Bioactive Phenolic and Isocoumarin Glycosides from the Stems of Homalium paniculiflorum

Two new phenolic glycosides (1 and 2) and two new isocoumarin glycosides (3 and 4), along with 14 known compounds (5–18), were isolated from the stems of Homalium paniculiflorum. Their structures were established on the basis of extensive spectroscopic analyses and chemical methods. All new compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 4 exhibited inhibitory activities with IC50 values of 30.23 ± 1.23 μM and 19.36 ± 0.19 μM, respectively.


Results and Discussion
The methanol extract of the stems of H. paniculiflorum was suspended in water and extracted successively with petroleum ether and EtOAc. The EtOAc extract was repeatedly subjected to silica gel CC, reversed-phase C18 silica gel CC, Sephadex LH-20 CC and semi-preparative HPLC, to yield 18 compounds, including two new phenolic glycosides (1 and 2) and two new isocoumarin glycosides (3 and 4), as shown in Figure 1. Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C27H26O10 by HRESIMS (m/z 533.1413 [M + Na] + , calcd. 533.1418), indicating 15 degrees of unsaturation. Its IR spectrum showed the presence of hydroxyl groups (3468 cm −1 ), an ester carbonyl group (1708 and 1676 cm −1 ) and phenyl groups (1620, 1518 and 1493 cm −1 ). The UV maxima at 262 and 218 nm indicated that 1 possessed aromatic rings. The 13 C-NMR and DEPT data (Table 1) revealed the presence of 27 carbon atoms, including 20 sp 2 carbon atoms, five sp 3 methines and two sp 3 methylenes, which were attributable to two benzoate groups, one benzyl alcohol group and one glucopyranosyl moiety. The above data revealed that the structure of 1 was similar to that of itoside H (16) [20], except that the hydroxyl group at C-2′ was substituted by a hydrogen atom, which was supported by the HMBC correlations of H-2′ to C-4′ (δC 134.2), C-6′ (δC 130.6) and C-7′ (δC 167.8), as well as the 1 H-1 H COSY correlations from H-2′ to H-6′. Detailed analysis of 2D-NMR (HSQC, HMBC and 1 H-1 H COSY) spectra confirmed the structure of 1 (see Figure 2). Furthermore, the coupling constant of the anomeric proton resonating at δH 4.79 (1H, d, J = 7.6 Hz, H-1″) suggested that the glucopyranosyl moiety was β-glucoside. In order to further confirm the structure of 1, the acid hydrolysis reaction of 1 was carried out. As a result, a β-D-glucose was produced as the sole sugar identified on the basis of the same Rf value on co-TLC and the almost identical optical value by comparing with that of an authentic sugar sample. Therefore, compound 1 was determined as 4-hydroxy-2-{[(benzoyl)oxy] methyl}phenyl-β-D-glucopyranoside-6-benzoate, as shown in Figure 1. Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C 27 H 26 O 10 by HRESIMS (m/z 533.1413 [M + Na] + , calcd. 533.1418), indicating 15 degrees of unsaturation. Its IR spectrum showed the presence of hydroxyl groups (3468 cm −1 ), an ester carbonyl group (1708 and 1676 cm −1 ) and phenyl groups (1620, 1518 and 1493 cm −1 ). The UV maxima at 262 and 218 nm indicated that 1 possessed aromatic rings. The 13 C-NMR and DEPT data (Table 1) revealed the presence of 27 carbon atoms, including 20 sp 2 carbon atoms, five sp 3 methines and two sp 3 methylenes, which were attributable to two benzoate groups, one benzyl alcohol group and one glucopyranosyl moiety. The above data revealed that the structure of 1 was similar to that of itoside H (16) [20], except that the hydroxyl group at C-2 was substituted by a hydrogen atom, which was supported by the HMBC correlations of H-2 to C-4 (δ C 134.2), C-6 (δ C 130.6) and C-7 (δ C 167.8), as well as the 1 H-1 H COSY correlations from H-2 to H-6 . Detailed analysis of 2D-NMR (HSQC, HMBC and 1 H-1 H COSY) spectra confirmed the structure of 1 (see Figure 2). Furthermore, the coupling constant of the anomeric proton resonating at δ H 4.79 (1H, d, J = 7.6 Hz, H-1") suggested that the glucopyranosyl moiety was β-glucoside. In order to further confirm the structure of 1, the acid hydrolysis reaction of 1 was carried out. As a result, a β-D-glucose was produced as the sole sugar identified on the basis of the same R f value on co-TLC and the almost identical optical value by comparing with that of an authentic sugar sample. Therefore, compound 1 was determined as 4-hydroxy-2-{[(benzoyl)oxy] methyl}phenyl-β-D-glucopyranoside-6-benzoate, as shown in Figure 1.    In the 1 H-NMR spectrum of 2 (see Figure S6 (Table 1) revealed the existence of 22 carbon atoms, including 16 sp 2 carbon atoms, five sp 3 methines and one sp 3 methylenes, which were attributable to one p-coumaroyl moiety, one benzaldehyde moiety and one glucopyranosyl moiety. The above data revealed that the structure of 1 was similar to that of 6 -O-(Z)-p-coumaroylsalicin [21]. Further comparisons of 1 H-NMR, 13 C-NMR and DEPT data of 2 with 6 -O-(Z)-p-coumaroylsalicin indicated that there were two major differences between their structures. Firstly, the hydroxymethyl group at C-1 in 6 -O-(Z)-p-coumaroylsalicin was substituted by an aldehyde group in 2, which was supported by the HMBC correlations of H-6 to C-7 (δ C 191.8), as well as H-7 to C-1 (δ C 127.5), C-2 (δ C 161.3) and C-6 (δ C 128.7). Secondly, the orientation of the olefinic bond between C-7 and C-8 was assigned as E, based on the typical coupling constant between H-7 and H-8 (J = 16.0 Hz). Detailed analysis of 2D-NMR (HSQC, HMBC and 1 H-1 H COSY) spectra confirmed the planar structure of 2, as shown in Figure 2. Furthermore, the coupling constant of the anomeric proton resonating at 5.05 (1H, d, J = 7.6 Hz, H-1") suggested that the glucopyranosyl moiety was β-glucoside. In order to further confirm the structure of 2, the acid hydrolysis reaction of 2 was carried out. As a result, a β-D-glucose was produced as the sole sugar identified on the basis of the same R f value on co-TLC and the almost identical optical value by comparing with that of an authentic sugar sample. Thus, compound 2 was established as 6-O-(E)-p-coumaroyl-β-D-glucopyranoside-2-benzaldehyde, as shown in Figure 1.  (Table 2) revealed the presence of 21 carbon atoms, including 13 sp 2 carbon atoms, six sp 3 methines and two sp 3 methylenes, which were attributable to one dihydroisocoumarin skeleton and one glucopyranosyl moiety. The above data revealed that the structure of 3 was similar to that of thunberginol G 3 -O-glucoside [22], except that the hydroxyl group at C-8 was substituted by a hydrogen atom, which was supported by the HMBC correlations of H-8 to C-1 (δ C 164.9), C-4a (δ C 140.0) and C-8a (δ C 124.8), as well as the 1 H-1 H COSY correlations from H-5 to H-8. Detailed analysis of 2D-NMR (HSQC, HMBC and 1 H-1 H COSY) spectra confirmed the planar structure of 3 (see Figure 2). Furthermore, the coupling constant of the anomeric proton resonating at 4.59 (1H, d, J = 7.6 Hz, H-1") suggested that the glucopyranosyl moiety was β-glucoside. In order to further confirm the structure of 3, the acid hydrolysis reaction of 3 was carried out. As a result, a β-D-glucose was produced as the sole sugar identified on the basis of the same R f value on co-TLC and the almost identical optical value by comparing with that of an authentic sugar sample. In addition, the absolute configuration of the aglycone of 3, only holding one chiral center at C-3, was determined as S, based on its specific optical rotation of [α] 24 D −153.0 (c 0.9, CH 3 OH), which was similar with that of (S)-3,4-dihydro-3-phenylisochromen-1-one ([α] 24 D −158.0), whose structure and absolute configuration had been determined by a combination of spectroscopic analyses and chemical methods [23]. Hence, compound 3 was determined as 3S-(4 -hydroxyl-3 -O-β-D-glucopyranosyl phenyl)-dihydroiso coumarin, as shown in Figure 1.  Table 2) of 3 were nearly identical to those of 4. Detailed analysis of 2D-NMR (HSQC, HMBC and 1 H-1 H COSY) spectra confirmed that 4 shared the same planar structure with 3 (see Figure 2). The specific rotation of 4, [α] 24 D +187.0 (c 0.10, CH 3 OH), suggested that its configuration should be different from that of 3 ([α] 24 D −89.2). In order to further confirm the structure of 4, the acid hydrolysis reaction of 4 was carried out. As a result, a β-D-glucose was produced as the sole sugar identified on the basis of the same R f value on co-TLC and the almost identical optical value by comparing with that of an authentic sugar sample. In addition, the absolute configuration of the aglycone of 4 was determined as R, in consideration of its converse optical rotation of [α] 24 D +146.0 (c 0.9, CH 3 OH) with that of the aglycone of 3, which was very similar with that of (R)-3,4-dihydro-3-phenylisochromen-1-one ([α] 24 D +168.5), whose structure and absolute configuration had been determined by a combination of spectroscopic analyses and chemical methods [24]. Accordingly, compound 4 was identified as the 3-epimer of 3, namely, 3R-(4 -hydroxyl-3 -O-β-D-glucopyranosylphenyl)-dihydro isocoumarin, as shown in Figure 1.
All new compounds were evaluated for their anti-inflammatory properties via examining the inhibitory activity on NO production induced by LPS in mouse macrophage RAW 264.  The above findings may be used as an explanation of the folk use of H. paniculiflorum, which was used as an anti-inflammatory drug in China [1,11]. These findings also suggest that the phenolic glycoside and isocoumarin glycoside with significant inhibitory activities on NO production isolated from H. paniculiflorum could be used for the development of new anti-inflammatory agents.

Plant Material
The

Acid Hydrolysis of Compounds 1-4
Compounds 1-4 (each 1.0-2.0 mg) were refluxed with 2 mL of 1 N HCl for 1 h at 100 • C. The reaction mixtures were extracted with EtOAc and the aqueous phase was compared to an authentic sugar sample by co-TLC (CHCl 3 -CH 3 OH-H 2 O-AcHO, 13:3:3:1, R f 0.46 for glucose). The identification of β-D-glucose in each aqueous layer was realized by comparing the optical rotation of the liberated glucose with that of an authentic sample of β-D-glucose ([α] 24 D +55.0).

Inhibitory Assay of NO Production
Murine macrophage cell line RAW264.7 was obtained from Cell Bank of Chinese Academy of Sciences. RAW264.7 cells were seeded in 96-well cell culture plates (1.5 × 10 5 cells/well) and treated with serial dilutions of the compounds with a maximum concentration of 100 µM in triplicate, followed by stimulation with 1 µg/mL LPS (Sigma, St. Louis, MO, USA) for 18 h. NO production in the supernatant was assessed by Griess reagents (Reagent A & Reagent B, respectively, Sigma). The absorbance at 570 nm was measured with a microplate reader (Thermo, Waltham, MA, USA). N G -Methyl-L-arginine acetate salt (L-NMMA, Sigma, Hongkong, China), a well-known nitric oxide synthase (NOS) inhibitor, was used as a positive control [25]. The viability of RAW264.7 cells was evaluated by the MTS assay simultaneously to exclude the interference of the cytotoxicity of the test compounds [26].

Conclusions
Phytochemical investigation on the stems of H. paniculiflorum resulted in the isolation of two new phenolic glycosides (1 and 2) and two new isocoumarin glycosides (3 and 4), along with 14 known compounds (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Their structures were established on the basis of extensive spectroscopic analyses and chemical methods. All new compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on NO production induced by LPS in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 4 exhibited significant inhibitory activities with IC 50 values comparable to that of L-NMMA.
Supplementary Materials: 1D and 2D NMR spectra of compounds 1-4 as Supplementary Materials are available online.