The Constituents of the Stems of Cissus assamica and Their Bioactivities

Fifty-five compounds were isolated from the fresh stems of Cissus assamica, including 14 benzenoids, 11 triterpenes, nine steroids, five tocopherols, five chlorophylls, four flavonoids, two benzoquinones, two tannins, and three other compounds. Their structures were constructed by 1D and 2D nuclear magnetic resonance (NMR) and mass spectral data, and were also identified by a comparison of their spectral data with those reported in the literature. Among these isolates, 1,2-bis-(5-γ-tocopheryl) ethane (51) was reported for the first time from natural sources. Some purified compounds were examined for their anti-inflammatory and anticancer bioactivities. The results indicated that betulinic acid (16) exhibited strong inhibition of superoxide anion generation with IC50 value of 0.2 ± 0.1 μM, while betulinic acid (16) and pheophytin-a (47) inhibited elastase release with IC50 value of 2.7 ± 0.3 and 5.3 ± 1.0 μM, respectively. In addition, betulinic acid (16) and epi-glut-5(6)-en-ol (18) exhibited potential cytotoxicity to non-small-cell lung carcinoma (NCI-H226) and colon cancer (HCT-116) cell lines with IC50 values in the range of 1.6 to 9.1 μM.


Structural Elucidation of Compound 51
1,2-Bis-(5-γ-tocopheryl)ethane (51) was isolated as a light yellow syrup. Its UV spectrum had an absorption maximum at 294 nm. The IR spectrum suggested the presence of hydroxyl (3444 cm −1 ) and an aromatic conjugated double bond (1458 and 1377 cm −1 ). The 13 C-NMR and DEPT spectra exhibited a benzene ring partial structure that has two oxygenated substituents at δ 117.0 (s), 122.1 (s), 123.4 (s), 124.1 (s), 145.5 (s), and 146.3 (s). The 1 H-NMR spectrum of 51 exhibited signals for two methyl and one methylene groups attached to a benzene ring at δ 2.13, 2.18, and 2.73. Comparing all the 1 H-and 13 C-NMR spectral signals carefully, the structure of 51 was similar to that of α-tocopherol (48) [56]. It indicated that they are very closely related analogues, differing only in the presence of a methylene group (δ H 2.73, δ C 26.7) in 51, instead of the methyl group (δ H 2.17, δ C 11.2) found in 48 (Table 1). To establish the structure of 51, 2D NMR including correlation spectroscopy (COSY), nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) experiments were conducted. In the HMBC experiment, the correlations observed for H-5a (δ H 2.18)/C-4, C-5, C-6 and H-6a (δ H 2.13)/C-1, C-5, C-6 indicate that two methyl groups are located in the ortho position on the benzene ring. Moreover, the correlation of the methylene proton at δ H 2.73 with C-3 in HMBC spectrum suggests that the location C-3a in the dimerization of alpha-tocopherol forms the dimer 51. Conclusively, the structure of 51 was assigned as 1,2-bis-(5-γ-tocopheryl)ethane, which had been reported by synthesis [58], but is reported from natural sources for the first time. The NMR spectra are presented Figures S1-S6.

Anti-Inflammatory Activity
Neutrophils are the most abundant white blood cells and participate in the development of the inflammatory reactions in human body; they are important factors in the immune defense against various diseases. Some cytotoxins-for example, the superoxide anion radical, bioactive lipids, granule proteases, and elastase-can be secreted when the different stimuli activate neutrophils. Moreover, they are also major contributors to tissue destruction in chronic inflammatory diseases. It has been proposed that inhibiting neutrophil activation is a method of enhancing inflammatory disorders [63][64][65][66]. Most of the purified compounds in this study were inspected for the inhibition of elastase release and superoxide anion generation by human neutrophils in response to N-formyl-L-methionyl-phenylalanine/cytochalasin B (fMLP/CB). Only compound 16 (Figure 1) displayed significant inhibition of superoxide anion generation, with an IC 50 value of 0.2 ± 0.1 µM ( Table 2). In addition, compounds 16 and 47 also exhibited an inhibitory effect on elastase release with an IC 50 value of 2.7 ± 0.3 and 5.3 ± 1.0 µM, respectively ( Table 2). The inhibitory effects of all the tested compounds on superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB are presented in Table S1. The cytotoxicity of compounds 16, 47, and LY294002 (a PI3K inhibitor, as a positive control) was examined in human neutrophils using an LDH release assay ( Figure S7). All these compounds did not induce LDH release, suggesting that the inhibitory effects did not result from cytotoxicity in human neutrophils. Table 2. Inhibitory effects of isolated compounds on superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB.

Cytotoxicity
In order to evaluate the growth inhibitory activity of the purified compounds against cancer cells, this study selected three different cell lines from malignant tumors including human nasopharyngeal carcinoma (NPC-TW01), non-small-cell lung carcinoma (NCI-H226), and colon cancer cell lines (HCT116). The results showed that betulinic acid (16) and epi-glut-5(6)-en-ol (18) (Figure 1) exhibited significant cytotoxicity with IC 50 values ranged from 1.6 to 9.1 µM (Table 3). Moreover, betulinic acid (16) exhibited powerful inhibitory activity against NCI-H226 and HCT116 with IC 50 values of 2.0 and 1.6 µM, respectively. Our study suggested the stem extracts of C. assamica and the purified compounds are potential candidates for the development of anti-cancer drugs. The preliminary growth inhibitory activity of all the tested compounds is presented in Table S2.

Materials
The fresh stems of C. assamica L. were collected from Taitung Hsien, Taiwan

Extraction and Isolation
The fresh stems of C. assamica L. (15 kg) were extracted with methanol (15 × 20 L) and refluxed for 8 h. The filtrate was evaporated under reduced pressure to yield a dark brown syrup (418 g). The residue was suspended in water and then partitioned with chloroform (5 × 2 L) and n-butanol (5 × 2 L) successively to afford chloroform (63 g), n-butanol (145 g) and water (210 g) soluble fractions respectively.

Preparation of Human Neutrophils
Human neutrophils study was approved by Chang Gung Memorial Hospital Institutional Review Board, Taoyuan, Taiwan. It was conducted according to the Declaration of Helsinki. Blood was obtained from healthy donors (20-32 years old) who provided written informed consent before blood was drawn. Briefly, neutrophils were isolated by dextran sedimentation, Ficoll-Hypaque gradient centrifugation, and hypotonic lysis of the erythrocytes [67].

Measurement of Superoxide Anion Generation and Elastase Release
The superoxide anion generation and elastase release were measured using the reduction of ferricytochrome c and elastase substrate, methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, respectively, as described previously [68][69][70]. Human neutrophils were suspended in HBSS containing ferricytochrome c (0.6 mg/mL) or elastase substrate (100 µM) at 37 • C and treated with DMSO or tested compounds for 5 min. The cells were then activated using fMLF (0.1 µM)/cytochalasin B (CB, 1 µg/mL for superoxide generation and 0.5 µg/mL for elastase release) and the change of absorbance was continually measured at 550 nm and 405 nm by a spectrophotometer (U-3010, Hitachi) to determine the superoxide anion generation and elastase release, respectively.

Detection of Cytotoxicity
Human neutrophils were treated with DMSO or tested compounds and incubated at 37 • C for 15 min. The supernatant was assayed to detect the released LDH using CytoTox 96 non-radioactive cytotoxicity assay (Promega, Madison, WI, USA). The results are presented in Figure S7.

Cell Lines
Human cancer cell lines, non-small cell lung carcinoma (NCI-H226) and colon cancer cell line (HCT116) were obtained from the American Type Culture Collection (Rockville, MD, USA). A nasopharyngeal carcinoma (NPC-TW01) cell line was purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). Tumor cells were maintained in proper medium supplemented with 10% fetal bovine serum (FBS) at 37 • C in a humidified atmosphere of 5% CO 2 .

Growth Inhibition Assay
The evaluation of cell growth and survival was carried out according to Hansen et al. [71] with some modifications.

Conclusions
In summary, 55 compounds were characterized from the fresh stems of C. assamica, including 14 benzenoids, 11 triterpenes, nine steroids, five tocopherols, five chlorophylls, four flavonoids, two benzoquinones, two tannins, and three other compounds. Among these isolates, 1,2-bis-(5-γ-tocopheryl)ethane was reported for the first time from natural sources. Furthermore, the inhibitory activity on superoxide anion generation and elastase release and the cytotoxicity on three cancer cells were analyzed. The present study suggests that the stems of C. assamica and several compounds of its isolation could be further developed as candidates for the treatment or prevention of cancer and various inflammatory diseases. Thus, the detailed mechanism of action of these compounds appears worthy of follow-up investigation.
Supplementary Materials: The following are available online. Tables S1 and S2: Anti-inflammatory and cytotoxic effects of all the tested compounds from C. assamica; Figures S1-S6: NMR spectra of compound 51; Figure S7