Diterpenoids from Saliva plebeia R. Br. and Their Antioxidant and Anti-Inflammatory Activities

A new skeleton of diterpenoid, 1,2,3,4,4α,9,10,10α-octahydro-(4α-hydroxyymethyl)-1,1-dimethyl-9-(1-methylethyl)-(2S,3S,4αR,9R,10αS)-2,3,5,7-phenanthrenetertrol, named plebeianiol A (1), along with four known diterpenoids (2–5), were isolated from Salvia plebeia R. Br. Their structures were determined on the basis of spectral analysis. In the bioactivity tests, compounds 1, 2 and 5 showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities with IC50 values of 20.0–29.6 µM. In addition, these three compounds had significant inhibitory effects on reactive oxygen species (ROS) production in lipopolysaccharide (LPS)-induced macrophages. Compounds 1–3 inhibited nitric oxide (NO) production in LPS-induced macrophages with IC50 values of 18.0–23.6 µM. These results showed that compounds 1, 2 had significant antioxidant and anti-inflammatory activities and might provide basis for the treatment of diseases associated with oxidative lesions and inflammation.


Introduction
Salvia plebeia R. Br. is one of the genera from the Salvia family. It is widely distributed in many provinces of China. As a traditional Chinese medicine (TCM), it has been used to treat various diseases, such as urinary tract infection and bronchitis [1]. Modern pharmacological studies have shown that it exhibits anti-inflammatory [2], antioxidant [3], antitumor [4], hepatoprotective [5] and antimicrobial [6] activities. Phytochemical studies of S. plebeia R. Br. revealed the presence of flavonoids [7], terpenoids [8] and lignans [9].
In the course of researching bioactive components from S. plebeia R. Br., we found that ethyl acetate (EtOAc) fraction of S. plebeia R. Br. showed significant inhibitory activity against the production of NO in LPS-induced RAW 264.7 macrophages. According to the guidance of bioactivity, a new skeleton of diterpenoid (1) and four known diterpenoids (2)(3)(4)(5) were isolated from the bioactive fractions of EtOAc fraction of S. plebeia R. Br. In the present study, we reported the isolation, structure elucidation, as well as antioxidant and anti-inflammatory activities of the isolated compounds in vitro.
The antioxidant activity of compounds 1-5 was evaluated by DPPH radical scavenging activity. The results showed that compounds 1, 2 and 5 had significant effects with IC50 values of 29.6, 28.8 and 20.0 µM, respectively ( Table 2). Compared with Vitmain C (Vit. C), these three compounds showed similar antioxidant effects. In order to further evaluate the antioxidant effects, the ability to scavenge ROS in LPS-induced RAW 264.7 macrophages was measured at various concentrations (10, 30 and 100 µM) ( Table 3). The results showed that compounds 1, 2 and 5 had significant effects along with the inhibition rates of 64.8%, 80.3% and 91.3% at the concentration of 100 µM. The results of intracellular ROS assay were similar to DPPH radical scavenging assay. The anti-inflammatory activity tests showed that compounds 1-3 inhibited NO production in LPS-induced macrophages with IC50 values of 18.0, 21.5 and 23.6 µM, respectively. In addition, RAW 264.7 macrophages were cultured with the obtained compounds at the concentration of 100 µM for 24 h and cell viability was evaluated using MTT assay. The cell viability was in the range of 91.6%-110.5% of the control. These results demonstrated that all compounds did not exhibit substantial cytotoxicity on RAW 264.7 macrophages. The anti-inflammatory experimental data and cell viability are shown in Table 4.

Plant Material
The air-dried herbs of S. plebeia R. Br. were purchased from the Pharmacy of Yi-Feng (Nanjing, China) in July 2013. The herb were collected from Nanning, Guangxi province, China (Production license: 20100109), and identified by Min-Jian Qin, China Pharmaceutical University (Nanjing, China). A voucher specimen (No. 20130702) was deposited in Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.

Extraction and Isolation
The air-dried herb of S. plebeia R. Br. (5.0 kg) was soaked in 80% EtOH (90.0 L) at room temperature for 12 h and extracted under refluxed four times (3 h each). The combined extracts were filtered and solvent was recycled under reduced pressure with a rotary evaporator at 65 °C to obtain a black crude extract (1000.0 g). The extract was suspended in water (3000 mL) and re-extracted by sequentially with petroleum ether (3 × 3000 mL), EtOAc (3 × 3000 mL) and n-BuOH (3 × 3000 mL), respectively. Each partitioned fraction was dried at 60 °C under reduced pressure with a rotary evaporator, and the results were 150.0 g (petroleum ether extract), 260.0 g (EtOAc extract) and 200.0 g (n-BuOH extract), in turn.

Antioxidant Assay
DPPH radical assay: DPPH radical scavenging assay was performed according to previous protocol with modifications [14]. DPPH was dissolved in ethanol at the concentration of 200 µM. One hundred microliters of compounds (5, 10, 20, 30, 50 µM) were added to the 96-well plates, and evaded the light preservation for 30 min after addition 100 µL DPPH. Then the absorbance was measured at 517 nm using Thermo Scientific Varioskan Flash. Vitamin C was used as the positive control. The inhibitory effect was expressed as scavenging rate of alleviation and was calculated as follows: A1 refers to the absorbance with various concentrations of test compounds and DPPH, A2 refers to the absorbance with various concentrations of test compounds and ethanol, A0 refers to the absorbance with distilled water and DPPH. Intracellular ROS assay: Intracellular ROS assay was performed according to the method reported previously [15]. RAW 264.7, a murine macrophage cell line, was obtained from the Pharmacognosy Laboratory of Prof. Mian Zhang, China Pharmaceutical University. Cells were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. The cells (1 × 10 5 cells/well) were pre-incubated in the 96-well plate for 12 h. Then, cells were cultured in normal medium as normal group, or 1 μg/mL LPS as control group, or 1 μg/mL LPS plus different concentration of compounds as test group. After the cells of each group were treated for 6 h, the medium was removed, and the cells were incubated with 50 μL 2,7-Dichlorofluorescein diacetate (DCFH-DA) (10 μM) for 20 min at 37 °C. After that, the cells were washed with PBS carefully three times to remove free DCFH-DA. The absorbance was determined at 525 nm using Thermo Scientific Varioskan Flash. Apocynin was used as the positive control. The inhibitory rate (%) was calculated according to the equation: B1 refers to the absorbance of control group and B2 refers to the absorbance of test group, B0 refers to the absorbance of normal group.

Anti-Inflammatory Assay
The production of inflammatory mediators-NO in LPS-stimulated RAW 264.7 macrophages were used to evaluate the anti-inflammatory activity of testing drugs. The nitrite concentration was an indicator of NO production, and measured according to the Griess reaction in the culture medium [16]. After the cells of each group were treated as described above for 24 h, 100 μL of supernatant were mixed with 100 μL of Griess reagents (Reagent A: including 100 µg sulfanilamide and 600 µL 85% H3PO4 in 10 mL double distilled water; Reagent B: including 10 µg naphthyl ethylenediamine hydrochloride and 10 mL double distilled water; mixed equal volumes of reagents A and B) and standing for 10 min. The absorbance at 540 nm was measured using Thermo Scientific Varioskan Flash. The NO concentrations were calculated by regression analysis of a standard curve with sodium nitrite as a standard. The ibuprofen as the positive medicine was measured at the concentration of 30 µM. Inhibitory rate (%) was calculated according to the formula: C1 refers to the absorbance of control group and C2 refers to the absorbance of test group, C0 refers to the absorbance of normal group.

Cell Viability Assay
The MTT assay was used to evaluate the cell viability, as previously reported [17]. RAW 264.7 macrophages were seeded at a density of 1 × 10 5 cells /mL in 96-well plates and cultured for 24 h with the test compound (100 µM) at 37 °C in a 5% CO2 incubator. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h at 37 °C and the resulted crystals were dissolved in DMSO. The optical density was measured at 490 nm using Thermo Scientific Varioskan Flash.