Stachydrine, a Bioactive Equilibrist for Synephrine, Identified from Four Citrus Chinese Herbs

**Background:** The citrus herbs Aurantii Fructus Immaturus (Zhishi), Aurantii Fructus (Zhiqiao), Citri Reticulatae Pericarpium Viride (Qingpi), and Citri Reticulatae Pericarpium (Chenpi) are widely used in traditional Chinese medicine for gastrointestinal and cardiovascular diseases. While synephrine and N-methyltyramine in these herbs produce adrenergic effects (raising blood pressure, constricting vessels), decoctions paradoxically show some contrary effects—transiently lowering blood pressure, providing cardiovascular protection, and exerting two-way regulation on gastrointestinal smooth muscle. This suggested the presence of cholinergic or anti-adrenergic compounds. The authors hypothesized that choline analogs, which share a structural fragment (a nitrogen atom linked to an oxygen atom by a 2–3 carbon alkane chain), might be responsible for these effects.

**Methods:** A thin-layer chromatography (TLC) method was developed using an improved Dragendorff reagent (bismuth nitrite, potassium iodide, 50% phosphoric acid) to detect choline analogs. Solvent system I (ethyl acetate–95% ethanol–formic acid, 10:4:5) was optimized. Choline chloride served as a positive control; γ-aminobutyric acid and synephrine were exclusion controls. Decoctions and 80% ethanol extracts of 39 herb samples (10 Zhishi, 9 Zhiqiao, 10 Qingpi, 10 Chenpi) from different Chinese producing areas were analyzed. Preparative TLC isolated compounds from mixed Zhishi samples, identified by HRMS and 1H/13C NMR. A TLC scanning (TLCS) method was established for quantitative analysis of stachydrine and choline (measured/reference wavelengths: 574/820 nm and 568/820 nm, respectively). Synephrine was quantified by HPLC-UV at 275 nm using a SinoChrom ODS2 column with methanol-phosphate buffer (67:33) mobile phase. Statistical analyses used bilateral t-tests.

**Key Results:** Three compounds were isolated from Zhishi: choline (1), stachydrine (2), and synephrine (3). Stachydrine (C7H14NO2, HRESIMS m/z 144.1010 [M]+) showed a reddish-brown spot with the improved Dragendorff reagent, while synephrine showed orange-red. The TLCS method for stachydrine showed LOD 1.0 μg (RSD 0.08%), LOQ 2.0 μg (RSD 0.18%), linearity y = 6565.7x + 5522.7 (r = 0.9996) over 2.0–14.0 μg, with average recovery 100.28% (RSD 3.08%). For choline: LOD 0.5 μg (RSD 0.08%), LOQ 0.8 μg (RSD 0.18%), linearity y = 12302.1x − 909.3 (r = 0.9987) over 1.0–4.0 μg, recovery 101.04% (RSD 2.31%). For synephrine HPLC: LOD 0.02 μg, LOQ 0.05 μg, linearity y = 4435448.7x + 2217.2 (r = 1.0000) over 0.5–32.0 μg, recovery 99.89% (RSD 3.19%). Content ranges: Zhishi—stachydrine 2.94–8.53 mg/g, choline 0.00–0.87 mg/g, synephrine 3.56–21.07 mg/g; Zhiqiao—stachydrine 1.43–5.13 mg/g, choline 0.00–0.21 mg/g, synephrine 0.00–2.42 mg/g; Qingpi—stachydrine 0.99–3.51 mg/g, choline 0.21–0.60 mg/g, synephrine 4.39–7.19 mg/g; Chenpi—stachydrine 0.86–3.26 mg/g, choline 0.00–0.20 mg/g, synephrine 1.86–3.80 mg/g. Statistical analysis confirmed stachydrine and synephrine contents were significantly higher in Zhishi than other herbs (p < 0.05 or p < 0.01). In Zhiqiao, stachydrine was the largest of the three ingredients (p < 0.05 or p < 0.01). All three ingredients decreased with prolonged growth time (p < 0.05), with synephrine decreasing most rapidly and stachydrine most slowly. The stachydrine/synephrine ratio increased from Zhishi (0.44 ± 0.16) to Zhiqiao (2.43 ± 1.03) (p < 0.05) and from Qingpi (0.34 ± 0.14) to Chenpi (0.67 ± 0.33) (p < 0.05).

**Clinical Implications:** The identification of stachydrine as a choline analog with high bioavailability (>90%) and extensive cardiovascular protective effects (rapid vascular relaxation via endothelial nitric oxide synthase, slowing heart rate, decreasing cardiac output, suppressing myocardial fibrosis, inhibiting platelet aggregation) provides a mechanistic explanation for the paradoxical effects of citrus herb decoctions. Stachydrine counteracts synephrine's adrenergic effects (vasoconstriction, increased platelet levels, uterine contraction), explaining why decoctions sometimes lower rather than raise blood pressure. The two compounds act as a 'bioactive equilibrists' pair, analogous to sympathetic/parasympathetic neurotransmitters. This has direct clinical relevance: the varying stachydrine/synephrine ratios across harvest times and producing areas explain differences in pharmacological effects among the four herbs. For example, Zhiqiao (harvested later) has stachydrine as the predominant alkaloid, correlating with its different clinical applications. The study also suggests that stachydrine's hepatoprotective, renal protective, and neuroprotective effects contribute significantly to the herbs' therapeutic profiles, and that quality markers for these herbs should be reevaluated to include stachydrine content.