Apigenin

High performance liquid chromatography was performed by using Agilent Chem station Rev. B.02-01-SR1 (260) and Agilent 1200 series binary gradient pump coupled with diode array detector (DAD; Agilent technologies, Germany). Reverse phase chromatographic analysis was carried out with a Zorbex-C8 analytical column (4.6 × 250 mm, 5 μm particle size, Agilent, USA), injection volume 20 μl and the gradient elution was conducted according to the method previously described with minor modifications [7 ]. Mobile phase consisted of acetonitrile-methanol–water-acetic acid in a ratio of 5:10:85:1 (solvent A) and acetonitrile-methanol-acetic acid in a ratio of 40:60:1(solvent B). Gradient method was 0–20 min for 0–50 % B, 20–25 min for 50–100 % B and then isocratic 100 % B till 30 min. Flow rate was maintained at 1 ml/min. Stock solutions of various phenolic standards i.e., phenolic acid (gallic acid), flavan-3-ol (catechin), flavonol flavonoids (quercetin, myricetin, kaempferol), hydroxycinnamate (caffeic acid), flavone aglycone (apigenin) and flavonol glycoside (rutin) were prepared in methanol and subsequently diluted to get a final concentration of 10, 20, 50, 100, 200 μg/ml of methanol. The data for peak area versus standard concentration was used to construct the calibration curve, the correlations were found to be significant at 0.05 level, results of which are summarized in Table 2. The respective limit of detection (LOD) and limit of quantification (LOQ) as determined by linear regression analysis of the calibration curve were calculated by using the expression 3.3 * (σ/b) and 10 * (σ/b) respectively where;
σ = Standard deviation of response
b = Slope of calibration curve
Prior to use, standard solutions, samples and mobile phases were all degassed and filtered through 0.45 μm membrane filter (Millipore). The absorption of samples was recorded at 257nm (rutin), 279 nm (gallic acid, catechin), 325 nm (caffeic acid, apigenin) and 368 nm (myricetin, quercetin and kaempferol).
Chromatographic operation was carried out at ambient temperature and in triplicate. Before starting the next analysis column was reconditioned for 10 min and the results were expressed as mg/g DW. Comparison of retention time and UV absorption spectra of extracts with those of standards was done for the identification of compounds.

E. coli DH5α and C41(DE3) were used as hosts for DNA cloning and whole-cell biotransformation, respectively. Primer STAR HS DNA polymerase and restriction endonucleases were obtained from Takara (Dalian, China). DNA and genomic DNA Extraction Kits were obtained from Thermo Scientific (Waltham, United States) and TIANGEN (Beijing, China), respectively. The plasmid miniprep purification kit was purchased from Sangon Biotech (Shanghai, China). Oligonucleotide synthesis and sequence analysis were achieved by Sangon Biotech (Shanghai, China). ALA and hemin were purchased from Sigma-Aldrich (St. Louis, MO, United States). Naringenin, eriodictyol, dihydrokaempferol, dihydroquercetin, kaempferol, quercetin, apigenin, luteolin, daidzein, and 7,3′,4′-trihydroxyisoflavone were purchased from Yuanye Bio-Technology (Shanghai, China). Other chemicals were purchased from Sangon Biotech (Shanghai, China) and were of the highest commercial grade available.