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A New Dolabrane Dinorditerpene from Ceriops tagal

DOI: 10.4236/oalib.1102957    886 Downloads   1,256 Views   Citations

ABSTRACT

A new dolabrane dinorditerpene, tagalsine X(1), was isolated from the leaves of Ceriops tagal, along with five known analogues (2-6). Their structures were established on the basis of spectroscopic data or comparison with the literatural data. Their cytotoxic activities against four human carcinoma cell lines (CNE-2, HCT-116, HepG2and A549) were also evaluated, which displayed that only compound 2 had significant cytotoxicity against those cell lines with IC50 values of 13.57, 42.32, 11.21 and 15.23 μM, respectively.

Subject Areas: Plant Science

1. Introduction

The plant Ceriops tagal (Rhizophoraceae), which has been used to treat malaria [1] , infected wounds and obstetric and hemorrhagic conditions [2] , is the only species of genus Ceriops distributed in southern coastal zone of China [1] . Its previous chemical studies have yielded a series of diterpenoids and triterpenoids [3] with antifouling [4] , antifeedant [5] and anticancer [6] activities. In this paper, we reported the isolation and structural elucidation of a new dolabrane dinorditerpene, tagalsine X(1), along with five known analogues (2-6), as well as their cytotoxic activities against four human carcinoma cell lines (CNE-2, A549, HepG2 and HCT-116).

2. Materials and Methods

2.1. Plant Material

The leaves of Ceriops tagal (Figure 1) were collected in Haikou City, Hainan Province of China, and authenticated by Prof. Weidong Han (College of Agriculture, Guangdong Ocean University). A voucher specimen (No. 20130513) was deposited in the Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical College, Zhanjiang, China.

2.2. General

Human carcinoma cell lines (CNE-2, A549, HepG2 and HCT-116) all came from ATCC. Column chromatographies (CC) were carried out byD101 macroporous resin (Beilianchem, China), silica gel (Qingdaohaiyang, China) and reversed-phase C18 silica gel (YMC, Japan). 1D and 2D NMR spectra were acquired on Bruker AV- 400 spectrometer.HR-ESI-MS was recorded by an Agilent 6210 LC/MSD TOF mass spectrometer. Analytical high-performance liquid chromatography (HPLC) were carried out on a Agilent 1200 series and a C18 reversed- phase column (Cosmosil, 4.6 mm × 250 mm, 5.0 μm). Preparative HPLC were carried out on a Gilson 305 pump, a Varian Prostar 345 UV detector and a C18 reversed-phase column (Cosmosil, 20 mm × 250 mm, 5.0 μm).

2.3. Extraction and Isolation

The air dried leaves of Ceriops tagal (10.0 kg) were powdered and extracted three times (24 h for each) with 95% EtOH at room temperature (3 × 30 L). The tannins were precipitated and filtrated after keeping the solution standing for 48 hours at room temperature. Crude extract (800 g) was yielded by concentrating the filtration under vacuum. The residue was suspended in water, and then partitioned with EtOAc. After removing the solvent, the EtOAc extract (300 g) was separated by D101 macroporous resin column chromatography (CC) using gradient ethanol aqueous solutions (60%, 80% and 95%) as eluants to give three fractions (A-C). Fraction B (80% ethanol, 100 g) was then subjected to silica gel CC eluting with n-hexane-ethyl acetate (100:0→10:1) to yield seven subfractions B1-B7. Subfraction B4 (H:E 9:1, 7.0 g) was chromatographed on ODS column eluting with CH3CN-H2O (1:9→7:3) to afford eight subfractionsB4a-B4h. B4b(CH3CN:H2O 3:7, 0.6 g) was purified repeatedly by prepared HPLC (CH3CN:H2O 4:6) to yield compound 1 (13.0 mg), 2 (14.8 mg), 3 (65.1 mg), 4 (28.3 mg), 5 (50.1 mg) and 6 (61.3 mg) (Figure 2).

Figure 1. The twigs of C. tagal with leaves and fruit.

Figure 2. The structures of compounds 1-6.

2.4. Cytotoxicity Assay by MTT Method

The cytotoxicity effects of compounds 1-6 were tested against four human carcinoma cell lines (CNE-2, A549, HepG2 and HCT-116) by the MTT method as described previously [7] . Generally, the cell suspensions were platedinto 96-well plates and cultured in RPMI-1640 at 37˚C, with 5% CO2 in incubator overnight. The test compound solutions (in 0.1% DMSO) at different concentrations were added to the corresponding wells. After exposure for 68 h, MTT was added to each well and the plates were incubated for 4 h. Finally, the supernatant was discarded and 200 μL of DMSO was added to the well to dissolve the blue-violet crystal, then the opticaldensity (OD) values were read on the microplate reader at 570 nm. All tests and analyses were carried out in triplicate. DMSO and doxorubicin were applied as the blank control and positive control, respectively.

3. Results and Discussion

3.1. Structure Elucidation

Compound 1, obtained as pale yellow amorphous powder in methanol, had the molecular formula C18H28O2 as established by its HR-ESI-MS at m/z 299.1992 [M + Na]+ (calcd for C18H28O2Na: 299.1982), which implied that 1 had five degrees of unsaturation. The 13CNMR and DEPT-135 spectra of 1 displayed a carbonyl carbon, and two olefinic carbons, as well as 15 aliphatic carbons including four methyls, five methylenes, three methines, and three quaternary carbons. The 1HNMRspectrum of 1 showed fourmethyls at δH 0.87 (3H, s), 0.92 (3H, s), 1.03 (3H, d, J = 5.2 Hz), 1.31 (3H, s), a pair of olefinic protons at δH 6.84 (1 H, dd, J = 10.1, 5.9 Hz) and6.13 (1H, d, J = 10.2 Hz), a methine at δH 2.8 (1H, dd, 13.3, 6.6)and a set of aliphatic protons ranging from δH 1.22 to 1.96. The above spectroscopic features revealed that compound 1 was a dolabrane-type dinorditerpenoid. The 13C NMR and 1H NMR spectra of compound 1 were very similar to those of tagalsin Q (5) [8] except for the positions at 3-5, 18 and 19, which suggested that their B/C rings were the same, while their A rings were different. All the 1H and C NMR signals of 1 were assigned as shown in Table 1 with the aid of 1H-1H COSY, HSQC and HMBC experiments. The bond between C-4 and C-18 changed from olefinic double-bond of tagalsin Q (5) into aliphatic single-bond of 1, which was further supported by the HMBC correlations between H-18 (δH 1.03) and C-3 (δC202.7)/C-5 (δC39.0) (Figure 3). The relative configuration of 1 was proposed on the basis of ROESY correlations (Figure 3). The β-orientation of H-8, H-10, H3-19 and H3-17, and the α-orientation of H3-18 and were H3-20 deduced from the presence of ROESY interactions between H-8/H-6β, H-6β/H-10, H-10/H3-19, H-19/H3-4, H-10/H-6β, H-6β/H-8, H-10/H-8, H-10/H-11β, H-11β/H-17 and H-18/H-6a, and the absence of ROESY interactions between H3-18/H-19, H3-18/H-10, H3-20/H-8 and H3-20/H-19. On the basis of the above results, compound 1 was identified as (4S*, 5S*, 8S*, 9S*, 10R*)-13S*-hydroxy-15, 16-dinorlabr-1(2)-en-3-one, and named tagals in X.

The known compounds were identified as (5S*, 8S*, 9S*, 10R*, 13S*)-2-hydroxy-16-nor-3-oxodolabr- 1,4(18)-dien-15-oic acid (2) [8] , (5S*, 8S*, 9S*, 10R*, 13S*)-3-hydroxy-16-nor-2-oxodolabr-3-en-15-oic acid (3) [8] , tagalsin P (4) [8] , tagalsin Q (5) [8] and (5S*, 8S*, 9S*, 10R*, 13S*)-3,16-dihydroxydolabr-3-ene-2,15- dione (6) [8] , respectively, on the basis of their 1H NMR and 13C NMR spectra analysis and comparison with those reported data in the related literatures (Figure 2).

Figure 3.The key HMBC and ROESY correlations of compound 1.

Table 1. NMR data of compound 1 (at 500 MHz in CDCl3, in ppm, J in Hz)a,b.

aAssignments were established by interpretation of the 1H-1H COSY, HSQC, and HMBC spectra; bOverlapped signals are reported without designating multiplicity.

3.2. Cytotoxicity Activity in Vitro

As can be seem from Table 2, compound 2 showed significant cytotoxicity against CNE-2, A549, HepG2 andHCT-116 cell lines with IC50 values of 13.57 ± 1.02, 42.32 ± 2.21, 11.21 ± 1.13 and 15.23 ± 1.42 μM, respectively, while the other five analogues had no obvious effect even with the concentration of 50 μM.

Table 2. Cytotoxicity of compounds 1-6 against four cancer cell linesa.

aAll results are expressed as mean ± SD, n = 3 for each group; bPositive control.

3.3. Discussion

Up to now, chemical examinations of the plant Ceriops tagal have resulted in the isolation of 27 dolabrane-type diterpenes [3] [9] , which could be divided into four sub-types: diterpene (twenty), 16-norditerpene (three), 15, 16-dinorditerpene (two) and ring A-seco-diterpene (two). Interestingly, dolabranes were regarded as a small group of natural products as there were only about fifty dolabranes isolated from plants, and as this papermentioned, most of them existed in Ceriops tagal. So we regarded dolabrane as chemoaxonomic marker of Ceriops tagal.

4. Conclusion

The present study attempts to explore the chemical constituents of the leaves of Ceriops tagal, and their cytotoxicity against four human carcinoma cell lines (CNE-2, HCT-116, HepG2 and A549). The result indicated that a new dolabrane dinorditerpene, tagalsine X, and five known analogues were isolated and identified. Among them, only compound 2 had significant cytotoxicity against the tested cell lines with IC50 values ranging from 11.21 to 42.32 μM, which deserved further studies on its exact mechanisms.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81503226) and the 863 Program (2013AA092902).

NOTES

*Corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Wu, X. , Liao, H. , Lu, H. and Zhang, C. (2016) A New Dolabrane Dinorditerpene from Ceriops tagal. Open Access Library Journal, 3, 1-6. doi: 10.4236/oalib.1102957.

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