Bioactive Compounds from the Stems of Clausena lansium

In view of the significant neuroprotective effect of Clausena lansium, we continued to separate the n-butanol and the water extracts from the stems of C. lansium in order to find the leading compounds with significant activity. Two new phenolic glycosides, Clausenolside A–B (1–2), one new pair of phenolic enantiomers (3a, 3b), and two new monoterpenoids, clausenapene A–B (4–5), together with twelve known analogues (6–17) were isolated from the stems of C. lansium. Compounds 1–17 were obtained from C. lansium for the first time. Compounds 3a, 3b, 4, 16, and 17 showed strong or moderate potential neuroprotective effects on inhibited PC12 cell injury induced by okadaic acid, and compound 9 exhibited strong potential hepatoprotective activities. Their structures were elucidated on the basis of spectroscopic analyses, including UV, IR, NMR experiments, and electronic circular dichroism (ECD) spectra.


Introduction
Clausena lansium (Lour.) Skeels (syn. Clausena wampi (Blanco) Oliv.; Clausena punctate (Sonn.) Rehd. & Wils.; Cookia punctate Sonn.; Cookia wampi Blanco; Quinaria lansium Lour.) is a minor member of the Rutaceae. It is an attractive shrub or small tree with somewhat grapelike fruit, similar to the citrus fruits and commonly called Wampee, False or Fool's Curry [1]. It grows in the southern area of mainland China and is cultivated in Taiwan, Fujian, Guangdong, Guangxi, Hainan, etc. It also occurs in Vietnam, the Philippines, Malaysia, Singapore, Miami, etc. [2]. In traditional Chinese medicine, the leaves and roots of C. lansium were used to treat coughs, asthma, dermatological diseases, viral hepatitis, and gastro-intestinal diseases. The fruit were used to treat digestive disorders and the seeds were used to treat acute and chronic gastro-intestinal inflammation, ulcers, and so on [3].
Various bioactive constituents including coumarins, carbazole alkaloids, and amide alkaloids have been isolated and identified from this plant [4][5][6]. Our research group has previously characterized a variety of new carbazole alkaloids, new amide glycosides, new coumarins, and new megastigmane glucoside from the leaves and stems of C. lansium, and several of these compounds showed selective neuroprotective and hepatoprotective effects [7][8][9][10][11][12]. However, the n-BuOH and the water extracts from the stems of C. lansium have not been investigated in detail. Herein, this paper reports on a further investigation of the water and n-BuOH extracts from the stems of C. lansium, which led to the isolation and characterization of two new phenolic glycosides (1)(2), one new pair of phenolic enantiomers (3a and 3b), two new monoterpenoids (4)(5), together with twelve known analogues (6-17) ( Figure 1). They were obtained from C. lansium for the first time. The determination of their absolute configurations occurred through spectroscopic analysis and electronic circular dichroism (ECD) experiments. Moreover, compounds 1-4 and 6-17 were assayed for their in vitro hepatoprotective and neuroprotective effects.

Purification and Characterization
Clausenolside A (1) was obtained as a white, amorphous solid. Its molecular formula was deduced as C 22 H 32 O 13 on the basis of its 13 13 C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra ( Figure S3) along with the heteronuclear singular quantum correlation (HSQC) correlations ( Figure S4) exhibited the presence of a benzene ring, a keto-carbonyl group, a methoxyl group, an oxygenated methine group, a methyl group, a glucosyl group, and a rhamnosyl group. On the basis of the NMR data analysis (Table 1), compound 1 was identified as a phenolic glycoside. In the heteronuclear multiple bond correlation (HMBC) spectrum ( Figure S5), Correlations from H-8 to C-7 and from H-9 to C-7 and C-8 indicated that the oxygenated methine group was attached to C-7 and C-9. The correlations of H-2/C-4 (δ C 150.6), C-6 (δ C 122.7), C-7 (δ C 200.3); H-6/C-7 (δ C 200.3), C-4 (δ C 150.6); H-5/C-1 (δ C 128.5), C-3 (δ C 148.7); and 3-OCH 3 / C-3 (δ C 148.7) demonstrated that the carbonyl was attached to C-1 and the methoxyl group was resonated at C-3. Correlations from H-1 to C-4 and from H-1" to C-6 indicated that the rhamnosyl group was linked with C-6 and the glucosyl group was linked with C-4 ( Figure 2). The aglycone (1a) and sugar moieties were produced by acid hydrolysis of 1. Sugar moieties were confirmed to be D-glucose and L-rhamnose by silylation followed with gas chromatography (GC) analysis. The absolute configuration of 1a was defined as 8S by comparison of the experimental ECD spectra and the calculated ECD data using the time-dependent density functional theory (TDDFT) method at the B 3 LYP/6-31G (d) level [13]. The calculated ECD spectrum of (8S) 1a ( Figure 3) matched the experimental spectrum of 1a and 1 very well, which indicated that the structure of 1a had not changed in the process of acid hydrolysis and the absolute configuration of 1 was elucidated as 8S. Thus, the structure of 1 was assigned as depicted.  [14], except that the methoxyl group and the hydroxymethyl group of 2 replaced the hydrogen proton of C-6 and the propen-2-en-1-ol of C-4 replaced 6. In the HMBC spectrum (Figure 2), the correlations from H-3 and H-5 to C-1 (δ C 133.9), C-2 (δ C 152.7), and C-7 (δ C 63.0); from H-7 to C-3 (δ C 103.5), C-4 (δ C 138.2), and C-5 (δ C 103.5) showed that the hydroxymethyl group was linked to C-4; the correlations from H-1 to C-1 (δ C 133.9), C-2 (δ C 67.6); from H-2 to C-1 (δ C 81.4), C-3 (δ C 60.1); from H-1" to C-2 (δ C 67.6) indicated that the propanetriol group was linked to C-1 and the β-glucopyranosyl unit was linked to C-2 . The correlations from OCH 3 to C-2 (δ C 152.7), C-6 (δ C 152.7) demonstrated that the methoxyl group had attached to C-2 and C-6. The aglycone (2a) and sugar moiety were produced by an acid hydrolysis of 2. Sugar moiety was confirmed to be D-glucose by silylation followed with GC analysis. Hence, the structure of 2 was assigned as shown.  Compound 3 (3a/3b) was obtained as a white powder. Its molecular formula C 10 H 12 O 5 was deduced from the HRESIMS (m/z 235.0573 [M + Na] + , calculated as C 10 H 12 NaO 5 , 235.0577) and the 13 C-NMR spectroscopic data, corresponding with five indices of hydrogen deficiency. The IR spectrum displayed absorptions characteristic of amino (3394 cm −1 ), amide (1667 cm −1 ), and of aromatic ring (1591, 1517, and 1465 cm −1 ) groups. According to 1 H and 13 C-NMR (Table 2), the plane structure of 3 was the same as 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one [15].

Neuroprotective Effect and Hepatoprotective Effect of Compounds 1-4 and 6-17
Compounds 1-4 and 6-17 were evaluated for their neuroprotective effect on PC12 cells induced by okadaic acid (OKA) in vitro using the MTT method. As shown in Figure 8, at 10 µM, 3a, 3b, 4, 16, 17 increased the cell survival rate of the okadaic acid-treated group, while other compounds were inactive. Compounds 1-4 and 6-17 were also tested for hepatoprotective activities against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 (human hepatocellular liver carcinoma cell line) cells, using the hepatoprotective activity drug bicyclol as the positive control. As shown in Figure 8, compound 9 exhibited hepatoprotective activity, while other compounds were inactive.
Clausena lansium (Lour.) Skeels, which belongs to the Rutaceae, has been cultivated in southern China and other warm areas of the world. Many chemical components, including carbazole alkaloids, coumarins, acyclic amides, cyclic amides, quinolones, phenyl glycosides, lactams and oxyneolignan were characterized from the stems, roots and leaves of C. lansium, and showed various biological activities. Some of the carbazole alkaloids, alkaloid glycosides, amides, and coumarins have exhibited potential anti-inflammatory activity, neuroprotective, hepatoprotective, and cytotoxicity activities [5,7,9,11,[32][33][34][35][36]. However, as for the constituents from the fruit, seeds and peels, the references were very few. Few alkaloids, amides, and monoterpenes were isolated from the seeds of C. lansium [37,38]. The 8-hydroxypsoralen which showed antioxidant and cytotoxic activities, and the two new monoterpenoid coumarins (clauslactone V-W) which showed α-glucosidase inhibitory activity were obtained from the peels of C. lansium [6]. Some monoterpenoid coumarins and seven carbazole alkaloids were also isolated from the peels of Clausena lansium (Lour.) Skeels, and claulansine J exhibited moderate antibacterial activity against Staphylococcus aureus [39,40]. Three new jasmonoid glucosides, two new sesquiterpenes, two new coumarins, and others were isolated from the fruit of C. lansium. One coumarin was active against S. aureus and S. dysenteriae, and also exhibited moderate antioxidant activity, while one sesquiterpene, (+)-(E)-a-santalen-12-oic-acid, showed an inhibitory effect on B. cereus [41].
In this paper, the stems of C. lansium were collected in the Liuzhou commercial cultivation in Guangxi, China. Liuzhou, located in northern Guangxi, is a subtropical monsoon climate. Light, temperature, and water are very rich in Liuzhou. Therefore, the chemical components of C. lansium collected in Liuzhou could vary and be rich. Compounds 1-17 were obtained from the BuOH and the water extracts from the stems of C. lansium for the first time. The results are basically the same as those reported in the genus Rutaceae and for C. lansium. Some A,D-seco-limonoids have been characterized from the stems of Clausena emarginata [42] and we think that the two new monoterpenoids, clausenapenes A and B, may be decomposition products from the limonoides. In our studies, compounds 1-4 and 6-17 were assayed for the hepatoprotective and neuroprotective effects in vitro, in order to discover potential lead compounds. Herein, compounds 3a, 3b, 4, 16, and 17 showed strong or moderate potential neuroprotective effects by inhibiting PC12 cell injury induced by okadaic acid, and compound 9 exhibited strong potential hepatoprotective activities. It indicated that it is worth studying the chemical compositions of the BuOH and the water extracts of the stems of C. lansium to find more lead compounds.

Plant Materials
The stems of C. lansium were collected in Liuzhou, Guangxi, China, in March 2013, and were from commercial cultivation. C. lansium was identified by Engineer Guangri Long, Forestry of Liuzhou. A voucher specimen has been deposited at the Herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (ID-S-2320). The 95% ethanol extract from the stems of C. lansium was stored in a refrigerator at −80 • C.

Acid Hydrolysis and GC Analysis of Compounds 1 and 2
Compound 2 (2 mg) was dissolved in 2 mol HCl-H 2 O (2 mL) and was then heated to 90 • C for 15 h. The reaction mixture was extracted with EtOAc. The aqueous layer was evaporated under vacuum, diluted repeatedly with H 2 O, and evaporated in vacuo to furnish a neutral residue. The residue was dissolved in anhydrous pyridine (1 mL), to which 2 mg of L-cysteine methyl ester hydrochloride was added. The mixture was stirred at 60 • C for 2 h, and after evaporation in vacuo to create dryness, 0.2 mL of N-trimethylsilylimidazole was added. The mixture was kept at 60 • C for another 2 h.
The reaction mixture was partitioned between n-hexane and H 2 O (2 mL each), and then the n-hexane extract was analyzed by GC under the following conditions: capillary column, HP-5 (30 m × 0.25 mm, with a 0.25 µm film; Dikma, Beijing, China); detection, FID; detector temperature, 280 • C; injection temperature, 250 • C; initial temperature 200 • C, then raised to 280 at 5 • C/min, final temperature maintained for 10 min; carrier, N 2 gas. From the acid hydrolysate of 2, D-glucofuranurono-6, 3-lactone was confirmed by comparison of the retention time of its derivative, with that of an authentic sugar derivatized in a similar way, which showed a retention time of 18.4 min. The constituent sugar of compound 1 was identified by the same method as 2. Retention times of authentic sample were detected at 18.4 min (D-glucose) and 14.7 min (L-rhamnose) for 1.

Hepatoprotective Activity Assay
Human HepG2 hepatoma cells were cultured in a DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C in a humidified atmosphere of 5% CO 2 + 95% air. The cells were then passaged by treatment with 0.25% trypsin in 0.02% EDTA. The MTT assay was used to assess the cytotoxicity of test samples. The cells were seeded in 96-well multiplates. After an overnight incubation at 37 • C with 5% CO 2 , 10 µM test samples and APAP (final concentration of 8 mM) were added into the wells and incubated for another 48 h. Then, 100 µL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and they were incubated for an additional 4 h. The resulting formazan was dissolved in 150 µL of DMSO after aspiration of the culture medium. The plates were placed on a plate shaker for 30 min and read immediately at 570 nm using a microplate reader [43]. The cell inhibitory rate (%) was calculated by (A sample − A blank )/(A untreated − A blank ) × 100. p-Values of <0.05, <0.01, and <0.001 were regarded as statistically significant.

Neuroprotective Activity Assays
Pheochromocytoma (PC12) cells were incubated in DMEM, supplied with 5% fetal bovine serum and 5% equine serum as a basic medium. PC12 cells in the logarithmic phase were cultured at a density of 5000 cells per well in a 96-well microtiter plate. After 24 h incubation, the medium of the model group was changed to DMEM or a basic medium with 50 nM OKA for 24 h. Test compounds dissolved in DMSO were added to each well for >1000-fold dilution in the model medium at the same time. Each sample was tested in triplicate. After the incubation at 37 • C in 5% CO 2 for 24 h, 10 µL of MTT (5 mg/mL) was added to each well and they were incubated for another 4 h. Then, the liquid in the wells was removed. Test compounds (100 µL) were added to each well. The absorbance was recorded on a microplate reader (Bio-Rad model 550, California, USA) at a wavelength of 570 nm [44]. Analysis of variance (ANOVA) followed by the Newman-Keuls post hoc test were performed to assess the differences between the relevant control and each experimental group. The cell inhibitory rate (%) was calculated by (A sample − A blank )/(A untreated − A blank ) × 100. p-Values of <0.05, <0.01, and <0.001 were regarded as statistically significant.

Conclusions
In summary, this work described the isolation and the structure identification of two new phenolic glycosides (1-2), one new pair of phenolic enantiomers (3a, 3b), and two new monoterpenoids (4)(5), together with twelve known analogues (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). They were obtained from the stems of C. lansium for the first time. In addition, compounds 3a, 3b, 4, 16, and 17 showed strong or moderate potential neuroprotective effects on inhibiting PC12 cell injury induced by okadaic acid, and compound 9 exhibited strong potential hepatoprotective activities. In traditional Chinese medicine, the leaves, fruit, seeds, and the roots of C. lansium were used as folk medicine for treating many kinds of diseases. It has been reported that the stems of C. lansium have a characteristic chemical composition including carbazole, amide, quinolone alkaloids, coumarins, and others, which have various biological activities such as neuroprotective, anti-inflammatory, hepatoprotective, and cyctoxicity capacities. Therefore, not only the roots and the leaves but also the stems are important medicinal materials, which indicate that the chemical compositions and the biological activities of the stems of C. lansium are worth studying in order to find other compounds with potential activity.

Conflicts of Interest:
The authors declare no conflict of interest.