Mounting evidence has shown that overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including superoxide anion and hydroxyl radicals, hydrogen peroxide, nitric oxide, nitrosonium, nitroxyl anion and peroxynitrite, can cause DNA damage resulting in cell malfunction [1], and this ultimately leads to cancer due accumulation of somatic mutations [2]. Furthermore, uncontrolled cell proliferation would eventually result in a net increase in the number of cancer cells, which is strongly associated with the development and progression of cancer [3]. In this respect, removing ROS/RNS generated in excess and restraining cancer cell proliferation are considered an effective means to fight cancer [4].

Recently, a great concern has been focused on searching for potential anticancer agents of natural origin [5]. Flavonoids, ubiquitously distributed in plants, are plant-derived polyphenolic molecules that are well known as effective antioxidants, preventing oxidative stress induced by ROS/RNS [6]. Moreover, the growing epidemiologic evidence has demonstrated that flavonoid consumption is associated with a decreased risk of colorectal cancer, which has recently been documented by a large case-control study [7]. Hitherto, a number of flavonoids, exemplified by isoquercitrin [8], quercetin [9], and catechin [10] have been shown to exert anti-colorectal cancer activity by inhibiting cell proliferation.

Bidens pilosa Linn. var. radiata Sch. Bip., belonging to Asteraceae family, originated from South America and can be found in almost all countries from tropical and subtropical regions [11]. And there are three common variants of this species: Bidens pilosa Linn. var. radiata Sch. Bip., Biden pilosa L. var. pilosa and Bidens pilosa L. var. minor [12]. They are usually employed in traditional medicines as a remedy to treat various diseases, such as hepatitis, stomach disorders, diabetes, hypertension, malaria, inflammation and digestive disorders [13]. Although Bidens species have been studied a lot to our best knowledge, the chemical constituents and their bioactivities from this variant remain largely unknown.

Recently, several compounds have been first isolated from this plant in our previous work, including flavonoids, flavonoid glycosides, phenols and phenylpropanoids [14]. Moreover, 3, 3′-dimethylquercetin was documented to exhibit significant cytotoxicity and apoptosis-inducing activity against RKO cells [15]. As part of our ongoing research on chemical constituents, a total of 6 compounds were successively obtained, mainly flavonoids or flavonoid glycosides. They were further evaluated for the antioxidant activity by using two complementary systems, namely DPPH and ABTS free radical scavenging assays, as well as their growth inhibitory effect against human colon cancer cell lines RKO by the MTT method.

Chemicals and reagents

Methanol, ethanol, petroleum ether, ethyl acetate, n-butylalcohol and potassium persulfate were purchased from Sinopharm Chemical Reagent, Co., Ltd. (Shanghai, China). 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), trypsin, and penicillin–streptomycin were obtained from Hyclone Laboratories, INC. (Logan, UT, US). The normal phase silica gel (200-300 mesh) was obtained from Qingdao Marine Chemical Co., China. Sephadex LH-20 (20-100 μm) was obtained from Pharmacia Fine Chemical Co., Ltd, Uppsala, Sweden and RP-18 silica gel was from Merck Co., Germany. Spectrophotometric measurements were performed on an Ultraviolet–visible spectrometer (Shimadzu UV-2008; Japan). All other chemicals were of analytical grade.

Plant material

The whole plant of BP was collected in Fujian province, China, in August 2009 and authenticated by Associate Professor Chengzi Yang, a taxonomist at the Department of Pharmacy, and a voucher specimen (no. 2009-BP) was deposited in the herbarium of Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, China.

Extraction and isolation

The whole plant material (9.0 kg) was dried at room temperature, powdered, and refluxed with 75 % ethanol for 2 × 3 h to obtain total extract (1033.0 g). The alcohol was removed under vacuum using a rotary evaporator. The residue was suspended in water, and partitioned sequentially with petroleum ether, EtOAc and n-BuOH to obtain a petroleum ether fraction, ethyl acetate fraction (EE-BP), n-BuOH fraction and the remainder, the water fraction. EE-BP was evaporated in vacuo. EE-BP (90.0 g) was subjected to column chromatography over silica gel (200–300 mesh) developed with petroleum ether–acetone (50:1–1:1). The flavonoid compounds were isolated and purified from the petroleum ether–acetone (3:1) fraction by repeated column chromatography including silica gel, Sephadex LH-20 and ODS-C18 column. TLC was detected with 10 % H₂SO₄-EtOH solution. Melting points were determined on X-4 apparatus and uncorrected. ¹H and ¹³C NMR spectra were recorded with TMS as internal standard on a Bruker AvanceTM 400 MHz. ESI-MS were recorded on a Bruker ApexⅡmicro mass spectrometer.

Determination of DPPH radical scavenging activity

The antioxidant activity of the six compounds was measured on the basis of the scavenging activities of stable DPPH free radical [16]. The values of IC₅₀, concentration of sample required to scavenge 50 % of free radicals were calculated from the regression equation, and percentage inhibition of free radical formation was assayed. BHT was used as positive control. All tests were carried out in triplicate. DPPH radical-scavenging activity (D) was calculated as in Eq 1.

D (%) = {(Ac – As)/Ac}100 …………………. (1)

where Ac and As are the absorbance of control and test samples, respectively.

ABTS radical scavenging assay

The radical scavenging activity of the six compounds against ABTS radical cation was measured using the method of Re et al [17] with some modifications. ABTS was dissolved in water to a concentration of 7 mmol/L. ABTS radical cation was produced by reacting ABTS stock solution with 2.45 mmol/L potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The ABTS radical cation solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm and equilibrated at 30 °C. An aliquot of each sample (0.1 mL) was mixed with 3.9 mL of diluted ABTS radical cation solution. The absorbance at 734 nm of the mixture was measured after reaction for 6 min. The ability of individual compound to scavenge ABTS radicals (%) and the IC₅₀ were calculated as described in the DPPH radical scavenging assay. BHT was used as positive control.

MTT assay

The human colon cancer RKO cell lines, used for the antitumor activity assay, were obtained from the American type culture collection (ATCC, Manassas, VA, USA), and they were cultured in DMEM supplemented with 10 % FBS, 100 units/ml penicillin, and 100 μg/mL streptomycin in a 37 °C humidified incubator with 5 % CO₂ atmosphere. The anti-proliferative effect of isolated compounds was measured using the standard MTT assay [18]. In brief, exponentially growing cells (about 1 × 10⁵ cells/well) were plated into 96-well plates and allowed to adhere for 24 h prior to extract addition. The extract was dissolved in 0.1 % DMSO then diluted with the medium and filtered using 0.22 μm syringe filters. The cells were then exposed to different concentrations of extract. The cells in the control wells received medium containing the same volume of DMSO (0.1 %). After the incubation, 20 μL of MTT reagent (5 mg/mL in PBS) was added and cells were incubated for an additional 4 h. The formazan produced by the viable cells was solubilized by addition of 100 μL DMSO. The suspension was placed on a micro-vibrator for 10 - 15 min and absorbance was recorded at 570 nm with the ELISA reader (BioTek ELx800, Winooski, VT, USA). The experiment was performed in triplicate. Growth inhibition (G) was calculated with respect to vehicle control as in Eq 2.

G (%) = {(Ac – At)/(Ac – Ab)}100 …………. (2)

where Ac, At and Ab are the absorbance of control and test and blank samples, respectively.

Statistical analysis

All data are presented as mean ± standard deviation (SD, n = 3). Statistical comparison was performed via one-way analysis of variance followed by Dunnett’s test using SPSS Software version18.0 (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.

Isolation and identification of compounds

By using various column chromatography purification methods, a total of 6 flavonoid compounds were eventually isolated and purified ().  They are flavonoids or flavonoid glycosides, identified and listed as follows:

(1)        Isoquercitrin, C₂₁H₂₀O₁₂, yellow needle crystal. mp: 225～227 °C, ESI-MS m/z 463.0 [M-H]-, ¹H NMR (400 MHz, DMSO-d6, δ, ppm, J/Hz). d: 12.65 (1H, s , 5-OH), 9.35 (2H, brs, 2×OH) , 7.56 (2H, d, J=8.1, H-2’ and H-6’), 6.81 (1H, dd, J=3.6, J=8.5, H-5’), 6.41 (1H, d, J=1.6, H-8), 6.21 (1H, d, J=2.1, H-6), 5.46 (1H, d, J=7.2, H-1’’); 13C-NMR (100 MHz, DMSO-d6, δ, J/Hz): 177.86 (C-4), 164.66 (C-7), 161.69(C-5), 156.78 (C-9), 156.61 (C-2), 148.92 (C-4’), 145.27 (C-3’), 133.75 (C-3), 122.06(C-1’), 121.61 (C-6’), 116.64 (C-5’), 115.66 (C-2’), 104.40 (C-10), 101.30 (C-6), 93.97(C-8), 78.03 (C-3’’), 76.95 (C-3’’), 74.54 (C-2’’), 70.38 (C-4’’), 61.21 (C-6’’) [19].

(2)        Vitexin, C₂₁H₂₀O₁₀, yellow needle crystal. mp: 258～259 °C, ESI-MS m/z 433[M+H]+; ¹H NMR (400 MHz, MeOD, δ, ppm, J/Hz): d: 7.61 (1H, d, J=8.0 Hz, H-6’), 7.54 (1H, s, H-2’), 6. 85 (1H, d, J = 8.1 Hz, H-5’), 6.71 (1H, s, H-3), 6.30 (1H, s, H-6), 4.62 (1H, d, J = 8.6 Hz, H-1’’); 13C NMR (100 MHz, MeOD, δ, J/Hz ): 182.0 ( C-4), 167. 2 (C-2), 162. 7 (C-7), 161.2 (C-5), 160.2 (C-4’), 156.1 (C-9), 132.3 (C-2’), 132.0 (C-6’), 120.5 ( C-1’), 114.9 (C-3’), 114.3 (C-5’), 104.3 ( C-8), 103.5 ( C-10) , 102.3 (C-3 ), 99.2 (C-6 ), 84.9 (C-5’’), 78.9 (C-3’’), 78.2 (C-1’’ ), 70.9 (C-2’’), 70.0 (C-4’’), 60. 8 (C-6’’) [20].

(3)        Astragalin, C₂₁H₂₀O₁₁, yellow powder. mp: 163～165 °C, ESI-MS m/z 449 [M+H]+; ¹H NMR (500 MHz, DMSO-d6, δ, ppm, J/Hz) d: 12.39 (1H, s, -OH), 10.94 (1H, s, -OH), 10.35 (1H, s, -OH),8.05 (2H, d, J=8.0 Hz, H-2’ and H-6’), 6.89 (2H, d, J=8.0 Hz, H-3’ and H-5’), 6.45 (1H, s, H-8), 6.22 (1H, s, H-6), 5.45 (1H, d, J=7.5 Hz, H-1”), 5.32~3.09 (10H, H-2”~6”, 4-OH). 13C NMR (DMSO-d 6, 125 MHz) δ, ppm, 177.8 (C-4), 164.1 (C-7), 161.4 (C-5), 159.5 (C-9), 156.3 (C-4’), 156.2 (C-2), 133.22(C-3), 130.8 (C-2’ and C-6’), 120.2 (C-1’), 115.1 (C-3’ and C-5’), 104.3 (C-1”), 100.9 (C-10), 98.9 (C-6), 93.5 (C-8), 77.4 (C-2”), 76.4 (C-5”), 74.3 (C-4”), 69.2 (C-3”), 60.6 (C-6”) [21].

(4)        5,6,7,4'-tetramethoxylflavone, C₁₉H₁₈O₆, yellow powder. mp: 142～143 °C, ¹H NMR (400 MHz, MeOD, d, ppm, J/Hz). d: 7.95 (2H, dd, J=8.1, 2.1 Hz, H-6’ and H-2’), 7.12(2H, dd, J=8.1, 2.3 Hz, H-5’ and H-3’), 7.06 (1H,s, H-3), 6.62 (1H, s, H-8), 3.92 (3H, s, 5- OCH3), 3.78 (3H, s, 7- OCH3), 3.41 (3H, s, 6-OCH3), 3.01 (3H, s, 4’-OCH3) [22].

(5)        5,3',4'-trihydroxy-3,7-dimethoxylfla- vone, C₁₇H₁₄O₇, yellow powder. mp: 218～220 °C, ¹H NMR (MeOD, 400 MHz, J/Hz). d (ppm) 7.59 (1H, dd, J=8.5, 2.1 Hz, H-6’), 7.05 (1H, d, J=8.5 Hz, H-5’), 6.41(1H, d, J=1.8 Hz, H-6), 6.18 (1H,d, J=1.8 Hz, H-8), 3.91 (3H, s, 7- OCH3), 3.76 (3H, s, 4’-OCH3) [23].

(6)        Quercetin, C₁₅H₁₀O₇, yellow powder, mp: 313～314 °C, ¹H NMR (400 MHz, MeOD, d, ppm, J/Hz). d: 7.72 (1H, d, J=2.1 Hz, H-2’), 7.65(1H, dd, J=8.5, 2.1 Hz, H-6’), 6.87 (1H, d, J=8.3 Hz, H-5’), 6.38 (1H, d, J=2.0 Hz, H-8), 6.18 (1H, d, J=2.0 Hz, H-6) [24].

DPPH and ABTS free radical scavenging effect

The isolated flavonoids or flavonoid glycosides were evaluated for their antioxidant effect using DPPH and ABTS free radical scavenging methods DPPH-radical scavenging activity is a rapid means used to compare the antioxidant capacity of different natural compounds. When treated with the isolated compounds, DPPH solution, initially deep violet in color, turned pale yellow in the presence of non-radical form of DPPH-H [25]. As seen in , compounds 1, 5 and 6 showed significant DPPH scavenging activity, with IC₅₀ values of 15.2 μmol/L, 43.1 μmol/L and 10.7 μmol/L, respectively. All of them were found to be more efficient than the positive control BHT, with IC₅₀ value of 58.6 μmol/L. However, compound 3 exerted moderate DPPH scavenging activity, with IC₅₀ value of 199.8 μmol/L. Furthermore, compounds 2 and 4 were documented to present the lowest scavenging activity. Their radical scavenging ability decreased in the following decreasing order: 6 > 1 > 5 > BHT > 3 > 2 and 4.

ABTS is converted to its radical cation (ABTS^•+) by reacting with a strong oxidizing agent (e.g., potassium permanganate, potassium persulfate). This radical cation is blue in color and absorbs light at 734 nm, which will be converted back to its colorless neutral form by hydrogen-donating antioxidant [26]. The inhibitive efficiency of ABTS^•+ by the assayed compounds is presented in . Our data showed that quercetin (compound 6) was the most effective in ABTS free radical-scavenging assay with IC₅₀ value of 17.5 μmol/L. Nevertheless, ABTS radical-scavenging activity of compound 3 was lower than BHT. Moreover, compounds 2 and 4 were also found to show the weakest scavenging activity. The radical scavenging ability decreased in the order: 6 > 1> 5≈BHT>3 > 2 and 4.

Anti-proliferative activity against RKO cells

MTT assay was conducted to evaluate the growth inhibitory effect of the compounds on the cell viability of human colon cancer cell line RKO. As provided in A, various concentrations (50, 100, 200 and 400 μmol/L) of compounds 1, 2, 3 and 6 exhibited a significant cytotoxic effect against RKO cells in a dose-dependent manner for 24 h (p < 0.05). The IC₅₀ values of the compounds 3 and 6 were 265.34 μmol/L and  452.48 μmol/L, respectively. However, lower cytotoxicity was observed in compounds 1 and 2 (both their IC₅₀ values were > 500 μmol/L). Compared with the four aforementioned compounds, compound 4 and 5 were shown to display more potent anti-proliferative activity, with IC₅₀ values of 39.08 and 17.68 μmol/L, respectively (p < 0.01), as shown in B. Taken together, our result suggested that the flavonoid compounds contained in BP were partially responsible for its biological/ pharmacological activities.

BP, as a rich source of flavonoids, is used to treat periappendicular abscess in traditional Chinese medicine, which is also employed as a folk medicine and a major ingredient of herbal tea in Taiwan believed to be able to prevent inflammation and cancer [15]. Recently, our consecutive phytochemical investigation led to 6 flavonoid compounds from this plant, including isoquercitrin (1), vitexin (2), astragalin (3), 5,6,7,4'-tetramethoxylflavone (4), 5,3',4'-trihydroxy-3,7-dimethoxylflavone (5) and quercetin (6), whose chemical structures are presented in .

The antioxidant capacities of these isolated compounds had been evaluated by using DPPH and ABTS free radical scavenging assays. The result from antioxidant evaluation indicated that quercetin was the most effective in both DPPH (IC₅₀ value of 10.7 μmol/L) and ABTS (IC50 value of 17.5 μmol/L) free radical-scavenging assays.

However, vitexin (2) and 5,6,7,4'-tetramethoxylflavone (4) displayed low radical-scavenging activity, with maximum level of inhibition below 50 %, as seen in . It was interesting to reveal the structure-activity relationship. Flavonoids exhibit their antioxidative functions through two major mechanisms viz. hydrogen atom transferring and electron donation [27]. The antioxidant activity of flavonoids is due to their molecular structure. The number and position of hydroxyl groups on the B and A rings are the key factors for the antioxidant activity. As to the C-glycoside, with glucose at C-6 or C-8 the antioxidant activity decreases due to steric hindrance by the glucose moiety [28]. From this point of view, the absence of hydroxyl groups in 5, 6, 7, 4'-tetramethoxylflavone (4) and the glucose at C-8 of vitexin (2) were responsible for the low radical scavenging activity, respectively. Moreover, the presence of an ortho-dihydroxy (3`, 4`-di OH) moiety on the B ring improve the radicals scavenging activity and the antioxidant potential of the flavonoids due to hydrogen bonding and electron delocalization which have influence on stabilization of the flavonoid phenoxyl radicals [29]. Therefore, the high contribution of an ortho-dihydroxy moiety on the B ring of isoquercitrin (1), 5,3',4'-trihydroxy-3,7-dimethoxylflavone (5) and quercetin (6) led to their significant antioxidant activity. Additionally, it was reported that the presence of a saccharide group at C-3 in the C ring had little influence on antioxidant activity [30]. Hence, astragalin (2) demonstrated relatively lower effect mainly due to the lack of an ortho-dihydroxy moiety.

The anti-proliferative activity of the isolated flavonoids was evaluated against RKO cells by using MTT method. Various concentrations (50, 100, 200 and 400 μmol/L) of isoquercitrin (1), vitexin (2), astragalin (3), and quercetin (6) exhibited a significant cytotoxic effect against RKO cells in a dose-dependent manner for 24 h. But all of them were much less effective than 5, 6, 7, 4'-tetramethoxylflavone (4) and 5, 3', 4'-trihydroxy-3, 7-dimethoxylflavone (5), with IC₅₀ value of 39.08 μmol/L and 17.68 μmol/L, respectively (p < 0.01). There is increasing evidence that methoxylated flavones have chemopreventive properties superior to the common unmethylated flavonoids or polyphenols. For example, 5, 7, 4′-trimethoxyflavone was about eight times more potent than apigenin [31], which is in agreement with our previous findings that 3, 3′-dimethylquercetin exhibited much more cytotoxicity than quercetin [15]. Thus, 5, 6, 7, 4'-tetramethoxylflavone and 5, 3', 4'-trihydroxy-3,7-dimethoxylflavone displayed more remarkable growth inhibitory effect than the other four flavonoids in our study.

BP is a noteworthy material containing a series of bioactive flavonoid compounds, thus suggesting that it can be utilized as a potential health supplement and an easily available source of natural antioxidants, as well as an effective material in pharmaceutical applications.