Anti-Inflammatory Principles from the Needles of Pinus taiwanensis Hayata and In Silico Studies of Their Potential Anti-Aging Effects

Pinus needle tea are very popular in Eastern countries such as Japan, Russia, Korea, and China. Pine needle tea is claimed to have significant anti-aging effects, but no clear evidence has supported this until now. In the present study, five undescribed compounds (1–5) as well as seventy-two known compounds were purified and characterized from the bioactive fraction of methanol extracts of P. taiwanensis needles. Most of the isolates were examined for their anti-inflammatory bioactivity by cellular neutrophil model and six compounds (45, 47, 48, 49, 50, and 51) exhibited a significant inhibition on superoxide anion generation and elastase release with IC50 values ranging from 3.3 ± 0.9 to 8.3 ± 0.8 μM. These anti-inflammatory ingredients were subjected to docking computing to evaluate their binding affinity on the ghrelin receptor, which played an important role in regulating metabolism, with anti-aging effects. Compounds 49, 50, and 51 formed a stable complex with the ghrelin receptor via hydrogen bonds and different types of interactions. These results suggest the flavonoids are responsible for the potential anti-aging effects of pine needle tea.


Introduction
Pinus genus (Pinaceae), comprising more than 100 species and are mainly distributed in the Northern Hemisphere, are generally evergreen trees while some are shrubs [1]. The pine wood is light and often used for furniture. However, various pharmacological effects of the needles on the pine shoots have been recorded in the ancient books of traditional Chinese medicine. In "The Divine Husbandman's Herbal Foundation Canon", pine needles promoted hair growth, prolonged life, and quenched thirst. In "Taiping Shenghui Fang", it was recorded that pine needles could be cooked with alcohol and displayed an anti-aging effect. Nowadays, pine needles are processed as tea and are popular in Asia. In Korea, the constituents of the P. densiflora needle tea as well as its bioactivity on a cellular neutrophil model. In addition, the isolates from P. taiwanensis needles with anti-inflammatory bioactivities were subjected to docking computing and investigated for their interaction with the ghrelin receptor.

General Experimental Procedures
The melting points were recorded on a WRX-4 melting-point apparatus without correction. Optical rotations were recorded on a Jasco P-2000 digital polarimeter. The ultra violet (UV) spectra were obtained by a Hitachi U-2001 UV/V is spectrometer. The infrared (IR) spectra were examined with a Jasco FT/IR-4100 spectrophotometer. 1 H-, 13 C-, and 2D nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV-400 NMR spectrometer. Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard. The δ H and δ C values were for the chemical shifts of the signals, respectively. High resolution electrospray ionization mass spectrometry (HR-ESI-MS) was conducted with a JEOL JMS-700 spectrometer (operated in the negative-ion mode).

Plant Material
The needles of P. taiwanensis were collected in Puli, Nantou, Taiwan and identified by Prof. Sheng-Yang Wang (Department of Forestry, National Chung-Hsing University, Taichung, Taiwan). The voucher specimen (PCKuo_2016003) was deposited in the herbarium of School of Pharmacy, National Cheng Kung University, Tainan, Taiwan.

Human Neutrophil Preparation
The study was conducted with the approval of the Institutional Review Board of Chang Gung Memorial Hospital (IRB No. 201800369A3). Blood samples were drawn from healthy human donors (20 to 30 years old), and neutrophils were isolated and purified according to the protocols described previously [40].

Superoxide Anion Generation Measurement
The assay for measuring superoxide anion generation was based on the SOD-inhibitable reduction of ferricytochrome c as described previously [40].

Elastase Release Assay
Degranulation of azurophilic granules was determined by measuring the release of elastase as previously described [40].

Statistical Analysis
The results are expressed as mean ± standard error of the mean (SEM). Computation of 50% inhibitory concentrations (IC 50 ) was performed using PHARM/PCS v.4.2 software. Statistical comparisons were made between groups using the Student's t-test. Values of p < 0.05 were considered to be statistically significant.

Molecular Docking Study
The in silico evaluation was conducted on AutoDock Vina software [41]. The crystal structure of the ghrelin receptor has been characterized [42], and a .PDB file was downloaded from the Protein Databank (PDB ID: 6KO5). The 3D structures of ligands were constructed in the Chem3D program. The hydrogen supplement, Gasteiger charge measurement for protein atoms, and selection of flexible torsions for ligands were conducted by AutodockTools (ADT ver. 1.5.6). The size of the grid was designed at 18.5 Å × 18.5 Å × 18.5 Å and a grid center at dimensions (x, y, and z, respectively): 9.7, -19.2, 14.6 was determined. The binding affinity energy was provided as docking scores and shown in kcal/mol. The best interaction was considered only the top-scoring pose. The visualization of the best docking interactions was analyzed in Biovia Discovery Studio client 2020 [43].
Compound 5 possessed the molecular formula C 11 H 14 O 5 determined from a deprotonated molecular ion peak in the negative mode HR-ESI-MS analysis (m/z 225.0767 Figure S29). In its IR spectrum, hydroxyl (3421 cm −1 ) and carboxyl (1572 cm −1 ) functionalities could be detected. The 1 H-NMR spectrum ( Figure S30 . Moreover, one carboxylic group was located at δ C 176.2 (C-10) in its 13 C-NMR spectrum ( Figure S31). The planar structure of 5 was established by the significant HMBC correlations ( Figure S32) of OCH 3 -3 to C-3; H-6 to C-2, C-4, C-7, and C-10; H-7 to C-4, C-5, and C-8; H-9 to C-7, and C-8, respectively. Other 2D spectra ( Figure S33-S35) furnished the full assignment of proton and carbon signals. The above evidence suggests the structure of 5 as 2-hydroxy-5-(3-hydroxypropyl)-3-methoxybenzoic acid (Figure 1), which was already reported as styraxinolic acid in the previous synthetic literature [48]. Nevertheless, the present research is the first report of 5 from natural sources.

Anti-Inflammatory Activity
Inflammation is one of the major self-defense mechanisms stimulated by bacteria, virus, wound, or various other environmental factors. It is a first response of the immune system against infection and irritation. Neutrophils belong to an abundant kind of macrophage and play a major role in inflammation, and are usually the first lymphocytes to reach the infected region [49]. Neutrophils secrete a series of cytotoxins such as superoxide anion and elastase in response to the activation of the immune system [50]. In recent years, various human diseases have been demonstrated to be related to neutrophil overexpression [51][52][53][54][55]. The relationship between inflammation and cancer has been established, and the authors pointed out that the formation of cancer cells was directly related to inflammation [49]. Therefore, new anti-inflammatory compounds are worthwhile for further study on cancer treatment. Forty-three isolated compounds were evaluated for the inhibition of superoxide anion generation and elastase release by human neutrophils in response to fMLF/CB [56] (see Supplementary Materials, Table S2). The significant inhibitory results (Table 2) demonstrated that only 45, 47, 48, 49, and 50 ( Figure 2) displayed a significant inhibition of superoxide anion generation, with IC 50 values ranging from 3.3 ± 0.9 to 7.7 ± 0.9 µM compared with the positive control LY294002 (IC 50 1.1 ± 0.3 µM). Moreover, 48, 50, and 51 ( Figure 2) revealed the significant inhibition of elastase release with IC 50 values ranging from 5.3 ± 0.2 to 8.3 ± 0.8 µM compared with the positive control LY294002 (IC 50 3.2 ± 1.0 µM) ( Table 2). Compounds 48 and 50 displayed both inhibition of superoxide anion generation and elastase release, indicating their multiple anti-inflammatory bioactivities. The needles of P. morrisonicola have been reported to have an anti-inflammatory effect in RAW 264.7 macrophages [13]. The authors proposed that epicatechin and p-coumaric acid identified in P. morrisonicola may be the active ingredients. In the present research, all the active compounds contained the flavone backbone similar to that of epicatechin and the p-coumaroyl moiety could also be observed in 49, 50, and 51. This indicates that the flavonoid and p-coumaroyl functional groups may contribute the anti-inflammatory bioactivity in the present study. These bioassay results suggest that flavonoids play key roles in Pinus species for anti-inflammation bioactivity. Table 2. Inhibitory effects of purified compounds on superoxide anion generation and elastase release by human neutrophils in response to fMLF/CB.

Molecular Docking Study
The age-related decline in GH levels is considered to be a symptom of neuroendocrine aging [57]. This phenomenon exists in several mammalian species such as humans, domestic dogs, and laboratory rodents [57]. In human, the GH levels in plasma begins to decrease with age after full physical maturation, and continues during the decades of life [57]. Ghrelin is identified as the endogenous ligand for the GHSR and is a main regulator of GH secretion [18,19]. Ghrelin is involved in various physiological and pathophysiological mechanisms in humans such as aging [24,25]. In addition, ghrelin may be thought to be related to anti-inflammatory activity. Immune cell activation was limited by ghrelin treatment through the inhibition of NF-κB activation and subsequent MCP-1 secretion [26]. A synthetic ghrelin analog growth hormone-releasing peptide-2 (GHRP-2) was reported to reduce the inflammatory factors in arthritic rats, and it supports that the immune cells were mediated by the activation of ghrelin receptors [27]. Neves et al. also proposed the regulation of inflammation as an anti-aging intervention [58]. Thus, according to the anti-inflammatory bioassay experimental data, 45, 47, 48, 49, 50, and 51 ( Figure 2) showed significant inhibitory effects, and were selected to determine their binding abilities to the ghrelin receptor. Before docking simulation, the native ligand (8QX) included in the 6KO5 PDB file was re-docked for validation. The interactions between 8QX and 6KO5 and the best pose of calculated results showed high similarity and repeatability with native data (data not shown). The results indicate the high accuracy of the existing simulation system and supported the further computing.
The lowest binding energy of each ligand was considered the best conformation. The binding affinities are listed in Table 3. Growth hormone-releasing peptide 6 (GHRP-6) was used as a positive control for docking to the binding pocket of the ghrelin receptor as in our previous report. Although AutoDock Vina is not constructed for docking between peptides and proteins, several successful results have been published in previous reports [59][60][61]. Therefore, in this study, GHRP-6 was first computed to determine the accuracy of the present docking model and the results coincided well ( Figure 3A). Compared with GHRP-6, the binding energies of 49, 50, and 51 were lower than −10.3 kcal/mol (Table 3). This suggests that 49, 50, and 51 could dock into the pocket of the ghrelin receptor similar or even better than that of GHRP-6. For 49, the hydrogen bonds could be observed between two carbonyl groups (C-4 and p-coumaroyl) and Arg283, C-5 hydroxyl and Gln120, C-7 hydroxyl and Tyr313, and C-4 hydroxyl and Cys304, respectively. Arg199 formed a conventional hydrogen bond with a carbonyl group of p-coumaroyl. In addition, 49 was linked to the Arg283, Arg102, Asp99, Phe279, Phe312, Leu181, and Pro200 residues of the ghrelin receptor via different effects such as the π-cation, π-anion, π-π T-shaped, and π-alkyl interactions. These allowed compound 49 and protein to form a stable complex ( Figure 3B). 50 was bound with Asp99, Arg102, Gln120, Arg283, Leu103, Asn305, and Arg199 through various hydrogen bonds, while other interactions (π-cation, π-anion, π-π T-shaped, and π-alkyl) were also observed with Asp99, Arg102, Arg283, Phe279, Phe312, Leu181, and Leu210 ( Figure 3C). 51 also established hydrogen or carbon hydrogen bonds with Tyr313, Ser123, Arg283, Arg102 and Asn305, together with other interactions (π-cation, π-anion, π-sigma, π-π T-shaped, and π-alkyl) to link with Asp99, Arg283, Arg102, Leu181, Phe312, and Pro200 residues of ghrelin receptor could be detected ( Figure 3D). Compared compounds 45, 47, and 48 with 49, 50, and 51, the former group possessed a flavonoid skeleton only while the latter group had sugar and coumaroyl moieties. It was reported that the coumaroyl group attached on the sugar was crucial for the binding affinity to the ghrelin receptor [34]. The gap structure of GHSR interacting with the acyl acid moiety of ghrelin resulted in the transformation of the ghrelin receptor into an active configuration [43]. Moreover, the binding pocket of GHSR is bifurcated by the salt bridge between Glu124 and Arg283, and this region is rich in hydrophobic amino acids [43]. According to our data, the docking scores of 49, 50, and 51 were higher than those of 45, 47, and 48, which suggested the better binding capability. The major structural characteristics were the coumaroyl groups rather than the sugar moieties and this could be further evidenced by examination of more compounds possessing coumaroyl functionalities. In this study, the active ingredients 49, 50, and 51 possessed not only anti-inflammatory bioactivity, but also the ghrelin receptor binding potential. This indicated that the claimed anti-aging effects of pine needle tea may be also based on these teaghrelin-like compounds.

Conclusions
A total of seventy-seven isolates comprising five undescribed compounds were purified from the methanol extracts of P. taiwanensis needles. Their structures were characterized through spectroscopic and spectrometric analyses. Forty-three purified compounds were examined for their anti-inflammatory activity by the inhibition of superoxide anion generation and elastase release on neutrophil model. The results suggest that 45, 47, 48, 49, 50, and 51 possess significant anti-inflammatory potentials. Further molecular docking computing results supported 49, 50, and 51 exhibiting a binding affinity to the active pocket of the ghrelin receptor. Therefore, the crude extracts and purified constituents of P. taiwanensis have the potential to be developed as new anti-inflammatory lead drugs or food ingredients.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.339 0/antiox10040598/s1, Appendix A: Complete extraction and isolation procedures, Appendix B: References of known compounds, Table S1: Preliminary bioactivity screening of needles of P. taiwanensis on superoxide anion generation and elastase release by human neutrophils in response to fMLF/CB, Table S2: Inhibitory effects of purified compounds on superoxide anion generation and elastase release by human neutrophils in response to fMLF/CB, Figure