The genus Rubus is one of the most species-rich (about 700 species) genera in the Rosaceae family, of which 194 species are found in the southwest of China and a few species have been used as medicinal herbs.^[1] Rubus idaeus Linnaeus is a Chinese herbal medicine that has been widely used in China to nourish the liver, reinforce the kidney, reduce urination, and improve vision.^[2] It’s not only a medicinal herb but also an ornamental. R. idaeus is a vine-like shrub that is 1~2 m high. The fruit of R. idaeus is nearly spherical, succulent and red with a diameter of 10~15 mm. Previous phytochemical investigations on R. idaeus have identified a diverse range of compounds, including flavonoids, phenolic acid, coumarins, tannins, lignans, terpenoids, alkaloids and steroids.^[3-8] Among these compounds, many anti-inflammatory compounds were reported such as anthocyanins and ellagitannins.^[9]

In order to search for significant bioactive natural products from the medicine-food plant R. idaeus, a new tetrahydrogenated naphthol syringic acid ester, named rubusno- licester (1), two new phenolic glycoside derivatives, 4-chloro-2,6-dimethoxylphenol-1-O-β-D-glucopyranoside (2) and salicylic acid-2-O-(6'-O-acetyl)-β-D-glucopyrano- side (3), together with one known salicylic acid glycoside derivative (4) and three known flavonoids derivatives (5~7), were isolated (Figure 1). Their structures were elucidated by HRESI-MS, NMR spectroscopy, and a comparison of optical rotation (OR). Herein, their isolation, structure elucidation and biological activities were reported.

Compound 1 was obtained as white powder and had the molecular formula C₁₉H₂₀O₆ based on the prominent signal at m/z 343.1185 [M－H]^－ in the HRESI-MS. The ¹H NMR spectrum of 1 showed the presence of five aromatic protons in the downfield at δ 7.34 (s, 2H, H-2',6'), 6.86 (d, J=8.4 Hz, 1H, H-9), 6.54 (dd, J=8.4, 2.4 Hz, 1H, H-8), and 6.50 (d, J=2.4 Hz, 1H, H-6). In the upfield, one oxygenated methine proton at 3.97~4.05 (m, 1H, H-2), two methoxyl groups at δ 3.89 (s, 6H, 3'/5'-OMe), and three methylene groups at δ 2.94 (dd, J=15.6, 5.2 Hz, 1H, H-1a), 2.59 (dd, J=15.6, 8.4 Hz, 1H, H-1b), 2.82~2.88 (m, 1H, H-4a), 2.70~2.78 (m, 1H, H-4b), 1.98~2.04 (m, 1H, H-3a) and 1.67~1.75 (m, 1H, H-3b) were also observed. In addition, 19 carbon signals were detected in the ¹³C NMR spectrum (Table 1) including signals of two methoxy groups, three methylene carbons, and one oxygenated methine carbon, twelve aromatic carbons and one ester carbonyl carbon. These ¹H NMR and ¹³C NMR spectroscopic features suggested that the structure of 1 comprised a syringic acid^[10] subunit and a (R)-1,2,3,4-tetrahydro-6-hydroxynaphthalen- 2-ol^[11] subunit. These assignments were confirmed by the following heteronuclear multiple-bond correlation (HMBC) data. The ¹H-¹H correlation spectroscopy (COSY) of H-1/ H-2, H-2/H-3, and H-3/H-4 combined with the HMBC from H-9 to C-1/5/7/10, H-6 to C-4/8/10 indicated the presence of the subunit (R)-1,2,3,4-tetrahydro-6-hydroxynaphthalen- 2-ol in 1 (Figure 2). The HMBC from H-2′/6′ to C-1'/3'/4'/7' (Figure 2) indicated the subunit syringic acid. Furthermore, in the HMBC spectrum, H-2 showed a correlation to C-7' (Figure 2). These data indicated that two subunits were joined via a C-2-O-C-7′ linkage. Compound 1 has the (2R)-form based upon the positive optical rotation of 1 and the reported similar compounds in literature.^[11] Thus, the structure of compound 1 was identified as the Figure 1. compound 1 was named as rubusnolicester.

Compound 2 possessed a molecular formula of C₁₄H₁₉- ClO₈, as deduced from HRESIMS (m/z 351.0846 [M＋H]^＋, calcd 351.0841). The ¹H NMR data revealed two aromatic protons at δ 7.01 (s, 2H, H-3,5), two methoxyl groups at δ 3.81 (s, 6H, 2/6-OMe), together with many protons arising from sugar moiety. The ¹³C NMR and 135 distortionless enhancement by polarization transfer (DEPT) data showed 14 resonances, including six aromatic carbons (δ 154.8, 154.8, 137.8, 128.2, 103.3 and 103.3), six O-bearing carbons (δ 106.2, 78.3, 77.9, 75.8, 71.4 and 62.7), and two methoxyl carbons (δ 56.8 and 56.8). These data closely resembled those of known leonuriside A^[12] except for the differences of chemical shift at C-4 (δ 128.2 for 2, 154.0 for leonuriside A). These data combined with the molecular formula of C₁₄H₁₉ClO₈ of 2 indicated that the OH group at C-4 in leonuriside A was placed by the Cl at C-4 in 2. The 2D structure of 2 was thus constructed. Detailed analysis of 2D NMR (HSQC (heteronuclear single quantum coherence), ¹H-¹H COSY and HMBC) spectra confirmed that the other part of the molecule was the same as those of leonuriside A. The glycone part of 2, a glucose moiety, was identified and characterized by the presence of six carbon signals (δ 154.8, 154.8, 137.8, 128.2, 103.3 and 103.3) and a signal for one anomeric proton doublet at δ 4.67 (J=7.2 Hz, H-1'), respectively. The large ³J_H-1'/2'=7.2 Hz, large ³J_H-2'/3'=9.2 Hz, large ³J_H-3'/4'=7.6 Hz and large ³J_H-4'/5'=8.4 Hz revealed the axial-axial relationship for these protons. These data suggested that the glycosyl moiety was β-glu- cose. Acid hydrolysis of 2 produced glucose as the sole sugar identified on the basis of PMP-labeling HPLC analysis with a ULTRON ES-OVM chiral column. The retention time of the PMP-labeling product of compound 2 (retention time: 44.87 min) was the same as the retention time of the PMP-labeling product of D-glucose standard (retention time: 44.87 min). The optical rotation value of the glucose from compound 2 ($[\alpha]_{\mathrm{D}}^{24}$＋51.6, H₂O) was also the same as the standard D-glucose ($[\alpha]_{\mathrm{D}}^{24}$＋52.0, H₂O). Thus, the structure of compound 2 was identified as 4-chloro-2,6- dimethoxylphenol-1-O-β-D-glucopyranoside (Figure 1).

Compound 3 was obtained as white amorphous powder, with the molecular formula C₁₅H₁₈O₉ from HRESIMS m/z 341.0875 [M－H]^－ (C₁₅H₁₇O₉, calcd 341.0878) combined with ¹H NMR and ¹³C NMR spectroscopic data. In the ¹H NMR spectrum, four aromatic protons at δ 7.59 (d, J=7.6 Hz, 1H, H-6), 7.48 (dd, J=8.4, 7.6 Hz, 1H, H-4), 7.17 (d, J=8.4 Hz, 1H, H-3) and 7.05 (dd, J=7.6, 7.6 Hz, 1H, H-5), one singlet methyl group at δ 1.96 (s, 3H, H-2''), together with many protons arising from sugar moiety were observed. The ¹³C NMR data showed 15 resonances, including two carbonyl carbons (δ 170.7 and 167.6), six aromatic carbons (δ 155.7, 133.7, 130.8, 122.3, 122.0 and 116.8), six O-bearing carbons, and one methyl carbon (δ 21.1), which are characteristic signals of a phenolic acid glycoside. The ¹H NMR and ¹³C NMR data (Table 1) were very similar to those of known compound 4, except for the presence of one acetyl group signal [δ_H 1.96 (3H) and δ_C 167.6/21.1] in 3 and the absence of a methoxyl group signal (δ_C 52.0) in 4. The location of the acetyl group at C-6' was confirmed by the HMBC correlations from H-6' to C-1'' (Figure 2). The HMBC correlations from H-1' to C-2 indicated that C-1' linked with C-2 by a glycosidic bond. The HMBC correlations from H-6 to C-1/7 and H-3 to C-1 indicated that the carboxyl group located at C-1. Detailed analysis of 2D NMR (HSQC, ¹H-¹H COSY and HMBC) spectra confirmed that the other part of the molecule was the same as those of 4. The glycone part of 3, a glucose moiety, was identified and characterized by the anomeric proton doublet at δ_H 4.91 (d, J=6.0 Hz, 1H, H-1')/δc 101.1. These data suggested that the glycosyl moiety was β- glucose. According to the biogenic synthesis pathway and the similar optical rotation value of compounds 3 and 4, the glycosyl moiety was assigned as D-glucose. Furthermore, it was also determined by the PMP-labeling HPLC analysis method. The retention time of the PMP-labeling product of compound 3 (retention time: 44.87 min) was the same as the retention time of the PMP-labeling product of D-glucose standard (retention time: 44.87 min). The optical rotation value of the glucose from compound 3 ($[\alpha]_{\mathrm{D}}^{24}$＋51.4, H₂O) was also the same as the standard D-glucose ($[\alpha]_{\mathrm{D}}^{24}$＋52.0, H₂O). This result indicated that the glycosyl of compound 3 was D-glucose (Figure 3). Thus, compound 3 was identified as salicylic acid-2-O-(6'-O-acetyl)-β-D-glucopyranoside, a new phenolic glycoside.

The structures of known compounds 4~7 were identified by comparison of their ¹H NMR and ¹³C NMR spectra and $[\alpha]_{\mathrm{D}}^{24}$ with those in the literature. The four known compounds were identified as 2-O-β-D-gluco- pyranosylbenzoate (4),^[13] catechin (5),^[14] epicatechin (6)^[14] and quercetin (7).^[14]

Compounds 1~7 were evaluated their anti-inflammatory activities via testing the inhibitory activities against the NO production induced by lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells in vitro. As a result, compounds 1 showed significant inhibitory activity with the IC₅₀ value of (12.28±1.25) μmol/L. The positive control, hydrocortisone, showed an inhibitory activity with the IC₅₀ value of (8.16±1.02) μmol/L. Compounds 3 and 4 showed moderate inhibitory activity with the IC₅₀ value of (25.24± 1.58) and (36.65±1.87) μmol/L, respectively. No cytotoxicity was observed in compounds 1~7 treated cells (cell viability>95%). The IC₅₀ values of other compounds higher than 40 μmol/L were regarded as inactive.

In this study, a new tetrahydrogenated naphthol syringic acid ester, rubusnolicester (1), two new phenolic glycoside derivatives, 4-chloro-2,6-dimethoxylphenol-1-O-β-D-gluco- pyranoside (2) and salicylic acid-2-O-(6'-O-acetyl)-β-D- glucopyranoside (3), together with one known salicylic acid glycoside derivative (4) and three known flavonoids derivatives (5~7), were isolated from the fruits of R. idaeus. Compound 2 represented the rare example of a chlorine-substituted phenolic glycoside derivative from natural products. The significant inhibitory activities on NO production of compound 1 could be used for the development of new anti-inflammatory agents.