P. hexandrum was collected from a forest (2,100 m) in HuiChuan, WeiYuan of Gansu Province, China, after the plant fruit ripened in September 2011. The plant was dried in shade under room temperature. The species was identified by Professor Yanling Qi (Gansu Provincial Academy of Agricultural Sciences, Lanzhou, Gansu, P.R.China). A voucher specimen (No.0209069) was deposited in the herbarium of College of Agronomy, Gansu Agricultural University, Lanzhou, Gansu, P.R. China.
Reagents and instrumentation
DPPH, 2, 4, 6-tris (2-pyridyl)-s-triazine (TPTZ), PADE, BADE, Polyneuridine, PODD, β-Sitosterol, and POD were purchased from Sigma Chemical Company (St. Louis, MO, USA). Ethyl acetate, ethanol, methanol, ASA, FeCl3 · 6H2O, and HCl were purchased from Guangfu Chemical Research Institute (Tianjin, P.R.China). UV-1810 (Beijing Persee General Instrument Co., Ltd, P.R.China) and Trace DSQ GC-MS (American Finnigan Company, USA) were used.
Preparation of extracts
Rhizome and petiole were washed, dried and grinded to powder. Then the powder was weighted (20.00 g) and soaked in ethyl acetate and ethanol (500 mg/mL) for 10 months at room temperature in dark, and was then filtered, evaporated and condensed to dryness under nitrogen to obtain extracts.
Although there are numerous methods for determining the antioxidant capacity of soluble natural extracts and insoluble food components , no perfect system is available to help us know the “true” antioxidant capacity of a complex medium [13, 14]. The DPPH and FRAP assays, despite their disadvantages, are still used by many researchers for rapid evaluation of antioxidant .
The free radical scavenging activity (FRSA) of DPPH was measured according to Ramadan et al.  and Nencini et al. . which is one of the few stable and commercially available organic nitrogen radical assays [18, 19]. Foti et al.  suggested it is an electron transfer reaction. The initial electron transfer occurs very quickly, while the subsequent hydrogen transfer occurs more slowly and depends on the hydrogen-bond accepting solvent [18, 19]. This reaction has been measured by the decoloration assay where DPPH has an absorption band at 515 nm which disappears upon reduction by an antiradical compound [18, 21]. The specific steps are as follows.
The extracts were diluted with 15% aqueous ethanol with concentration of 10 mg/mL, and then 50 μL of the diluted extracts was added with 950 μL of 10−4
M DPPH methanol solution. Then the mixture was shaken and kept in dark for 30 min at room temperature. The decreased absorbance of DPPH solution was evaluated at 515 nm by a spectrophotometer UV-1018, and 500 μM 15% aqueous ethanol ASA was tested as a positive control. The test was carried out in triplicate, and the capability to scavenge the DPPH radicals was calculated as:
0 and A were the absorbance of DPPH without and with sample, respectively.
The FRAP test was first introduced by Benzie et al.  for measuring the total antioxidant activity, which was initially developed to assay plasma antioxidant capacity but can also be used on other fluids. In the FRAP test, reductants (antioxidants) in the sample reduce ferric-tripyridyltriazine complex (Fe3+- TPTZ), in stoichiometric excess, to a blue ferrous form (Fe2+), with an increase in absorbance at 593 nm . The specific steps were described by Tsao et al.  as follows.
The working FRAP reagent was prepared ex tempore by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 10 mmol/L TPTZ in 40 mmol/L HCl, and 20 mmol/L FeCl3 · 6H2O at 10:1:1 (v/v/v).
The 300μL FRAP reagent and the 10μL standard samples (FeSO4
O, 500 μM) or test samples (10 mg/mL 15% aqueous ethanol) were added and mixed well. The reaction temperature was 37°C and the absorbance readings were taken at 593 nm immediately and 4 min later using a spectrophotometer UV-1018. And 500 μM 15% aqueous ethanol ASA was tested as a positive control. All tests were carried out in triplicate. The FRAP value of the test samples was calculated on the basis of 500 μM Fe2+
O) as follows:
was the absorbance of the sample minus the absorbance of the blank at the 4th minute.
The samples were the rhizome extracts of ethyl acetate and ethanol (500 mg/mL). Trace DSQ GC-MS from American Finnigan Company was used. The GC conditions were: 1 N NOWAX quartz capillary column: 30 m × 0.32 cm × 0.25 mm; column temperature: 50–190°C; procedure temperature: 5°C/min; carrier gas: He; vaporizer temperature: 280°C; the MS conditions were: Ion source: EI; temperature: 200°C; ionizing voltage: 70 eV; electric current of collection: 300 μA; electric current of emission: 1 mA; resolution: 600; mass: 10–600.
Evaluation of antioxidant assays
Reagents PADE, BADE, Polyneuridine, PODD, β-Sitosterol, POD, and including the positive control ASA were dissolved in 15% aqueous ethanol and tested at the same concentration of 500 μM. Antioxidant capacity was expressed IC50 (μM) and FRAP (μM) and the test methods were mentioned above (as the DPPH and FRAP assays).
The results were presented as mean ± SD of triplicate determinations. Statistical analysis was performed by SPSS 11.5. One-way analysis of variance (ANOVA) was utilized to evaluate differences.