New Diphenol and Isocoumarins from the Aerial Part of Lawsonia inermis and Their Inhibitory Activities against NO Production

Lawsonia inermis Linn (Lythraceae), also known as henna, is a small shrub or tree distributed throughout Taiwan’s Lanyu Island, in North Africa, and in Australia. Its leaves are used as a folk medicine for the treatment of external hemorrhage and fingernail abscesses. Investigation of the ethyl acetate (EtOAc)-soluble fractions from methanol extract of the aerial part of Lawsonia inermis has led to the isolation of a new diphenol, (Z)-4,4′-(prop-1-ene-1,3-diyl)diphenol (1), two new isocoumarin carbonates, inermiscarbonates A (2) and B (3), and six known compounds, 4′-hydroxyflavanone (4), apigenine (5), kampferol (6), luteolin (7), quercetin (8), and (-)-catechin (9). Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. Compounds 1 and 4–9 were evaluated for the inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated product of nitrite in RAW 264.7 cells with IC50 values of 5.63, 15.72, 8.67, 6.67, 6.17, 7.61, and 14.52 μg/mL, respectively.


Isolation and Structural Elucidation
The MeOH extract of the aerial part of Lawsonia inermis was concentrated to give a brown-green residue, which was suspended in water and partitioned with EtOAc and H2O, successively. The combined ethyl acetate (EtOAc)-soluble fraction was purified by repeated silica gel column chromatography and normal phase semipreparative high-performance liquid chromatography (HPLC) to obtain a new diphenol, (Z)-4,4′-(prop-1-ene-1,3-diyl)diphenol (1), two new isocoumarin carbonates, inermiscarbonates A (2) and B (3), and six known flavonoids, 4-9. The identification of the known compounds was established through direct comparison with the published physical and spectral data.
Compound 1 was obtained as a pale yellow powder. Its molecular formula was calculated as C15H14O2 from the analysis of its high resolution electron spray mass spectrometry (HR-ESI-MS) data, corresponding to nine degrees of unsaturation. The UV spectrum exhibited conjugated absorption at λ (log ε) 285 (4.32) and 296 (4.24) nm. Its IR spectrum showed absorption bands for

Isolation and Structural Elucidation
The MeOH extract of the aerial part of Lawsonia inermis was concentrated to give a brown-green residue, which was suspended in water and partitioned with EtOAc and H 2 O, successively. The combined ethyl acetate (EtOAc)-soluble fraction was purified by repeated silica gel column chromatography and normal phase semipreparative high-performance liquid chromatography (HPLC) to obtain a new diphenol, (Z)-4,4 -(prop-1-ene-1,3-diyl)diphenol (1), two new isocoumarin carbonates, inermiscarbonates A (2) and B (3), and six known flavonoids, 4-9. The identification of the known compounds was established through direct comparison with the published physical and spectral data.

Inhibitory Activity against Nitric Oxide Production
Nitric oxide (NO) is derived from the oxidation of L-arginine by NO synthase (NOS), and is a mediator in the inflammatory response involved in host defense [13]. In inflammation and carcinogenesis conditions, there is an increased production of NO by inducible NO synthase (iNOS) [14]. The anti-inflammatory effects of the compounds isolated from the Lawsonia inermis were also evaluated for the suppression of lipopolysaccharide (LPS)-induced NO generation in murine macrophage. In this study, the inhibitory activity of three new compounds (1-3) and six flavonoids (4-9) toward NO production was evaluated by the measurement of nitrite/nitrate in LPS-stimulated RAW 264.7 cells. To search for the appropriate concentrations for the above assay, these nine compounds were first tested for their cytotoxic activity against the RAW 264.7 cells, and no significant cytotoxic activities were observed under all tested concentrations. From the results of our anti-inflammatory tests, the following conclusions could be drawn: (a) The high cell viability (>92%) indicated that the inhibitory activities of compounds 1 and 4-9 on LPS-induced NO production did not result from their cytotoxicities; (b) Compounds 1, 6, and 7 exhibited inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide production in RAW 264.7 cells with IC 50 values of 5.63, 6.67, and 6.17 µg/mL, respectively (Table 4). (1) is the most effective among the isolated compounds against LPS-induced NO generation, with IC 50 = 5.63 ± 3.64 µg/mL.

General
UV spectra were obtained with a Shimadzu Pharmaspec-1700 UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan). Infrared spectra were obtained with a Shimadzu IR prestige-21 Fourier transform (FT) infrared spectrophotometer (Shimadzu). 1D-and 2D-NMR spectra were recorded with a Bruker DRX-500 FT-NMR spectrometer (Bruker, Bremen, Germany). Mass spectrometric (HR-EI-MS) data were generated at the Mass Spectrometry Laboratory of the Chung Hsing University (Taichung, Taiwan). Column chromatography was performed using LiChroCART Si gel (5 µM; Merck, Darmstadt, Germany), and TLC analysis was carried out using aluminum pre-coated Si plates (Merck & Co., Inc.), and the spots were visualized using a UV lamp at λ = 254 nm.

Chemicals
The solvents used to open column isolation (Sephadex LH 20 and silica gel column) in the study, such as n-hexane, chloroform, ethyl acetate, acetone, and methanol were ACS grade. The HPLC grade n-hexane, ethyl acetate, and acetone for HPLC isolation, and the deuterated solvent for NMR measurement (CDCl 3 , CD 3 OD) were purchased from the branch of Merck in Taipei

Extraction and Isolation
The dried aerial part (5.0 kg) of Lawsonia inermis was extracted three times with MeOH (50 L each) for 7 days. The extract was concentrated under reduced pressure at 35 • C, and the residue (440 g) was partitioned between EtOAc and H 2 O (1:1) to provide the EtOAc-soluble fraction (fraction A; 132.5 g). Fraction A (132.5 g) was purified by column chromatography (CC) (6.0 kg of SiO 2 , 70-230 mesh; n-hexane/EtOAc/methanol gradient) to afford 14 fractions: A1-A14.

Cell Culture
A murine macrophage cell line RAW 264.7 (BCRC No. 60001) was purchased from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) in a CO 2 incubator (5% CO 2 in air) at 37 • C and subcultured every 3 days at a dilution of 1:5 using 0.05% trypsin-0.02% EDTA in Ca 2+ -, Mg 2+ -free phosphate-buffered saline (DPBS).

Cell Viability
Cells (2 × 10 5 ) were cultured in 96-well plates containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with compounds 1-9 in the presence of 100 ng/mL LPS (lipopolysaccharide) for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 µL of 0.5 mg/mL MTT for 2 h at 37 • C, testing for cell viability. The medium was then discarded, and 100 µL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Measurement of Nitric Oxide/Nitrite
NO production was indirectly assessed by measuring the nitrite levels in the cultured media and serum determined by a colorimetric method based on the Griess reaction. The cells were incubated with different concentrations of samples in the presence of LPS (100 ng/mL) at 37 • C for 24 h. Then, cells were dispensed into 96-well plates, and 100 µL of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min, and the absorbance was measured at 540 nm with a Micro-Reader (Molecular Devices, SpectraMax ® M2e, Sunnyvale, CA, USA). By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured from absorbance at 540 nm.

Statistical Analysis
The data is expressed as means ± standard errors (SE). The IC 50 values were calculated from the dose curves using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc., Chicago, IL, USA, 2002). Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe's multiple range tests).