Anti-oxidative effects of the biennial flower of Panax notoginseng against H2O2-induced cytotoxicity in cultured PC12 cells
© Choi et al; licensee BioMed Central Ltd. 2010
Received: 5 April 2010
Accepted: 28 October 2010
Published: 28 October 2010
Radix notoginseng is used in Chinese medicine to improve blood circulation and clotting; however, the pharmacological activities of other parts of Panax notoginseng have yet to be explored. The present study reports the anti-oxidative effects of various parts of Panax notoginseng.
Various parts of Panax notoginseng, including the biennial flower, stem-leaf, root-rhizome, fiber root and sideslip, were used to prepare extracts and analyzed for their anti-oxidation effects, namely suppressing xanthine oxidase activity, H2O2-induced cytotoxicity and H2O2-induced ROS formation.
Among various parts of the herb (biennial flower, stem-leaf, root-rhizome, fiber root and sideslip), the water extract of the biennial flower showed the strongest effects in (i) inhibiting the enzymatic activity of xanthine oxidase and (ii) protecting neuronal PC12 cells against H2O2-induced cytotoxicity. Only the water extracts demonstrated such anti-oxidative effects while the ethanol extracts did not exert significant effects in suppressing xanthine oxidase and H2O2-induced neuronal cytotoxicity.
The present study demonstrates the biennial flower of Panax notoginseng to have neuroprotection effect on cultured neurons and the underlying protection mechanism may involve anti-oxidation.
Radix Notoginseng (Sanqi, the root of Panax notoginseng) is a Chinese herbal medicine used in China to promote blood circulation, remove blood stasis, induce blood clotting, relieve swelling and alleviate pain [1, 2]. Moreover, Panax notoginseng is beneficial for coronary heart disease, cerebral vascular disease as well as learning and memory improvement [3–7]. These therapeutic effects are attributed to its active ingredients, namely saponins [8, 9], flavonoids  and polysaccharides [11, 12].
Saponins isolated from Radix Notoginseng increase the blood flow of coronary arteries , prevent platelet aggregation , decrease oxygen consumption by heart muscles , restore learning impairment induced by chronic morphine administration  and protect neuronal cell death against oxidative stress . Flavonoids increase the coronary flow, reduce myocardial oxygen consumption and lower arterial pressure . A flavonol glycoside called quercetin 3-O-β-D-xylopyranosyl-β-D-galactopyranoside (RNFG) from the root and rhizome of Panax notoginseng is promising in treating Alzheimer's disease through inhibiting amyloid-β aggregation and amyloid-β-induced cytotoxicity in cortical neuron cultures. Such neuroprotection effect was mediated by the suppression of apoptosis triggered by amyloid-β . Moreover, polysaccharide extracted from the root-rhizome of Panax notoginseng is also considered to be an active constituent with immuno-stimulating activities in vitro [11, 12, 19].
While the therapeutic effects of the root of Panax notoginseng have been demonstrated, the pharmacological effects of other parts of Panax notoginseng are largely unknown. The present study examines the anti-oxidation effects of other parts of Panax notoginseng.
Plant materials and preparation
Fresh Panax notoginseng from Wenshan in Yunnan Province (China) was identified morphologically during harvest. Voucher specimen (number 03-6-8) of Panax Notoginseng was confirmed by genetic analysis  and deposited at the Department of Biology, Hong Kong University of Science and Technology. For water extraction, the biennial flower, stem and leaf, root-rhizome, fiber root and/or sideslip (10 g) were boiled in 80 ml of water for two hours twice. The extract was then dried by lyophilization with an extraction efficiency of 15-18%. For ethanol extraction, biennial flower (10 g) was sonicated in 100 ml of 30%, 50%, 70% and 90% ethanol for 30 minutes twice. The extract was dried by rota-evaporation at 60°C with an extraction efficiency of 5-8%. The water and ethanol extracts were re-dissolved in water to 100 mg/ml stock concentration.
Rat pheochromatocytoma PC12 cell line was obtained from ATCC (CRL-1721; USA). The cells were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 6% fetal bovine serum and 6% horse serum at 37°C in a water-saturated 7.5% CO2 incubator. Reagents for cell cultures were purchased from Invitrogen Technologies (USA).
In vitro xanthine oxidase activity
Xanthine oxidase activity assay was described previously . In brief, the herbal extracts (0.1 mg/ml) were pre-mixed with 0.05U/ml xanthine oxidase for 20 minutes. Then 0.4 mM xanthine and 0.24 mM hydroxyl amine were incubated for 20 minutes at 37°C. Reactions were stopped by adding 0.1% SDS to the mixture and measured at 550 nm absorbance. Vitamin C at various concentrations (0, 17.6, 35.2, 52.8 and 88 μg/ml) served as the positive control of anti-oxidation. All the chemicals were purchased from Sigma (USA).
Cell viability test
Cultured PC12 cells in 96-well-plate (5000 cells/well) were pre-treated with various extracts (1 mg/ml) for 24 hours. After washed with PBS and replaced by fresh culture medium, the cultures were treated with 13.6 μg/ml hydrogen peroxide (H2O2) for 24 hours. Cell viability test was performed with the addition of thiazolyl blue tetrazolium bromide (MTT) (Sigma, USA) in PBS at a final concentration of 5 mg/ml for four hours. After the solution was removed, the purple precipitate inside the cells was re-suspended in DMSO and then measured at 570 nm absorbance . H2O2 at various concentrations (0, 1.7, 3.4, 6.8 and 13.6 μg/ml) served as a control for the cytotoxicity test.
Determination of ROS formation
The reactive oxygen species (ROS) level in cell cultures was determined according to the method by Zhu et al. . Cultured PC12 cells in 96-well-plate were pre-treated with the water and ethanol extracts of biennial flower (1 mg/ml) for 24 hours, and then the cells were labeled by 100M dichlorofluorescin diacetate (DCFH-DA, Sigma, USA) in HBSS for one hour at 25°C. Cultures were treated with 13.6 μg/ml H2O2 for one hour. The amount of intracellular H2O2-induced ROS was detected by fluorometric measurement with excitation at 485 nm and emission at 530 nm (SPECTRA max® GEMINI XS, Molecular Devices Corporation, USA).
Individual data were expressed as mean ± standard deviation (SD). A post-hoc Dunnett's test was used to obtain corrected P values in group comparisons. Statistical analyses were performed with one-way ANOVA (version 13.0, SPSS, USA). Data were considered as significant when P < 0.05 and highly significant when P < 0.001.
Anti-oxidative effects of Panax notoginseng's biennial flower
Comparison of anti-oxidative effects by water and ethanol extracts
The present study, for the first time, demonstrated the anti-oxidative effects possessed by the water extract of the biennial flower of Panax notoginseng through the suppression of H2O2-induced ROS formation and neuroprotection against H2O2 insult. More importantly, it was the biennial flower instead of the root-rhizome that showed the strongest effects. These results support the multi-functional roles of Panax notoginseng and warrant further studies to explore other pharmacological effects of the plant. In terms of identifying the possible active ingredient(s) from the biennial flower, the anti-oxidation effects of different ethanol extracts were shown to be significantly less potent than that of the water extract, suggesting that the majority of active compounds might be preferentially water soluble. However, a continuous work of activity-guided fractionation is required to purify and identify the candidates from the water extract of biennial flower. In this case, the high solubility of those active compounds in water will facilitate the preparation of health food supplements and drinks that could be developed from the biennial flower. Indeed, this new application will increase the economic value of Panax notoginseng.
Neuronal action of Panax notoginseng on the brain possesses various aspects. Saponins derived from the herb have been shown to prevent the neuronal cell death against hypoxia condition. The mechanism was related to the improvement of energy metabolism . The therapeutic effect of saponins derived from Panax notoginseng was further supported by promoting the absorption of hematoma in hemorrhagic apoplexy at super-early stage in rat , and protecting the neuron against insults and promoting functional rehabilitation in patients after cerebral hemorrhage . In addition, the co-treatment of icariin and sponins derived from Panax notoginseng exerted significant prophylactic and therapeutic effects in rat models of Alzheimer's disease in vivo , as well as ameliorated the learning and memory deficit and blood viscosity by protecting neurons from oxidative stress in ischemic brain . For neurotrophic effects, the phosphorylated neurofilament- and MAP2-expressing neurites could be extended in SK-N-SH cells by the treatment of saponins and Panax notoginseng extracts, suggesting the possible axonal and dendritic formation activity . Therefore, the multi-functional effects of saponins from Panax notoginseng might be a good candidate in mediating the anti-oxidation activities because of the high extractability of saponins by water. This speculation was in accordance to our previous finding that the amounts of four active constituents, notoginsenoside R1, ginsenoside Rg1, Rb1 and Rd, by water extraction were higher than that of 30% and 70% ethanol extractions . In addition to saponins, a flavonol glycoside, named RNFG, isolated from Panax notoginseng also possesses the neuroprotective effect against amyloid-β-induced apoptosis and cytotoxicity at cellular level, and which improves the learning and memory process in rats . Interestingly, this compound also exerts a significant anti-oxidative activity by lowering the amount of reactive oxygen species (ROS) induced by H2O2 in cultured PC12 cells. Based on the above findings, it should be very interesting to know if the biennial flower contains RNFG, and which could have neuroprotective effect in cell cultures and in animal study. Therefore, the identification and isolation of the possible active ingredients (saponins, flavonoids, flavonol glycoside or others) will be essential to extend and support the multi-functional usages of Radix Notoginseng in future.
The present study demonstrates the biennial flower of Panax notoginseng to have neuroprotection effect on cultured neurons and the underlying protection mechanism may involve anti-oxidation.
Dulbecco's modified eagle medium
high performance liquid chromatography
3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide
reactive oxygen species
sodium dodecyl sulfate
This research was supported by grants from the University Grants Committee (AoE/B-15/01), Research Grants Council of Hong Kong (HKUST 6419/06M, 662608, N_HKUST629/07) and Croucher Foundation (CAS-CF07/08.SC03) to KWKT.
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