Triterpenes and Pheophorbides from Camellia ptilosperma and Their Cytotoxicity, Photocytotoxicity, and Photodynamic Antibacterial Activity

Phytochemical investigation of the leaves of Camellia ptilosperma S. Y. Liang et Q. D. Chen led to the isolation of ten undescribed compounds, including six new triterpenes (1–6) and four new pheophorbide-related compounds (7–10). Meanwhile, the cytotoxic activity of the six triterpenes against six cancer cell lines was evaluated by MTT assay. Compound 2 showed potent cytotoxicity toward HepG2 cells with an IC50 value of 2.57 μM. Compounds 4 and 5 exhibited cytotoxicity against MDA-MB231 cells, with IC50 values of 11.31 and 5.52 μM, respectively. Additionally, the cytotoxicity of four new pheophorbides against these cancer cells was evaluated both in the presence and absence of light treatment. Compound 7 exhibited exceptional photocytotoxicity against Hela, MCF-7, and A549 cells, with IC50 values of 0.43 μM, 0.28 μM, and 0.92 μM, respectively. Compound 10 demonstrated significant photodynamic cytotoxic activity against BEL-7402 and HepG2 cells with IC50 values of 0.77 μM and 0.33 μM, respectively. The photodynamic antibacterial activity of 7–10 was also tested for S. aureus, E. coli, K. pneumoniae, and P. aeruginosa under direct illumination. Compounds 8 and 10 exhibited sensitivity to E. coli and demonstrated a photodynamic antibacterial effect, with a MIC value of 0.625 μM.


Introduction
Yellow camellia, a member of the Theaceae family, is an evergreen shrub or dungarunga that was first discovered in Fangchenggang, Guangxi, China in 1933.To date, more than 43 species, including 5 variants, of yellow camellias have been identified, with their primary distribution spanning southwest China and northern Vietnam [1].Distinguished from common tea plants with their red, pink, and white flowers, yellow camellia has unique golden-yellow flowers due to which it is honored as 'flora panda' and 'camellia queen'.The leaves and flowers of yellow camellias are commonly utilized in the preparation of a popular tea known as Jin-Hua-Cha among local communities.Furthermore, local communities have long employed yellow camellias as traditional remedies for conditions such as hypertension, sore throat, and cancer prevention.In 2010, the Chinese Ministry of Health included yellow camellia in the national list of new food resources, leading to the continued development of functional foods based on yellow camellia, and currently available products include oral solutions [2] and instant teas [3] with yellow camellia.Camellia ptilosperma, a renowned species of yellow camellia, was discovered in 1982 in Chongzuo, Guangxi.Unlike other yellow camellia, which bloom from September to November, C. ptilosperma blooms from July to the following April [4].
The chemical composition of C. ptilosperma is currently unknown.In view of its potential medicinal and economic value, there is a need for phytochemical research on it.According to the literature reports, the leaves of plants in Theaceae contain mainly polyphenols, terpenes, flavonoids, tannins, and chlorophylls [19][20][21][22].Although polyphenols, flavonoids, and tannins have been extensively studied [23][24][25][26][27], there have been no relevant reports on pheophorbide in the last 5 years.Chlorophyll and pheophorbide belong to the porphyrin group and are potential photosensitizers [28][29][30].In the presence of light, they can produce free radicals or reactive oxygen species, which have a powerful killing effect on bacteria, microorganisms, and viruses [31,32].Many triterpenoids have shown anti-tumor activity [33][34][35].Therefore, this study focuses on the triterpenoids and pheophorbides in C. ptilosperma.In the biological activity experiment section, the cytotoxic activity of triterpenoids against cancer cells, photocytotoxicity against cancer cells, and the photodynamic antibacterial activity of pheophorbides were evaluated.
Compound 9 was obtained as a dark green powder with the molecular formula of (C 38 H 42 N 4 O 6 ) deduced by HR-ESI-MS analysis (m/z 651.3179, [M+H] + , calcd.651.3183).Its 1 H and 13 C NMR data (Tables 3 and 4) were similar to those of compound 7, the main distinction being the substituent groups and their configuration at position C-13 2 .The relative up-field shift of H-17 at δ H 4.17, as mentioned in the structural elucidation of 7, indicated the OH-13 2 as β-configuration.Also, an ethyl formate unit with α-orientation was assigned to C-13 2 based on the NMR data analyses.Thus, compound 9 was designated to be 13 3 -ethoxypheophorbide-a ethyl ester.
Compound 10 was obtained as a dark green powder and had a molecular formula of C 37 H 38 N 4 O 8 as determined by its HR-ESI-MS results (m/z 667.2759, [M+H] + , calcd.667.2768).Compounds 10 and 8 showed similar 1 H and 13 C NMR data (Tables 3 and 4) and 2D NMR data.The main differences between 10 and 8 were observed from the chemical shift changes at C-13 (δ C 112.3 for 10 vs. δ C 127.1 for 8) and C-13 1 (δ C 160.5 for 10 vs. δ C 192.0 for 8), indicating that compound 10 should have a six-membered lactone ring in its skeleton structure.Furthermore, the downfield shift of H-17 (δ H 4.67), which arose from the inductive effect of OH-15 1 , suggested that OH-15 1 was β-oriented.Thus, compound 10 was designated as 7-formyl-15 1 -hydroxypurpurin-7-lactone ethyl methyl diester.
The compounds 1-6 isolated from the leaves of C. ptilosperma in the present study were tested in terms of their cytotoxicity against six human cancer lines, namely Hela, MCF-7, BEL-7402, A549, HepG2, and MB-231, by MTT assay (Table 5).Compound 2 showed potent cytotoxicity toward HepG2 cells with an IC 50 value of 2.57 ± 0.29 µM.In particular, the inhibitory effect of compound 2 was comparable to that of the positive control drug.Compounds 4 and 5 exhibited moderate cytotoxicity against MDA-MB-231 cells, with IC 50 values of 11.31 ± 3.05 and 5.52 ± 0.13 µM, respectively.All the compounds were found to exhibit lower or no inhibitory activity against Hela, MCF-7, BEL-7402, and A549 cancer cells.The results indicated that triterpenoids were highly selective in inhibiting tumor cells.Preliminary SAR (structure-activity relationship) analysis indicated that ursane-type triterpenes were more cytotoxic than oleanane-type triterpenes, and the presence of benzoyloxy and angeloyloxy groups reduced cytotoxicity against these cancer cells.Overall, among the isolated ursane-type compounds, the presence or absence of methoxy or hydroxyl groups at the position of C-11 and hydroxyl or carbonyl groups at the position of C-22 has no significant effect on cytotoxicity.Compared to the reported triterpenoids isolated from camellias [39][40][41][42], the triterpenoids isolated from C. ptilosperma in this study showed more potent cytotoxicity against hepatocellular carcinoma and lung cancer cells.
The cytotoxicity for compounds 7-10 in the absence of direct illumination was assayed against six tumor cell lines, Hela, MCF-7, BEL-7402, A549, HepG2, and MB-231, by the MTT method (Table 6).Compounds 8 and 9 exhibited limited or negligible cytotoxic activity against all tested cell lines.In contrast, compound 7 exhibited moderate inhibitory activity against MCF-7 cells, yielding an IC 50 value of 5.26 ± 0.71 µM, while compound 10 demonstrated moderate cytotoxicity against BEL-7402 and HepG2 cells, with IC 50 values of 7.68 ± 1.87 and 3.77 ± 0.49 µM, respectively.The findings for compounds 7 and 10, observed in the absence of direct illumination, indicate that the cytotoxicity of pheophorbide-related compounds may involve mechanisms distinct from the previously reported photodynamic action.Furthermore, the photocytotoxicity of compounds 7-10 was assessed against the above tumor cell lines when exposed to light radiation (Figure 5).Four compounds demonstrated heightened inhibition of proliferation in all tested cell lines when subjected to illumination, and this effect intensified with longer light exposure times.Notably, compound 7 exhibited exceptional photocytotoxicity against Hela, MCF-7, and A549 cells, with IC 50 values of 0.43 ± 0.15 µM, 0.28 ± 0.05 µM, and 0.92 ± 0.21 µM, respectively.Compound 10 demonstrated significant photodynamic cytotoxic activity against BEL-7402 and HepG2 cells with IC 50 values of 0.77 ± 0.34 µM and 0.33 ± 0.04 µM, respectively.Conversely, compounds 8 and 9 exhibited limited photodynamic activity across all tested cell lines, despite a noticeable improvement in inhibition effects under light radiation.These results suggest that certain pheophorbide-related compounds derived from C. ptilosperma have the potential to serve as potent photosensitizers for photodynamic therapy (PDT).Pheophorbide is one of the classes of porphyrins.Porphyrins are a family of heteromeric macrocyclic organic compounds containing four pyrrole rings linked by naturally occurring methine bridges, capable of generating monoclinic oxygen in the presence of light and oxygen, effectively killing tumor cells.[43][44][45].Both natural and synthetic porphyrins exhibit photocytotoxicity.Thomas et al. [46] designed and prepared an N-fused porphyrin (NCP), displaying an IC50 value of 6 µM.Hynek et al. [47] synthesized porphy- Pheophorbide is one of the classes of porphyrins.Porphyrins are a family of heteromeric macrocyclic organic compounds containing four pyrrole rings linked by naturally occurring methine bridges, capable of generating monoclinic oxygen in the presence of light and oxygen, effectively killing tumor cells.[43][44][45].Both natural and synthetic porphyrins exhibit photocytotoxicity.Thomas et al. [46] designed and prepared an N-fused porphyrin (NCP), displaying an IC 50 value of 6 µM.Hynek et al. [47] synthesized porphyrin derivatives containing methyl, isopropyl, and phenyl groups, and strong photocytotoxicity against Hela cells with an IC 50 value of 0.45 µM was shown.However, the porphyrins isolated in this study were less effective in inhibiting tumor cells than other types of photocytotoxic compounds, such as metal complexes [48][49][50].This also suggested that compounds 7-10 can be further structurally modified to increase their photocytotoxicity.Preliminary SAR (structure-activity relationship) analysis suggested that the length of side chain substituents, differences in functional groups, and chiral carbon configurations did not reflect a significant degree of photocytotoxicity.
Two Gram-positive bacteria (S. aureus and E. coli) and two Gram-negative bacteria (K.pneumoniae and P. aeruginosa) were selected to evaluate the antibacterial activity of compounds 7-10 using MIC values.In the absence of light exposure, compounds 7-10 exhibited no activity against the four bacteria at a concentration of 100 µM.However, when the bacteria were exposed to compounds 7-10 with 30 min of photo-irradiation, four of the pheophorbides displayed limited antibacterial activity against S. aureus and E. coli (Table 7).Notably, compounds 8 and 10 exhibited sensitivity to E. coli and demonstrated a photodynamic antibacterial effect, with a MIC value of 0.625 µM.Conversely, none of the tested compounds displayed any activity for the two Gram-negative bacteria, whether subjected to photo-irradiation or not.

General Experimental Procedures
HR-ESI-MS was measured on a Waters G2-XS Q-TOF mass spectrometer.All mass spectrometric data were obtained in positive ion mode using an ESI ion source, with a scan range from 100 to 1000 (m/z).NMR spectra were recorded on a Bruker AVANCE III HD 600 MHz spectrometer with TMS as the internal standard.Analytical HPLC was carried out on an SSI 1500 HPLC system equipped with a Model 201 UV detector and a Welch XB-C18 column (5 µm, 4.6 × 250 mm, 1.0 mL/min).Semipreparative HPLC was performed on a Laballiance HPLC system with a Welch XB-C18 column (5 µm, 10 × 250 mm, 4 mL/min) and a Model 500 UV detector.Silica gel (100-200 and 300-400 mesh, Qingdao Marine Chemical Inc., Qingdao, China), neutral alumina (100-200 mesh, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and Sephadex LH-20 (20-150 µm, GE Healthcare, Boston, TX, USA) were used for column chromatography.Thin-layer chromatography (TLC) was performed on precoated silica gel G plates (Qingdao Marine Chemical Inc., Qingdao, China) and detected by heating after spraying a solution of 5% H 2 SO 4 in EtOH.Photodynamic cytotoxicity and antibacterial activity evaluation used a 10 W halogen tungsten lamp (Philips, Amsterdam, The Netherlands) as the light source.All reagents used in the extraction and column chromatography process were analytically pure and in the HPLC analysis and preparation were of chromatographic purity.

Plant Material
The leaves of C. ptilosperma were picked in Daxin County, Chongzuo City, Guangxi Zhuang Region (China) in August 2019.A voucher specimen (No. 20190804-7014) representing this plant has been deposited at the Guangxi Institute of Botany, Chinese Academy of Sciences.

Cytotoxicity Assays
The cytotoxic activity of compounds 1-6 against Hela, MCF-7, BEL-7402, A549, HepG2, and MDA-MB-231 cancer cell lines was evaluated using the MTT assay according to the reported method [51,52] with doxorubicin as a positive control drug.MCF-7 and BEL-7402 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and Hela, A549, HepG2, and MDA-MB-231 cells were cultured in DMEM medium containing 10% fetal bovine serum, all of which were incubated in a constant temperature incubator at 5% CO 2 at 37 • C. A549 in the logarithmic growth phase were inoculated into 96-well plates at a density of 4 × 10 4 cells/mL.MCF-7, HepG2, BEL-7402, MDA-MB-231, and Hela were inoculated at a density of 5 × 10 4 cells/mL, with 100 µL in each well.After the cells were attached to the wall, the drug treatment was performed.Different concentrations (0.1, 0.5, 1.0, 5.0, 1.0, and 20.0 µM) of doxorubicin (dissolved in PBS) and compounds 1-6 (1.0, 2.0, 5.0, 10.0, 25.0, and 50.0 µM, dissolved in 0.1% DMSO/PBS) were administered.Three parallel tests were conducted for each concentration and the cells were cultured for 48 h.An amount of 10 µL of MTT was added to each well, and the cells were incubated.After incubation for 4 h at 37 • C, the medium was aspirated and 150 µL of DMSO solution was added and shaken for 10 min, the plate was shocked using an enzyme marker, and the absorbance value was detected at 490 nm to calculate the cell survival rate.The entire experiment was repeated three times.

Photocytotoxicity Assay
The cytotoxicity of compounds 7-10 with or without photo-irradiation was evaluated against the same six cancer cell lines by the MTT method.For the photodynamic cytotoxicity assay, a halogen tungsten lamp was employed as the irradiation source.The lamp was positioned immediately above the 96-well plate, maintaining a separation distance of 10 cm.The concentrations of the compounds 7-10 were set at 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 µM.Immediately after the addition of different concentrations of these compounds, the cells were exposed to continuous light for 0, 60, 120, 210, and 300 s.The subsequent experimental steps were the same as described in Section 3.4.1.

Photodynamic Antibacterial Activity Assay
The MIC (minimal inhibitory concentration) value was identified as the lowest concentration of the compound that inhibited visible bacterial growth following the incubation period.It was determined using the double dilution method.S. aureus, E. coli, K. pneumoniae, and P. aeruginosa were inoculated on Mueller-Hinton agar medium and incubated at 37 • C for 24 h.The concentration of the bacterial suspension was adjusted to 1.0 × 10 6 CFU/mL by the use of sterile saline solution and then the bacterial solution was inoculated on a 96-well plate with 100 µL per-well.
Compounds 7-10 and positive control drugs were diluted to attain the final concentrations within the range of 0.625-10.0µM.These different concentrations of solutions and positive control drugs were inoculated into 96-well plates with an inoculation volume of 50 µL per well, while for the blank control, only 50 µL of culture medium was added.The solutions were then incubated at 37 • C for 24 h.All the above operations were carried out under light-protected conditions.Bacterial growth in the treated group was determined by comparing the characteristics of bacterial growth in the blank control group and recording the minimum concentration corresponding to bacterial growth.
In the photodynamic antibacterial test, the samples were exposed to continuous light irradiation for a duration of 30 min using a halogen tungsten lamp positioned 10 cm above the samples after administration.The other procedures were the same as in the light avoidance condition.The experiment was repeated three times.

Statistical Analysis
Data from the cytotoxicity assays were evaluated according to their means and standard deviations.The cytotoxic concentration at 50% was determined to compare with the control obtained from nonlinear regression.These analyses were performed using SPSS ® Statistics 18.0 (IBM software, Armonk, NY, USA).

Conclusions
In summary, ten new compounds were isolated from the leaves of Camellia ptilosperma, including six triterpenes and four pheophorbides.The structures of the undescribed compounds were based on NMR and HR-ESI-MS spectroscopic data.
Meanwhile, the cytotoxic activity of the six triterpenes against six cancer cell lines was evaluated by MTT assay.Among them, compounds 1, 4, and 5 showed no significant

Figure 4 .
Figure 4. Key HMBC correlations of compounds 7 and 10.Compound 8 was isolated as a dark green amorphous solid and showed a molecular formula of C 37 H 38 N 4 O 7 based on HR-ESI-MS analysis (m/z 673.2635, [M+Na] + , calcd.