Cytotoxicity Effect of Constituents of Pinus taiwanensis Hayata Twigs on B16-F10 Melanoma Cells

Pinus taiwanensis Hayata (Pinaceae) is an endemic plant in Taiwan. According to the Chinese Materia Medica Grand Dictionary, the Pinus species is mainly used to relieve pain, and eliminate pus and toxicity. In this study, nineteen compounds were isolated from the ethyl acetate layer of the ethanolic extract of P. taiwanensis Hayata twigs using bioassay-guided fractionation, and their anti-melanoma effects were investigated through a B16-F10 mouse melanoma cell model. The structures of the purified compounds were identified by 2D-NMR, MS, and IR, including 1 triterpenoid, 9 diterpenoids, 2 lignans, 4 phenolics, 1 phenylpropanoid, 1 flavonoid, and 1 steroid. Among them, compound 3 was found to be a new diterpene. Some of the compounds (2, 5, 6, 17, 18) showed moderate cytotoxicity effects. On the other hand, the anti-melanoma effect was no better than that from the original ethyl acetate layer. We presumed it resulted from the synergistic effect, although further experimentation needs to be performed.


Introduction
Melanoma is initially formed from a malignant transformation of melanocytes in the basal layer of epidermal cells. When the skin is exposed to the sun for a long time, the melanocytes continue to proliferate and accumulate, forming the common nevus on the skin. It is usually a benign nevus at first. However, a nevus that abnormally darkens in color or changes in shape and size is the precursor of melanoma. Current known risk factors for melanoma include long-term exposure to ultraviolet radiation, structural and quantitative moles, genetic mutations, familial inheritance, past skin cancer, radiation therapy, chemical carcinogens, and immunodeficiency [1]. UV radiation is an established risk factor for melanoma. As people become more frequently exposed to natural and/or artificial UV radiation, public concerns of melanoma rise [2]. The median age of people diagnosed with melanoma is 52 years old, which is younger than most solid tumors [3]. There are 22.8 melanomas per 100,000 people per year. In 2021, there was an estimate of 106,110 diagnoses, and 7180 deaths from melanoma [4]. Characteristics of metastasis melanoma include asymmetry, a blue-white veil, structureless areas, and atypical vascular structures [5]. Compared to other cancers, melanoma is fairly common [4].
Pinus taiwanensis Hayata belongs to Pinaceae. It is endemic to Taiwan, and is widely distributed in the mountainous area at an altitude of 700-3500 m. It is a large evergreen tree with a straight trunk of length up to 35 m and a diameter of 80 cm. The bark is gray-brown, longitudinally grooved, and irregularly flaky. The twigs grow horizontally. The dark green leaves are rigid in quality with two needles in a bunch, each 8 to 11 cm long and often possessing 4 to 7 fat grooves [6]. Chinese ancient writing says that Pinus was the top-grade drug in Shen Nong Ben Cao Jing. However, as far as we know, the research publications for P. taiwanensis Hayata are still limited. The biological activities of P. taiwanensis Hayata include anti-inflammatory [7], antioxidant [8], anti-lipemic [9], anti-glycemic [9], and antitumor effects [10,11]. The major components found are terpenoids [12], steroids [13], flavonoids [7], and alkanes [13]. In the preliminary stage of this study, we used ultra-highperformance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-MS/MS) to analyze the signals of the twig extracts of P. taiwanensis Hayata, and found thousands of mass-to-charge ratios (m/z) that had never been reported previously [7,13]. These observations indicate that there might be lots of unknown components worthy of exploration. Moreover, the crude extract of P. taiwanensis Hayata showed obvious cytotoxic effects on B16-F10 melanoma cells. Therefore, the aim of this study is to isolate the potential novel components from the twigs of P. taiwanensis Hayata, and to evaluate their cytotoxicity against a melanoma B16-F10 cell model.

Bioassay-Guided Compound Isolation from Pinus taiwanensis Hayata Twigs
The twigs of P. taiwanensis Hayata were soaked in ethanol at a ten times volume-toweight ratio, extracted three times, and concentrated to obtain a crude extract. The crude extract of twigs of P. taiwanensis Hayata (CE) was analyzed through UHPLC-MS/MS. As shown in the chromatogram (Figure 1), thousands of m/z were never reported in the studies of P. taiwanensis Hayata. The crude extract was subsequently partitioned to obtain the ethyl acetate (EA) layer, n-butanol (BU) layer, and water (H) layer of the extract.
We performed the analysis on the B16-F10 cells and the results showed that the EA layer was the most effective layer (data no shown). Then, we continued with the MTT assay. The results of the cytotoxicity test of crude extracts on B16-F10 cells are shown in Figure 2. The CE of P. taiwanensis Hayata at 50 µg/mL significantly reduced the cell survival rate to 23.18% ± 10.85% (mean ± standard deviation). Moreover, the extract of the EA layer was the most cytotoxic among three partitions, with a cell survival rate of 14.10% ± 1.95% at the concentration of 50 µg/mL. Based on the obvious cytotoxic effect shown in the EA layer, it was selected for further analysis.
The 13 C-NMR spectrum showed two quaternary carbons of δ C 79.1 and 80.7, which may be affected by the hydroxy group and appeared in the lower field (downfield) position. The HMBC experiment ( Figure 5, Supplementary Figure S4) showed that δ H 0.95 was related to δ C 79.1 (C-13), δ C 32.1 (C-15), and δ C 17.1 (C-17), δ H 0.94 was related to δ C 79.1 (C-13), and δ H 1.88 and 6.07 were related to δ C 79.1 (C-13). In conclusion, isopropyl was located on C-13 and δ C 79.1 was in the C-13 position. Furthermore, the relationship between δ H 2.41, 2.50, 2.05, 6.07, 1.06, and δ C 80.7 (C-9) suggests that the position of δ C 80.7 is C-9. According to the results of HMBC experiments, δ H 6.07 was related to δ C 79.1 (C-13), 80.7 (C-9), 32.1 (C-15), and 24.2 (C-7), which confirmed that the double bond of δ C 144.1 and 127.0 is positioned at C-8 and C-14.    46.3 ------C-4, C-9, C-10, C-18, C-   As for the stereochemistry, it could be explained by the NOESY spectrum ( Figure 5, Supplementary Figure S5). There was NOESY association between H-20 and H-19, which showed that -COOH was located in the equatorial direction. H-20 was related to H-11, which meant that 9-OH was a pseudo-axial. H-12 was related to H-17, which indicated that 13-OH was located on the axial. Based on the above information, compound 3 was identified as a new compound named 9α-13β-Dihydroxyabiet-8(14)-enoic acid, that had not been documented in the literature. The structure is shown in Figure 4.

Evaluation of Cytotoxicity
The toxicity effect of pure compounds 1-19 (except 3) isolated from P. taiwanensis Hayata twigs on B16-F10 cells is shown in Figure 6. At a concentration of 50 µM, compounds 2, 5, 6, 17, and 18 showed a statistic cytotoxic effect. In order to explore its possible causes, the compounds were once again mixed in the ratio of weights of the purified compounds. Compounds 6, 7, 8, 9, and 11 were mixed back as fraction 4 , while compounds 10, 12, 15, 16, 17, and 18 were mixed back as fraction 5 . The cell survival rates at different concentrations of fraction 4 were 24.77% at 100 µg/mL, 42.40% at 50 µg/mL, and 82.57% at 25 µg/mL. The cell survival rates of fraction 5 were 12.38% at 100 µg/mL, 21.20% at 50 µg/mL, and 46.64% at 25 µg/mL. The results showed that fraction 4 and fraction 5 had more cytotoxic effects than pure compounds. The cytotoxic effects on B16-F10 melanoma cells were increase when remixed, which could be attributed to the synergistic effect. 19.1(q) 1.06 (s) C-1, C-5, C-9, C-10

Evaluation of Cytotoxicity
The toxicity effect of pure compounds 1-19 (except 3) isolated from P. taiwanensis Hayata twigs on B16-F10 cells is shown in Figure 6. At a concentration of 50 μM, compounds 2, 5, 6, 17, and 18 showed a statistic cytotoxic effect. In order to explore its possible causes, the compounds were once again mixed in the ratio of weights of the purified compounds. Compounds 6, 7, 8, 9, and 11 were mixed back as fraction 4′, while compounds 10, 12, 15, 16, 17, and 18 were mixed back as fraction 5′. The cell survival rates at different concentrations of fraction 4′ were 24.77% at 100 μg/mL, 42.40% at 50 μg/mL, and 82.57% at 25 μg/mL. The cell survival rates of fraction 5′ were 12.38% at 100 μg/mL, 21.20% at 50 μg/mL, and 46.64% at 25 μg/mL. The results showed that fraction 4′ and fraction 5′ had more cytotoxic effects than pure compounds. The cytotoxic effects on B16-F10 melanoma cells were increase when remixed, which could be attributed to the synergistic effect.

Discussion
The study of secondary metabolism on P. taiwanensis Hayata is rare, and therefore it is necessary to distinguish this plant from the site of phytochemistry. Moreover, the great cytotoxicity on B16-F10 cells made P. taiwanensis Hayata more valuable to discuss. This was the first metabolites research focusing on the twigs of the P. taiwanensis Hayata. As a result, 19 compounds were isolated, including a new diterpenoid. Moreover, 14 of these 19 compounds had not been found in other parts of P. taiwanensis Hayata. The extracts of twigs of P. taiwanensis Hayata were separated and purified before 19 compounds were obtained in the EA layer. Structures of these compounds included 1 triterpenoid, 9 diterpenoids, 2 lignans, 4 phenolics, 1 phenylpropanoid, 1 flavonoid, and 1 steroid [34]. Diterpene was the main component, and it showed the same tendency as other Pinus plants. The compounds L-α-pinene, DL-limonene, bornyl acetate, and D-longifolene had previously been found in P. taiwanensis Hayata [12]. Among them, 9α,13β-dihydroxyabiet-8(14)-enoic acid (3) was found as a new compound. The above results showed that the low-polarity part of the composition of P. taiwanensis Hayata twigs was dominated by diterpenoids. This result provided an understanding of the composition of secondary metabolites of P. taiwanensis Hayata twigs.
According to the previous studies, less than 100 compounds were identified in all parts of P. taiwanensis Hayata, and 70 compounds matched the m/z signals detected in UHPLC-MS/MS analyses. On the other hand, the ion signal of a few numbers of substances had not been found in the UHPLC-MS/MS chromatogram, which may be absent or minor in the P. taiwanensis Hayata twigs. This was confirmed with the 19 main compounds isolated from the twigs of P. taiwanensis Hayata, and only 5 components were repeated from the previous research [7]. The 19 main substances isolated from the P. taiwanensis Hayata twigs were detected by UHPLC-MS/MS as well, but the rest of the thousands of m/z signals represented that most of the compounds were not explored yet, which are worthy of future study. UHPLC-MS/MS is suitable for the exploration of plant metabolites, and could be an important tool to investigate the composition of natural products.
In the diterpenoids 2 to 10 from P. taiwanensis Hayata, the difference in isopropyl chemical shifts on C-13 can be found, and they were grouped into three types. Abietane type (compounds 2-9) could determine the attached functional group by the H-15 isopropyl group. If the isopropyl group was connected to the benzene ring next to it, the chemical shift of 15-H of the isopropyl group was about 2.8 ppm (e.g., compound 2, dehydroabietic acid). If the isopropyl group was attached with a double bond, the 15-H hydrogen spectrum shift of the isopropyl group was 2.4 ppm (e.g., compound 6, 12α-hydroxyabietic acid), and if the isopropyl group was attached with -OH, its chemical shift was 1.8 ppm (e.g., compound 3, 9α-13β-dihydroxyabiet-8(14)-enoic acid). The literature corroborated this phenomenon [35].
Due to the richness and diversity of natural plant compounds, natural products and their derivatives account for more than half of the development of anti-cancer drugs. In this experiment, P. taiwanensis Hayata, which is unique to Taiwan, was used as the research subject. Despite the results showing that the pure isolated compounds were not as effective as pre-existing drugs, the toxic effects on B16-F10 melanoma cells were retained when remixed during a cytotoxicity test, which could be attributed to the synergistic effect. The toxicity of the crude extract on B16-F10 melanoma cells is worthy of further investigation.
Regarding the uniqueness of this Taiwan endemic species, more activity screening tests can be performed on the pure compounds isolated from this plant in hopes that more special structures and mechanisms of action can be purified and isolated in the future.  230.02 g of crude extract. The crude extract was then suspended in water and partitioned with ethyl acetate and n-butanol. Thereafter, the ethyl acetate layer (170.19 g), the n-butanol layer (31.80 g), and the water layer (25.02 g) were obtained.

Melanin Content Assay
The B16-F10 melanoma cells were seeded with 1 × 104 cells/well in 3 mL of medium in 6-well culture plates and incubated overnight. The cells were treated with different concentrations of the substance, compound, or fluorouracil for 72 h in the presence of 100 nM of α-MSH. At the end of the treatment, the cells were washed with PBS and lysed with 150 µL of 1 N NaOH (Merck, Darmstadt, Germany) containing 10% DMSO for 1 h at 80 • C. The absorbance was measured by a microplate reader at 405 nm. The melanin content of control cells was assigned as 100%, and the melanin content of compound-treated cells was calculated relative to the control group.

MTT Cell Viability Assay
The MTT assay was used to determine the cytotoxicity effects of the crude extract and compounds on B16-F10 cells. B16-F10 cells were treated with crude extracts or compounds for 48 h at 37 • C, then stained with MTT solution and further incubated for 2 h. Afterwards, supernatants were removed and 400 µL of DMSO was added to each well of the plate. The positive control was fluorouracil [36]. The absorbance of the solution was analyzed using a microplate reader (Molecular Devices, San Jose, CA, USA) under a wavelength of 570 nm. All experiments were carried out three times and the formula used to calculate the survival rate was as follows: (Absorbance value in the dosing place/Resting absorbance value) × 100%

Statistical Analyses
Statistical analysis of the data was performed with one-way analysis of variance (ANOVA), and Dunnett's post-test was employed for multi-groups comparisons using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). The results were expressed as mean ± SD of triplicates and differences were considered statistically significant when p < 0.05.

Conclusions
The constituents of Pinus taiwanensis Hayata twigs were diterpenoids, including a new compound, 9α,13β-dihydroxyabiet-8(14)-enoic acid (3). The cytotoxicity test of B16-F10 melanoma cells was performed on the 19 purified compounds. However, the isolated pure compounds showed limited cytotoxic effects, even lower than that of the crude extract at a concentration of 50 µM. Interestingly, the cytotoxicity was restored after we remixed the compounds, which may be due to potential synergistic effects. This will be explored in more detail in the future.