Two New Flavones from Tridax procumbens Linn

Two new flavones, 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β-D-glucopyranosyl flavone (1) and 6,8,3′-trihydroxy-3,7,4′-trimethoxyflavone (2) were isolated from Tridax procumbens Linn., together with the four known compounds puerarin (3), esculetin (4), oleanolic acid (5) and betulinic acid (6). The structures of the two new flavones were elucidated based on chemical analysis and spectral methods (IR, 1D and 2D NMR, ESI-MS, HR-ESI-MS). The antioxidant activity of the two new flavones were evaluated by two methods, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and ferric reducing antioxidant power (FRAP) assays, and the data showed that compounds 1 and 2 have certain antioxidant activity, with the antioxidant activity of compound 2 being stronger than that of compound 1.


Antioxidant activity
The antioxidant activities of compounds 1 and 2 were evaluated using the DPPH and FRAP assays. The DPPH assay has been widely used to test the free radical scavenging ability of plant components. In the present study, it was found that both the two new compounds had DPPH free-radical scavenging activity. The DPPH scavenging rates are shown in Figure 4(A), and the IC 50 values are shown in Table 2. From these data, it can be concluded that the sequence of antioxidant activity of these compounds was as follows: Trolox (reference) > compound 1 > compound 2. The FRAP assay can reflect the antioxidant activity through the variation of absorbance at a certain wavelength. The absorbances of the two new compounds are shown in Figure 4(B), and TEAC values are shown in Table 2. These TEAC values demonstrated that the sequence of reducing power of these compounds was as follows: Trolox > compound 1 > compound 2.

General
1D and 2D NMR spectra (chemical shifts in ppm, coupling constants in Hz) were recorded on a Bruker DPX 400 instrument. The 1 H NMR spectra were reported in delta (δ) units, parts per million (ppm) downfield from the internal standard. Coupling constants are reported in Hertz (Hz). ESI mass spectra were obtained on a Waters Q-Tof Micro TM mass spectrometer coupled to a Waters 2795 system that included an online photodiode array detector. HR-ESI-MS spectra were determined on a Waters Q-Tof MicroTM ESI-MS. The compounds were detected from their UV absorbance and by spraying on TLC plates with FeCl 3 -K 3 [Fe(SCN) 6 ] reagent.

Plant aaterial
The whole plants of Tridax procumbens was collected in Sanya city, Hainan Province, China, in September 2007. The plant was identified by Prof. Shi-man Huang of Hainan University. A voucher specimen of this plant (ZJFU-20070910-10) has been deposited in our laboratory.

Extraction and isolation [10]
Air-dried and powdered whole plants of Tridax procumbens (5 kg) was extracted with 70% EtOH   13 C-NMR and 2D experiments see Table 1.

Process of acid hydrolysis
The process of acid hydrolysis was: compound 1 (30 mg) was dissolved in 2% sulfuric acid aqueous solution (30 mL) in a 100 mL besker by heating of electric furnace. Solution was heated for one hour in boiling condition and then filtered. the filter residue was aglycone. The concentrated hydrolysate was identified by paper chromatography by D-glucose as authentic sample and B:A:W (4:1:5) as the solvent system. The sugars was identified as D-glucose. Aglycone was yellow powder, mp 235-236 °C.

DPPH and FRAP assays
3.6.1. DPPH assay The DPPH radical scavenging activity was evaluated according to the literature [11] with the slight modification. Briefly, each sample (100 µL of various concentrations) was added to ethanolic DPPH solution (100 µL, 2.5 × 10 -2 mg/mL). After mixing gently and standing at 24 °C for 30 min, the absorbances were measured at 530 nm using a VERSAmax microplate reader spectrophotometer. The percentage of DPPH which was scavenged was calculated using the following formula: Scavenging %= 1-(Ap-Ac) /Amax × 100%.
3.6.2. FRAP assay [12] FRAP assay was carried out in a microtiter plate. Each sample (80 μL) with appropriate dilution if necessary was added to FRAP reagent [13] (150 μL, 10 parts of 0.3 mol/L sodium acetate buffer at pH 3.7, 1 part of 0.01 mol/L TPTZ solution, and 1 part of 0.02 mol/L FeCl 3 . 6H 2 O solution), and 70% ethanol (80 μL) plus FRAP reagent (150 μL) was used as a blank, then the wavelength at 593 nm was read after 10 min. Fresh working solutions of Trolox were used for calibration, and the total antioxidant capacity were expressed as TEAC (Trolox Equivalent Antioxidant Capacity) values, it was calculated when the absorbency at 0.4 TEAC value means mg TE/mg flavone.

Conclusions
In our present study, two new flavones 1-2, together with four known compounds 3~6 were separated from the aerial parts of Tridax procumbens, and their structures were identified by chemical analysis and extensive spectral evidence, including IR, 1D and 2D NMR, ESI-MS, HR-ESI-MS data. The antioxidant activity of the two new flavones were evaluated by the DPPH and FRAP assays, and the data showed that compounds 1 and 2 have certain antioxidant activity. The antioxidant activity of compound 2 was weaker than that of Trolox, the positive control, but stronger than that of compound 1.