PREPARATIVE SEPARATION AND PURIFICATION OF ALKYLAMIDES FROM ZANTHOXYLUM BUNGEANUM MAXIM BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY

Abstract

High-speed counter-current chromatography (HSCCC) was successfully applied to preparative isolation and purification of alkylamides, the tingling-taste components, from pericarp extract of Zanthoxylum bungeanum Maxim (Dahongpao). With the lower phase of n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v) eluted at a flow rate of 1.5 mL · min⁻¹, and the revolution speed at 850 rpm, 0.19 g of alkylamides in purity of 98.0% by the relative peak area of HPLC was obtained from 1.0 g of extract in one run within 2 hr. On the basis of MS, IR, and ¹H NMR analysis, the product of alkylamides was confirmed to be composed of hydroxy-α -sanshool, hydroxy-β-sanshool, and hydroxy-ϵ-sanshool. Furthermore, using the HSCCC product as external standard, alkylamide in the extract was determined to be in total of 21.41%, among which hydroxy-α-sanshool, hydroxy-β-sanshool, and hydroxy-ϵ-sanshool were in 15.69%, 4.76%, and 0.96%, respectively.

Keywords:

• alkylamide
• HSCCC
• hydroxy-α, β and ϵ-sanshools
• separation
• tingling-taste
• zanthoxylum

ACKNOWLEDGMENTS

This work was funded by National Natural Science Foundation of China (No. 31171755), by Beijing Municipal Education Commission Science and Technology Development Plan Key Project and Beijing Municipal Natural Science Foundation (no. KZ201010011011), and by Beijing Municipal Science and Technology Innovation Platform Construction Project (No. 201149-50).

Notes

K _D (A), K _D (B), and K _D (C) refer to the K _D values of compound A, B, and C in Figure 3 by HPLC, which were identified to be hydroxy-ϵ-sanshool, hydroxy-α-sanshool, and hydroxy-β-sanshool in turn.

^a Retention of the stationary phase, S_f = V_s/V_c, where V_s is the volume of stationary phase retained in column when hydrodynamic equilibrium reached, V_c is the total column volume.

^b The sum of relative peak areas (%) of compound A (hydroxy-ϵ-sanshool), B (hydroxy-α-sanshool), and C (hydroxy-β-sanshool) in HPLC (See Figure 3a).

^c No peak (a flat UV trace) given when the alkylamides eluted out.

^d A heart cut from the center of the peak collected.