www.mdpi.com/journal/ijms Anti-Inflammatory Components from the Root of

Two new norsesquiterpenoids, solanerianones A and B (1–2), together with nine known compounds, including four sesquiterpenoids, (−)-solavetivone (3), (+)-anhydro-β-rotunol (4), solafuranone (5), lycifuranone A (6); one alkaloid, N-trans-feruloyltyramine (7); one fatty acid, palmitic acid (8); one phenylalkanoid, acetovanillone (9), and two steroids, β-sitosterol (10) and stigmasterol (11) were isolated from the n-hexane-soluble part of the roots of Solanum erianthum. Their structures were elucidated on the basis of physical and spectroscopic data analyses. The anti-inflammatory activity of these isolates was monitored by nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells. The cytotoxicity towards human lung squamous carcinoma (CH27), human hepatocellular carcinoma (Hep 3B), human oral squamous carcinoma (HSC-3) and human melanoma (M21) cell lines was also screened by using an MTT assay. Of the compounds tested, 3 exhibited the strongest NO inhibition with the average maximum inhibition (Emax) at 100 μM and median inhibitory concentration (IC50) values of 98.23% ± 0.08% and 65.54 ± 0.18 μM, respectively. None of compounds (1–9) was found to possess cytotoxic activity against human cancer cell lines at concentrations up to 30 μM.


Introduction
Solanum erianthum D. Don (Solanaceae) is a shrub or small tree with stellate tomentose. It is native to South America and widespread in tropical Asia and Oceania [1]. The leaves are used for the treatment of cancer and malaria in Nigeria [2]. In Taiwan, the leaves are used as maternal tonic and to treat lumbar neuralgia; the stems and roots are used to cure rheumatism and cold; the roots are also used for the treatment of stomachache, abdominal pain, fracture, bruises, and chronic granular leukemia [3,4].
Flavonoids, steroidal alkaloids, amides and fatty acids were isolated from the leaves of S. erianthum [5][6][7][8]. The presence of some steroidal alkaloids was detected in the fruits, stem barks, xylem, roots and leaves of this plant by thin-layer chromatography (TLC) [9]. Sesquiterpenes and monoterpenes were the main components in the essential oil from the fruits and leaves of S. erianthum, respectively [10,11]. However, there has been no previous report on the chemical constituents of the root of this species. During preliminary screening, the MeOH extract of the root of S. erianthum was shown to be able to inhibit NO release without affecting the cellular viability in lipopolysaccharide (LPS)-activated Raw 264.7 cells and display a selective cytotoxic activity against Hep 3B cell line. In the present study, we set out to isolate the active principles from the root extract and to assess the bioactivity of the pure isolates. We now report the isolate of two new solanerianones A and B (1-2), and nine known compounds, including four sesquiterpenoids, (−)-solavetivone (3), (+)-anhydro-βrotunol (4), solafuranone (5) and lycifuranone A (6); one alkaloid, N-trans-feruloyltyramine (7); one fatty acid, palmitic acid (8); one phenylalkanoid, acetovanillone (9), and two steroids, β-sitosterol (10) and stigmasterol (11) from an n-hexane-soluble fraction of the root of S. erianthum ( Figure 1). However, owing to the paucity of plant extracts, some compounds could not be obtained in sufficient quantities for bioassay. Herein, we describe the structure elucidation of these two new compounds, anti-inflammatory activity of compounds 3-8 and cytotoxicity evaluation of compounds 1-9 against four human cancer cell lines.

Anti-Inflammatory Activities
NO, overproduced by activated macrophages via inducible NO synthase (iNOS), is suggested to be a significant pathogenic factor in various inflammatory tissue injuries. In order to elucidate the anti-inflammatory action of the root of S. erianthum, the present study was designed to isolate its active constituents and examine their effects on NO production, detected as nitrite in the culture medium, induced by LPS through iNOS expression in RAW264.7 cells, to reflect the degree of anti-inflammatory activity. By the guidance of the bioassay, the n-hexane-soluble fraction was isolated to exhibit a significant bioactivity without affecting the cellular viability at a concentration of 100 μg/mL; the inhibition being 76.78% ± 0.34% and with an IC 50 value of 72.80 ± 1.50 μg/mL ( Figure 4A). This finding prompted us to investigate the active principles from this fraction, and led to the isolation and identification of two novel norsesquiterpenoids along with eight known compounds. The effects of 3-8 on the inhibition of NO production in LPS-activated RAW264.7 cells were evaluated. E max (%) and IC 50 (μM) values of iNOS inhibitory activity were obtained at the concentration range of 3.0 to 100 μM. Results are shown in Table 2. Among the isolates, solavetivone (3) which was the major compound in n-hexane-soluble fraction exhibited a significant activity, with the E max and IC 50 values of 98.23% ± 0.08% and 65.54 ± 0.18 μM, respectively. As shown in Figure 4B, it inhibited LPS-induced NO production in a concentration-dependent manner. (+)-Anhydro-β-rotunol (4) was similar to 3 belonging to spirovetivene sesquiterpenoid, but it showed a mild effect. It suggested that the stereochemistry of the double bond between C-9 and C-10 of 4 decreased the iNOS inhibitory activity. Solafuranone (5) and lycifuranone A (6) contain the same carbon skeleton which may be biologically converted from 3 [16], and they seem not to be linked to NO production at the tested concentrations, although it did not affect cell viability. N-trans-Feruloyltyramine (7) exhibited moderate iNOS inhibitory activity, with E max value of 33.33% ± 1.69%. The iNOS inhibitory activity of palmitic acid (8) showed weak effect, with E max value of 13.22% ± 1.11%. Under the same conditions, the maximum inhibitory effects of positive controls aminoguanidine (a selective iNOS inhibitor) and N ω -nitro-L-arginine (a nonselective iNOS inhibitor) were 80.97% ± 0.63% and 42.19% ± 0.94%, respectively.   (3) 98.23 ± 0.08 65.54 ± 0.18 (+)-Anhydro-β-rotunol (4) 8.77 ± 1.24 >100 Solafuranone (5) 0 ± 0 >100 Lycifuranone A (6) 0 ± 0 >100 N-trans-Feruloyltyramine (7) 33.33 ± 1.69 >100 Palmitic acid (8) 13 .

General
Silica gel 60 F 254

Plant Material
The roots of S. erianthum were collected from Tainan, Taiwan, in July 2009 and identified by Dr. Yu-Chang Chen. A voucher specimen (YCC 0971) has been deposited in the Herbarium of the College of Pharmacy, China Medical University, Taichung, Taiwan.

Extraction and Isolation
Dried roots (12.8 kg) of S. erianthum were sliced and extracted with cold MeOH three times. After removal of the solvent under vacuum, the extract was partitioned into an n-hexane-soluble fraction (fraction A, 47.8 g), a EtOAc-soluble fraction (fraction B, 54.0 g), an n-BuOH-soluble fraction (fraction C, 335.0 g), and a water-soluble fraction (fraction D, 431.8 g).

Anti-Inflammatory Activity Assay
3.6.1. Cell Culture RAW 264.7 cells, a transformed murine macrophage cell line, obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan), were maintained by twice-weekly passage in DMEM supplemented with 10% fetal calf serum (FCS; HyClone Laboratories, Logan, UT, USA) and penicillin-streptomycin.

Evaluation of NO Product by Nitrite Measurement
Nitrite measurement was based on our previously published technique [23]. Cell aliquots (5 × 10 5 cells/mL) were grown to confluence on 24-well plates for 24 h. The medium was changed to serum-free DMEM for another 4 h to render the attached cells quiescent. To assess the effects on LPS-induced NO production, compounds, two positive controls aminoguanidine (a selective iNOS inhibitor; 100 μM) and N ω -nitro-L-arginine (a non-selective NOS inhibitor; 100 μM) or vehicle (0.1% DMSO) were added in the presence of LPS (200 ng/mL) to the cells for another 24 h. The culture supernatant was subsequently collected for nitrite assay as a reflection of NO production [24]. Briefly, an aliquot of supernatant was mixed with an equal volume of Griess reagent (prepared by adding 1 part 0.1% napthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance at 550 nm was measured by a microplate spectrophotometer (Bio-Tek Instrument, Inc., Winooski, VT, USA). Fresh medium was used as the blank. The nitrite concentration was determined by reference to a standard curve, using sodium nitrite diluted in the stock culture medium. Results are expressed as percentage of inhibition calculated versus vehicle plus LPS-treated cells.

Cell Viability Assay
A redox indicator, alamarBlue, was used to measure the cytotoxicity as has been described previously [25]. After culture supernatant was removed for nitrite measurement as described above, a solution of 10% alamarBlue in DMEM was added to each well containing RAW 264.7 cells. The plates were incubated at 37 °C in a humidified 5% CO 2 for 1 h. Following incubation, the absorbance of the alamarBlue was read spectrophotometrically at dual wavelengths of 570 and 600 nm against the blank prepared from cell-free wells. The absorbance in cultures treated with LPS plus vehicle was regarded as indicating 100% cell viability.

Mitochondrial Reductase Activity
Cells were seeded at a density of 5 × 10 4 cells per well onto 12-well plates 48 h before being treated with test compounds. The cells were incubated with 30 μM of test compounds for 24 h. The control cultures were treated with 0.1% dimethyl sulfoxide (DMSO). After treatment, the cells were washed with phosphate-buffered saline (PBS). Cellular mitochondrial reductase activity of live cells was determined by measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). At each end point, the treatment medium was replaced with fresh serum-free medium containing 2.4 × 10 −4 M MTT at pH 7.4. Cells were incubated with MTT medium for 1 h at 37 °C. After solubilization in DMSO, absorbance was measured at 550 nm.

Statistical Analysis
For each experimental series, data are given as mean ± S.E., with n representing the number of independently performed experiments. Comparisons of the concentration and treatment effects were conducted with ANOVA, followed by post hoc comparisons by Newman Keuls test as appropriate. The average IC 50 value was determined by data fitting with GraFit (Erithacus Software, Surrey, UK). A p value of less than 0.05 was considered to indicate a statistically significant difference.

Conclusions
Solanerianones A and B (1-2) were new norsesquiterpenoids which contain a carbon skeleton similar to (−)-solavetivone (3) except that isopropenyl group at C-2 was replaced by an acetyl group. Compounds 3-7 and 9 were first isolated from S. erianthum. The spirovetivene compound 3, the major active constituent in n-hexane-soluble fraction of the root of this plant, significantly inhibited NO production of RAW264.7 cells without any cytotoxicity. It was reported to have fungitoxicity [12], antimicrobial activity [26] and weak cytotoxicity [16], but its anti-inflammatory activity was studied first. Other spirovetivene compounds were reported to show spasmolytic activity [27] and an induction effect on brain-derived neurotrophic factor mRNA expression [28]. On the other hand, compound 7 exhibited moderate iNOS inhibitory activity. These findings suggest that naturally occurring iNOS inhibitors 3 and 7 may provide a rationale for the potential anti-inflammatory effect of S. erianthum.
The yields of compounds 1 and 2 were too low to study the anti-inflammatory activities. Compound 2 will be prepared from 3 by oxidative cleavage of the methylene group following the Tamariz technique [29] in the future. However, it is difficult to prepare compound 1 from 3. Therefore compound 1 should be prepared by total synthesis unless a proper precursor is obtained.