Production of Two Isomers of Sphaeralcic Acid in Hairy Roots from Sphaeralcea angustifolia

**Background:** Sphaeralcea angustifolia (Cavanilles) G. Don (Malvaceae) is used in Mexican traditional medicine as an anti-inflammatory and for treating gastrointestinal diseases. Previous work identified β-sitosterol, α- and β-amyrin, trans-cinnamic acid, and scopoletin as active anti-inflammatory compounds. Cell suspension cultures produced scopoletin, tomentin, and sphaeralcic acid, with sphaeralcic acid showing potent anti-inflammatory effects in acute and chronic mouse models. Hairy root cultures offer advantages of fast growth, biosynthetic stability, and potential to produce novel compounds not found in wild plants.

**Methods:** Two previously established hairy root lines (SaTRN12.2, line 1; SaTRN7.1, line 2) of S. angustifolia were cultured in phyto-regulator-free MS medium with 3% sucrose at 26°C with a 16h light/8h dark photoperiod and orbital agitation at 110 rpm. Roots were subcultured every three weeks. Dried roots (173 g SaTRN12.2 and 100 g SaTRN7.1) were extracted thrice by maceration with dichloromethane-methanol (9:1). Scopoletin and sphaeralcic acid were quantified by HPLC at λ=344 nm and λ=357 nm respectively. Compounds were isolated using open column chromatography on silica gel with gradient elution systems. NMR spectra were recorded on Agilent DD2-600 at 600 MHz (¹H) and 150 MHz (¹³C). FAB-MS and HRFAB-MS were performed on a JEOL MS-700 mass spectrometer. For the gastroprotective study, female ICR mice (35-40 g) were divided into groups of six, fasted 24h, then pre-treated with vehicle (1% Tween-20), omeprazole (20 mg/kg), or dichloromethane-methanol extract (100 mg/kg) of SaTRN7.1. Gastric lesions were induced with absolute ethanol (0.10 mL/10g body weight) 1h after treatment. After 1h, mice were anesthetized and sacrificed; stomachs were removed and ulcer area measured by planimetry using ImageJ.

**Key Results:** Line 1 (SaTRN12.2) produced scopoletin (0.0022 mg/g) and sphaeralcic acid (0.22 mg/g). Line 2 (SaTRN7.1) produced scopoletin (0.011 mg/g) and sphaeralcic acid (3.07 mg/g), representing an 85-fold higher yield than previously reported cell suspension cultures (0.0359 mg/g). Both lines contained stigmasterol and β-sitosterol. Two novel compounds were isolated from line 2: iso-sphaeralcic acid (compound 6, C₁₆H₁₆O₄, green powder, λmax=222, 261, 345 nm) and 8-methyl-iso-sphaeralcic acid (compound 7, C₁₇H₁₈O₄, yellow solid, λmax=214, 262, 347 nm). In the gastroprotective assay, vehicle-treated mice had an average ulcer index of 0.664. Omeprazole (20 mg/kg) reduced the ulcer index to 0.403 (39.30% protection). The SaTRN7.1 extract (100 mg/kg) reduced the ulcer index to 0.052 (92.11% protection, p<0.05 vs. omeprazole by Student's T test).

**Clinical Implications:** The SaTRN7.1 hairy root line demonstrates stable, enhanced production of anti-inflammatory compounds including sphaeralcic acid at levels suitable for biotechnological scale-up. The 92.11% gastroprotective effect of the extract supports the traditional use of S. angustifolia for gastrointestinal illness and exceeds the protection provided by omeprazole in this model. The two newly identified sphaeralcic acid isomers (iso-sphaeralcic acid and 8-methyl-iso-sphaeralcic acid) warrant further evaluation in acute and chronic inflammation models. Hairy root culture technology offers a sustainable, scalable platform for producing these bioactive compounds without harvesting wild plants.