Anti-Inflammatory Principles From The Needles Of Pinus Taiwanensis Hayata And In Silico Studies Of Their Potential Anti-Aging Effects

Abstract: Pinus needle tea is very popular in Eastern countries such as Japan, Russia, Korea, and China. Pine needle tea is claimed to have signifificant anti-aging effects, but no clear evidence has supported this until now. In the present study, fifteen 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. 

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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 signifificant inhibition on superoxide anion generation and elastase release with IC50values ranging from 3.3 ± 0.9 to 8.3 ± 0.8 µM. These anti-inflflammatory 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.

1. Introduction 

Pinus genus (Pinaceae), comprising more than 100 species and mainly distributed in the Northern Hemisphere, are generally evergreen trees while some are shrubs [1]. Pine wood is light and often used for furniture. However, various pharmacological effects of needles on 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 antioxidant and anti-bacterial bioactivities were extensively investigated [2,3]. Since the early last century, more than seven hundred compounds have been identified from Pinus needles. Most of the reported literature are related to the essential oil of P. densiflflora [4,5], P. halepensis [6,7], P. nigra [4,8], and P. sylvestris [4,9]. The characterized components of Pinus needles are mostly benzenoids, diterpenoids, flavonoids, lignans, monoterpenoids, and sesquiterpenoids [10]. The needles of P. densiflora and P. morrisonicola have been reported for their antioxidant bioactivity [11–14]. In addition, the ethyl acetate extract of needles of P. morrisonicola inhibited the protein and mRNA expression of NO and iNOS in LPS-induced RAW 264.7 macrophage, exhibiting a signifificant anti-inflflammatory bioactivity [13]. The supercritical flfluid extract of P. densiflora needles displayed the inhibitory effect on LPS-induced NO production by downregulating the expression of iNOS, and reducing the expression of IL-6 and IL-1β and activation of STAT1 and STAT3 proteins in macrophages induced by LPS [15]. The ethanol extract of P. thunbergii needles showed a signifificant anti-inflflammatory effect in macrophages and suppressed arachidonic acid-induced ear edema and inhibited myeloperoxidase enzymatic activity [16]. Moreover, the fermented P. morrisonicola needles showed their excellent antioxidant and anti-inflflammatory bioactivities by modulating the NF-κB signaling pathway [17]. 

Ghrelin is a peptide hormone consisting of twenty-eight amino acids and was originally discovered in the stomach [18,19]. It is an endogenous ligand for the growth hormone secretagogue (GHSR), which is a member of the β-branch in class A GPCRs (G ProteinCoupled Receptors) [18,19]. Ghrelin is the only peptide hormone that causes hunger to promote appetite [20]. Many studies have suggested that ghrelin plays an important role in regulating metabolism, energy balance, memory, cardiovascular, and gastrointestinal functions in the human body [21–23]. Ghrelin is also involved in various physiological and pathophysiological mechanisms in the human body such as aging [24,25], and it may be related to anti-inflflammatory activity [26,27]. In 2014, the unique acylated flflavonoid tetra glycosides named teaghrelins, were first identified in Chin-shin oolong tea by our group and demonstrated their promoting activity in growth hormone (GH) release [28]. Two other similar compounds were purified in Shy-jih-chuen oolong tea and their bioactivity on the ghrelin receptor was also verified [29]. The four teaghrelins induced hunger through the same regulatory pathway as ghrelin. Not limited to tea, compounds with similar structures or bioactivities to teaghrelins have also been explored in Polygonum multiflflorum (Hes houwu) [30], Ginkgo biloba [31], Morus alba [32], and Cistanche tubulosa [33]. These results support us in the search for natural anti-aging principles by screening the tea ghrelin-like compounds that possess anti-inflflammatory effects. In addition to directly performing the promoting activity in growth hormone release in cellular models, molecular modeling of some potential compounds would be more effificient in exploring possible candidates [34]. Computational methods have been applied to the development and evaluation of pharmacological hypotheses. Molecular docking is one of the most commonly utilized techniques and anticipates the conformation and affinity of ligand binding to the active pocket with high accuracy [35,36]. Docking methods effectively search high-dimensional spaces for possible interaction and use a scoring function that properly ranks the candidates [37]. 

In 2018, three compounds were purified from the needles of P. morrisonicola and exhibited signifificant vasorelaxant activity, and among these constituents, one was reported with a tea ghrelin-like structure [38]. In Taiwan, P. taiwanensis needles are usually dried and baked and then processed as a tea due to their famous bioactivities. P. taiwanensis Hayata is one of the native and endemic species in Taiwan. It is also called Taiwan red pine, or huangshan pine. There are two needles in a fascicle with 8–11 cm long and slightly stiff and straight, and it grows across the whole island from low to high altitudes and often form pure forest [39]. In a preliminary examination, the ethyl acetate layer of methanol extracts of P. taiwanensis needles displayed a signifificant inhibition on superoxide anion generation and elastase release with IC50 values of 0.8 ± 0.2 and 1.0 ± 0.1 µg/mL, respectively (Table S1). Therefore, in the present study, the bioactive constituents of P. taiwanensis needles were investigated and the purified compounds were evaluated for their anti-inflflammatory bioactivity on a cellular neutrophil model. In addition, the isolates from P. taiwanensis needles with anti-inflflammatory bioactivities were subjected to docking computing and investigated for their interaction with the ghrelin receptor. 

2. Materials and Methods

2.1. General Experimental Procedures

ultraviolet (UV) spectra were obtained by a Hitachi U-2001 UV/V is a spectrometer. The infrared (IR) spectra were examined with a Jasco FT/IR-4100 spectrophotometer. 1H-, 13C-, 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 δH and δC 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).

2.2. 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.

2.3. Extraction and Isolation

2.4. Spectral and Physical Data of 1–5

2.4.2. 1-[(70 R,80 S)-70 ,90 -Dihydroxy-70 -(4-hydroxy-3-methoxyphenyl)propan-80 -yloxy]-2- hydroxybenzoic Acid (2) Colorless syrup; [α]25 D + 5.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 284 (3.59), 253 (3.80) nm; ECD (c 3.51 × 10−4 M, MeOH) λmax (∆ε) 248 (+1.33), 224 (+1.03), 214(+1.10) nm; IR (neat) νmax 3425, 2927, 1541, 1384, 1271 cm−1;1H and 13C NMR; HRESIMS m/z 349.0936 ([M − H]− calcd for C17H17O8, 349.0923).

2.4.3. (13E,12R)-12-Hydroxyagathic Acid (3)

Colorless powder; mp: 263 ◦C (dec.); [α]25 D + 30.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 225 (sh) (3.83) nm; IR (neat) νmax 3450, 2937, 1648, 1252 cm−1;1H-NMR (CD3OD, 400 MHz) δ 0.63 (3H, s, CH3-20), 1.09 (1H, ddd, J = 13.6, 13.6, 3.6 Hz, H-3a), 1.21 (3H, s, CH3-18), 1.21 (1H, m, H-1a), 1.42 (1H, m, H-5), 1.51 (1H, m, H-2a), 1.59 (2H, m, H-11),1.79 (1H, m, H-1b), 1.91 (1H, m, H-2b), 1.91 (1H, m, H-6a), 1.98 (1H, m, H-7a), 2.01 (1H, m,H-6b), 2.08 (3H, s, CH3-16), 2.12 (1H, m, H-9), 2.14 (1H, m, H-3b), 2.43 (1H, m, H-7b), 4.03 (1H, dd, J = 9.2, 2.8 Hz, H-12), 4.53 (1H, s, H-17a), 4.91 (1H, s, H-17b), 5.88 (1H, br s, H-14); 13C-NMR (CD3OD, 100 MHz) δ 13.5 (CH3-20), 14.9 (CH3-16), 21.2 (C-2), 27.6 (C-6), 29.6 (CH3-18), 31.5 (C-11), 39.4 (C-3), 40.0 (C-7), 40.3 (C-1), 41.2 (C-10), 45.3 (C-4), 53.1 (C-9), 57.6 (C-5), 75.5 (C-12), 106.9 (C-17), 117.8 (C-14), 150.2 (C-8), 159.5 (C-13), 167.2 (C-15), 181.5 (C-19); HRESIMS m/z 349.2024 ([M − H]− calcd for C20H29O5, 349.2015).

2.4.4. 5-Isopropyl-3-oxocyclohex-1-ene-1-carboxylic Acid (4)

Colorless tabular crystal; mp: 235 ◦C (dec.); [α]25 D + 28.6 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 237 (sh) (3.40) nm; IR (neat) νmax 3456, 2925, 1635, 1395 cm-1;1H-NMR (CD3OD, 400 MHz) 0.88 (3H, d, J = 6.8 Hz, CH3-9), 0.98 (3H, d, J = 6.8 Hz, CH3-10), 1.84 (1H, m, H-4a), 2.02 (1H, m, H-4b), 2.09 (1H, m, H-5), 2.31 (1H, hept, J = 6.8 Hz, H-8), 2.50 (1H, dddd, J = 19.2, 9.2, 4.8, 2.4 Hz, H-6a), 2.70 (1H, dddd, J = 19.2, 5.2, 5.2, 1.2 Hz, H-6b), 6.32 (1H, dd, J = 2.4, 1.2 Hz, H-2); 13C-NMR (CD3OD, 100 MHz) δ 19.0 (C-9), 20.9 (C-10), 24.3 (C-4), 27.0 (C-6), 27.1 (C-8), 53.7 (C-5), 128.7 (C-2), 160.6 (C-1), 174.8 (C-7), 205.7 (C-3); HRESIMS m/z 181.0855 ([M − H]− calcd for C10H13O3, 181.0865).

2.4.5. Styraxinolic Acid (5)

Colorless syrup; UV (MeOH) λmax (log ε) 308 (2.89), 237 (sh) (3.37), 222 (sh) (3.70) nm; IR (neat) νmax 3421, 2926, 1572, 1395, 1268 cm-1;1H-NMR (CD3OD, 400 MHz) 1.82 (2H, tt, J = 8.0, 6.8 Hz, H-8), 2.61 (2H, t, J = 8.0 Hz, H-7), 3.56 (2H, t, J = 6.8 Hz, H-9), 3.83 (3H, s, OCH3-3), 6.86 (1H, d, J = 2.4 Hz, H-4), 7.31 (1H, d, J = 2.4 Hz, H-6); 13C-NMR (CD3OD, 100 MHz) δ 32.7 (C-7), 35.6 (C-8), 56.6 (OCH3-3), 62.3 (C-9), 116.3 (C-4), 120.1 (C-1), 122.8 (C-6), 131.9 (C-5), 149.2 (C-3), 150.7 (C-2), 176.2 (C-10); HRESIMS m/z 225.0767 ([M − H]−calcd for C11H13O5, 225.0763).

2.5. Anti-Inflflammatory Bioactivity Examination

2.5.1. 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 purifified according to the protocols described previously [40]. 

2.5.2. 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].

2.5.3. Elastase Release Assay

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

2.5.4. Statistical Analysis The results are expressed as mean ± standard error of the mean (SEM). Computation of 50% inhibitory concentrations (IC50) 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. 

2.6. 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].