LAUSR

Panax vietnamensis Ha et Grushv. (Araliaceae) is a perennial endemic species of mountainous regions of Vietnam and northern China. A unique feature of the plant is a high content of triterpene glycosides, especially ocotillol derivatives [1]. Its distribution area is alarmingly decreasing because of its broad medicinal use. Therefore, studies on the development of alternate reproduction methods of P. vietnamensis are critical. Previously, root cells of P. vietnamensis were demonstrated by us to be capable of forming callus and suspension cultures that produced triterpene glycosides [2]. Phenolic compounds from the intact plant and its cell cultures have not been characterized although phenolic compounds from subterranean organs of P. vietnamensis and two types of cell cultures (callus and suspension) were studied by us during our research. Aerial organs of P. vietnamensis were collected in 2018 on Ngoc Linh Mountain, Quang Nam Province, Vietnam; 72% moisture. Callus culture of P. vietnamensis cells was produced from roots of intact plants using Murashige−Skoog medium [3] containing 2,4-diphenoxyacetic acid (0.5 mg/mL), α-naphthaleneacetic acid (1 mg/mL), and kinetin (0.05 mg/mL) and cultivation in Petri dishes in the dark at 26°C for three months (seven passages) [2]. Suspension culture of P. vietnamensis cells was produced in a bioreactor (20-L) from virulent calluses in the same medium without adding agar at 26°C in the dark. Column chromatography (CC) used polyamide, reversed (RP-SiO2) and normal-phase silica gel (SiO2), Sephadex LH-20, and cellulose (Sigma-Aldrich, St. Louis, MO, USA). Preparative HPLC used a Summit liquid chromatograph (Dionex, Sunnyvale, CA, USA) [4]; analytical HPLC, an LC-20 Prominence liquid chromatograph (Shimadzu, Columbia, MD, USA) [5]. Spectrophotometric studies used an SF-2000 spectrophotometer (OKB Spectr, St. Petersburg, Russia). Mass spectrometric studies used an LCMS-8050 TQ mass spectrometer (Shimadzu, Columbia, MD, USA) and the previously described conditions [6]. NMR spectra were recorded on a VXR 500S NMR spectrometer (Varian, Palo Alto, CA, USA). Roots of P. vietnamensis (2.5 kg) were ground in a blender. The resulting matter was extracted with EtOH (96%, 10 L, 2×) at 50°C in an ultrasonic bath (100 W, 35 kHz) for 3 h. The EtOH extract was filtered and evaporated to dryness under vacuum at 40°C. The dry solid (161 g) was suspended in H2O (1 L). The resulting suspension was extracted with CHCl3 and n-BuOH. The BuOH fraction (42 g) was placed onto polyamide (600 g) and eluted sequentially with H2O (fraction 1), EtOH (40%, fraction 2), EtOH (90%, fraction 3), and NH4OH solution (0.5%) in EtOH (90%) (fraction 4). Fraction 1 (1.4 g) was separated over RP-SiO2 (CC, 2 × 40 cm, H2O–MeCN eluent, 100:0→50:50) and Sephadex LH-20 (CC, 1 × 80 cm, 50% EtOH eluent) to afford astragalin (kaempferol-3-O-glucoside, 16 mg, 1) [7] and trifolin (kaempferol-3-O-galactoside, 10 mg, 2) [7]. Fraction 2 (0.8 g) was chromatographed over SiO2 (CC, 2 × 30 cm, EtOAc−Me2CO eluent, 90:10→70:30) and Sephadex LH-20 (CC, 1 × 80 cm, 70% EtOH eluent) to isolate dianthoside (maltol O-glucoside, 11 mg, 3) [8]. Fraction 3 (9.4 g) was separated using Sephadex LH-20 (CC, 3 × 80 cm, Me2CO−H2O eluent, 50:50→10:90), RP-SiO2 (CC, 1 × 40 cm, H2O−MeCN eluent, 100:0→30:70) and prep. HPLC [gradient mode (%B), 0−60 min, 5−30%; 60−90 min, 30−70%] to afford 5-O-caffeoylquinic acid (65 mg, 4) [9], 3-O-caffeoylquinic acid (20 mg, 5) [10], 5-O-feruloylquinic acid (15 mg, 6) [11], 3-O-feruloylquinic acid (8 mg, 7) [11], 3-O-feruloyl-5-O-caffeoylquinic acid (11 mg, 8) [12], and a mixture of 9−11 (108 mg). Compounds 9−11 were separated using CC over cellulose (4 × 100 cm, 15% HOAc