Abietane Diterpenoids Isolated from Clerodendrum bracteatum and Their Antioxidant and Cytotoxic Activities

Two new abietane diterpenoids (1,2), along with five known diterpenoids (3–7), were first isolated and purified from the stems of Clerodendrum bracteatum. The structures of the new compounds were established by extensive analysis of mass spectrometric and 1-D, 2-D NMR spectroscopic data. Their antioxidant activities were determined on DPPH radical scavenging and ABTS. The in vitro cytotoxic activities of the compounds were evaluated against the HL-60 and A549 cell lines by the MTT method.


Introduction
The genus Clerodendrum is a diverse genus with about 580 species of small trees, shrubs, or occasionally perennial herbs, mostly in the tropics and subtropics of the world, including Africa and southern Asia. A few species are found in South America, northern Australia, and eastern Asia [1]. The whole plant has been used for the treatment of bleeding, rheumatism, hemorrhoids, and lung cancer. Previous phytochemical investigations on this genus resulted in the isolation of various types of compounds, including flavonoid compounds, phenylpropanoid glycosides, sesquiterpenoids, diterpenoids, triterpenoids, alkaloids, and so on, which exhibited a broad range of biological activities, such as antioxidant, antitumor, antibacterial, and anti-inflammatory [2,3].

Plant Material
Woody branches and healthy stems of C. bracteatum were collected in July 2014 from the mountain of Dulongjiang, Yunnan Province, People's Republic of China. The plant was identified by Dr. Chunhui Dai in Zhejiang Academy of Traditional Chinese Medicine. A voucher specimen (201418) has been deposited in the Key Laboratory for Genetic Improvement and Quality Control of Medical Plants of Zhejiang Province, Hangzhou Normal University.

Extraction and Isolation
Cut and air-dried stems (9 kg) of C. bracteatum were extracted under reflux with 90% ethanol (3 × 90 L) at 70 • C. The ethanol extracts were combined and evaporated to dryness under vacuum at 50 • C to afford a gummy residue (630 g). Part of the crude extract (500 g) was suspended in water (1 L) at 50 • C and fractionated with EtOAc (3 × 2 L) and n-BuOH (3 × 2 L) successively to yield the EtOAc (81 g) and n-BuOH (90 g) fractions, respectively.

Cytotoxicity Assay
The inhibitory effects of the compounds against HL-60 and A549 cells were determined using a MTT assay [10]. The cells (5000-10,000 per well) were cultivated in 96-well plates for 24 h. The medium was then replaced with new medium containing different concentrations of the compounds, and using cisplatin as a positive control. After incubation for 24 h, the medium was replaced by 100 µL of MTT, and the cells were further incubated for another 4 h at 37 • C to allow MTT formazan formation. Following incubation, the medium was replaced by acidic isopropanol (100 µL) to dissolve the formazan in each well. The absorbance was detected by a microplate reader (Multiskan Spectrum, Thermo Electron Corporation, Vantaa, Finland) at 570 nm. The concentration giving 50% inhibition (IC 50 ) was calculated by NDST software, and each assay was performed in triplicate.

Free Radical Scavenging Assay and ABTS Test
The DPPH radical scavenging activity of the compound was determined according to the method ofŚlusarczyk et al. with slight modifications [11]. Briefly, 0.2 mM solution of DPPH in methanol was prepared and 2.5 mL of this solution were added to 2.5 mL of compound solution in methanol at different concentrations. Then, 30 min later, the absorbance was measured at 517 nm in the UV spectrophotometry. A calibration curve was prepared using different Trolox concentrations (standard Trolox solutions ranging from 10 to 320 µM). The percentage inhibition activity was calculated as follows: (A 0 -A t )/A 0 × 100%, where A 0 is the absorbance of the control and A t is the absorbance in the presence of samples.
The ABTS + free radical scavenging assay was determined according to the method described by Wang with some modification [12]. ABTS+ radical cation was produced by mixing 7 mM ABTS + solution with 2.45 mM potassium persulfate, and the mixture was stored at room temperature and in the dark for 12 h. Then, the ABTS + solution was diluted with ethanol until its absorbance at 734 nm was 0.70. Next, 5 µL of sample solution was mixed with 2 mL of diluted ABTS + radical solution and allowed to react for 6 min. The absorbance was measured at 734 nm by UV spectrophotometry. The scavenging activity was expressed as IC 50 (the concentration of the tested sample required to scavenge 50% of ABTS), calculated by linear regression analysis. The experiment was conducted in triplicate.

Statistical Analysis
We described all values as the mean ± SD and analyzed by Graphpad Prism 6.0. To analyze the statistical significance among multiple groups, we used one-way analysis of variance (ANOVA) followed by Tukey post hoc test. p-values < 0.05 were considered to indicate statistical significance.

Conclusions
Two new (1,2) and five known abietane diterpenoids were isolated from C. bracteatum. These structures were identified by using spectroscopic methods. All the isolated compounds were evaluated for their cytotoxic and antioxidant activities. The results of the present study help to learn the potency of C. bracteatum as a potential source of natural antioxidants and suggests that C. bracteatum. might be explored as a viable source of potent antioxidants for the protection of food from oxidation. However, further research is needed to identify individual components that form an antioxidative system and develop their applications for food and pharmaceutical industries.