Phytochemical Investigation of Tradescantia Albiflora and Anti-Inflammatory Butenolide Derivatives

Phytochemical investigation of the whole plant of Tradescantia albiflora Kunth led to the isolation and characterization of a butanolide, rosmarinosin B (1), that was isolated from natural sources for the first time, a new butenolide, 5-O-acetyl bracteanolide A (2), and a new apocarotenoid, 2β-hydroxyisololiolide (11), together with 25 known compounds (compounds 3–10 and 12–28). The structures of the new compounds were elucidated by analysis of their spectroscopic data, including MS, 1D, and 2D NMR experiments, and comparison with literature data of known compounds. Furthermore, four butenolides 4a–4d were synthesized as novel derivatives of bracteanolide A. The isolates and the synthesized derivatives were evaluated for their preliminary anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in RAW 264.7 cells. Among them, the synthesized butenolide derivative n-butyl bracteanolide A (4d) showed enhanced NO inhibitory activity compared to the original compound, with an IC50 value of 4.32 ± 0.09 μg/mL.


Introduction
Tradescantia albiflora Kunth (Commelinaceae) is native to tropical rainforests.It has been used as a traditional medicine for treating hyperuricemia and gout in Taiwan.Previous research described the inhibitory activity against xanthine oxidase (XO), which plays a central role in metabolic disorders such as hyperuricemia and gout, of the methanol extract and compounds isolated from the leaves of T. albiflora [1].However, none of the isolated compounds showed significant inhibitory activity.
Our continuing investigation on the bioactive compounds from T. albiflora has now led to the extraction, purification, and structural elucidation of three new naturally occurring compounds, together with 25 known compounds.The butenolide bracteanolide A (4) was the most abundant compound among the isolates, and it has been reported to show inhibitory ability against lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in RAW 264.7 cells.This inhibition was associated with its selective suppression on inducible NO synthase (iNOS) induction [2], indicating its potential to treat inflammatory diseases caused by NO production.For this reason, bracteanolide A (4) was used as a starting material for the preparation of butenolide derivatives.In addition, the isolates and four newly synthesized derivatives were evaluated for their preliminary anti-inflammatory activity against LPS-stimulated NO production in RAW 264.7 cells.
The 1 H-NMR spectrum (Table 1) displayed signals characteristic of the presence of two hydroxyl groups attached on the benzene ring (δ H 8.63, brs) as determined by D 2 O exchange experiment, a highly deshielded oxymethine (δ H 7.40, s, H-5), a trisubstituted benzene ring indicated by an ABX-pattern for three aromatic protons [δ H 7.15 (d, J = 2.1 Hz), 7.11 (dd, J = 8.3, 2.1 Hz), and 6.94 (d, J = 8.3 Hz)], a conjugated olefinic proton (δ H 6.49, s, H-3), and an acetyl group (δ H 2.15, 3H, s).Additionally, twelve carbon signals were displayed in the 13 C-NMR spectrum.The assignments of two ortho-hydroxyl groups attached on the benzene ring were confirmed by two deshielded signals of aromatic carbons at δ C 150.7 and 146.8.The IR peak at ν max 1757 cm −1 for γ-lactone functionality together with the carbon signals at δ C 171.0, 162.8, 112.8, and 93.5 indicated the presence of the oxygenated unsaturated butenolide moiety.This moiety was also deduced from the HSQC correlations from H-3 (δ H 6.49 s) to C-3 (δ C 112.8) and from H-5 (δ H 7.40 s) to C-5 (δ C 93.5).The HMBC correlation from H-5 (δ H 7.40, s) to the acetyl carbon (δ C 170.0) indicated that the acetoxyl group was attached on the C-5 in butenolide moiety.The overall 1D and 2D NMR data suggested the structural similarities between 2 and bracteanolide A (4), except that the hydroxy group on C-5 in 4 was replaced by the O-acetyl group.This assignment was also confirmed by a highly deshielded oxymethine signal (δ H /δ C 7.40/93.5).Compound 2 was consequently determined as new butenolide, named 5-O-acetyl bracteanolide A.
Compound 11 was obtained as a colorless oil with [α] 25 D + 27.6, and its high resolution electron ionization mass spectrometry (HREIMS) peak at m/z 212.1043 determined the molecular formula as C 11 H 16 O 4 , indicating four degrees of unsaturation.The IR spectrum displayed the presence of hydroxyl (3406 cm −1 ) and lactone (1741 cm −1 ) functionalities.
The 1 H-NMR spectrum (Table 2) indicated the presence of an olefinic proton signal at δ H 5.78 (1H, s), two oxymethine signals at δ H 3.80 and 3.04, a methylene signal at δ H 2.38 and 1.43, and three methyl signals at δ H 1.58, 1.33, and 1.18 (each 3H, s). 13     In this study, the phytochemical investigation on the bioactive compounds from T. albiflora led to the isolation of 28 compounds including a butanolide rosmarinosin B (1) and four butenolides, 5-O-acetyl bracteanolide A (2), 4-(3',4'-dihydroxyphenyl)furan-2(5H)-one (3), bracteanolide A (4), and bracteanolide B (5).Among the isolates, the butanolide bracteanolide A (4) was highly abundant in this plant (1.31 mg/g extract).It has been reported to exhibit inhibitory activity against LPS-activated NO production in RAW 264.7 cells by suppressing iNOS expression selectively [2], which is the potential target for the treatment of the inflammatory diseases caused by NO production.In order to screen the naturally anti-inflammatory butenolides and their related derivatives, four new butenolide derivatives 4a-4d were synthesized by modification from bracteanolide A (4) at C-4, focusing on changing the hydroxy group into alkoxy groups (Scheme 1).Then, the isolated compounds 2-8 and four new butenolide derivatives 4a-4d were evaluated for their preliminary anti-inflammatory activity against NO production in RAW 264.7 cells, a reliable indicator in investigating inflammatory activity [19].In this study, the phytochemical investigation on the bioactive compounds from T. albiflora led to the isolation of 28 compounds including a butanolide rosmarinosin B (1) and four butenolides, 5-O-acetyl bracteanolide A (2), 4-(3',4'-dihydroxyphenyl)furan-2(5H)-one (3), bracteanolide A (4), and bracteanolide B (5).Among the isolates, the butanolide bracteanolide A (4) was highly abundant in this plant (1.31 mg/g extract).It has been reported to exhibit inhibitory activity against LPS-activated NO production in RAW 264.7 cells by suppressing iNOS expression selectively [2], which is the potential target for the treatment of the inflammatory diseases caused by NO production.In order to screen the naturally anti-inflammatory butenolides and their related derivatives, four new butenolide derivatives 4a-4d were synthesized by modification from bracteanolide A (4) at C-4, focusing on changing the hydroxy group into alkoxy groups (Scheme 1).Then, the isolated compounds 2-8 and four new butenolide derivatives 4a-4d were evaluated for their preliminary anti-inflammatory activity against NO production in RAW 264.7 cells, a reliable indicator in investigating inflammatory activity [19].All compounds evaluated displayed lower anti-inflammatory activity than the positive control dexamethasone (Table 3), which has been reported to decrease iNOS-dependent NO production [20].Dexamethasone is a highly effective anti-inflammatory and immunosuppressant corticosteroid.Unfortunately, the long-term use may cause serious systemic side effects, ranging from weight gain, diabetes, hypertension, immunosuppression, psychological disturbances, fragile skin, muscle weakness, osteoporosis, and Cushing's syndrome [21].All compounds evaluated displayed lower anti-inflammatory activity than the positive control dexamethasone (Table 3), which has been reported to decrease iNOS-dependent NO production [20].Dexamethasone is a highly effective anti-inflammatory and immunosuppressant corticosteroid.Unfortunately, the long-term use may cause serious systemic side effects, ranging from weight gain, diabetes, hypertension, immunosuppression, psychological disturbances, fragile skin, muscle weakness, osteoporosis, and Cushing's syndrome [21].
As shown in Table 3 and Figure S22, compounds 4, 4b, 4d, 6, and 7 showed inhibitory potential against NO production, which was not associated with their cytotoxicity against RAW 264.7 cells (Figure S23).The results suggested that the disappearance of the hydroxy group (3) or the presence of methyl (5), ethyl (4a), i-propyl (4c), and acetyl groups (2) resulted in a decreased activity; the presence of n-propyl group did not affect the activity.The data also revealed that the catechol group might have contributed to the activity even if the result of compound 8 was unsatisfactory (the IC 50 value was above 50 µg/mL).Among compounds evaluated, compound 4d with an n-butyl group showed enhanced anti-inflammatory activity (IC 50 value of 4.32 ± 0.09 µg/mL) compared to the original compound (Table 3).

Plant Material
The greenhouse-grown plant material was obtained from Dr. T.-F.K. (Department of Post-Baccalaureate Veterinary Medicine, Asia University).A voucher specimen (TAIF-PLANT-199332) has been retained at the Herbarium of Taiwan Forestry Research Institute, Taipei, Taiwan.

Preparation of Butenolide Derivatives 4a-4d
Bracteanolide A (20 mg) was dissolved in the corresponding alcohol (10 mL).Concentrated hydrochloric acid (3 drops) was added and the solution was refluxed and stirred for 12 h.The solvent was evaporated under vacuum to produce a yellow residue that was diluted with distilled water and fractionated twice with dichloromethane.The organic extracts were combined and dried over magnesium sulfate to give butenolide derivatives 4a-4d, in approximately 70% yield.

Cell Culture
A murine macrophage cell line RAW264.7 (BCRC No. 60001) was obtained from the Bioresources Collection and Research Center of the Food Industry Research and Development Institute (Hsinchu, Taiwan).Cells were maintained in Dulbecco s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma) in an incubator containing 5% CO 2 at 37 • C and subcultured every 3 days using 0.05% trypsin-0.02%EDTA in Ca 2+ -, Mg 2+ -free phosphate-buffered saline (DPBS).

Cell Viability
Raw 264.7 cells (5 × 10 4 cells/well) were seeded into 96-well plates and incubated for 24 h.Then, cells were treated with different concentrations of samples in the presence of 100 ng/mL LPS.After incubation overnight, the cells were washed twice with DPBS and incubated with 100 µL MTT (0.5 mg/mL) for 3 h.The medium was removed, and MTT formazan was dissolved by 100 µL dimethyl sulfoxide (DMSO).Then, absorbance at 570 nm was read using a microplate reader.

Measurement of Nitric Oxide/Nitrite
NO production was indirectly measured by determining the nitrite levels in the cultured medium using a colorimetric assay based on the Griess reaction.Cells were treated with different concentrations of samples in the presence of LPS (100 ng/mL) and incubated for 24 h.Then, each supernatant (100 µL) was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 5% phosphoric acid) and incubated for 5 min, and the absorbance at 540 nm was measured using a microplate reader.

Conclusions
In summary, phytochemical investigation of the whole plant of Tradescantia albiflora Kunth has led to the isolation and characterization of a butanolide, rosmarinosin B (1), that was isolated from natural sources for the first time, a new butenolide, 5-O-acetyl bracteanolide A (2), and a new apocarotenoid 2β-hydroxyisololiolide (11), together with 25 known compounds (compounds 3-10 and 12-28).The isolated compounds 2-8 and four new synthetic butenolide derivatives 4a-4d, which were synthesized from bracteanolide A (4), were evaluated for their preliminary anti-inflammatory activity against LPS-stimulated NO production in RAW 264.7 cells.Among them, the new synthetic butenolide derivative n-butyl bracteanolide A (4d) exhibited better NO inhibitory activity than the original compound bracteanolide A (4).
C-NMR and DEPT experiments revealed the presence of 11 carbon signals, indicating a lactone carbon at δ C 170.7, a pair of conjugated carbon at δ C 180.3 and 114.0, one oxygenated quaternary carbon at δ C 85.3, two oxymethines at δ C 81.7 and 67.9, a methylene at δ C 43.6, and three methyls at δ C 25.7, 25.2, and 18.7.
7) of 11 by comparing the NMR data of 12.The planar structure of 11 was confirmed by HMBC correlations shown in Figure 2. The di-axial orientations of H-2 and H-3 were deduced from the coupling constant of 9.3 Hz between them.The NOESY correlations between H-2 and Me-9; H-3 and Me-11; Me-10 and Me-11 established the relative configuration of 11.Thus, compound 11 was determined as a new apocarotenoid, named 2β-hydroxyepiloliolide.additional hydroxy group was assigned at C-2 (δC 81.7) of 11 by comparing the NMR data of 12.The planar structure of 11 was confirmed by HMBC correlations shown in Figure 2. The di-axial orientations of H-2 and H-3 were deduced from the coupling constant of 9.3 Hz between them.The NOESY correlations between H-2 and Me-9; H-3 and Me-11; Me-10 and Me-11 established the relative configuration of 11.Thus, compound 11 was determined as a new apocarotenoid, named 2βhydroxyepiloliolide.

Table 1 . 1 H-and 13 C-NMR spectral data of compounds 1 and 2 (δ in ppm, J in Hz). 1 a 2 b No. δ H δ C δ H δ C
a Measured in methanol-d 4 .b Measured in acetone-d 6 .
An additional hydroxy group was assigned at C-2 (δ C 81.