Mass balance and stability study of ligustilide as a bioactive marker of Angelica sinensis and other Apiaceaous botanicals

Angelica sinensis is one of the 15 most commonly used Traditional Chinese Medicines (TCMs), predominantly in formulations for the treatment of irregular menstrual cycles and premenstrual syndrome [1]. The bioactive marker persistently associated with observed bioactivities is the monomeric phthalide, ligustilide, a compound known for its instability and rapid chemical degradation [2,3]. The first rigorous approach to achieving a mass balance quantification of ligustilide using quantitative ¹H NMR in sealed tubes (MB-qHNMR) was developed. The method also provides insights into the (partial) structure and nature of the degradation products. The influence of temperature, light, and oxygen on ligustilide stability in plant material, essential oil, and isolated ligustilide was assessed. Four ligustilide-rich species were investigated: A. sinensis, Ligusticum porteri, L. striatum, and L. sinense. Essential oils were gently obtained by supercritical CO₂ fluid extraction. Pure ligustilide was isolated from the essential oil of A. sinensis by a two-step procedure involving countercurrent separation (CCS) [4] and prep-HPLC, yielding qHNMR purities of 93% and 98%, after each step. After 6 months, a remarkable influence of all investigated factors on stability and degradation products (“tertiary metabolome”) became evident. Ligustilide shows the best stability within the original plant material, with no change in content after one year. In crude essential oil, ligustilide stability was influenced mostly by temperature. Isolated ligustilide is the least stable form. MB-qHNMR provides a viable approach to analyzing botanical markers in crude extracts, active fractions and as isolated compounds.

Keywords: Angelica sinensis, ligustilide, stability, countercurrent separation, centrifugal partition chromatography, high-speed countercurrent chromatography, quantitative ¹HNMR.

References:

[1] Schinkovitz A, Pro SM, Main M, Chen SN, Jaki BU, Lankin DC, Pauli GF. Dynamic Nature of the Ligustilide Complex. J Nat Prod 2008; 71: 1604 – 1611.

[2] Gödecke T, Yao P, Napolitano JG, Nikolić D, Dietz BM, Bolton JL, Breemen RB, Farnsworth NR, Chen SN, Lankin DC, Pauli GF. Integrated standardization concept for Angelica botanicals using quantitative NMR. Fitoterapia 2012; 83: 18 – 32.

[3] Lu Y, Liu S, Zhao Y, Zhu L, Yu S. Complexation of Z-ligustilide with hydroxypropyl-β-cyclodextrin to improve stability and oral bioavailability. Acta Pharm 2014; 64: 211 – 222.

[4] Pauli GF, Pro SM, Friesen JB. Countercurrent Separation of Natural Products. J Nat Prod 2008; 71: 1489 – 1508.

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