2.2. Extraction and Purification of Andrographolide

The dry leaves of A. paniculata were mixed with 95% aqueous ethanol solution and then ultrasound extracted twice at 45°C for 1 h. Then, the ethanol extracts were combined, filtered, and concentrated. The crude mixture was reextracted with petroleum ether twice and followed by ethyl acetate extraction. The residue was obtained after the evaporation of ethyl acetate portion and used for high-speed countercurrent chromatography (HSCCC, TBE-300A, Tauto Biotechnique Company, Shanghai, China) separation and HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, USA) analysis [21]. The purity of andrographolide was 98.8%.

The structure of andrographolide was identified by NMR. Andrographolide was dissolved in 0.5 mL of CD₃OD. The experiment was performed on a Bruker AVANCE™ III spectrometer (14.1 Tesla), with a Larmor frequency of 150 MHz for ¹³C and 600 MHz for ¹H. ¹H NMR of andrographolide (CD₃OD, 600 MHz) is as follows: δ 6.86-6.88 (m, 1H), 5.03 (d, J = 6.0 Hz, 1H), 4.91 (d, J = 6.0 Hz, 1H), 4.69 (s, J = 6.0 Hz, 1H), 4.49 (dd, J = 10.2 Hz, 6.6 Hz, 1H), 4.18 (dd, J = 10.2 Hz, 1.8 Hz, 1H), 4.14 (d, J = 10.8 Hz, 1H), 3.38-3.44 (m, 2H), 2.57-2.68 (m, 2H), 2.43-2.47 (m, 1H), 2.03-2.08 (m, 1H), 1.94-1.96 (m, 1H), 1.84-1.89 (m, 2H), 1.80-1.83 (m, 2H), 1.36-1.43 (m, 1H), 1.30-1.35 (m, 2H), 1.24 (s, 3H), and 0.77 (s, 3H). ¹³C NMR of andrographolide (CD₃OD, 150 MHz) is as follows: 172.6, 149.4, 148.8, 129.8, 109.2, 80.9, 76.1, 66.7, 65.0, 57.4, 56.3, 43.7, 40.0, 39.0, 38.1, 29.0, 25.7, 25.2, 23.4, and 15.5.