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Two Anti-inflammatory Steroidal Saponins from Dracaena angustifolia Roxb

Two new steroidal saponins, named drangustosides A–B (1–2), together with eight known compounds 3–10 were isolated and characterized from the MeOH extract of Dracaena angustifolia Roxb. The structures of compounds were assigned based on 1D and 2D NMR spectroscopic analyses, including HMQC, HMBC, and NOESY. Compounds 1 and 2 showed anti-inflammatory activity by superoxide generation and elastase release by human neutrophils in response to fMLP/CB.


Introduction
The genus Dracaena (Agavaceae) includes more than 50 species found in tropical and subtropical regions of the eastern hemisphere. Dracaena angustifolia Roxb. is a native shrub or small tree in southern Taiwan, widely planted in Australia, India, Malaysia, and Philippines [1]. The decoction of the underground parts of this plant is used as a tonic and for the treatment of asthma, diarrhea, and inflammation [2]. Previous phytochemical investigations on the genus Dracaena have reported the presence of a variety of components, including steroidal saponins [3][4][5][6][7], flavonoids [5,[8][9][10], and phenolic compounds [8,11]. The pharmacological investigation indicated that the C 27 steroidal saponins present on the genus Dracaena showed broad biological activities, such as anti-inflammatory, antifungal, antimicrobial, antiviral, analgesic, antioxidative, cytotoxic, and hypoglycemic properties [3][4][5]11]. D. angustifolia Roxb. has been studied, and this revealed the presence of several steroidal saponins with antifungal [4], antituberculosis [12], antiproferative [7], and cytotoxic activities [13]. In our continuing phytochemical investigation on D. angustifolia Roxb., we have now further identified two new steroidal saponins, drangustosides A-B (1-2), one known steroidal saponin, alliospiroside A (3) [14], and seven benzenoids 4-10 in the MeOH extract of D. angustifolia Roxb., and report herein the structural determination of these substances using extensive spectroscopic methods. Neutrophils play a significant role in the pathogenesis of several inflammatory diseases. The production of vast amounts of superoxide anion and elastase by activated neutrophils can cause tissue damage and contribute to the development of a wide spectrum of airway inflammatory diseases [15,16]. The anti-inflammatory activity of new compounds was also evaluated as inhibitory activities against formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion production and elastase release in human neutrophils.

Results and Discussion
The MeOH extracts of the whole plant of D. angustifolia Roxb. was extracted successively with EtOAc and n-BuOH. Compounds 1-10 ( Figure 1) were obtained from the EtOAc fraction by using a series of chromatographic techniques on silica gel, Sephadex LH-20, and RP-HPLC. The structures of the two new steroidal saponins 1-2 were elucidated as follows: Compound 1 was obtained as a white amorphous solid. Its molecular formula was established as C 45 H 72 O 17 based on an [M+Na] + ion peak at m/z 907.4667 in its HRESIMS. The IR spectrum of 1 indicated the existence of hydroxyl groups (3,389) and the characteristic absorption bands of a (25S)-spiroketal at 987, 918, 898, and 843 (intensity 918 > 898) cm −1 [14]. The 1 H-NMR spectrum ( Table 1) 5.73 (br s), and 5.82 (br s)], two of which were considered to be L-rhamnosides. Based on the chemical shifts and coupling constants, the three monosaccharides were considered as a combination of one D-glucoside and two L-rhamnosides. The 13 C-NMR spectrum (Table 1) exhibited 45 carbon atoms, 27 of which belonged to the aglycone carbons while the remaining were due to three hexose sugar units. The 13 C-NMR and DEPT spectra revealed C 27 signals including four methyl, nine methylene, ten methine, and four quaternary carbons. Among the four quaternary carbon signals, the signal at δ C 110.0 was identified as an acetal carbon (C-22) and the signal at δ C 139.3 was assigned as a diakyl substituted olefinic carbon (C-5). The aforementioned data suggested that 1 is possibly a spirostanol glycoside with the aglycone being a 27-carbons skeletonal aglycone along with three sugar moieties [4].   Comparison of the 1 H and 13 C-NMR signals (Table 1) of the aglycone moiety of 1 with those of alliospiroside A (3) [14], indicated that the structures of the aglycone parts of 1 and 3 were almost superimposable. The only significant differences were seen in the 13 C-NMR signals of C-1 and C-3 in the ring A portion. Generally, a carbon attached to an -O-glycoside group exhibits the higher 13  These results lead to the conclusion that the -O-glycoside is unambiguously located at C-3 in compound 1. Comparing the NMR data of C-1 and C-3 between 1 and 3, the -O-glycosidation at C-3 in compound 1 and at C-3 in compound 3 agrees with the general knowledge. The coupling constant of H-1 (dd, J = 11.6, 3.9 Hz) can be assigned to an α-axial orientation. H-1 and H-3 have a NOESY correlation, therefore revealing that H-3 was located with the same α-axial orientation. In general, the difference in chemical shifts between axial and equatorial protons among on H 2 -23, H 2 -24, and H 2 -26 can be used to resolve the absolute configuration of C-25 [17,18]  D-Glucose and L-rhamnose in a 1:2 ratio were obtained upon acidic hydrolysis of 1 with HCl in 1,4-dioxane [19], as indicated by chiral HPLC methodology [20]. Analysis of the NMR data indicated the presence of a trisaccharide unit connected to an aglycone moiety, with three anomeric protons [δ    (3,389) and the characteristic absorption bands of (25S)-spiroketal at 987, 920, 897, and 840 (intensity 920 > 897) cm −1 [14]. The 1 H-and 13 C-NMR spectroscopic features of the aglycone moiety of 2 (Table 1) were very similar to those of 1, which was suggested that compounds 2 and 1 possessed the same (1,3,22R,25S)-spirost-5-ene-1,3-diol aglycone. The 1 H-and 13 C-NMR spectra of 2 exhibited two anomeric protons signals at δ H 6.36 (br s) and 5.06 (d, J = 7.3 Hz), as well as the corresponding anomeric carbon resonances at δ C 102.3 and 100.7, respectively, that established the existence of two sugar units. The differences in the 1 H and 13 C-NMR spectra of 1 and 2 showed that 2 had one less rhamnoside unit than 1. In addition, the molecular ion mass spectrum of 2 was 146 atomic units lower than that of 1, and only two anomeric proton signals were observed at δ H 5.06 (d, J = 7.3 Hz) and 6.36 (br s). Acid hydrolysis of 2 yielded a L-rhamnose and a D-glucose in 1:1 ratio. The TOCSY spectrum of 2 showed that the proton at δ H 5.06 (H-1′) was coupled to the signals at δ  [28], respectively, by comparison with the spectroscopic data reported in the literature for these compounds.
Inhibition of superoxide generation and elastase release by human neutrophils in response to fMLP were utilized to measure the anti-inflammatory activity of compounds 1 and 2 (Table 2). LY294002, a phosphatidylinositol-3-kinase inhibitior, was used as a positive control for inhibition of superoxide anion generation and elastase release with IC 50 values of 2.00 ± 0.59 and 4.94 ± 1.69 μM, respectively. Compound 1 showed the highest anti-inflammatory activity against superoxide generation (IC 50 = 18.55 ± 0.23 μM) and elastase (IC 50 = 1.74 ± 0.25 μM).

General Procedures
The infrared (IR) spectra were measured on a Mattson Genesis II spectrophotometer using a KBr matrix. The optical rotations were measured on a JASCO P-1020 polarimeter equipped with a sodium lamp (589 nm). 1 H-and 13 C-NMR spectra were recorded on a Bruker DRX-500 spectrometer with CD 3 OD as the solvent. The HRESIMS data were collected using a Finnigan MAT95S mass spectrometer. The GC-MS was performed using a Thermo Finnigan TRACE GC Ultra instrument. Sephadex LH-20, and silica gel (Merck 70-230 mesh and 230-400 mesh) were used for the column chromatography. The preparative HPLC was performed using a reverse phase column (Cosmosil 5C 18 -AR-II column, 5 μm particle size, 250 mm × 20 mm i.d.) and the stereoselective HPLC analysis was performed using a normal phase column (Chiralpak AD-H column, 5 μm particle size, 250 mm× 10 mm i.d.) on a Shimadzu LC-6AD series apparatus with a RID-10A Refractive Index detector. The MPLC was performed using a reverse phase column (Buchi MPLC glass column, C 18 , 460 mm × 36 mm i.d.) on a Buchi pump module C-601 series apparatus without detector.

Plant Material
The leaves of D. angustifolia Roxb. used in this experiment was collected on the mountains of Nantou County, Taiwan

Acid Hydrolysis of 1-2
Compound 1 or 2 (2 mg each) were heated with 1N HCl (dioxane-H 2 O, 1:1, 2 mL) at 90 °C for 4 h, and then evaporated under reduced pressure to give a residue. The residue was partitioned with CH 2 Cl 2 and H 2 O three times. The aqueous (pH = 2.0) was neutralized with aqueous 1N NaOH and then evaporated under reduced pressure. The aqueous layer (0.3 mg; pH = 7.0) was dissolved in MeOH (0.5 mL) and then analyzed by stereoselective HPLC under the following conditions: column: Chiralpak AD-H; solvent system: n-hexane-ethanol-TFA (7:3:0.1, v/v); flow rate: 0.5 mL/min; inject volume: 20 μL; detector: refractive index. D-Glucose and L-rhamnose for compounds 1 (t R = 14.31 and 15.80 min, respectively) and 2 (t R = 14.36 and 15.89 min, respectively) were determined by comparison of the respective retention times of D-glucose (t R = 14.33 min) and L-rhmanose (t R =15.85 min) standards.

Superoxide Generation and Elastase Release by Human Neutrophils
Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Blood was drawn from healthy human donors (20-32 years old) by venipuncture using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Neutrophils were isolated using a