Anti-Aging Constituents from Pinus morrisonicola Leaves

Pinus morrisonicola Hayata is a unique plant species found in Taiwan. Previous studies have identified its anti-hypertensive, anti-oxidative, and anti-inflammatory effects. In this study, a bioactivity-guided approach was employed to extract 20 compounds from the ethyl acetate fraction of the ethanol extract of Pinus morrisonicola Hayata’s pine needles. The anti-aging effects of these compounds were investigated using HT-1080 cells. The structures of the purified compounds were confirmed through NMR and LC-MS analysis, revealing the presence of nine flavonoids, two lignans, one coumarin, one benzofuran, one phenylic acid, and six diterpenoids. Among them, PML18, PML19, and PML20 were identified as novel diterpene. Compounds 3, 4, and 5 exhibited remarkable inhibitory effects against MMP-2 and showed no significant cell toxicity at 25 μM. Although the purified compounds showed lower activity against Pro MMP-2 and Pro MMP-9 compared to the ethyl acetate fraction, we speculate that this is the result of synergistic effects.


Introduction
The skin is considered the largest organ in the human body. Skin aging is a complex process that can be caused by either intrinsic natural aging or external factors [1]. Intrinsic aging is associated with decreased mitotic activity, increased duration of the cell cycle and migration time, which can result in poor wound healing and metabolism. These effects can result in a reduction in dermis thickness, a decrease in the number of fibroblasts, and impaired functionality of sebaceous and sweat glands. Additionally, they lead to a reduction in the microvasculature in the skin, which lowers skin vascular reactivity, temperature regulation, and nutrient supply, resulting in pale or sallow skin [2]. The most important environmental factors contributing to skin aging are UV radiation and smoking, which increase the expression of matrix metalloproteinases (MMPs) in the skin [3,4]. MMPs, are responsible for degrading extracellular matrix proteins, such as collagen, fibronectin, elastin, and proteoglycans [5]. UV radiation-induced MMP expression plays a crucial role in the mechanism of photoaging through a series of signaling pathways [6]. MMPs are regulated by the transcription factor AP-1, which is significantly increased after UV exposure, resulting in increased mRNA and protein expression of MMPs. Increased level of MMPs leads to excessive degradation of the extracellular matrix, causing structural breakdown of the skin, wrinkles, and loss of elasticity, which in turn results in aging [6][7][8].
Pinus morrisonicola Hayata is a plant in the family of Pinaceae and the genus of Pinus. It is an endemic species in Taiwan and is mainly distributed in mountainous areas at altitudes of 300 to 2000 m, and is a tall evergreen tree with a trunk height of up to 30 m and a diameter of 1.2 m. The bark is dark gray and has a scale-like cracked appearance. The leaves are needle-shaped, with five needles in a bundle, and are 4 to 10 cm long [9]. The main components of P. morrisonicola are flavonoids (chrysin, apigenin) [10], stilbenes (pinosylvin and its derivatives) [11], terpenoids (pinene, terpinene) [12], and steroids (βsitosterol) [13]. A previous study has demonstrated that P. morrisonicola extract exhibits good cell toxicity against GBM8901 glioblastoma cells [14], and the potential for antihypertensive, anti-oxidative, and anti-inflammatory effects [15][16][17]. Based on its extracts' significant antioxidant and anti-inflammatory properties and the fact that flavonoids, the main component of P. morrisonicola, have shown anti-aging effects [18], it is expected to find potential anti-aging compounds from P. morrisonicola. Therefore, the purpose of this study is to isolate potential anti-aging compounds from P. morrisonicola.

Bioasasay-Guided Compound Isolated from Pinus morrisonicola Hayata Leaves
The leaves of Pinus morrisonicola Hayata were extracted with ethanol in a 10-times volume to obtain the crude extract (PML), which was then liquid-liquid partitioned to obtain the ethyl acetate layer (PMLEF), n-butanol layer (PMLBF), and water layer (PMLWF). The zymography method was then employed to assess the inhibitory activities of crude extract and three layers against Pro MMP-9, Pro MMP-2, and MMP-2. At 100 g/mL, PML showed significant inhibitory effects only on MMP-2 (0.43 ± 0.10), while PMLEF exhibited the best inhibitory activity against Pro MMP9 (0.47 ± 0.16), Pro MMP2 (0.54 ± 0.17), and MMP2 (0.16 ± 0.01) ( Figure 1). Based on the significant cell activity of PMLEF, further isolation of its active components will be conducted.
Pinus morrisonicola Hayata is a plant in the family of Pinaceae and the genus o It is an endemic species in Taiwan and is mainly distributed in mountainous altitudes of 300 to 2000 m, and is a tall evergreen tree with a trunk height of up and a diameter of 1.2 m. The bark is dark gray and has a scale-like cracked appe The leaves are needle-shaped, with five needles in a bundle, and are 4 to 10 cm l The main components of P. morrisonicola are flavonoids (chrysin, apigenin) [10], s (pinosylvin and its derivatives) [11], terpenoids (pinene, terpinene) [12], and ster sitosterol) [13]. A previous study has demonstrated that P. morrisonicola extract good cell toxicity against GBM8901 glioblastoma cells [14], and the potential f hypertensive, anti-oxidative, and anti-inflammatory effects [15][16][17]. Based on its e significant antioxidant and anti-inflammatory properties and the fact that flavono main component of P. morrisonicola, have shown anti-aging effects [18], it is exp find potential anti-aging compounds from P. morrisonicola. Therefore, the purpos study is to isolate potential anti-aging compounds from P. morrisonicola.

Bioasasay-Guided Compound Isolated from Pinus morrisonicola Hayata Leaves
The leaves of Pinus morrisonicola Hayata were extracted with ethanol in a 1 volume to obtain the crude extract (PML), which was then liquid-liquid partiti obtain the ethyl acetate layer (PMLEF), n-butanol layer (PMLBF), and wate (PMLWF). The zymography method was then employed to assess the inhibitory a of crude extract and three layers against Pro MMP-9, Pro MMP-2, and MMP-2. g/mL, PML showed significant inhibitory effects only on MMP-2 (0.43 ± 0.10 PMLEF exhibited the best inhibitory activity against Pro MMP9 (0.47 ± 0.16), Pro (0.54 ± 0.17), and MMP2 (0.16 ± 0.01) ( Figure 1). Based on the significant cell act PMLEF, further isolation of its active components will be conducted.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 13 (C) (D)  Figure S18). NMR spectrum showed one carboxylic acid at δC 178.9 (C-18), one ester group at δC 175.7 (C-15), and one double bond signal at δH 5.68, suggesting the presence of four rings (Figures S10 and S11, Table 1). The 1 H-NMR, 13 C-NMR and DEPT spectra indicated that compound 20 has two methyls, eight methylene, four methine, and six quaternary carbons. 1  In terms of stereochemistry, the proton at δH 0.75 (C-20) was relatively upfield, suggesting its axial orientation was influenced by the carboxylic acid at C-18, as it did not show any correlation with δH 1.20, but with δH 1.45, 1.75 in the NOESY spectrum. ( Figure  5D, S10 and S13) Therefore, it was confirmed that H-20 and -COOH were in the axial direction (β-orientation), while H-9 was located in the equatorial direction. Furthermore, δH 4.18 was also observed to be in the β-orientation, as it showed correlation with δH 1.45 and δH 1.87 in the NOESY spectrum ( Figures 5D and S13). Thus, the structure of compound 20 was determined to be Morrisonicolene.   (Figures 4A and S6). According to the above NMR analysis, the planar structure of compound 18 is shown in Figure 4. The methyl group (H-24) on C10 of compound 18 is affected by the isotropic acid on C4 at high magnetic fields (δ H 0.75) ( Figure S1). The δ H 0.57 determined from NOESY spectra correlates with δ H 1.83 at C-11, indicating that -COOH and H-9 are in the β-orientation ( Figure S4). The proton δ H 3.91 of H-14 is correlated with δ H 1.44, 1.75 of H-12 and δ H 4.96 of H-15, indicating that the hydroxyl group of C-13, C-14 and the ethoxy group of C-15 are all in the β-direction ( Figure S4). Therefore, the three-dimensional structure of compound 18 is shown in Figure 4.  Figures 5B, S1, S4 and S7). Therefore, it can be determined that the ethoxy group at C-16 in PML18 is a β-form, while ethoxy group at C-16 in PML19 (δ H 4.83) is an α-form. Therefore, the structures of compounds 18 and 19 were determined as 15β,16β-diethoxy,13,14dihydroxy-labd-8(21)-en-22-oic acid (18) and 15β,16α-diethoxy,13,14-dihydroxy-labd-8(21)en-22-oic acid (19).   Table 1). The 1 H-NMR, 13 C-NMR and DEPT spectra indicated that compound 20 has two methyls, eight methylene, four methine, and six quaternary carbons. 1 (Figures 5C and S12). HMBC experiment showed that δ H 4.18 was correlated to ester group δ C 175.7 (C-15) and quaternary carbon δ C 79.2 (C-13), and C-13 was correlated to δ H 4.18 (H-16) Figure S15).

Structure Analysis of PML20
In terms of stereochemistry, the proton at δ H 0.75 (C-20) was relatively upfield, suggesting its axial orientation was influenced by the carboxylic acid at C-18, as it did not show any correlation with δ H 1.20, but with δ H 1.45, 1.75 in the NOESY spectrum. (Figures 5D, S10 and S13) Therefore, it was confirmed that H-20 and -COOH were in the axial direction (β-orientation), while H-9 was located in the equatorial direction. Furthermore, δ H 4.18 was also observed to be in the β-orientation, as it showed correlation with δ H 1.45 and δ H 1.87 in the NOESY spectrum ( Figures 5D and S13). Thus, the structure of compound 20 was determined to be Morrisonicolene.

Anti-Aging Activity Test of Flavonoid Compounds
Due to the potential anti-aging effects of flavonoids, our focus was on screening the flavonoid compounds in the active fraction for their anti-aging activity [18]. The cell viability of compounds 3, 4, 5, 7, and 8 was evaluated through MTT assay at a concentration of 25 µM for 24 h, showing no cytotoxicity. Subsequently, the effects of these flavonoid compounds 3, 4, 5, 7 and 8 at a concentration of 25 µM on MMP-2, Pro MMP-2, and Pro MMP-9 were evaluated using zymography. As per the results, as shown in Figure 6, compounds 3, 4, and 5 exhibited significant inhibitory effects on MMP-2, with inhibition rates of 0.46 ± 0.05, 0.63 ± 0.08, and 0.60 ± 0.07, respectively. Among them, compound 3 demonstrated a particularly remarkable inhibitory effect. None of the five compounds exhibited significant inhibitory effects on Pro MMP-2 and Pro MMP-9. (Figure 6) The inhibitory activity on MMPs indicated the potential anti-aging effect of these compounds.

Discussion
In this study, a total of 20 compounds were isolated and purified from PMLEF by active fractionation method. Apart from compounds 1 and 2, the remaining constituents

Discussion
In this study, a total of 20 compounds were isolated and purified from PMLEF by active fractionation method. Apart from compounds 1 and 2, the remaining constituents were discovered for the first time in Pinus morrisonicola Hayata [10]. Compounds 8 and 9 have been identified in other Pinus species [23]. In our study, we revealed flavonoids with sugar moieties and coumaroyl group or feruloyl group, besides the commonly reported hydroxy group-containing flavonoids in P. morrisonicola. Additionally, our findings demonstrated the presence of diterpenes, which is consistent with our previous finding on Pinus taiwanensis Hayata [31], while previous studies predominantly reported monoterpenes in Pinus species.
Both PML and PMLEF exhibited significant inhibitory effects on MMP-2, Pro MMP-2, and Pro MMP-9 in the Zymographic assay. However, no compound displayed significant inhibition of Pro MMP-2 and Pro MMP-9, which contradicted the results obtained from fractions 3-7 of PMLEF. The purified compounds 3, 4, 5, 7, and 8 obtained from the activity-guided fractionation did not show comparable effects to PML or PMLEF. Hence, we speculate that PML possesses multiple compounds working synergistically to inhibit Pro MMP-2 and Pro MMP-9. In the case of MMP-2, compounds 3, 4, and 5 demonstrated significant effects ( Figure 6); this suggests that glucopyranoside (compounds 3, 4, 5) exhibits better activity than arabinoside (compounds 7, 8). The result in Figure 6 indicated that both compounds 7 and 8 mildly inhibited MMP-2 activity, without obvious differences between them. Considering the structural difference between compounds 7 and 8, which lies in the cis or trans configuration of the double bond, it can be concluded that the orientation of the double bond does not affect their inhibitory activity against MMP-2. Furthermore, compound 3 showed stronger inhibition of MMP-2 compared to compound 4, while the structural dissimilarity between compounds 3 and 4 lies only in the substitution of the coumaroyl group with the feruloyl group at the 3 position. Thus, the presence of a methoxy group at the 3 position is likely to decrease the inhibitory effect on MMP-2 (Figures 4 and 6).
According to the MMPs assay, the EA layer of PML and its fractions showed remarkable activity on Pro MMP-9, Pro MMP-2 and MMP-2. However, compounds 3, 4 and 5 only revealed an inhibitory effect on MMP-2. Therefore, we considered the EA layer of PML or its fraction to have more potential to serve as anti-aging cosmetics owing to the multicomponent effect. In the previous study, chrysin could increase collagen I secretion and decrease the degradation of collagen I to repair oxidation damage. In addition, chrysin has been presented to inhibit melanin synthesis by reducing tyrosinase activity and suppressing the expression of melanogenic proteins [32] (Table 2). Choi et al. indicated that apigenin reduced the expression of collagenase [33], and Park et al. demonstrated that apigenin exhibited anti-aging and anti-inflammatory effects through the inhibition of nitric oxide (NO) production and cytokine expression in RAW264.7 cells and inhibited the expression of high-affinity IgE receptor and cytokines in RBL-2H3 cells [34] (Table 2). Loliolide reduces the activity of senescence-associated β-galactosidase (SA-β-gal) and decreases the levels of p21 protein, exerting an inhibitory effect in human dermal fibroblasts [35] (Table 2). Moreover, loliolide exhibits significant antioxidant and anti-inflammatory activities, as well as photoprotective effects, by improving collagen synthesis, reducing intracellular reactive oxygen species (ROS) levels, and inhibiting apoptosis in UVB-irradiated human keratinocytes and the expression of matrix metalloproteinases. It also reduces ROS, NO, lipid peroxidation, and cell death in UVB-irradiated zebrafish [36] (Table 2). Pinoresinol showed the antioxidant and anti-UV radiation through SPF value, UV absorption capacity, and the DPPH assay [37] (Table 2). At last, PML4, PML5 and PML11 all demonstrated antioxidants in DPPH radical-scavenging activity [38][39][40] (Table 2). Based on our research and previous study, we believe that PML and PMLEF might have the potential to be developed as versatile cosmetic ingredients. Table 2. Main effects of isolation compound from Pinus morrisonicola on cosmeceutical activity.

Number
Compound Name Cosmeceutical Activity Reference

Extraction and Partition
A total of 9.8 kg of dried Pinus morrisonicola Hayata leaves were soaked in 10 times volume of ethanol and extracted three times. The resulting extract was concentrated under evaporator to obtain a crude extract (692 g). The crude extract was then partitioned with water, ethyl acetate, and n-butanol, and obtained an ethyl acetate layer (293 g), a n-butanol layer (297 g), and a water layer (102 g).

MTT Cell Viability Assay
HT-1080 cells were seeded by 5 × 10 5 cells/mL per well in 24-well plates for 24 h, and treated with crude extract or pure compound for another 22 h. Then, cells were cultured with MTT solution for further 2 h. After that, supernatants were removed and 400 µL DMSO was added to the plate. Mixtures were transferred to 96-well plate and detected by ELISA reader (MRX microplate reader, Vodickova, Czech Republic) under the wavelength of 550 nm. Cell viability was calculated as follows: (Treating absorbance value)/(Resting absorbance value) × 100% (1)

Zymography
HT-1080 cells were placed at a density of 5 × 10 5 cells/mL in 24-well plate and incubated at 37 • C for 24 h to allow attachment. After treatment with samples, the cells were incubated for an additional 24 h at 37 • C. The reactions were then terminated and cell supernatant was mixed with sample loading dye in a 1:1 volume ratio and thoroughly mixed. The mixture was then subjected to electrophoresis on a 10% polyacrylamide gel (containing 1% gelatin) in running buffer at 130 V and 90 mA. The gel was then washed two times with 2.5% Triton X-100 at room temperature for 30 min each. The gel was then incubated in reaction buffer at 37 • C for 24 h, and further 30 min to immobilize the proteins on the gel by fixing solution. The gel was then stained uniformly using Brilliant Blue G-Colloidal Concentrate, and destained using destain solution to optimize the results. Finally, the gel was photographed using CCD in an imaging analysis system (Vilber Lourmat, France), and the image was analyzed using imaging analysis software (Bio-1D version 99). The brightness of the vehicle was used as the reference value of 1, and the brightness of the other bands was expressed in relative multiples.

Statistical Analyses
The data results of this experiment are expressed as Mean ± SD. Statistical analysis was performed using One Way ANOVA followed by the Student-Newman-Keuls Test. A p value less than 0.05 indicates a significant difference.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Sample Availability: Samples of the compound 18, 19, and 20 is available from the authors.