- Open Access
Saponin accumulation in the seedling root of Panax notoginseng
© Wang et al; licensee BioMed Central Ltd. 2011
Received: 4 September 2010
Accepted: 24 January 2011
Published: 24 January 2011
Panax notoginseng is an important Chinese medicinal plant. Dammarene-type triterpenoid saponins are main pharmacologically effective compounds in P. notoginseng. This study aims to investigate the formation and accumulation of saponins in P. notoginseng roots during germination and juvenile stage.
P. notoginseng seeds were collected and stored in wet sand. After germination, the seedlings were transplanted into a soil nursery bed and cultivated for one year. During this period, samples were collected every month and the concentrations of ginsengnosides Rg1, Re, Rb1, Rd and notoginsengnoside R1 were determined by HPLC.
There was little saponin in the P. notoginseng seed. The chemical composition of seed was different from that of root. After germination, Rb1, Rg1, Re, Rd and R1 appeared successively in the seedling root. And in the five-month-old root, all these five main saponins came into existence. The accumulation of saponins in P. notoginseng root was affected by seasons.
The accumulation of saponins showed a time-dependent increase after germination of P. notoginseng.
Panax notoginseng (Burk.) F. H. Chen. (Sanqi), a species belonging to the Araliaceae family, is an important medicinal plant for its haemostatic and restorative properties . Much chemical and pharmacological research on P. notoginseng has been carried out, indicating that dammarene-type triterpenoid saponins are not only the main chemical components but also the main pharmacologically effective components. They exert various effects on the cardiocerebral vascular system, central nervous system and endocrine system [2–7]. To date, over 70 dammarene-type triterpenoid saponins have been isolated from the whole plant of P. notoginseng[8–10], with the major saponins isolated from the root being ginsenosides Rg1 (Rg1), Re (Re), Rb1 (Rb1), Rd (Rd) and notoginsenoside R1 (R1).
Although detailed chemical studies have been carried out on mature root and leaf of P. notoginseng, very little information is available on the chemical composition of its seed or on saponin accumulation during root development. The present study aims to investigate the formation and accumulation of saponin constituents in P. notoginseng during germination and juvenile stages, particularly the developmental changes of Rg1, Re, Rb1, Rd and R1 in the seed and seedling root.
Solvents and chemicals
Methanol (MeOH) was purchased from Tianjing chemical Ltd (China). Acetonitrile (MeCN) was purchased from Merck (Germany). Standard Rg1, Rb1, Re, Rd and R1 were isolated from the root of P. notoginseng and their chemical structures were determined with nuclear magnetic resonance (NMR) and mass spectrometry (MS).
Mature seeds were collected from a 3-year-old P. notoginseng in November 2005 from the farm of Miaoxiang Ltd (Wenshan County, Yunnan Province, China). After removal of the pericarp, the seeds were stored in wet sand; after germination, the seedlings were transplanted into a soil nursery bed under a sheltering net with 80% shadowiness. During this period, 50 g seed or 20 individual seedling roots were sampled at intervals of one month until seedlings grew to 12 months old. The samples were dried at 45°C and powdered. Concentrations of Rg1, Re, Rb1, Rd and R1 were analyzed with a high-performance liquid chromatography (HPLC).
After removal of the pericarp, 100 g of fresh seeds was crushed and extracted at room temperature with MeOH (300 ml) for three times. The concentrated MeOH extract was partitioned between water and petroleum ether. The aqueous part was concentrated under reduced pressure as a crude seed extract (0.3 g).
A Waters Alliance HPLC (USA) equipped with Alliance separation module 2695 and photodiode array detector 2996 was used in the analysis. A reversed-phase column (Waters Symmetry C-18, 3.9 × 150 mm i.d., 5 μm) was used. The gradient elution system consisted of water (A) and acetonitrile (B). Separation was achieved using the following gradient: 0-20 min: 20%-22% B, 20-45 min: 22%-46% B, 45-55 min: 46%-55%, 55-60 min: 55%-90% B. The column temperature was set at 25°C. The flow rate was 1 ml/min. The UV detection wavelength was 203 nm. The mean values of three replicates were calculated.
Recovery rates of five main saponins in P. notogingseng
mean (SD) (mg)
Regression equations of five main saponins in P. notoginseng
Test range (μg)
y = 584809x-47740
y = 645054x+46049
y = 616766x-36027
y = 456796x+125610
y = 614439x-14119
Limits of detection and limits of quantitation
The standard solutions were diluted with 70% aqueous methanol to provide appropriate concentrations. When the ratio of the testing peak signal-to-noise (S/N) was 4, the limit of detection (LOD) for each analyte was determined; when the S/N ratio was 10, the limit of quantitation (LOQ) was determined.
Sample preparation for HPLC analysis
For seed samples of germination test, 1.0 g of powder was weighed accurately and extracted ultrasonically for 30 minutes in 70% methanol in a 10 ml volumetric flask. After cooled down and made up the lost volume with methanol, the sample solution was obtained by filtering the supernate with a nylon filter membrane (0.45 μm) prior to the HPLC analysis. For seedling root samples, 50 mg of powder was weighed accurately and extracted in 70% methanol ultrasonically in a 5 ml volumetric flask. The other steps were similar to those of the seed sample. Injection volumes of seed and seedling sample solutions for HPLC were 100 μl. As to HPLC analysis of seed extract, the raw extract was dissolved in MeOH (10 mg/ml) and filtered with 0.45 μm nylon filter membrane and 10 μl of solution was injected for HPLC analysis.
Linear regression was performed with Excel 2003 (Microsoft, USA). RSDs were also calculated with Excel 2003 (Microsoft, USA).
Under the HPLC conditions used in this study, all five saponins were baseline separated and their calibration curves exhibited good linear regressions. The method validation analysis demonstrated that the analytical method developed in this study for all five saponins was accurate and precise.
Saponin concentrations (%) in seed and seedling root of P. notoginseng during seed germination and juvenile stage
Saponins in the seed
Saponin accumulation in the root
Dammarene-type triterpenoid saponins are main secondary metabolites of Panax notoginseng. The present study demonstrates a temporal and spatial distribution of saponins during the germination process and the growth of young plants. Our results show that ginsenosides Rg1, Re, Rb1, Rd and notoginsenoside R1 were not detected in P. notoginseng seed. The formation of saponins in root is a gradual process. The synthesis and accumulation of saponins began after germination and continued with the growth of seedling. Saponin synthetases were activated after seed began to germinate. In young roots, saponin constituents formed and accumulated mainly between July and October, the most vigorous period of growth. This periodic change is, as in adult plant, closely related to the growth pattern of Panax notoginseng; the formation and accumulation of saponins were affected by seasons . As a plant grows up, more and more saponins accumulate in the root. Our previous work revealed that, in a 3-year-old root, the concentrations of Rg1, Rb1, Rd, Re and R1 reached 4.11%, 4.12%, 0.82%, 0.83% and 1.14% respectively . All these findings suggest that saponins may not serve as the nutrient storage in the seed. The protective functions of saponins in other plants are reported [13, 14]. The role of this kind of secondary metabolites in P. notoginseng requires further investigation.
The accumulation of saponins showed a time-dependent increase after germination of P. notoginseng.
This work was funded by the Science & Technology Bureau of Yunnan Province, China (Grant: 2008IF006).
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