1. Introduction

Juncus effusus L.(Juncaceae) is a perennial herbaceous plant distributed in the wetlands and coastal marshes of South China.Its stem pith has long been used as a sedative, anxiolytic, antipyretic and diuretic agent for the treatment of fidgetiness, insomnia and edema in traditional Chinese medicine [1, 2].Previous phytochemical investigations on this genus have reported the existence of secondary metabolites such as phenanthrenoids [1-17], dihydro-dibenzoxepins [13, 18], benzocoumarins [19], phenylpropane glycerides [6, 20, 21], flavonoids [7], triterpenes [22], and phenolic acid derivatives [1].Phenanthrenoids are characteristic constituents of the genus Juncus, exhibiting a wide range of biological activities like anti-algal [3, 8-10, 12, 13, 15], cytotoxic [1, 2], antitumor [17], anti-inflammatory [2, 4], anxiolytic [5, 14], and antioxidant [7] activities.Phenanthrenoid dimers were previously reported from this genus, however, their number is still few [2, 7-9, 12].In our efforts on searching for bioactive natural products from Chinese medicinal plants, an investigation of the whole plant of J.effusus was carried out, which led to the isolation of two phenanthrenoid dimers named dijuncuenins A and B (1, 2, Fig. 1).These two compounds possessed linkages of C-14-C-3' and C14-C-8', respectively, between two phenanthrenoid halves, both of which have not been reported before.This paper describes the isolation and structure elucidation of compounds 1 and 2, and their plausible biosynthetic pathways as well.

2. Experimental 2.1. General experimental procedures

Optical rotations were measured on a Rudulph Autopol Ⅵ Automatic polarimeter.IR and UV data were recorded on a Nicolet Magna FTIR-750 and a Shimadzu UV-2550 spectrophotometer, respectively.NMR spectra were recorded on a Bruker AM-400 (Bruker, Ettlingen, Germany) and a Bruker Avance Ⅲ (Bruker, Ettlingen, Germany) 500 NMR spectrometer with TMS as internal standard.HRESIMS were measured on a Waters Xevo QTof mass detector.Analytical HPLC was applied on a Waters 2695 instrument coupled with a 2998 PDA, a Waters Acquity ELSD, and a Waters 3100 SQDMS detector.Chiral HPLC analysis was detected on a JASCO 2000 instrument by using Daicel columns with 2-propanol/hexane as the eluents.TLC was performed on pre-coated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Industrials).MCI gel CHP20P (75-150 μm, Mitsubishi Chemical Industries, Japan), Silica gel (Qingdao Marine Chemical Industrials), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC).Preparative HPLC was performed on a Varian PrepStar system with an Alltech 3300 ELSD using a Waters Sunfire RP C₁₈, 5 μm, 30 × 150 mm column.All solvents used for CC and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd.) and gradient grade (Merck KGaA, Germany), respectively.

2.2. Plant material

The whole plant of J.effusus was collected in Jinxiu County, Guangxi Province, China, and identified by Professor Jing-Gui Shen of Shanghai Institute of Materia Medica. A voucher specimen (No.20120618) was deposited at the herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

2.3. Extraction and isolation

The air-died and powdered whole plant of J.effusus (10 kg) was extracted with 95% EtOH (4 × 30 L) at room temperature (each 72 h).The combined extracts were concentrated to yield a dark brownish residue (605 g).The residue was suspended in water and then partitioned with petroleum ether (PE) and CH₂Cl₂ successively to yield a PE (85 g) and a CH₂Cl₂ extract (150 g).The CH₂Cl₂ extract was applied to a MCI gel column eluted with EtOH-H₂O in gradient (50:50 to 95:5) to obtain six fractions (Fr.1-Fr.6).Fr.5 was separated over silica gel CC (200-300 mesh) eluted gradiently with PE-acetone (15:1 to 0:1) to afford nine subfractions (Fr.5A-Fr.5I).Fr.5I was purified by CC over Sephadex LH-20 gel and then preparative HPLC (MeCN-H₂O, 50-60%, 0-100 min, 25.0 mL/min) to yield 1 (20 mg) and 2 (3 mg).

Dijuncuenin A (1): Yellow powder; 0 (c 0.35, MeOH); UV (MeOH) λ_max (log ε) 262 (4.74), 214 (4.80) nm; IR (KBr, cm^-1): ν_max 3513, 3378, 2957, 1617, 1576, 1474, 1441, 1392, 1278, 1220, 1100, 1054, 908; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 529.3 [M-H]^-; HRESIMS m/z 529.2355 [M-H]^- (calcd. for C₃₆H₃₃O₄, 529.2379).

Dijuncuenin B (2): Yellow powder; 0 (c 0.35, MeOH); UV (MeOH) λ_max (log ε) 262 (4.83), 215 (4.70) nm; IR (KBr, cm^-1) ν_max 3404, 3077, 2954, 2922, 2835, 1658, 1614, 1464, 1379, 1268, 1213, 1072, 1059, 882, 804, 755; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 527.2 [M-H]^-; HRESIMS m/z 527.2231 [M-H]^- (calcd. for C₃₆H₃₁O₄, 527.2222).

3. Results and discussion

Compound 1 had the molecular formula of C₃₆H₃₄O₄ as determined by HRESIMS (m/z, 529.2355, [M-H]^-) with 20 degrees of unsaturation. The IR absorption bands at 3513 and 3378 cm^-1 indicated the presence of hydroxyl groups. The ¹H NMR spectrum (Table 1) displayed signals attributable to four ortho-coupled aromatic protons (δ_H 8.45, 7.84, 7.72, 7.28), four singlet aromatic protons (δ_H 8.76, 7.70, 7.30, 6.67), a vinyl group (δ_H 6.53, 5.25, 5.09), an oxygenated aliphatic methine (δ_H 5.23, dd, J=7.8, 1.9 Hz), and four methyls (δ_H 2.52, 2.48, 2.21, 2.11). The ¹³C NMR spectrum (Table 1) exhibited 36 resonances including 18 sp² quaternary (three oxygenated), nine sp² methine, one oxygenated sp³ methine, four methylene (three sp³ and one sp²), and four methyl carbons. These data suggested the existence of a phenanthrene unit and a 9, 10-dihydrophenanthrene unit in the molecule. These two units accounted for all the 20 degrees of unsaturation. Therefore, a C-C linkage was assigned between the two units. In the ¹H-¹H COSY spectrum (Fig. 2), the proton at δ_H 5.23 (H-13) was observed to correlate with the methylene at δ_H 2.83 and 2.99 (H₂-14), indicating that they might be derived from the vinyl substituent of one unit. The HMBC correlations from H₂-14 to C-6 (δ_C 142.8), C-20 (δ_C 151.8), and C-4'(δ_C 129.7), and from H-13 to C-5 (δ_C 118.4) (Fig. 2) revealed the connections between C-14 and C-3' and between C-13 and C-6, respectively. In addition, the ROESY correlations of H₃-11↔H-10, H-3↔H-4↔H-5, H-8↔H₃-12, H₃-11'↔H₂-10', H₂-14↔H-4'↔H-13'↔H₃-12', and H-8'↔H₂-9' (Fig. 2), with the aid of the HMBC correlation from H-9 to C-8 (δ_C 129.0), determined the substitution pattern of 1: two methyls at C-1 and C-7, and a hydroxyl group at C-2 in one phenanthrene unit; two methyls at C-1' and C-6', two hydroxyl groups at C-2' and C-7', and a vinyl group at C-5' in the other 9, 10-dihydrophenanthrene unit. The planar structure of 1 was thus constructed while the stereochemistry of C-13 still remained uncertain. The specific optical rotation of 1 was recorded as zero. When compound 1 was run on HPLC with optically active stationary phase, two peaks with a ratio of 1:1 were observed, suggesting that 1 was racemic. Accordingly, the structure of 1 was established as the first phenanthrenoid dimer with a 14-3' linkage and named dijuncuenin A.

Compound 2 was obtained as yellow powder and had a molecular formula of C36H32O4 as determined by HRESIMS (m/z 527.2231, [M-H]^-). The IR absorption band at 3404 cm^-1 indicated the presence of hydroxyl groups. The 1D NMR data analysis (Table 1) of 2 indicated that it consisted of two phenanthrene units. In comparison to 1, a detailed 2D NMR analysis revealed that 2 possessed the same phenanthrene unit of 1 while the other unit and the connection site between two units were different. In the ¹H NMR spectrum, the signals observed for a vinyl group (δ_H 7.35, dd, J=18.0, 11.3 Hz; 5.64, dd, J=11.3, 2.0 Hz; 5.20, dd, J=18.0, 2.0 Hz), and an oxygenated aliphatic methine (δ_H 5.60, dd, J=9.0, 1.9 Hz) suggested that 2 might possess a similar linkage to that of 1. In the HMBC spectrum, the key correlations from H₂-14 (δ_H 3.63, m) to C-7' (δ_C 153.5, oxygenated), C-8' (δ_C 119.8), and C-8a0 (δ_C 130.1), as well as correlations from H-9' (δ_H 7.98, d, J=9.6 Hz) and H-100 (dH 7.86, d, J=9.6 Hz) to C-8a', confirmed a linkage between C-14 and C-8'. The correlations from H₂-140 (δ_H 5.65, 5.25) to C-5', and from H₃-120 (δ_H 2.51, s) to C-5' (δ_C 136.1), C-6' (δ_C 126.6), and C-7', assigned a vinyl, a methyl and a hydroxyl group at C-5', C-6', and C-7', respectively. In addition, the ROESY correlations of H₃-11↔H-10, H-3↔H-4↔H-5, H₃-12↔H-8, H-13↔H₃-12, H₃-11'↔H-10', H-3'↔H-4', and H-9'↔H₂-14 further confirmed the substitution pattern of 2. Similarly, compound 2 was proved to be racemic due to the optical rotation value and the HPLC behavior on a chiral column. Therefore, the structure of 2 was established as the first 14-80 linked phenanthrenoid dimer and named dijuncuenin B.

The plausible biosynthetic pathways of 1 and 2 are proposed from three known compounds (Scheme 1). Compound 1 could be derived from a coupling reaction between dehydrojuncuenin A and juncusol, both of which are metabolites and also isolated in this study. The mechanism might involve the attack of a hydroxyl radical at C-13 of dehydrojuncuenin A first, and the loss of the hydrogen free radical from juncusol at C-3 at the same time, and consequently the linking between C-14 of dehydrojuncuenin A and C-3 of juncusol. Compound 2 was presumably to have a similar mechanism to that of 1, but with a different linkage formed between C-14 of dehydrojuncuenin A and C-8 of dehydrojuncusol.

Compounds 1 and 2 were evaluated for their neuroprotective activity against Aβ_25-35-induced neurotoxicity in cultured SHSY5Y cells by MTT method. Unfortunately, they did not show any positive effect at concentrations of 1 μmol/L and 10 μmol/L.

4. Conclusion

Phenanthrenoid dimers have been reported rarely from the plants of the genus Juncus so far. The dimerization pattern between two monomeric halves could be classified into two types. One features a cage-like carbon framework with complicated stereochemistry, and the other is characteristic by a single C-C' linkage between two halves, such as the reported 3-3', 7-7', 8-3', 8-8', and 8-11' linkages. Our investigation of two new phenanthrenoid dimers, dijuncuenins A (1) and B (2) with novel 14-3' and 14-8' linkages, respectively, enriches the dimerization patterns of phenanthrenoids of the genus Juncus. Our findings suggest that additional novel dimeric phenanthrenoids are to be expected from the Juncus plants since there are a variety of possible linkage patterns between two halves.

Acknowledgments

We are thankfulfor thefinancial support ofthe NationalScience & Technology Major Project "Key New Drug Creation and Manufacturing Program" (No. 2012ZX09301001-001). Our thanks are also given to the National Natural Science Foundation of China (Nos. 81302657, 81473112, 81573305), Youth Innovation Promotion Association CAS and for grants from the Chinese Academy of Sciences (No. KSZD-EW-Z-004-01), and the State Key Laboratory of Drug Research (No. SIMM1501ZZ-03).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.04.008.