Separation of nine novel triterpene saponins from Camellia japonica seeds using high-performance countercurrent chromatography and reversed-phase high-performance liquid chromatography.

INTRODUCTION: Camellia japonica L. (Theaceae) is an evergreen shrub, which is cultivated as a popular ornamental tree in Korea, China, and Japan and its seeds have been used as a source of cooking oil, in cosmetics and as a traditional medicine. Intensive phytochemical works have revealed that oleanane-type saponins are the characteristic compounds of the seeds of C. japonica. OBJECTIVE: The purpose of the present study is to isolate and determine oleanane-type saponins from C. japonica using high-performance countercurrent chromatography (HPCCC) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) and spectroscopic evidences, respectively. METHODOLOGY: HPLC electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) was applied to profile the saponin composition of an enriched saponin extract of C. japonica seeds. The enriched saponin extract was separated by HPCCC using a dichloromethane/methanol/isopropanol/water (9:6:1:4, v/v/v/v) system and RP-HPLC. The structures of the isolates were determined utilising ESI-Q-TOF-MS, one-dimensional and two-dimensional NMR and optical rotation. RESULTS: HPCCC on enriched saponin extract of C. japonica yielded four saponin fractions in the order of the number of sugars attached to the triterpene aglycone, and preparative RP-HPLC on each saponin fraction led to the isolation of nine novel saponins, namely camoreoside A-I, along with six known ones. CONCLUSIONS: This study indicates that combination of HPLC-ESI-Q-TOF-MS analysis and HPCCC coupled with RP-HPLC are excellent tools for discovering saponins from natural sources.

Self-sterility in Camellia oleifera may be due to the prezygotic late-acting self-incompatibility.

In this report, self-sterility in Camellia oleifera was explored by comparing structural and statistical characteristics following self-pollination (SP) and cross-pollination (CP). Although slightly delayed pollen germination and pollen tube growth in selfed ovaries compared to crossed ovaries was observed, there was no significant difference in the percentages of pollen that germinated and pollen tubes that grew to the base of the style. There was also no difference in morphological structure after the two pollination treatments. However, the proportions of ovule penetration and double fertilization in selfed ovules were significantly lower than in crossed ovules, indicating that a prezygotic late-acting self-incompatible mechanism may exist in C. oleifera. Callose deposition was observed in selfed abortive ovules, but not in normal. Ovules did not show differences in anatomic structure during embryonic development, whereas significant differences were observed in the final fruit and seed set. In addition, aborted ovules in selfed ovaries occurred within 35 days after SP and prior to zygote division. However, this process did not occur continuously throughout the life cycle, and no zygotes were observed in the selfed abortive ovules. These results indicated that the self-sterility in C. oleifera may be caused by prezygotic late-acting self-incompatibility (LSI).

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi.

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 µM IBA and 0.3 µM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 µM BAP and 0.1 µM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks' acclimatization.