Cytotoxic and Hypoglycemic Activity of Triterpenoid Saponins from Camellia oleifera Abel. Seed Pomace

One new and three known triterpenoid saponins were isolated and identified from Camellia oleifera seeds through IR, NMR, HR-ESI-MS and GC-MS spectroscopic methods, namely oleiferasaponin A3, oleiferasaponin A1, camelliasaponin B1, and camelliasaponin B2. The structure of oleiferasaponin A3 was elucidated as 16α-hydroxy-21β-O-angeloyl-22α-O-cinnamoyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3β-O-[β-d-galactopyranosyl-(1→2)]-[β-d-xylopyranosyl-(1→2)-β-d-galactopyranosyl-(1→3)]-β-d-gluco-pyranosiduronic acid. Camelliasaponin B1 and camelliasaponin B2 exhibited potent cytotoxic activity on three human tumour cell lines (human lung tumour cells (A549), human liver tumour cells (HepG2), cervical tumour cells (Hela)). The hypoglycemic activity of oleiferasaponin A1 was testified by protecting pancreatic β-cell lines from high-glucose damage.


Introduction
Triterpenoid saponins are vital plant secondary metabolites that have been applied to cosmetics [1,2], agriculture [3], and medicine [4,5] for their diverse biological and pharmacological activities. Camellia oleifera was named for its seeds with plentiful edible oil. Tea seed pomace-the byproduct of oil manufacture-contains about 8% saponins, which have historically been wasted without full use [6]. In recent years, some research works concerning the extraction, structures, and activity identification of saponins obtained from Camellia oleifera seed have been published. There are 11 novel triterpenoid saponin compounds obtained from Camellia oleifera seed [6][7][8][9][10][11][12][13]. Meanwhile, their cell protective activity [14], antioxidant activity [10,15], anti-fungal activity [16,17], cytotoxic activity [11][12][13]18] have been reported, indicating that the different activities depend on the different compound structures. Approximately 30 types of saponin were indicated by liquid chromatography-mass spectrometry (LC-MS) analysis in the seed pomace of Camellia oleifera [17]. Therefore, it is significant to continue extracting, identifying, and exploring the biological and pharmacological activities of saponins in Camellia oleifera seed pomace.
One new triterpenoid saponin (oleiferasaponin A 3 ) and three known saponins (oleiferasaponin A 1 , camelliasaponin B 1 , and camelliasaponin B 2 ) were isolated from the tea seed pomace of Camellia oleifera in our study. We observed that camelliasaponin B 1 and camelliasaponin B 2 significantly inhibited the proliferation of human lung cancer cells (A549), human liver cancer cells (HepG2), cervical cancer cells (Hela), especially A549 cell lines. In addition, a glucose-stimulated insulin secretion (GSIS) experiment
There are some reports about the structure-activity relationships of triterpenoid saponins [21,22]. 0.93, indicating that the glycosidic chain group at C-3 and Ang group at C-21 are β-configured. The absolute configuration of sugars of oleiferasaponin A 3 was confirmed by acid hydrolysis and GC-MS analysis, which revealed one unit of D-glucuronic acid (GlcA), two units of D-galactose (Gal) and one unit of D-xylose (Xyl) [13,20]. Synthesizing the above analysis of all the proton and carbon signals, we established the structure of oleiferasaponin A 3 as 16α-hydroxy-21β-O-angeloyl-22α-O-cinnamoyl-23α-aldehyde- The other three known compounds were oleiferasaponin A 1 (22-

Anti-Proliferative Activity
Oleiferasaponin A 1 , oleiferasaponin A 3 , camelliasaponin B 1 and camelliasaponin B 2 obtained from Camellia oleifera seed pomace were tested against three human tumour cell lines (A549, Hela, HepG2) using cell proliferation bioassay (SRB). Camelliasaponin B 1 and camelliasaponin B 2 at the concentration of 20 µM exhibited effective anti-proliferative activity on the human tumour cell lines tested (Figure 3)-the inhibition ratios were more than 50%. Camelliasaponin B 1 and camelliasaponin B 2 at the concentration of 10 µM significantly inhibited the proliferation of human lung cancer cells (A549) (Figure 3)-the inhibition ratios were 94.44% and 79.12%, respectively. Our results indicated that camelliasaponin B 1 and camelliasaponin B 2 possessed potent cytotoxic activity. There are some reports about the structure-activity relationships of triterpenoid saponins [21,22].
The structures of camelliasaponin B1 and camelliasaponin B2 are similar, except for the orientation of C-22 angeloyl. Compared to previously reported results [11][12][13], it seems that the main groups contributing cytotoxicity are the C-22 Ang group and the C-28 free hydroxy group. As a result, the cytotoxic activity is a combined effect of sugar moieties and aglycone, rather than an isolated structural effect. Oleiferasaponin A1 and oleiferasaponin A3 did not show cytotoxic activity.

Hypoglycemic Activity
Diabetes mellitus (DM) is the third most prevalent disease globally, and manifests as a disorder of blood glucose caused by metabolic disorder, which can induce cardiovascular system diseases and cancer, then threatening human health and life. Many studies regarding the cytotoxic activity of triterpenoid saponins have been reported [11][12][13], while few have been conducted concerning hypoglycemic activity [23]. Oleiferasaponin A1 and oleiferasaponin A3 did not exhibit cytotoxic activity on three human tumour cell lines (A549, Hela, HepG2), so we carried out a hypoglycemic activity study for further exploration of structure-activity relationship. Oleiferasaponin A1 and oleiferasaponin A3 were tested for their protective effect on RIN-m5f (islet-β cells) injured by high glucose. The insulin content of RIN-m5f cells upon treatment under 16.7 mmol/L glucose are shown below (Figure 4). With higher oleiferasaponin A1 concentration, the insulin levels of RIN-m5f (islet-β cells) was enhanced, which indicates that oleiferasaponin A1 has potential hypoglycemic activity against the damage induced by high glucose, and oleiferasaponin A1 may be a therapeutic agent for hyperglycemia treatment. Regarding the oleiferasaponin A3 group, no improvement effect on insulin levels was found in RIN-m5f (islet-β cells) injured by high glucose, even at a concentration of 100 μM. The difference of bioactivity between oleiferasaponin A1 and oleiferasaponin A3 is due to the different structure, including aglycone and sugar moieties (Figure 1). Compared with oleiferasaponin A3, we infer that the trans-2-hexenoyl group of oleiferasaponin A1 at C-22 may influence the activities, cooperating with sugar moieties. The structures of camelliasaponin B 1 and camelliasaponin B 2 are similar, except for the orientation of C-22 angeloyl. Compared to previously reported results [11][12][13], it seems that the main groups contributing cytotoxicity are the C-22 Ang group and the C-28 free hydroxy group. As a result, the cytotoxic activity is a combined effect of sugar moieties and aglycone, rather than an isolated structural effect. Oleiferasaponin A 1 and oleiferasaponin A 3 did not show cytotoxic activity.

Hypoglycemic Activity
Diabetes mellitus (DM) is the third most prevalent disease globally, and manifests as a disorder of blood glucose caused by metabolic disorder, which can induce cardiovascular system diseases and cancer, then threatening human health and life. Many studies regarding the cytotoxic activity of triterpenoid saponins have been reported [11][12][13], while few have been conducted concerning hypoglycemic activity [23]. Oleiferasaponin A 1 and oleiferasaponin A 3 did not exhibit cytotoxic activity on three human tumour cell lines (A549, Hela, HepG2), so we carried out a hypoglycemic activity study for further exploration of structure-activity relationship. Oleiferasaponin A 1 and oleiferasaponin A 3 were tested for their protective effect on RIN-m5f (islet-β cells) injured by high glucose. The insulin content of RIN-m5f cells upon treatment under 16.7 mmol/L glucose are shown below (Figure 4). With higher oleiferasaponin A 1 concentration, the insulin levels of RIN-m5f (islet-β cells) was enhanced, which indicates that oleiferasaponin A 1 has potential hypoglycemic activity against the damage induced by high glucose, and oleiferasaponin A 1 may be a therapeutic agent for hyperglycemia treatment. Regarding the oleiferasaponin A 3 group, no improvement effect on insulin levels was found in RIN-m5f (islet-β cells) injured by high glucose, even at a concentration of 100 µM. The difference of bioactivity between oleiferasaponin A 1 and oleiferasaponin A 3 is due to the different structure, including aglycone and sugar moieties (Figure 1). Compared with oleiferasaponin A 3 , we infer that the trans-2-hexenoyl group of oleiferasaponin A 1 at C-22 may influence the activities, cooperating with sugar moieties.

Plant Material
Tea seed pomace (Camellia oleifera) was collected from a factory in Shucheng, Anhui province, China. The plant material was identified by one of the authors (Associate Prof. X.F. Zhang), and was deposited in State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University.

General
HPLC was run on Agilent 1260 HPLC (Agilent, Palo Alto, CA, USA). IR (infrared) spectra was recorded on Nicolet iN10 (Thermo Scientific Instrument Co., Boston, MA, USA) with KBr pellets. NMR spectra was measured on an AVANCE III (600 MHz) spectrometer (Bruker, Fallanden, Switzerland) using methanol-d 4 (Sigma-Aldrich St. Louis, MO, USA) as solvent. HR-ESI-MS were determined on an electrostatic field orbital trap mass spectrometer (Thermo Scientific, Bremen, Germany) using an ESI source.

Plant Material
Tea seed pomace (Camellia oleifera) was collected from a factory in Shucheng, Anhui province, China. The plant material was identified by one of the authors (Associate Prof. X.F. Zhang), and was deposited in State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University.

Acid Hydrolysis and GC-MS Analysis
Oleiferasaponin A 3 was dissolved in 1 M HCI (Guoyao chemical reagent Co. Ltd, Beijing, China) (1 mL) for 3 h at 90 • C, then extracted with chloroform (Guoyao chemical reagent Co. Ltd, Beijing, China). The aqueous phase was evaporated under N 2 flow. The residue was dissolved in 0.2 mL pyridine (Aladdin Industrial Co. Shanghai, China) containing L-cysteine methyl ester hydrochloride (10 mg/mL) and reacted at 70 • C for 1 h, then evaporated under N 2 flow again. After concentrated, 0.2 mL trimethylsilylimidazole (Aladdin Industrial Co. Shanghai, China) was added for derivatization reaction, and reacted at 70 • C for another 1 h. The reaction mixture was partitioned between n-hexane and water. The organic phase was analysed by GC-MS (Agilent, Palo Alto, CA, USA) (injector temperature at 280 • C; the initial oven temperature was 160 • C for 1 min, linearly increased to 200 • C at 6 • C/min, then a further linear increase to 280 • C at 3 • C/min and held for 5 min). The standard sugar samples were subjected to the same reaction and GC-MS conditions.

Cell Culture
Human lung tumour cell (A549) lines, human liver tumour cell (HepG2) lines, and cervical tumour cell (Hela) lines were obtained from Qingdao Marine Biomedical Research Institute Limited by Share Ltd Testing Center (Qingdao, Shandong, China). Cells were cultured in DMEM complete medium supplemented with 10 % fetal bovine serum, 2 mM l-glutamine, 100 U·mL −1 penicillin, and 100 µg·mL −1 streptomycin at 37 • C in a 5% CO 2 humidified atmosphere. The culture medium was refreshed every other day. After 80% of the cells were fused, cells were kept in logarithmic phase by trypsinization and subculturing.

Cell Viability Assay
Human tumour cell lines in logarithmic phase were seeded in a 96-well plate at 4 × 10 3 cells per well (180 µL per well), and incubated for 24 h. After 24 h, negative control without additions; solvent control was supplied with 0.1% DMSO; positive control with 1 µM adriamycin; 20 µM, 10 µM saponins were added to trial group, all incubated for 72 h. Then, 50% (m/v) ice-cold trichloroacetic acid was added to the medium for fixed cells. After staining by sulforhodamine B, tris solution (150 µL per well) was added to culture medium. Absorbance values were measured at 540 nm using an enzyme-linked immunosorbent reader (SpectraMax i3, Molecular Devices, San Francisco, CA, USA). The inhibition rate of cell proliferation was calculated as: Inhibition rate (%) = [(OD 540 (control group) − OD 540 (trial group)) / OD 540 (control group)] × 100% (1)

Hypoglycemic Activity Assay
Pancreatic β-cell lines (RIN-m5f) were obtained from Qingdao Marine Biomedical Research Institute Limited by Share Ltd. Testing Center (Qingdao, Shandong, China). Cells were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% glutamine. Then, pancreatic β-cell lines (RIN-m5f) were seeded in a 96-well plate (1 × 10 4 cells per well). Cells were set in four groups: normal group with 5.5 mmol L −1 glucose; injured group with 16.7 mmol L −1 glucose; trial group with 16.7 mmol L −1 glucose and different concentrations (25, 50, 100 µM) of oleiferasaponin A 1 ; positive control group with 16.7 mmol/L glucose and 100 µM ZnSO 4 . Each group was set three parallels and incubated for 48 h. Next, the medium was removed, cleaning twice with polybutylene succinate (PBS). The cells were incubated in medium with 5.5 mmol L −1 glucose for 1 h. Then, the medium was replaced by medium with 33.3 mmol L −1 glucose and incubated for 2 h. The supernatant was collected for insulin content detection using ELISA kit (CEA448Ra, Cloud-Clone Corp, Houston, TX, USA).
Supplementary Materials: HR-ESR-MS and NMR spectra data of oleiferasaponin A 3 can be accessed online.