Single‐Step Preparative Separation of Barbinervic Acid and its Epimer (Rotungenic Acid), Along with Two Other Pentacyclic Triterpene Acids from the Leaves of Diospyros kaki Using HSCCC

Abstract

The leaf of Diospyros kaki is a traditional Chinese medicine, which has been used for treatment of stroke or syndrome and as a hypotensive drug. The pentacyclic triterpene acids, isolated from the leaves of Diospyros kaki, are their main bioactive compounds, which are very difficult and tedious to be obtained by conventional chromatography methods, especially for the separation of barbinervic acid and its epimer (rotungenic acid). In the present report, for the first time a one step preparative high speed countercurrent chromatography (HSCCC, with a 1000 mL coil column) method was developed for the separation and purification of four bioactive pentacyclic triterpene acids from this plant with a solvent system composed of n‐hexane–ethyl acetate–methanol–water (3:6:4:2, v/v/v/v). In a single operation, 49.6 mg of barbinervic acid (3α,19α,24‐trihydroxy‐urs‐12‐en‐28‐oic acid) and its epimer, 32.2 mg of rotungenic acid (3β,19α,24‐trihydroxy‐urs‐12‐en‐28‐oic acid) along with 11.0 mg of 24‐hydroxy ursolic acid (3β,24‐dihydroxy‐urs‐12‐en‐28‐oic acid), which was reported for the first time from the leaves of Diospyros kaki, and 18.0 mg of ursolic acid (3β‐hydroxy‐urs‐12‐en‐28‐oic acid) were obtained from 750.0 mg of the extract of the leaves of Diospyros kaki. The chemical structure of the four compounds was elucidated by EI‐MS, ESI‐MS, ¹H NMR, and ¹³C NMR.

Keywords:

• Preparative chromatography
• High speed countercurrent chromatography
• Diospyros kaki
• Pentacyclic triterpene acid
• Barbinervic acid
• Ursolic acid

Acknowledgment

Financial support from National Natural Science Foundation of China (No. 20176049) is gratefully acknowledged.