A New Saponin from Tea Seed Pomace (Camellia oleifera Abel) and Its Protective Effect on PC12 Cells

A new triterpenoid saponin, oleiferasaponin A1, was isolated from tea seed pomace (Camellia oleifera Abel). The structure of oleiferasaponin A1 was elucidated on the basis of chemical and physicochemical evidence and was found to be 22-O-cis-2-hexenoyl-A1-barrigenol 3-O-[β-D-galactopyranosyl(1→2)] [β-D-glucopyranosyl(1→2)-α-L-arabinopyranosyl(1→3)]-β-D-glucopyranosiduronic acid. PC12 cells injured with H2O2 were used as the model to test the protective effects of oleiferasaponin A1. The results indicated that oleiferasaponin A1 can potentially prevent the H2O2-induced cell death of PC12 cells.


Introduction
The Camellia plant, Camellia (C.) oleifera Abel, has been widely cultivated as an economic or ornamental plant in many parts of China, including the Hunan, Jiangxi, Anhui, Henan, Zhejiang and Fujian provinces. The seeds of this plant are used for oil manufacture, while the byproduct, tea seed pomace, is normally discarded as waste or is used for fuel and feed after treatment in traditional Chinese industries. However, there is about 8% saponin in tea seed pomace [1]. Saponin has been commercially utilized as a foam-stabilizing and emulsifying agent [1] and is extensively used in aquaculture to eliminate unwanted fish and harmful insects in prawn ponds [2]. In addition, it is used as a medicine for the treatment of intestinal disorders [3] and burn injuries [4].
The chemical constituents of the seeds of Camellia sinensis var. sinensis have been studied extensively, and many compounds such as the theasaponins A 1 -A 3 , F 1 -F 3 [5], A 4 , A 5 , C 1 , E 8 -E 9 , G 1 , H 1 [6], A 6 , A 7 , B 5 [7], E 1 , E 2 [8] and E 3 -E 7 [9] have been reported over the years. However, very few studies have been performed on characterizing the chemical constituents and biological activities of saponin obtained from the tea seeds of C. oleifera. Huang et al. identified a new compound, sasanquasaponin [3], which has a triterpenoid structure that is similar to the structures of some ginseng saponins [10]. Furthermore, the protective effect of this compound on endothelial cell injury has been studied. Kuo et al. detected the presence of camelliasaponin B 1 in a saponin mixture that was obtained from the methanol extract of tea (C. oleifera) seed pomace and studied the antifungal activities of this mixture [11]. Tea saponin has also been reported to exert many pharmacological effects, including antihyperlipidemic [12], antiallergic [13], and cardioprotective effects [14]. This paper deals with the isolation and structure elucidation of a new saponin, oleiferasaponin A 1 , from tea seed pomace of C. oleifera, as well as its protective effect on PC12 cells.

Results and Discussion
The methanol extract of tea seed pomace (Camellia oleifera Abel) was dissolved in water and was separated by a nanofiltration membrane; it was then successively subjected to purification through a macroporous resin column, a silica gel column, and repeated high pressure liquid chromatography (HPLC) to yield the new compound.
The new compound was visualized by spraying with 1% (w/v) Ce(SO 4 ) 2 in 10% (v/v) aqueous H 2 SO 4 , followed by heating at 120 °C, and it displayed purplish black spots on a thin-layer chromatography (TLC) plate, suggesting that it possessed the basic triterpenoid skeleton [15]. The IR spectrum showed absorption bands at 3386, 1717, 1655, 1077, and 1047 cm −1 due to hydroxyl; α, β-unsaturated ester; carboxy; and ether functions. The data were very similar to those of sasanquasaponin I [13] and camelliasaponin B 1 [16]. In addition, the signal of C-4 was markedly shifted downfield ( Δ δC = 17.2 ppm) and that of C-5 was shifted upfield ( Δ δC = −6.4 ppm), and HMBC correlations from -CHO (δppm 9.48) to C-4 showed that the 23-methyl was substituted by a -CHO moiety. Furthermore, the position of the acyl group and the structure of the oligoglycoside moiety were confirmed on the basis of HMBC correlations. Long-range correlations were observed between the following proton and carbon pairs: H-22 and C-1′′′′′, H-1′ and C-3, H-1′′ and C-2′, H-1′′′ and C-3′, and H-1′′′′ and C-2′′′. On the basis of the above mentioned evidence, the chemical structure of oleiferasaponin A 1 was determined to be 22   (Figure 2). These results show that oleiferasaponin A 1 has potential cytoprotective activity against H 2 O 2 -induced damage.

General
The following spectrometric instruments were used to obtain physical data: IR spectra were obtained on a Nicolet FTIR-8700 spectrometer (Thermo Scientific Instrument Co., Boston, USA); mass spectrometry was performed using a Thermo Scientific LTQ Orbitrap XL instrument (Thermo Electron, Bremen, Germany) equipped with an ESI source operated in the negative-ion modes; 1 H-NMR and 13 C-NMR spectra were obtained on an AVANCE AV 400 (400/100 MHz) spectrometer (Bruker, Fallanden, Switzerland), with tetramethylsilane as an internal standard. The following materials and equipment were used for membrane separation and chromatography: 3000-Da nanofiltration membrane (SJM, Hefei, China), AB-8 macroporous resin column (Bonc, Cangzhou, China), ordinary-phase silica gel column (200-300 mesh; Anhui Liangchen Silicon Material Co. Ltd., Huoshan, China) and a Varian Prostar HPLC instrument (Model 325) (Varian, Mulgrave, Australia).

Plant Material
Tea seed pomace (Camellia oleifera) was collected in October 2010 from a factory in Shucheng, Anhui, China.

Extraction and Isolation
The tea seed pomace (20 kg) was cut and extracted three times with methanol under reflux for 3 h. Evaporation of the solvent under reduced pressure provided a brown syrup (2.2 kg). The methanol extract (1.5 kg) was dissolved in water and purified using a nanofiltration membrane. The concentrated solution (1.1 kg) was subjected to AB-8 macroporous resin column chromatography with stepwise gradients of water and ethanol (100:0, 70:30, 30:70, and 0:100, v/v) to afford four subfractions. The third subfraction (590 g) was further subjected to ordinary-phase silica gel column chromatography [

Measurement of Cell Viability
PC12 cells were cultured as previously described [17]. Cells were added into the wells of a 96-well culture plate at a density of 10 5 cells/mL for incubation before the cell viability experiments. The cells were permitted to adhere to plates for 16 h after seeding. Then, different volumes of the fresh compound stock were added to the plates to achieve final concentrations of 5, 25, and 125 μM. After 2 h incubation with the compound, 5 mM H 2 O 2 was added into the plates. The cells were subjected to stress for 48 h before the experimental analyses.
The antioxidant properties of oleiferasaponin A 1 on cell viability were assessed by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [18]. In brief, the MTT assay was performed by the addition of MTT solution (5 μg/mL) to each well, and 2 h later, the culture was dissolved in DMSO. The absorbance of MTT was measured using a microplate absorbance reader at 570 nm. The data are presented as the percentage versus the blank control, which represents 100% cell viability.

Statistical Analysis
The mean value and standard deviation in this experiment were calculated by Excel 2007 (Microcal Software Inc., Northampton, MA, USA). Data were subject to statistics analysis by using the software package SPSS Statistics 17.0 for Windows (release 17.0.1; SPSS Inc., Chicago, IL, USA, 2008). ANOVA was carried out to determine significant difference (** p < 0.01).