Five Withanolides from the Leaves of Datura metel L. and Their Inhibitory Effects on Nitric Oxide Production

Four new withanolides named dmetelins A–D (compounds 1–4), along with the known compound 7α,27-dihydroxy-1-oxo-witha-2,5,24-trienolide (5) were isolated from the leaves of Datura metel L. (Solanaceae). Their structures were elucidated on the basis of detailed analysis of 1D and 2D NMR and mass spectrometry data. All the compounds were evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Compounds 1, 4 and 5 showed significant inhibitory activities, and compounds 2 and 3 showed moderate inhibitory activities with IC50 values of 17.8, 11.6, 14.9, 33.3 and 28.6 μM, respectively.


Introduction
Withasteroids are a group of structurally diverse steroidal compounds with a C 28 steroidal lactone skeleton, in which a characteristic feature is the presence of an α,β-unsaturated δ lactone ring in the side chain. They are presented primarily in the Solanaceae family, which includes Datura, Acnistus, Dunalia, Jaborosa, Physalis and Withania [1]. The isolation and synthesis of withanolides have OPEN ACCESS received considerable attention due to their significant biological activities, which include antitumor [2], cytotoxic [3,4], immunosuppressive [4], anti-inflammatory [5,6], and chemoprevention properties [7].
Flos daturae (baimantuoluo in Chinese), the dry flowers of Datura metel L. (Solanaceae), known as "Yangjinhua", have been widely used in Traditional Chinese Medicine for the treatment of coughs, asthma, rheumatism, pain, and convulsions for centuries [8]. It has also been reported that it displayed the most promising effects in treatment of psoriasis and were used in a clinical application at the First Affiliated Hospital of Heilongjiang University of Chinese Medicine (Heilongjiang, China) [9,10]. Withanolides have been studied for treating psoriasis as the main constituents of the effective part of flowers of D. metel [11,12]. Baimantuoluolines A-J, and baimantuoluosides A-H were also isolated and reported [8,[13][14][15][16][17][18]. However, the dry flowers of D. metel have the disadvantages of long florescence and low yield in comparison to its leaves. At the same time, the leaves of this herb, which can be regenerated every year, were typically discarded. In order to expand the available resources, our group found that the leaves of D. metel have some similarities in chemical constituents with its flowers and significant advantages in terms of high and stable yield. As a result, four new withanolides were isolated and named dmetelins A-D (compounds 1-4), together with one known withanolide, 7α,27-dihydroxy-1-oxowitha-2,5,24-trienolide (5) [19] (Figure 1). The structures of compounds 1-5 were determined by the interpretation of spectroscopic analysis, including 1D and 2D NMR spectroscopy. All isolates were identified as major active constituents having inhibitory effects of NO production in LPS-activated macrophage cell line, RAW 264.7 murine macrophages. Herein, we report the isolation, structural elucidation, and NO inhibitory effects of these isolates.
Since NO acts as an inflammatory mediator, inhibitors of NO production may exhibit therapeutic potential for the treatment of inflammation with overproduction of NO. Withanolides display great anti-inflammatory activity [22][23][24], so the compounds were evaluated for their effects on the inhibition of NO production in LPS-activated RAW264.7 cells [25]. In our experiment, the assayed compounds were first dissolved in DMSO, and the final concentration 0.2% (v/v) in cell culture supernatants did not show any effect on the assay systems. The effect of compounds 1-5 on cell viability was evaluated by the MTT assay to ascertain the absence of cytotoxicity (over 90% cell survival) to macrophage cells at the concentration of 100 μM. The inhibitory effects of withanolides ( Figure 4A

General
Optical rotations were measured using a Perkin-Elmer 341 polarimeter. UV spectra were obtained on a Shimadzu UV-1601 instrument. IR spectra were recorded on a Shimadzu FTIR-8400S. NMR spectra were recorded in MeOH using TMS as an internal standard on Bruker DPX 400 instrument (400 MHz for 1 H NMR and 100 MHz for 13 C NMR). Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HRESIMS was recorded on IonSpec Ultima 7.0 FTICR; GC analysis was performed on Agilent 7890A-5975C gas chromatograph equipped with a DB-1701 column (60 m × 0.25 mm × 0.25 m, film thickness); detection Triple-Axis Detector; Preparative HPLC (Waters, Delta 600-2487) was measured by Hypersil-ODS II (10 μm, 20 × 300 mm, Yilite, Da Lian, China); Column chromatography (CC) was carried out using silica gel of 200-300 mesh size (Qingdao Marine Chemical Ltd, Qingdao, China); All the solvents were of analytical grade and were purchased from TianJinFuYu Company Ltd. (Tianjin, China)

Plant Material
The leaves of D. metel were collected at Lingao County, Hainan Province, People's Republic of China, in September 2012, and was identified by Prof. Zhenyue Wang (Department of Chinese Medicine Resources, Heilongjiang University of Chinese Medicine). A voucher specimen (2012184) has been deposited at the Herbarium of Heilongjiang University of Chinese Medicine, P. R. China.

Acid Hydrolysis of Compound 3 and GC Analysis
Compound 3 (2.0 mg) was refluxed with H 2 O (2 mL) and 2 N aqueous HCl (1 mL) at 80 °C on a water bath for 3 h. After that time, the reaction mixture was extracted with ethyl acetate (3 × 5 mL). The aqueous layer was neutralized with 2 M NaHCO 3 and then evaporated to dryness. The residue of sugar was dissolved in 1 mL anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride (1.5 mg) stirred at 60 °C for 1 h. HMDS-TMCS (150 μL hexamethyldisilazane-trimethylchlorosilane, 3:1) was added and the mixture was kept at 60 °C for another 30 min. The precipitate was centrifuged off, and the supernatant was concentrated under a N 2 stream. The mixture was partitioned between n-hexane and H 2 O (0.1 mL each), and the hexane extracted (1 μL) was analyzed on an Agilent 7890A-5975C gas chromatograph equipped with a DB-1701 column (60 m × 0.25 mm × 0.25 m, film thickness) under the following conditions: firstly temperature was maintained at 50 °C, secondly raising to 190 °C at the rate of 40 °C/min, thirdly raising the temperature to 200 °C at the rate of 0.5 °C/min, fourthly raising the temperature to 210 °C at the rate of 1 °C/min, finally, raising the temperature to 280 °C at the rate of 20 °C/min. The carrier gas was He (1.0 mL/min), injector temperature: 250 °C; and split ratio: 1/20. By comparison of the retention times of authentic samples of D-glucose, the absolute configurations of the sugar residues were gave to be D-glucose (t R = 17 min).

Cell Culture
Raw 264.7, a murine macrophage cell line was purchased from the Cell Bank of the Chinese Academic of Sciences, (Shanghai, China) and maintained in supplemented with 10% Fetal Bovine Serum (Hyclone, Logan City, UT, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified incubator with 5% CO 2 .

Cell Viability Assay
Cell viability was assessed using the MTT assay as described previously [26,27]. In brief, RAW 264.7 cells were seeded into a 96-well plate at a density of 1.0 × 10 5 cells per well and incubated at 37 °C for 24 h. The cells were then treated with various concentrations of the samples. The maximum concentration of vehicle (dimethylsulfoxide, DMSO) in the culture media was 0.2% (v/v). After an additional 24 h incubation at 37 °C, 100 μL of MTT (0.5 mg/mL in PBS) was added to the wells, and the incubation continued for another 2 h. The medium was then discarded and 150 μL DMSO was added. The resulting color was assayed at 540 nm using a microplate reader (Molecular Devices, San Francisco, CA, USA).

The Determination of NO Production from RAW 264.7
To evaluate the effective of the tested materials on LPS-induced NO production, Raw 264.7 macrophages in 10% FBS-DMEM were plated at densities of 1.0 × 10 5 cell/wells in 96-well plates and grew overnight, after incubation, 20 μL serially diluted drugs (DMSO+serum-free DMEM as solvent) were applied to the cells for 4 h, and incubated in the medium with 20 μL LPS (lipopolysaccharide) of 1 μg/mL in the presence or absence of test samples for 24 h. Then, cells were dispensed into 96-well plates. One hundred μL of each supernatant was mixed with the same volume of Griess reagent [28] (1% sulfanilamide in 5% H 3 PO 4 and 0.1% N-1-naphthyletylenediamide dihydrochloride) and incubated at room temperature, away from the light, for 10 min. The absorbance was measured at 540 nm using an ELISA reader (PerkinElmer, Waltham, MA, USA), and the concentration of nitrite was calculated by comparison with a sodium nitrite standard curve. For this assay, N-monomethyl-L-arginine (L-NMMA) was used as positive control. Three independent experiments were performed, with each one in triplicate.

Statistical Analysis
The IC 50 values, the sample concentrations resulting in 50% inhibition of NO production, were determined by using nonlinear regression analysis (Sigma Plot 8.0; SPSS Inc. Chicago, IL, USA). The data are presented as mean ± S.D. of more than three independent experiments.

Conclusions
This study demonstrated that the leaves of D. metel were rich in withanolides, showing a similar structure as compounds found in its flowers. Moreover, five withanolides, including four new compounds 1-4, were isolated and identified, and these compounds were found to be responsible for the ability to inhibit NO production by activated macrophages. According to our results, further phytochemical and pharmacological studies of leaves D. metel are clearly worthwhile.