Chemical Constituents of Hedyotis diffusa and Their Anti-Inflammatory Bioactivities

Seven new anthraquinones with rare 2-isopropyldihydrofuran (1–3) and 2,2-dimethylpyrano (4–7) moieties together with thirty-four known compounds were isolated from the extracts of whole Hedyotis diffusa plants. Their structures were elucidated and established by various spectroscopic and spectrometric analytical methods. Among these isolates, selected compounds were examined for their anti-inflammatory activity. The results showed that rare substituted anthraquinones displayed potent inhibitory activity with IC50 values ranging from 0.15 ± 0.01 to 5.52 ± 1.59 µM on the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release cellular models. Meanwhile, the proposed drug target of the active anthraquinone was studied by computer modeling. The binding affinity between the anti-inflammatory anthraquinone and elastase was evaluated by molecular docking. These results provided the scientific insight into the medicinal values of Hedyotis diffusa and vision of development as lead compounds.

Neutrophils, the largest type of macrophages, account for 50 to 60% of the total circulating white blood cells and play a major role in inflammatory response [24]. In addition to phagocytosis and enzyme secretion against pathogenic bacteria, neutrophils also secrete some peroxides, such as superoxide anion. Moreover, neutrophils are involved in other immune responses during inflammation, such as the production of elastase. The main function of elastase is to hydrolyze elastin, which can decompose injured cells and invading pathogenic bacteria in the infected area, and to complete the entire protection of host cells and tissues through apoptosis [25,26]. Therefore, inhibition of elastase secretion and superoxide anion formation can effectively reduce cell inflammation. A human neutrophil cell model activated by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB) to inhibit superoxide anion generation and elastase release was used as a screening platform for anti-inflammatory activity in our previous study [27,28]. In the preliminary screening of a series of Chinese herbal medicines with heat-clearing and detoxification potentials, the H. diffusa ethanol extracts displayed 71.71 and 38.26% inhibitory activities at 10 µg/mL in fMLP/CB-induced superoxide anion generation and elastase release assay, respectively. Moreover, the ethanol extract of H. diffusa also has an inhibitory effect on hepatitis C virus and Dengue virus, with IC 50 and EC 50 values against hepatitis C virus of 131.1 and 49.5 µg/mL, respectively (unpublished data). At a sample concentration of 25 µg/mL, the methanol extract of H. diffusa reduced the RNA expression of Dengue virus by 30.0 ± 8.1% (unpublished data). According to these experimental screening data, the chemical components of the ethanol extract of H. diffusa were thoroughly separated and identified. The anti-inflammatory activity of the major chemical components and their molecular docking with elastase were also investigated. This research is expected to provide an important reference for the development of anti-inflammatory lead compounds, healthy foods, and cosmetic products.

General Experimental Procedures
A Jasco P-2000 digital polarimeter (Jasco, Tokyo, Japan) with 589 nm filter was used to measure the optical rotations of purified compounds. The ultraviolet (UV) spectra were determined on a Hitachi U-0080-D UV/Vis spectrometer (Hitachi, Tokyo, Japan) with a 1.0 cm length cell. The infrared (IR) spectra were recorded on a PerkinElmer FT-IF spectrum RX I (PerkinElmer, Waltham, MA, USA) using KBr pellets. Circular dichroism (CD) spectra were determined on the Jasco J-720 spectropolarimeter (Jasco, Tokyo, Japan). One-dimensional and two-dimensional NMR spectra were recorded on the Bruker Avance III 400 or Avance III HD 700 NMR spectrometer (Bruker, Billerica, MA, USA) using CDCl 3 , acetone-d 6 , or methanol-d 4 as solvent with tetramethylsilane as an internal standard. HR-ESI mass spectra were acquired from the Bruker APEX II mass spectrometer. Preparative high performance liquid chromatography (HPLC) was carried out on a Shimadzu LC-8A instrument (Shimadzu, Kyoto, Japan) equipped with UV-VIS detector (Shimadzu SPD-10A, Kyoto, Japan) and a Cosmosil 5C18-MS-II column (20 × 250 mm, Nacalai Tesque Kyoto, Japan). Column chromatography was performed on Geduran Si 60 (40-63 µm, Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) was carried out using precoated Kieselgel 60 F 254 plates (Merck), in which compounds were visualized by UV light or spraying with anisaldehyde solution followed by heating at 120 • C.

Plant Material
Dried whole herbs of H. diffusa were purchased from Chuang Song Zong Pharmaceutical Co. Ltd., Pingtung, Taiwan, in September 2013. The plant materials were authenticated by Prof. Chang-Sheng Kuoh, Department of Life Science, National Cheng Kung University (NCKU), Tainan, Taiwan. A voucher specimen (TSWu 2015-001-001) was deposited at School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Extraction and Isolation
The whole herbs of H. diffusa (5.2 kg) were refluxed with 95% ethanol (3 × 10 L) to give ethanol extract (370 g) after evaporation under reduced pressure. This crude extract was suspended in water and partitioned successively with ethyl acetate (EtOAc) to afford the EtOAc soluble fraction (110 g), water soluble layer (220 g), and precipitate (40 g).

Superoxide Anion Generation Measurement
The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c. Neutrophils (6 × 10 5 cells/mL) were equilibrated in the presence of 0.6 mg/mL ferricytochrome c at 37 • C for 2 min and incubated with each test compound or vehicle (0.1% DMSO, negative control) for 5 min. Cells were incubated with cytochalasin B (CB, 1 µg/mL) for 3 min. Neutrophils were then activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 100 nM). The changes in the absorbance of ferricytochrome c reduction at 550 nm were continuously monitored in a double-beam, sixcell positioner spectrophotometer (Hitachi U-3010, Tokyo, Japan) with constant stirring. A phosphatidylinositol 3-kinase (PIK3) inhibitor, LY294002, was used as a positive control [27].

Elastase Release Assay
Degranulation of azurophilic granules was determined by elastase release as described previously. Elastase substrate used in experiments was MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide. After supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM), neutrophils (6 × 10 5 /mL) were equilibrated at 37 • C for 2 min and incubated with test compounds or vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated by 100 nM fMLP and 0.5 µg/mL CB, and absorbance changes at 405 nm were continuously monitored to measure release of elastase. LY294002 was used as a positive control [27].

Statistical Analysis
The results are expressed as the mean ± standard error of the mean (SEM). 50% Inhibition concentration (IC 50 ) was calculated using a computer (PHARM/PCS v4.2). Student's t test was used for statistical comparison among each group. Values of p less than 0.05 were considered statistically significant.

Molecular Docking Study
An AutoDock Vina software (v.1_1_2) was used for the in silico evaluation [29]. The crystal structure of the Human neutrophil elastase was downloaded from the Protein Databank (PDB ID: 1H1B). The 3D structures of ligands were constructed in the Chem3D program. AutodockTools (ADT v1.5.6) carries out the hydrogen supplementation, Gasteiger charge measurement of protein atoms, and selection of ligand flexible torsions. Center at 18.6, 11.8, and 22.8 (x, y, z) of grid box was determined. The binding affinity energy was provided as docking scores and shown in kcal/mol. Biovia Discovery Studio client 2020 analyzed the visualization of the best docking interactions [30].

Structural Elucidation of Compounds 1-7
Compound 1 (Figure 1) Figure S1). The absorbance maxima at 245 and 275 nm in its UV spectrum were the typical feature of an anthraquinone-type compound [31]. The IR spectrum showed absorption bands at 1668, 1585, and 1447 cm −1 that indicated the presence of conjugated carbonyl groups and aromatic ring functionalities. The 1 H NMR spectrum of 1 (Table 1, Figure S2 ). In addition, five corresponding carbon signals in the 13 C and HSQC NMR spectra ( Figure S7), including two olefinic carbons (δ 143.4 (s, C−3 ) and 112.4 (t, C−4 )), one oxygenated methine (δ 87.7, d, C−2 ), one methylene (δ 36.1, t, C−1 ), and one methyl (δ 17.1, q, C−5 ) indicated the appearance of the isopentenyl dihydrofuranyl moiety in 1. The observed 2 Jand 3 J-HMBC correlations (Figure 2 and Figure S4) of the isopentenyl moiety from H−1 α (1H, δ 3.93) to C−3, from H−1 β (1H, δ 3.54) to C−3 , and from H−4 β (δ 4.96, 1H, s) to C−3 /C−5 established that this moiety was fused at C−3 and C−4 of the anthraquinone. The absolute configuration at C−2 of 1 was determined by the CD spectrum ( Figure S8), which showed a positive Cotton effect at 298 nm. This result is consistent with the positive value of R-dihydrocolumbianetin reported in the literature [32], which therefore determines the configuration of C−2 as R. Based on these above data of 1, its chemical structure was established as shown in Figure 1 and named trivially as diffusaquinone A.

Molecular Docking Study
Molecular docking is a popular computing technique that can accurately predict the conformation and affinity between the ligand and the active pocket [33,34]. The docking method provides the high-dimensional space for possible interactions and evaluates the ranking of candidates based on a scoring function [35]. To illustrate the binding ability between anthraquinones and human neutrophil elastase, compounds 1, 5, and GW475151 were selected for molecular docking studies based on the above-mentioned experimental results. The binding affinity is shown in kcal/mol according to the computing results, and the calculated complex with the lowest energy was designed as the best docking configuration. GW475151 is an elastase inhibitor and is used as a native binding ligand [36]. It binds to elastase via Gly219 by hydrogen bond, and other amino acid residues Val216 and Cys191 by alkyl and amide-π-stacked interactions, and van der Waals (Figure 3). A stable complex is formed with binding energy of −6.0 kcal/mol (Table 4). Compounds 1 and 5 display even lower binding energy, and it indicates that they connect to proteins easier than GW475151 (Table 4). Several interactions are completed between 1 and elastase, including hydrogen bonds between Val216 and A-ring and carbonyl group of 1; moreover, other interactions π-sigma, π-π T-shaped, alkyl, π-alkyl are linked to elastase by Leu99B, His57, Arg217A, and Phe215. 5 is bound with His57, Val99, and Val216 through hydrogen bond and linked with Phe215, Leu99B, Arg217A, and Phe192 via π-sigma, π-π T-shaped, alkyl, and π-alkyl effects, respectively. These interactions promote 5 and elastase to establish a stable unit, therefore resulting in a significant binding affinity with the receptor. All these computing results coincide well with the experimental data of the bioactivity examination. Therefore, it is speculated that the inhibition of elastase release may be related to the binding of anthraquinones with elastase, and further pharmacological mechanisms need to be verified.

Conclusions
In summary, a total of forty-one compounds, including seven new anthraquinones, were isolated from EtOAc layer of the ethanolic extract of Hedyotis diffusa. Among all the isolated compounds, anthraquinones and iridoids glycosides were the main components. Some anthraquinones, especially with 2-isopropyldihydrofuran or 2,2-dimethylpyrano moiety, showed promising anti-inflammatory activities for inhibiting superoxide anion generation and elastase release. Compound 1 with a 2-isopropyldihydrofuran moiety displayed the most potent anti-inflammatory activity for superoxide anion generation and elastase release with IC 50 values 0.92 ± 0.22 µM and 0.71 ± 0.22 µM, respectively. Compound 5 containing the 2,2-dimethylpyrano ring moiety inhibited superoxide anions generation and elastase release with the IC 50 values of 0.15 ± 0.01 µM and 0.20 ± 0.02 µM, respectively. These additional functional groups can significantly improve the anti-inflammatory effects of anthraquinones. The calculation results of molecular docking also confirmed the good binding affinity of 1 and 5 with neutrophil elastase. These functional groups are usually observed in natural products such as coumarins, acridones, and flavonoids but are rarely found in the anthraquinone skeleton. Such substructures can be used as a reference for medicinal chemistry derivation of anti-inflammatory lead compounds and as new candidates for the adjuvant therapy in the future.