Coriaria nepalensis is the only species in the Coriariaceae family and is distributed in the southern and southwestern of China. The dried root is used in the treatment of numbness, toothache, traumatic injury, and acute conjuncticitis in the traditional Chinese medicine [1]. Sesquiterpene lactones are the characteristic bioactive constituents of family Coriariaceae and possessed many interesting variations in the structure [2-7]. So far, there are only 17 similar sesquiterpene lactones separated from the species. In our previous study, we reported a series of sesquiterpenes and sesquiterpene lactones with anti-convulsant activities from C. nepalensis [8]. Continuing our investigation on the less polar fractions from the same plant led to the isolation of one new sesquiterpene lactone, corialactone E (1), one new neolignan, coriarianeolignan A (2), three known apocarotenoids, (6R, 9R)-9-hydroxy-4-megastigmen-3-one (3) [9], blumenol A (4) [10], and 3S, 5R-dihydroxy-6, 7-megstigmadien-9-one (5) [11], and one known neolignan, 1-(4-hydroxy-3, 5-methoxyphenyl)-2-(3, 5-dimethoxy-4-aldehyde-phenoxy)-propane-1, 3-diol (6) [12] (Fig. 1). In the present paper, we reported details of the isolation, structure elucidation of compounds 1-6 and their bioactivities.

The air-dried roots of C. nepalensis (52 kg) were smashed and extracted with 95% EtOH. The EtOH extract (10.5 kg) was suspended in H₂O and partitioned successively with petroleum ether, CHCl₃, EtOAc and n-BuOH. The 45% EtOH soluble portion (210 g) of the CHCl₃ extract (420 g) was then subjected to a polyamide column chromatography with a gradient of aqueous EtOH to yield 4 fractions. Fr1 (130 g) eluted by 45% EtOH was subjected to a silica gel column chromatography, a polyamide column chromatography, and an ODS column chromatography, purified by a Sephadex LH-20 and preparative HPLC to afford compounds 1 (5 mg), 2 (5 mg), 3 (20 mg), 4 (22 mg), and 5 (26 mg).

Compound 1 was obtained as colorless needles with [α]_D²⁰ +15.9 (c 0.09, MeOH). The molecular formula (C₁₅H₁₈O₆) with 7 degrees of unsaturation was determined by HR-ESIMS at m/z 317.1003 [M + Na]⁺) and NMR data (Table 1). The IR spectrum of 1 showed the absorption of lactone carbonyl group (1738 cm^-1). Fifteen carbon signals including one carbonyl carbon (δ_C 171.6) were observed in the ¹³C NMR (DEPT) data (Table 1). In comparison to the known compound apotutin [3], which is a typical sesquiterpene lactone separated from Coriaria nepalensis, the ¹H NMR data of compound 1 showed two proton signals for one AB spin-spin coupling system at δ_H 3.76 and δ_H 3.96, which is a characteristic functional group in the series of sesquiterpene lactones separated from Coriaria nepalensis. The C-11 (δ_C 56.5) and C-12 (δ_C 52.9) signals in the ¹³C NMR data confirmed the presence of epoxide ring of C-11/O/C-12. The C-6 (δ_C 85.4) and C-8 (δ_C 92.0) signals in the ¹³C NMR data also indicated the skeleton of compound 1 was mostly similar with apotutin. The ¹H NMR data of compound 1 showed the proton signals at δ_H 1.31, δ_H 4.00, and δ_H 3.53, which indicated that one methyl of isopropyl in compound 1 transformed into an oxygenated methylene. Structure units assigned above were further confirmed by the signals of C-10 (δ_C 20.1), C-8 (δ_C 92.0), and C-9 (δ_C 76.2) in the ¹³C NMR data. Meanwhile, the proton signals at δ_H 4.17 and δ_H 4.14, and the C-14 (δ_C 60.8) signals indicated the opening of epoxide ring of C-13/O/C-14, which is another characteristic functional group in the series of sesquiterpene lactones separated from Coriaria nepalensis, and transformed into hydroxymethyl in compound 1. Analysis of the ¹H-¹H COSY and HSQC correlations allowed for the assignment of two spin systems: CH₂(2) -CH(3) -CH(4) -CH(5) and CH(11) -CH(12) units (Fig. 2A and Figs. S4-S5 in Supporting information), which were further identified as characteristic fragments in a skeleton similar with the known compound apotutin. The C-3 (δ_C 77.4) and C-9 (δ_C 76.2) signals revealed that C-3 and C-9 were two oxygenated carbons, and the correlation of H-9a with C-3 in the HMBC spectrum indicated that C-3 and C-9 was connected by an ether bond (Fig. 2A). The C-15 (δ_C 171.6) signal indicated the presence of lactone carbonyl group, and the correlation of H-5 with C-15 in the HMBC spectrum confirmed that C-5 was connected with C-15 (Fig. 2A). The HR-ESIMS showed that compound 1 had six oxygen atoms with 7 degrees of unsaturation. So far the ether bonds of C-3/O/C-9, C-6/O/C-8, and C-11/O/C-12, the lactone carbonyl connecting C-5 and C-15, and the hydroxymethyl at C-13 have already showed six oxygen atoms and 6 degrees of unsaturation in compound 1. The C-13 (δ_C 91.7) signal indicated that C-13 was also an oxygenated carbon, so the only connecting way of oxygenated C-13 was to form an ester bond between C-13 and C-15. The structure shown above was further confirmed by the HMBC spectrum (Fig. S6 in Supporting information). In the HMBC spectrum, the correlations of H₃-10 with C-4, C-8 and C-9, of H₃-7 with C-1, C-2, C-6 and C-13, of H-2α with C-1, C-3, C-4, C-6 and C-7, of H-2β with C-1, C-7 and C-13, of H-4 with C-2, C-5, C-6, C-8 and C-10, of H-9b with C-8 and C-10, of H-5 with C-1, C-3, C-4, C-6, C-11 and C-15, of H-11 with C-1, C-6, C-12 and C-13, of H-12 with C-1, C-6, C-11 and C-13, of H-9a with C-3, C-4 and C-8, of H-14b with C-12, of H-14a with C-12 and C-1, and of H-3 with C-1 and C-8 verified the planar structure of 1.

The epoxide rings at C-11, C-12 and C-14 used to be considered as the characteristic function groups of this type of sesquiterpenes. However, in our previous study, we elucidated a series of four new sesquiterpene lactones (corialactone B, corialactone C, corialactone D, and corianol) and these compounds all possessed new variations in the epoxide rings. For example, the epoxide rings in corialactone B, corialactone C and corialactone D were opened and transformed into methylene (C-11), olefinic (C-12 and C-13), and aldehyde (C-14) groups in corialactone B, oxygenated methine (C-11), olefinic (C-12 and C-13), and hydroxymethyl (C-14) groups in corialactone C, and methylene (C-11), oxygenated methine (C-12), and terminal olefinic (C-13 and C-14) groups in corialactone D, respectively. Besides, in corianol, the connection of C-3 and C-11 formed a new oxygen bridge, which was also found from this family for the first time [8]. In this study, the epoxide ring of C-13/O/C-14 in compound 1 was also opened and transformed into hydroxymethyl (C-14) group. In addition, the lactone ring located at C-3 was opened and formed a new lactone ring connecting C-13 and C-5. Besides, oxygenated C-3 and C-9 connected and formed a new ether ring. The newly formed lactone ring located at C-13 and C-5 produced shielding effect, so the signals of C-12 (δ_C 52.9), C-6 (δ_C 85.4), and C-5 (δ_C 50.0) were observed at a higher field compared with those of apotutin (δ_C 57.2, δ_C 89.9, and δ_C 55.62, respectively). For the influence of the newly formed cyclic ether of C-3/O/C-9, the signals of C-3 (δ_C 77.4) and C-4 (δ_C 50.5) were also observed at a higher field comparing with those of apotutin (δ_C 83.6 and δ_C 53.5, respectively).

The relative configuration of 1 was elucidated based on the ROESY spectrum (Fig. S7 in Supporting information). Due to the fused ring structure of compound 1, the relative configuration was rather complex. As shown in Fig. 2B, in the ROESY spectrum, H₃-7 showed correlation with H-2α and H-14, indicating that H₃-7 and H-2α were in the same α-orientation, and H-14 was the same side with H₃-7. H-9a showed correlations with H₃-7 and H₃-10, indicating that the newly formed cyclic ether of C-3/O/C-9, H₃-10, and the cyclic ether of C-6/O/C-8 were all at the same side with H₃-7. Thus, the H-3 and H-4 adapted β-orientation. As elucidated in the literature [6-8], the relative configuration of H-5 was determined as α-orientation on the basis of its small coupling (J = 4.0 Hz) with H-4 and its REOSY correlations with H₃-10 and H-4. Moreover, as shown in Fig. 2B and according to the literature [6-8], H-11 showed correlations with H-5 and H-12, indicating that H-11 and H-12 were all in β-orientation. The absolute configuration of 1 was established by the sector rule [13]. Based on the assigned relative configuration and the positive Cotton effect at 208 nm in the CD spectrum (Fig. S11 in Supporting information), the absolute configuration of 1 was determined to be 1R, 3R, 4S, 5S, 6R, 8S, 11S, 12R, and 13S (Fig. 3). Hence, the structure of 1 was established as shown and named to be corialactone E.

Compound 2 was obtained as light yellow solid with [α]_D²⁰ +0.04 (c 0.08, MeOH). Its IR spectrum showed the presence of hydroxy (3387 cm^-1) and aromatic (1688 cm^-1, 1589 cm^-1) functionalities. The molecular formula (C₁₉H₂₂O₈) with 9 degrees of unsaturation was determined by HR-ESIMS at m/z 401.1218 [M + Na]⁺. The NMR data (Table 2) indicated that there are both guaiacylglycerol-8-yl and 3'5'-dimethoxy-4'-oxyphenyl groups in 2. The ¹H NMR data of 2 showed signals attributed to one oxymethylene at δ_H 3.72 and δ_H 3.36, together with two signals attributed to two oxymethines at δ_H 4.92 and δ_H 4.29. The ¹H NMR data of 1 also showed one group of 1, 3, 4-tri-substituted benzene ring signal at δ_H 6.91, δ_H 6.64, and δ_H 6.76, together with one group of 1, 3, 4, 5-tetra-substituted benzene ring signal at δ_H 7.15. The ¹³C NMR and DEPT spectra (Figs. S13-14 in Supporting information) of 2 showed 19 carbon resonances including twelve aromatic carbons. The ¹H-¹H COSY correlations (Figs. S15-16 in Supporting information) indicated the presence of CH(7)-CH(8)-CH₂(9) units. The ¹H NMR and ¹³C NMR data of 2 resembled those of the known compound, (+)-(7S, 8S)-4-hydroxy-3, 3', 5'-trimethoxy-8', 9'-dinor-8, 4'-oxyneolignan-7, 9-diol-7'-oic acid [14], except for substitution of CHO-1' in 2 by COOH-1' in (+)(7S, 8S)-4-hydroxy-3, 3', 5'-trimethoxy-8', 9'-dinor-8, 4'-oxyneolignan-7, 9-diol-7'-oic acid, which was indicated by the chemical shifts for C-1' and C-4' significantly shifted by Δδ_C 9.7 and 3.6, respectively. In the HMBC spectrum (Fig. S17 in Supporting information), the correlations of H-7 with C-2, C-6, C-8, and C-9, of H-2'/6' with C-3'/5', C-4' and CHO, and of OMe-3'/5' with C-3'/5' verified the location of the substitution groups and the 8, 4'-oxy linkage in 2. The HMBC spectrum also showed correlations of aldehyde hydrogen between C-10 and C-2'/6', which indicated the presence of one aldehyde group (Fig. 4).

It has been previously reported that for syringoylglycerols and guaiacylglycerol derivatives, the coupling constant (J value) between H-7 and H-8 is ≤ 5 Hz for the erythro isomer and ≥ 7 Hz for the threo isomer [15]. Thus, the coupling constant between H-7 and H-8 (J = 5.0 Hz) indicated the 7, 8-erythro configuration of compound 2. The absolute configuration was established by the CD spectrum. The positive Cotton effect at 246 nm in the CD spectrum indicated that the absolute configuration was 7R, 8S [16-20]. Hence, the structure of 2 was established to be coriarianeolignan A, and its structure was shown in Fig. 1.

Three known apocarotenoids, (6R, 9R)-9-hydroxy-4-megastigmen-3-one (3) [9], blumenol A (4) [10], and 3S, 5R-dihydroxy-6, 7-megstigmadien-9-one (5) [11], and one known neolignan, 1-(4-hydroxy-3, 5-methoxyphenyl)-2-(3, 5-dimethoxy-4-aldehyde-phenoxy)-propane-1, 3-diol (6) [12], were identified by comparing their spectroscopic data (NMR, UV, IR, and MS) with known compounds.

Compounds 1, 3, and 5 were also tested in neurotoxic assay against PC12 cells (pheochromocytoma 12 cells) and cytotoxic assay against human cancer cell lines including HCT-8 (human colon cancer), HepG2 (human hepatoma cancer), BGC823 (human gastric cancer), A549 (human lungepithelia cancer) by using MTT method [21]. All these compounds were inactive (10 μmol/L) in neurotoxic assay. Compound 5 showed cytotoxic activity on SKOV3 (human ovarian cancer) cells with IC₅₀ values of 4.67 μmol/L, and the other compounds were inactive at 10 μmol/L concentration in cytotoxic assay. In the preliminary anti-convulsant activity assay in vivo, compounds 3 and 5 were tested by electroshock experiments [22]. After intraperitoneal administration to mice at a dose of 5 mg/kg, compound 5 showed anti-convulsant activity by 34%. However, compound 3 induced convulsions at the same dosage. The positive control (sodium phenobarbital) showed 100% anticonvulsant activities at a dose of 20 mg/kg.

In conclusion, one new sesquiterpene lactone (1), one new neolignan (2), and four known compounds (3-6) were isolated from the ethanolic extract of roots of C. nepalensis. The unique structure of newly formed ether ring of C-3/O/C-9 and lactone ring connecting C-13 and C-5 of compound 1 is of interesting for synthetic chemists and biologists. Compound 5 showed cytotoxic activity on SKOV3 (human ovarian cancer) cells with IC₅₀ values of 4.67 μmol/L. In vivo system, compound 3 showed anti-convulsant activity by 34% at the dose of 5 mg/kg.

Acknowledgments

Financial support from the National Natural Science Foundation of China (No. 21132009) and the National Science and Technology Project of China (No. 2012ZX09301002-002) is acknowledged. We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for measuring the IR, UV, NMR, MS, and CD spectra.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2017.10.009.