Separation and Purification of Two Minor Compounds from Radix isatidis by Integrative MPLC and HSCCC with Preparative HPLC

Abstract

Radix isatidis has been widely used as a Chinese traditional medicine for its anti-virus and anticancer activities where the minor components may contribute to these beneficial pharmaceutical effects. In order to enrich the target minor compounds effectively and rapidly, extraction, medium-pressure liquid chromatography (MPLC), high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (pre-HPLC) were integratively used for separation and purification of two target minor compounds indole-3-acetonitrile-6-O-β-D-glucopyranoside (target 1) and clemastanin B (target 2) in the present study. Radix isatidis was dried, pulverized and extracted with 50% methanol at room temperature, then concentrated and subjected to pretreatment with D-101 macroporous resin chromatography and extraction by MPLC. The first target compound was separated by MPLC at the purity raised to 70–80%, but without the second minor compounds which were irreversibly adsorbed by C18 solid support. Therefore, the second target compound in the crude extract was directly separated by HSCCC at purity of 80–90%. Finally these refined samples were further separated by pre-HPLC to obtain a high purity at 98–99%. The chemical structure identification of each target compound was carried out by IR, ESI-MS and 1H NMR.

Evaluation of the Antiwrinkle Activity of Enriched Isatidis Folium Extract and an HPLC–UV Method for the Quality Control of Its Cream Products

“…Moreover, all UV spectra with their λmax at approximately 270 and 330 nm of peaks 1–4 agree with flavonoid characterization [ 25 ]. Based on the ESI-MS/MS and literature data, peaks 1–4 were assigned as isovitexin, isovitexin-3″-O-glucopyranoside, isoscoparin, and isoscoparin-3″-O-glucopyranoside ( Figure 3 ) [ 15 , 16 ].…”

“…Some previously developed analytical methods to control the Isatidis Folium quality are thin-layer chromatography, high-performance liquid chromatography-ultraviolet (HPLC-UV), HPLC-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS), HPLC-electrospray ionization (ESI)-MS, nuclear magnetic resonance, and Fourier Transform (FT)-Raman spectroscopy [ 16 , 17 , 18 , 19 ]. However, none of these established methods can be used to identify and quantify the bioactive compounds in our enriched Isatidis Folium extract because of many components with a high amount in the crude extract (such as alkaloids and glycosides) were removed during the purification process.…”

Application and comparison of high‐speed countercurrent chromatography and high‐performance liquid chromatography in semi‐preparative separation of decarboxymethyl oleuropein aglycone (3,4‐DHPEA‐EDA), a bioactive secoiridoid from extra‐virgin olive oil

“…Although some authors have reported high purities (>90%) using HSCCC , this type of separations frequently require a pre‐separation step employing macroporous resins when the crude extracts are complex . In addition, HPLC is orthogonal and complementary to HSCCC, and can often provide straightforward resolution of otherwise difficult separations by HSCCC . However, when considering these comparisons it should be noted that there is room for further optimization of both methodologies, in terms of both throughput and efficiency.…”

An effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum

“…Moreover, its merits of low cost, high sample loading, high efficiency, and various available modes [12,13] makes it suitable for the separation of minor components. Seven studies on the separation of minor compounds from natural products by HSCCC have been conducted over the last 5 years [14][15][16][17][18][19][20]. For compounds with similar polarities, it is sometimes not easy to find a perfect solvent system that performs at high purity in a one-step separation by HSCCC due to limitations in theoretical plates.…”