Two New Seco-Labdane Diterpenoids from the Leaves of Callicarpa nudiflora

Two new seco-labdane diterpenoids, nudiflopene N (1) and nudiflopene O (2), and four known compounds were isolated from the leaves of Callicarpa nudiflora. The structures of the new compounds were established by 1D-, 2D-NMR, and HR-ESI-MS spectral analyses. Compounds 1–3 showed inhibitory activities on lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells, and new compounds 1–2 exhibited more potent inhibitory activity than compound 3. The cytotoxicity of compounds 1–3 against human hepatocellular carcinoma HepG2 cells and human gastric carcinoma SGC-7901 cells were evaluated, while all of them exhibited no cytotoxicity.


Introduction
The genus Callicarpa comprises about 190 species and is widely dispersed across tropical and subtropical Asia and Oceania. Callicarpa nudiflora Hook. & Arn. (Verbenaceae) is a shrub or small tree mainly found in Guangdong, Guangxi, Hainan, China, with the medicinal materials in Wuzhishan, Hainan Province representing the best specimens [1]. C. nudiflora is a traditional Chinese medicinal herb for eliminating stasis in order to subdue swelling and hemostasis. In the Li nationality, C. nudiflora is called "Bu fa" and is usually used to treat traumatic bleeding in Hainan [2]. The roots and leaves of C. nudiflora can be used as medicine with the effects of antifungal, antibacterial, pro-coagulation, antiinflammatory, detoxification, blood circulation, swelling, and evacuation of wind; it can also be used to treat inflammation induced by pyogenic bacteria and acute infectious hepatitis [3][4][5]. The leaves of C. nudiflora are used medicinally to stop bleeding, relieve pain, dispel blood stasis, and reduce swelling [6][7][8]. Phytochemical studies have shown that the main chemical constituents of C. nudiflora are flavonoids, terpenoids, and lignans [9][10][11][12]. Diterpenoids are the characteristic compounds of C. nudiflora, and some of them showed anti-inflammatory activities by inhibiting NO production [13]. In the present study, we reported the isolation and structural elucidation of two new seco-labdane diterpenoids along with four known compounds. In addition, compounds 1-3 were evaluated for their anti-inflammatory activities and cytotoxicity.

NO Inhibitory Activities
NO was considered as a key inflamm flammation. RAW264.7 is derived from cells, which have a strong ability to phago tory, immune, and phagocytic response natural products using LPS-induced RA been widely used. Some research evidenc activity, so compounds 1-3 were assaye cells. Compounds 1-3 inhibited LPS-indu values of 34.43 ± 1.37, 29.87 ± 2.50, and 66  Table 1). The 1 H and 13 C NMR spectra (see Supplementary) of 2 showed high similarity to those of 1, implying a structurally similar diterpenoid for 2. According to their NMR data, the signals for the methoxy group in compound 1 were replaced by the ethoxy group in 2, which was supported by the DEPT experiments and 2D NMR spectra. The further interpretation of 2D NMR data led to the assignments of all the proton and carbon signals. The configuration of 2 was also identical with compound 1 based on the NOESY spectrum, optical rotation data, and ECD spectrum (see Supplementary). Therefore, the structure of compound 2 was elucidated and gave a successive name nudiflopene O.
Compounds 1 and 2 are derivatives of nudiflopene H (3). To prove that compounds 1 and 2 are not artifacts, we extracted the medicinal materials again with ethyl acetate and analyzed its constituents with HPLC-DAD. The results showed that compounds 1 and 2 can be found in the HPLC chromatogram of the ethyl acetate extract based on the retention time and UV spectrum (see Supplementary). Therefore, compounds 1 and 2 are not artifacts.

NO Inhibitory Activities
NO was considered as a key inflammatory mediator that may be helpful to treat inflammation. RAW264.7 is derived from Abelson murine leukemia virus-induced tumor cells, which have a strong ability to phagocytize antigens and play key roles in inflammatory, immune, and phagocytic responses. At present, the anti-inflammatory activity of natural products using LPS-induced RAW264.7 macrophages as screening model has been widely used. Some research evidenced that such diterpenoids showed NO inhibitory activity, so compounds 1-3 were assayed for their NO inhibitory effects in RAW 264.7 cells.

Cytotoxic Effects against Cancer Cells
Compounds 1-3 were evaluated for their cytotoxicity against HepG2 and SGC-7901 cells. All of them had no activity against these two cell lines.

Plant Material
The air-dried leaves of Callicarpa nudiflora were collected from Wuzhishan of Hainan Province, China in October 2015 and identified by Prof. Xi-Feng Sheng (Hunan Normal University, China). A voucher specimen (No. LHZZ-2015) has been deposited in the Department of Pharmacy, School of Medicine, Hunan Normal University.

Extraction and Isolation
The air-dried leaves of C. nudiflora (8.5 kg) were extracted twice with EtOH-H2O (80:20, v/v), and the concentrated liquid was dispersed in water and using petroleum ether, ethyl acetate, and n-butanol to extract twice to obtain three portions of petroleum ether (170 g), ethyl acetate (258 g), and n-butanol.

Cytotoxic Effects against Cancer Cells
Compounds 1-3 were evaluated for their cytotoxicity against HepG2 and SGC-7901 cells. All of them had no activity against these two cell lines.

General Experimental Procedures
Optical rotations were recorded on a Bellingham-Stanley ADP 440+ polarimeter (Bellingham-Stanley Ltd., Tunbridge Wells, UK). ECD spectra were obtained on a JASCO J-715 CD spectrometer (JASCO Corporation, Tokyo, Japan). 1 H NMR, 13  Mass spectra were obtained on a Bruker micro TOF mass spectrometer (ESI-MS) (Billerica, MA, USA). High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 Series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a four-pump with an in-line degasser, autosampler, oven, and Diode-array detector (DAD). The semi-preparative HPLC was performed using a YMC ODS-A chromatographic column (250 × 10 mm, µm).

Plant Material
The air-dried leaves of Callicarpa nudiflora were collected from Wuzhishan of Hainan Province, China in October 2015 and identified by Prof. Xi-Feng Sheng (Hunan Normal University, China). A voucher specimen (No. LHZZ-2015) has been deposited in the Department of Pharmacy, School of Medicine, Hunan Normal University.

Extraction and Isolation
The air-dried leaves of C. nudiflora (8.5 kg) were extracted twice with EtOH-H 2 O (80:20, v/v), and the concentrated liquid was dispersed in water and using petroleum ether, ethyl acetate, and n-butanol to extract twice to obtain three portions of petroleum ether (170 g), ethyl acetate (258 g), and n-butanol. II, respectively, were added. The absorbance was read with a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 550 nm. The experiment was performed three times. SPSS 16.0 and GraphPad Prism 6.01 software were used for statistical analysis.

Cytotoxicity Assay
The cytotoxicity assay was carried out using the CCK-8 method. HepG2 and SGC-7901 cells were cultured in a cell temperature incubator and a DMEM at 37 • C in 5% CO 2 , respectively. The cells of the logarithmic growth phase were seeded into 96-well plates with a density of 4000 cells/well in 200 µL medium, respectively. The cells were treated with the tested compounds at various concentrations (0, 10, 20, 40 and 80 µM), with sorafenib and cisplatin as positive control. Each of three parallel holes were located, then incubated for 24 h. Subsequently, the 96-well plate was taken out, and 10 µL of CCK-8 was added to the experimental and control wells; at the same time, two other individual wells were taken as blank controls, and only 10 µL of CCK-8 in 0.1 mL of DMEM was added to each well. Then incubation under the same conditions was conducted for 4 h. The optical density (OD) was measured at 450 nm using a Bio-Tek Synergy (Bio-Tek Instruments, USA). The experiment was repeated 3 times. Finally, the impact of drugs on cell growth inhibition rate and IC 50 values was calculated.

Conclusions
In this study, two new seco-labdane diterpenoids, nudiflopene N (1) and nudiflopene O (2), along with four known compounds, were isolated from the leaves of Callicarpa nudiflora Hook. & Arn. The structures of the new compounds were elucidated by spectroscopic analysis. Compounds 1-3 from this plant were evaluated for their NO inhibitory activity and cytotoxicity against human hepatocellular carcinoma HepG2 cells and human gastric carcinoma SGC-7901 cells. All of them showed good inhibitory activity on the LPS-induced NO production in RAW 264.7 cells, and compounds 1-2 showed more potent activity than compound 3.
Author Contributions: H.Z. conceived, designed, and supervised the research project and edited the manuscript; X.G. and L.Z. performed the experiments and prepared the manuscript; Y.Z., Y.X. and S.Y. performed the bioactivity assay; and X.S. and H.X. provided suggestions on the preparation of the manuscript. All authors have read and agreed to the published version of the manuscript.