Ultrasonic-Assisted Extraction and Evaluation of Biological Activities of Flavonoids from Flemingia philippinensis Merr et Rolfe

Purpose: To develop a simple and rapid method for extracting total flavonoids from the roots of Flemingia philippinensis and to investigate the antioxidant and anti-tumor activities of the extracts of the materials from various locations in China. Methods: The total flavonoids in F. philippinensis were obtained by ultrasonic-assisted conventional solvent extraction method, and the extraction conditions were optimized by single factor and orthogonal test. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and anti-tumor activities, using 3-(4,5- dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, of the extract were evaluated. The contents of flemiphilippinin A, auriculasin, 5,7,3',4'-tetrahydroxy-6,8 –diprenylisoflavone and dorsmanins I were also determined. Results: Optimal extraction conditions were as follows: extraction time, 40 min; methanol concentration, 85 %; and solvent to solid ratio, 40 mL/g; and number of extraction, once. Total flavonoid content varied greatly (3.7 - 14.35 %) among the 19 samples collected from different origins in China. Flemiphilippin A, 5,7,3',4'- tetrahydroxy-6,8 -diprenylisoflavone, auriculasin and dorsmanins I showed varying DPPH radical scavenging activities with effective half maximal concentration (EC50) of 18.36, 23.59, 57.25 and 63.54 μg/mL, respectively.  Flemiphilippinin A (5 μg/mL) also exhibited some level of antitumor activity against human hepatocellular carcinoma cell (BEL-7402), human lung epithelial (A-549) and human ileocecal adenocarcinoma cell (HCT-8) with inhibition of 91.13 ± 1.6, 91.22 ± 3.23, and 79.77 ± 3.57 %, respectively. Conclusion: Total flavonoids can be extracted efficiently from F.  philippinensis by ultrasonic-assisted extraction method. Flemiphilippinin A has a potential for use in medicine as an antioxidant and antitumor drug.


INTRODUCTION
Flemingia philippinensis Merr. et Rolfe, which belongs to the plant family Leguminosae, is mainly distributed in tropical areas of China. The traditional usage of the roots of F. philippinensis is for the treatment of rheumatism, arthropathy, leucorrhea, menalgia, menopausal syndrome, chronic nephritis, improving bone mineral density [1][2][3]. Modern pharmacological studies show that the extract from the roots of F. philippinensis exhibits anti-oxidative and anti-inflammatory activities due to its isoflavones [4]. The estrogenic and antiestrogenic bioactivities of the methanol extract were confirmed by the effects on the proliferation of MCF-7 cells and induction of β-galactosidase activity [5]. Flavonoids are the main constituents of F. philippinensis [3,[6][7][8][9][10][11][12], and many of them have pharmacological activities. Total flavonoids can be used to evaluate the quality of the herb [13,14].
To the best of our knowledge, there are no reports on total flavonoids content to evaluate the quality of the herb F. philippinensis and explore the antitumor activity. In this study, ultrasonicassisted extraction of total flavonoids and antioxidant and antitumor activity of the flavonoids isolated from F. philippinensis were carried out.  [8][9][10].

EXPERIMENTAL Chemicals and apparatus
All the cell lines were purchased from the Cell Culture Center of Institute of Basic Medical Science, Chinese Academy of Medical Sciences. 1,1-diphenyl-β-picrylhydrazyl (DPPH) and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide were purchased from Sigma Chemical Co. while Medium RPMI-1640 was purchased from Gibico Co. All other reagents were of analytical grade.

Total flavonoid extraction
The roots were dried at < 60 o C in an oven, powered to 40-mesh, weighed (0.5027 g) and placed in a 25 mL volumetric flask to which methanol was added to make up to volume. The flask was sealed, shaken well, and then extracted ultrasonically. The flavonoid content of the filtrate was determined spectrophotometrically at 258 nm (UV-1700 UV spectrophotometer, Shimadzu Corporation, Japan). The flavonoid content was calculated with reference to genistein standard. The flavonoid yield (Y) was defined as the ratio of total flavonoid in the filtrate to total flavonoid in raw material.

Extraction design
The effect of sonication time (A), methanol concentration (B), solvent volume (C) and extraction times (D) on the yield of total flavonoids were investigated.
A three-level, four-factor, orthogonal design was employed, in which 9 experiments (L 9 3 4 ) were involved, and the flavonoid yield (Y) was used as response in evaluating the extraction ( Table 2). The factors and levels studied were determined based on the single factor experiments, such as A (20, 40 and 60 min), B (70, 85 and 100 %), C (10, 20 and 30 mL) and D (1, 2 and 3 times).

DPPH radical scavenging activity of extract
DPPH radical scavenging activity was measured according to previous studies method [15]. Methanol solution of DPPH 2 mL of 0.2 mM radicals was incubated with a series of different concentrations of the extract or flavonoids. The absorbance of the mixture was measured at 517 nm after 30 min of incubation at room temperature in the dark. Ascorbic acid (Vc) was used as the control and methanol as the blank.
The scavenging effect was calculated using equation 1.
where AS and AB stand for absorbance obtained for a sample (the extract, flavonoid or Vc) and the blank, respectively. DPPH radical scavenging activity was plotted against sample concentration to obtain the EC 50 , which represents concentration of extract required to scavenge 50 % of DPPH radicals.

Antitumor test
Human hepatoma cell line BEL-7402, human lung carcinoma cell line A-549 and human colorectal cancer cell line HCT-8 were cultured in RPMI-1640 medium with 10 % new-born bovine serum (NBS), and incubated at 37 o C in a humidified atmosphere that contained 5 % CO 2 .
For the experiment, cells were seeded into 96well plates at a density of 5 × 10 3 cells/well. After 24 h, triplicate wells were treated with media and agents. Fluorouracil (5-FU) was used as positive control. After 72 h incubation at 37 o C in 5 % CO 2 , the drug-containing medium was removed and replaced by 100 μL fresh medium with 0.5 mg/mL MTT solution. After 4 h incubation, the medium with MTT was removed and 200 μL DMSO was added to each well. The plates were gently agitated until the color reaction was uniform and the OD570 was determined using a microplate reader (Wellscan MK3, Lab systems Dragon).

Statistical analysis
Each experimental value is expressed as the mean ± standard deviation (n = 3). Statistical analysis was performed using Graphpad prism 5.0 and data analyzed using one way analysis of variance (ANOVA) for comparison between groups followed by Dunnett's multiple comparison test at a significant level of p < 0.05.

Optimization of extraction conditions
As shown in Figure 1, the optimal conditions were as follows: sonication time, 40 min; methanol concentration, 85 %; solvent volume, 20 mL and number of extraction, once for 0.5 g root powder.
The results of orthogonal design experiments were shown in Table 2. In order to keep consistent with single factor test, the extraction conditions were optimized as follows: sonication time, 40 min; methanol concentration 85 %; solvent volume was 20 mL and number of extraction, once, at room temperature for 0.5 g of sample no. 4. The effect of the factors was in the rank order: B > A > D > C in the ranges studied.

Total flavonoid content
The total flavonoid content in 19 samples were determined by calibration curve of genistein (Y = 0.1726X-0.0756 with r 2 ＝0.9996) and repeated three times for each under the optimal conditions. The results in Table 3 showed that the total flavonoid content were of great difference  between 19 samples in which the lowest was 3.75 % from Yunnan and the highest was 14.35 % from Daoxian Hunan. In addition, the content of total flavonoids in the xylem was different from that in phloem, the result showed that total flavonoids in xylem was much more than in phloem.

Antioxidant activity
The antioxidant activity of 85 % methanol extract of F. philippinensis (specimen nos. 1 -19) and flavonoid compounds isolated from Zhongshan guangxi sample is reported in Figure 2.
The extracts of 19 samples exhibited DPPH radical scavenging activities in the range 12.20 ± 1.41 % to 42.69 ± 1.07 % at concentration of 25 µg dry matter/mL, which suggested that the activities remarkably consisted with the content of their total flavonoids extracted under the optimal condition.

Antitumor activity
The results for antitumor activities are presented in Table 3.
Flemiphilippinin A exhibited significant cytotoxic activity to three human cancer cell lines (HCT-8, A-549 and BEL-7402) in vitro, while three others showed no inhibitory activity against the three cancer cell lines. The antitumor activity of flemiphilippinin A against the three human cancer cell lines were quantitatively assessed further which was indicated in terms of IC 50 (ug/mL) values, which tested concentration were 0.005, 0.05, 0.5, 5 ug/mL. The results showed that flemiphilippinin A exhibited a potent inhibitory effects on the indicated cell lines, especially for HCT-8, with an IC 50 value of 1.17, which higher than the positive control 5-FU with IC 50 value 1.4. The IC 50 values of flemiphilippinin A inhibiting on A-549 and BEL-7402 were more than 5.0, which were higher than those of 5-FU, respectively, with IC 50 value of 1.8 and 0.5.

DISCUSSION
In the present study, total flavonoid was extracted and analyzed antioxidant activity using scavenging DPPH radical activity. The antioxidant and antitumor activities of the four   Ultrasonic-assisted extraction is a common and valid method to extract phytochemical compounds, including volatile constituents [16], flavonoid [17] and polysaccharides [18], etc. The orthogonal test design is a method that utilizes the orthogonal experiment tables to analyze experiment results scientifically. It provides a reasonable amount of information for testing the optimal combination from a fewer number of assays, therefore reducing more experiments associated with the analysis [19]. Under the optical extraction condition optimized with orthogonal design (L 9 3 4 ), total flavonoids concentration was in agreement with those evaluated by HPLC [20].
In the main peaks of the fingerprints, 14 peaks were identified by comparison of their retention times and UV absorption spectra with those of standard compounds which were separated from F. philippinensis. The identified peaks belonged to flavonoid. From the HPLC results, the most flavonoid compounds in this material were extracted under these extraction conditions. The total content of each flavonoid in the fingerprint was consistent with the result for total flavonoids. Therefore, using this method to extract and analyze total flavonoids in F. philippinensis is rational and feasible.
A preliminary study on antioxidant and antitumor activities of twelve flavonoids, isolated from F. philippinensis, suggested that there are only four flavonoids with antioxidant activities and one with antitumor activities. The results showed that the prenylated flavonoids (eg. flemiphilippinin A) had higher antioxidant activity than the prenylated ones (e.g., erythrinin B and genistin), this may be related to the hydroxyl number and threedimensional structure of the molecule, which can easily capture the free radicals caused by steric hindrance effect due to different prenyl groups. The underlying molecular phenomena of the radical scavenging activity of flavonoids can be explained by the ease of hydrogen atom abstraction and the ease of the termination of the flavonoid aroxyl radicals [21].

CONCLUSION
Among the materials tested, the plant material from Daoxian Hunan demonstrated the strongest radical scavenging, as well as the highest flavonoid contents. Flemiphilippinin A has high antioxidant and antitumor activities at low concentrations. The findings of this work also indicate that F. philippinensis is a potential new source of natural antioxidants and that flemiphilippinin A can be developed as an antioxidant and antitumor agent with great commercial prospects in the pharmaceuticals industry.