with IC50 of 34.6, 12.5, 40.5, 31.7 μmol L-1 respectively.


Introduction
Kosteletzkya virginica (L.) Presl.(Malvaceae) is a perennial root herb native to the saline tidal marshes in the United States east coasts from Texas to the Delaware, and was first introduced to China in 1992-1993. 1As a halophytic species with potential for agroecotechnology in Jiangsu Province, China, 2 K. virginica produces a relatively high yield of seeds.The hulled seeds having a high protein and fat content (25-35% protein, 20-30% oil composed largely of unsaturated fatty acids, high potassium and low sodium), 3 whereas the oil can be used as an edible oil or biodiesel. 4Mucilage from seed is suitable for industrial use as candy or gum.Considering these properties, K. virginica has a great potential for being developed to food (feed) or oil crops, and has been served as a candidate species of the current development and utilization of saline flats in the east of China such as Liaoning, Jiangsu, and Shandong Provinces.In addition, the fleshy root of this plant is used in American Indian traditional medicine for the treatment of the upper respiratory tract inflammation. 5 our study, we have isolated and identified the structures of a new naphthalene (1) and twelve known compounds (2-13).The antioxidant, cytotoxic and antiinflammatory activities of compounds 1-7 were also evaluated.

General comments
Melting points were measured using a XT-4 Boetius micro-melting point apparatus and were uncorrected.Infrared (IR) spectra were recorded on a Nexus 870 FT-IR spectrometer.HR-ESI-MS spectra were measured on Agilent 6530 UPLC/Q-TOF/MS spectrometer.NMR data were acquired on a Bruker DRX500 or 300 NMR spectrometer with 1 H and 13 C NMR observed at 500 or 300 and 125 or 75 MHz, with chemical shifts (δ) given in ppm with reference to the solvent, and coupling constants (J) in Hz.Silica gel (200-300 mesh) and Sephadex LH-20 for column chromatography were purchased from Qingdao Marine Chemical Factory, and Pharmacia Biotech, respectively.HPLC analyses were performed with a YMC ODS-5 μm (150 × 4.6 mm) column, an Agilent pump 1100 and a ELSD detector Alltech 500.All other chemicals used in this study were of analytical grade.TLC analyses were carried out on silica gel 60 F 254 (Merck) plates.The compounds were monitored by spraying 1% vanillin-H 2 SO 4 reagent, followed by heating at 105°C for 1-2 min.Bioassays were performed with an Infinite M200 microplate reader which was purchased from TECAN.

Plant material
In this study, tubers of K. virginica were collected at Yancheng, Jiangsu province, China, in May, 2010, and identified by Prof Yuan Changqi.A voucher specimen (20101001) was deposited in Jiangsu for Research and Development of Medicinal Plants, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences.

DPPH assays
A solution of DPPH • radical (0.16 mmol L -1 ) in absolute ethanol was prepared.Compounds 1-7 were dissolved at a concentration of 5 mmol L -1 in DMSO and diluted into several concentrations.100 μL of DPPH • solution, 20 μL of samples and 80 μL distilled water were added into wells of a 96-well plate, making the final concentrations of 500, 250, 125, 62.5 and 31.25 μmol L -1 .The absorbance (A i ) was measured at 517 nm after the 96-well plate was heated at 25 °C for 15 min.The absorbance of 20 μL of DMSO and 100 μL of absolute ethanol were measured respectively as A 0 and A j .All experiments were conducted in triplicate and Vitamin C (VC) was used as the standard antioxidant.The radical scavenging activity of each sample was calculated by the DPPH • inhibition percentage (%I DPPH ) according to the following equation: [6][7] The antioxidant activities of the tested compounds were expressed as IC 50 , defined as the concentration corresponding to %I DPPH equal to 50%.Vol. 26, No. 4, 2015   Measurement of NO production and cell inhibition in LPSactivated murine macrophages cells (RAW264.7)RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).In order to measuring NO production, the cells were dispensed in wells of a 96-well plate (2.5 × 10 5 cells/well).24 h later, different concentrations of compounds 1-7 were added to the cells for 30 min.Then the cells were stimulated with LPS (0.1 μg mL -1 ) for 24 h under the conditions of 37 °C and 5% CO 2 .50 μL of the supernatant was harvested and mixed with 50 μL of Griess reagent.After culturing 15 min at room temperature, the absorbance was then read at 540 nm.Then, a [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] (MTT) assay can be used for measuring cell inhibition. 8Indomethacin was tested as a positive control.

Cytotoxicity assays
HL-60 was maintained in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% heat-inactivated bovine serum, 100 units mL -1 penicillin and 100 μg mL -1 streptomycin.Lovo were grown under the same conditions described above, except for the basal medium (DMEM medium instead of RPMI-1640).The media were changed every two day.All the above cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .
The effect of compounds 1-7 on the viability of tumor cells was determined with the MTT assay.The cells were plated at 10,000 cells per well in 100 μL of complete culture medium and treated with various concentrations of the compounds in 96-well microtiter plates.Each concentration of the compound was repeated in six wells.After incubation for 72 h at 37 °C in a humidified incubator, cell viability was determined.MTT (5 mg mL -1 ) was added to each well and incubated for 4 h.Then 100 μL of the solubilization solution (10% sodium dodecyl sulfate (SDS) in 0.012 M HCl) were added into each well, and the plates were standed overnight in the incubator.Absorbance was recorded on a microplate reader at a wavelength of 570 nm (reference wavelength: 690 nm).The 50% inhibitory concentration (IC 50 ) was determined by interpolation from dose-response curves.Cis-platin was tested as a positive control.