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

The pine needles were extracted with methanol and partitioned with hexanes, ethyl acetate, and water to obtain three soluble layers, respectively. The anti-inflammatory fraction, the ethyl acetate layer, was subjected to continuous conventional chromatographic technique combination, and five undescribed compounds were characterized including two new lignans, 1-[(70 R,80 S)-70 ,90 -dihydroxy-70 -(4-hydroxyphenyl)propan-80 -yloxy]benzoic acid (1), 1-[(70 R,80 S)-70 ,90 -dihydroxy-70 -(4-hydroxy-3-methoxyphenyl)-propan-80 -yloxy]-2-hydroxybenzoic acid (2), one new diterpenoid, (13E,12R)-12 -hydroxyagathic acid (3), one monoterpenoid, 5-isopropyl-3-oxocyclohex-1-ene-1-carboxylic acid (4), and one phenylpropane, styraxinolic acid (5). The chemical structures of these new compounds were constructed with the assistance of the NMR spectral elucidation and MS spectrometric analysis. Moreover, seventy-two known compounds, comprising one steroid, β-sitosterol (6); one sesquiterpenoid, (-)-oplopan-4-one-10-α-O-β-D-glucoside (7); one coumarin, umbelliferone (8); one alkaloid, indole-3-aldehyde (9); four diterpenoids,

Click Here To Get Anti-Aging Cistanche Cream

3-methoxyphenyl)-1-propanone (64), 3-hydroxy-1-(4-hydroxyphenyl)-1-propanone (65),2-(4-hydroxyphenyl)acetic acid (66), phenylacetic acid (67), is vanillic acid (68), benzoicacid (69), vanillin (70), p-hydroxyacetophenone (71), sodium salicylate (72), vanillic acid4-O-α-L-rhamnoside (73), trans-ferulic acid (74), sodium p-coumarate (75), p-coumaricacid (76), trans-methyl p-coumarate (77), respectively, were identified by the examination of their physical and spectroscopic data with those previously published (references of known compounds were provided in Supplementary Materials Appendix B).

3.1. Structural Elucidation of Compounds 1–5

Compound 1 was isolated as an optically active colorless syrup, and the molecular formula was assigned as C16H16O6 by HR-ESI-MS analysis ([M − H]−, m/z 303.0855, calcd. for C16H15O6, 303.0869, Figure S1). The IR spectrum indicates the presence of a hydroxyl (3412 cm−1 ) and a conjugated carbonyl group (1598 cm−1 ). The 1H-NMR data (Figure S2) showed the signals for two para-substituted aromatic moieties [δH 6.72 (2H, d, J = 8.4 Hz, H-30 , -50 ), 6.86 (2H, d, J = 8.8 Hz, H-2, -6), 7.24 (2H, d, J = 8.4 Hz, H-20 , -60 ), and 7.83 (2H, d, J = 8.8 Hz, H-3, -5)], two oxygenated methines [δH 4.48 (1H, m, H-80 ), and 4.85 (1H, d, J = 5.6 Hz, H-70 )], and two methines [δH 3.81 (1H, dd, J = 12.0, 4.0 Hz, H-90 a), and 3.86 (1H, dd, J = 12.0, 5.6 Hz, H-90 b)]. The 13C and DEPT NMR spectra (Figure S3) of 1 displayed sixteen carbons, corresponding to one methylene group, two oxygenated carbons, twelve aromatic carbons, and one conjugated carbonyl (Table 1). The 2 J- and 3 J- HMBC correlations from H-2, -6 to C-1 and 4; from H-3, 5 to C-1 and 7; from H-20, 60 to C-40 and 70 ; from H-70 to C-80 and 90 ; and from H-80 to C-1, respectively, were observed in the HMBC spectrum of 1 (Figure S4). Moreover, a large coupling constant between H-70 and H-80 (J = 5.6 Hz) supported the relative configuration of 1 at C-70 /C-80 as threo [44]. The absolute configurations at C-70 and C-80 of 1 were determined by electronic circular dichroism (ECD) analysis. The positive Cotton effect at 230 nm (∆ε + 0.17) revealed an 8S configuration for 1, according to the published literature [37,38] and therefore 70 R was also determined. Other 2D spectra (Figure S5–S7) furnished the full assignment of proton and carbon signals. Accordingly, the structure of 1 was assigned as 1-[(70 R,80 S)-70 ,90 -dihydroxy- 7 0 -(4-hydroxyphenyl)propan-80 -yloxy]benzoic acid as shown in Figure 1. 

The molecular formula of 2 was assigned as C17H18O8 on the basis of HR-ESI-MS analytical data (m/z 349.0936 [M − H]−, Figure S8). The absorption in the IR spectrum (3425 and 1541 cm−1 ) indicated the hydroxyl and conjugated carbonyl functionalities, respectively. In comparison of the NMR spectra of 1 and 2, it could observe that they possessed different aromatic moieties. Two sets of ABX-coupled aromatic ring and one methoxy group could be detected in the 1H- (Figure S9) and 13C-NMR (Figure S10) data of 2 (Table 1), and it suggested that compound 2 possessed two trisubstituted rather than para-disubstituted aromatic moieties. The signifificant HMBC correlations (Figure S11) from H-3 to C-1, C-5, and C-7; from H-6 to C-4; from OCH3-30 to C-30 ; from H-60 to C-40 and C-70; from H-70 to C-10 , C-20 , C-80 and 90 ; from H-80 to C-1, respectively, established that the structure of 2 was also a neolignan skeleton. Through a combination of a large coupling constant (J7,8 = 5.2 Hz) and positive Cotton effect at 230 nm (∆ε + 1.14), the absolute configuration of 2 was assigned as the three- and (70 R,80 S)-form, the same as 1 [44,45]. Other 2D spectra (Figure S12–S14) furnished the full assignment of proton and carbon signals. These fifindings concluded the structure of 2 as 1-[(70 R,80 S)-70 ,90 -dihydroxy-70 -(4-hydroxy-3- methoxyphenyl)propan-80 -yloxy]-2-hydroxybenzoic acid (Figure 1). 

Compound 3 was obtained as a colorless powder and its molecular formula was assigned as C20H30O5 on the basis of HR-ESI-MS analytical data (m/z 349.2024 [M − H]−, Figure S15). Compound 3 showed absorption peaks at 3450 (OH), and 1648 (carboxylic acid) cm−1 in its IR spectrum. It was evidenced by the 13C-NMR spectrum (Figure S16) in which two carboxylic functionalities were observed at δC 167.2 (C-15) and 181.5 (C-19). In its 1H-NMR (Figure S17), the resonances at δH 4.03 (1H, dd, J = 9.2, 2.8 Hz, H-12) and δC 75.5 (C-12) indicated the presence of a secondary alcohol group. The terminal methylene group could be established due to the proton resonances at δH 4.53 (1H, s, H-17a) and 4.91 (1H, s, H-17b), and the carbon signals at δC 106.9 (C-17) and 150.2 (C-8), respectively. Two methyl groups at δH 0.63 (3H, s, CH3-20) and 1.21 (3H, s, CH3-18) were connected to the quarternary carbons (C-10 and C-4) evidenced by the HMBC correlations (Figure S18). The shielding effect of the carboxylic group at C-4 resulted in the upfield shift of CH3-20 (δH 0.63), suggesting its β-configuration [46]. In addition, the chemical shift of H-17a (δH 4.53) appeared in the upfield region, suggesting the 12R configuration [47]. In its HMBC spectrum, the correlations from H-12 to C-9, C-14, and C-16; from H-17 to C-7 and C-9; from CH3-16 to C-12 and C-14; from CH3-18 to C-3, C-4 and C-19; from CH3-20 to C-1, C-5,C-9 and C-10, respectively, constructed the planar structure of 3 as previously reported for 12-hydroxyagathic acid [46]. However, the C-13 configuration of 3 was determined as E by the NOESY analytical data (Figure S19), which displayed the NOE effects among H-5/H-9, H-5/H-18, and H-12/H-14. Moreover, the NOE between H-14 and CH3-16, which should be recorded in 12-hydroxyagathic acid [46], was not detected in 3. Other 2D spectra (Figure S20–S21) furnished the full assignment of proton and carbon signals. Conclusively, the structure of 3 was established as (13E,12R)-12-hydroxyagathic acid as shown (Figure 1).

The HR-ESI-MS spectrum of 4 exhibited an [M − H]− ion peak at m/z 181.0855 (Figure S22), consistent with the pseudomolecular formula of C10H13O3. The absorption peaks at 3456 and 1635 cm-1 in its IR spectrum displayed hydroxyl and conjugated carbonyl groups, respectively. Two methine protons at δH 2.09 (1H, m, H-5) and 6.32 (1H, dd, J = 2.4, 1.2 Hz, H-2), two methylene groups at δH 1.84 (1H, m, H-4a), 2.02 (1H, m, H-4b), 2.50 (1H, dddd, J = 19.2, 9.2, 4.8, 2.4 Hz, H-6a) and 2.70 (1H, dddd, J = 19.2, 5.2, 5.2, 1.2 Hz, H-6b), and one set of isopropyl protons at δH 0.88 (3H, d, J = 6.8 Hz, CH3-9), 0.98 (3H, d, J = 6.8 Hz, CH3-10) and 2.31 (1H, hept, J = 6.8 Hz, H-8) appeared in the 1H-NMR spectrum of 4 (Figure S23). In addition, one conjugated carbonyl carbon at δC 174.8 (C-7), and one carboxyl carbon at δC 205.7 (C-3) could be observed in its 13C- and DEPT NMR spectra (Figure S24). The observed HMBC correlations (Figure S25) from H-2 to C-7; from H-4 to C-3; from H-6 to C-1, C-2, and C-5; from CH3-9 to C-5, and CH3-10; from CH3-10 to C-8, respectively, constructed the structure of 4 as 5-isopropyl-3-oxocyclohex-1-ene-1-carboxylic acid (Figure 1). Other 2D spectra (Figure S26–S28) furnished the full assignment of proton and carbon signals. However, the stereochemistry at C-5 remained undetermined. 

Compound 5 possessed the molecular formula C11H14O5 determined from a deprotonated molecular ion peak in the negative mode HR-ESI-MS analysis (m/z 225.0767 [M − H]−, Figure S29). In its IR spectrum, hydroxyl (3421 cm−1 ) and carboxyl (1572 cm−1 ) functionalities could be detected. The 1H-NMR spectrum (Figure S30) revealed two long-range coupling aromatic protons at δH 6.86 (1H, d, J = 2.4 Hz, H-4) and 7.31 (1H, d, J = 2.4 Hz, H-6), one methoxy group at δH 3.56 (2H, t, J = 6.8 Hz, H-9), and one set of propanol protons at δH 1.82 (2H, tt, J = 8.0, 6.8 Hz, H-8), 2.61 (2H, t, J = 8.0 Hz, H-7) and 3.56 (2H, t, J = 6.8 Hz, H-9). Moreover, one carboxylic group was located at δC 176.2 (C-10) in its 13C-NMR spectrum (Figure S31). The planar structure of 5 was established by the significant HMBC correlations (Figure S32) of OCH3-3 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 fifirst report of 5 from natural sources. 

3.2. Anti-Inflflammatory Activity 

Inflflammation is one of the major self-defense mechanisms stimulated by bacteria, virus, wounds, 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 inflflammation, and are usually the fifirst 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 [51–55]. The relationship between inflflammation and cancer has been established, and the authors pointed out that the formation of cancer cells was directly related to inflflammation [49]. Therefore, new anti-inflflammatory 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 signifificant inhibitory results (Table 2) demonstrated that only 45, 47, 48, 49, and 50 (Figure 2) displayed a signifificant inhibition of superoxide anion generation, with IC50 values ranging from 3.3 ± 0.9 to 7.7 ± 0.9 µM compared with the positive control LY294002 (IC50 1.1 ± 0.3 µM). Moreover, 48, 50, and 51 (Figure 2) revealed the signifificant inhibition of elastase release with IC50 values ranging from 5.3 ± 0.2 to 8.3 ± 0.8 µM compared with the positive control LY294002 (IC50 3.2 ± 1.0 µM) (Table 2). Compounds 48 and 50 displayed both inhibitions of superoxide anion generation and elastase release, indicating their multiple anti-inflflammatory bioactivities. The needles of P. morrisonicola have been reported to have an anti-inflflammatory 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 flflavonoid and p-coumaroyl functional groups may contribute the anti-inflflammatory 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 purifified compounds on superoxide anion generation and elastase release by human neutrophils in response to fMLF/CB.

Figure 2. Structures of anti-inflammatory principles 45, 47, 48, 49, 50, and 51. 

3.3. 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 begin 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 the anti-inflflammatory 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 inflflammatory 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 inflflammation as an anti-aging intervention [58]. Thus, according to the anti-inflflammatory bioassay experimental data, 45, 47, 48, 49, 50, and 51 (Figure 2) showed signifificant 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 fifile 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 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 [59–61]. Therefore, in this study, GHRP-6 was fifirst 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-40 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 to compounds 45, 47, and 48 with 49, 50, and 51, the former group possessed a flflavonoid 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 a better binding capability. The major structural characteristics were the coumaroyl groups rather than the sugar moieties and this could be further evidenced by the examination of more compounds possessing coumaroyl functionalities. In this study, the active ingredients 49, 50, and 51 possessed not only anti-inflflammatory 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 tea ghrelin-like compounds.

4. Conclusions

A total of seventy-seven isolates comprising fifteen undescribed compounds were puri- verified 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-inflflammatory activity by the inhibition of superoxide anion generation and elastase release on the neutrophil model. The results suggest that 45, 47, 48, 49, 50, and 51 possess signifificant anti-inflflammatory 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-inflflammatory lead drugs or food ingredients.

Supplementary Materials: The following are available online at, 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 S1–S35: HRMS and NMR spectra of new compounds

1–5. Author Contributions: Conceptualization, P.-C.K. and J.T.C.T.; Methodology, P.-C.K. and T.-L.H.; Investigation, Y.-C.L., A.M.K. and M.-L.Y.; Resources, S.-Y.W.; Data curation, Y.-C.L. and G.-H.Y.; Writing—original draft preparation, P.-C.K. and Y.-C.L.; Writing—review and editing, P.-C.K. All authors have read and agreed to the published version of the manuscript. 

Funding: This study was sponsored by the Ministry of Science and Technology, Taiwan (MOST). The research was supported in part by the Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University (NCKU).

Institutional Review Board Statement: The study was conducted with the approval of the Institutional Review Board of Chang Gung Memorial Hospital (IRB No. 201800369A3). Informed Consent Statement: The statement can be provided upon request. 

Data Availability Statement: Original data can be obtained from the corresponding author upon request. 

Acknowledgments: The authors are also thankful to Chang Gung Memorial Hospital (CMRPD1B0281~3, CMRPF1D0442~3, CMRPF 1F0011~3, CMRPF1F0061~3 and BMRP450 awarded to T.-L.H.) for the partial financial support of the present research. The authors gratefully acknowledge the use of NMR equipment belonging to the Instrument Center of National Cheng Kung University. 

Conflicts of Interest: The authors declare no conflict of interest. 

References 

1. Xie, Q.; Liu, Z.; Li, Z. Chemical composition and antioxidant activity of essential oil of six Pinus taxa native to China. Molecules 2015, 20, 9380–9392. [CrossRef] 

2. Kim, K.Y.; Chung, H.J. Flavor compounds of pine sprout tea and pine needle tea. J. Agric. Food Chem. 2000, 48, 1269–1272. [CrossRef] 

3. Park, G.; Paudyal, D.P.; Hwang, I.; Tripathi, G.R.; Yang, Y.; Cheong, H. Production of fermented needle extracts from red pine and their functional characterization. Biotechnol. Bioprocess Eng. 2008, 13, 256. [CrossRef]

4. Idžojti´c, M.; Kajba, D.; Franji´c, J. Differentiation of F1 hybrids P. nigra J. F. Arnold × P. sylvestris L., P. nigra J. F. Arnold × P. densiflora Siebold et Zucc., P. nigra J. F. Arnold × P. thunbergiana Franco and their parental species by needle volatile composition. Biochem. Syst. Ecol. 2005, 33, 427–439. [CrossRef] 

5. Kim, Y.S.; Shin, D.H. Volatile components and antibacterial effects of pine needle (Pinus densiflflora S. and Z.) extracts. Food Microbiol. 2005, 22, 37–45. [CrossRef] 

6. Dob, T.; Berramdane, T. Essential oil composition of Pinus halepensis Mill. from three different regions of Algeria. J. Essent. Oil Res. 2007, 19, 40–43. [CrossRef] 

7. Nam, A.M.; Tomi, F.; Gibernau, M.; Casanova, J.; Bighelli, A. Composition and chemical variability of the needle oil from Pinus halepensis growing in Corsica. Chem. Biodivers. 2016, 13, 380–386. [CrossRef] 

8. Sezik, E.; Üstün, O.; Demirci, B.; Ba¸ser, K.H.C. Composition of the essential oils of Pinus nigra Arnold from Turkey. Turk. J. Chem. 2010, 34, 313–325.