HPLC Separation: Ginsenosides from American Ginseng

Section Overview

• Introduction to Ginsenosides
• Separation of Ginsenosides from Panax Quinquefolius(American Ginseng)
• Separating Ginsenosides by HPLC using a C18 Column
• Ginsenoside Standards

Introduction to Ginsenosides

Ginsenosides are mainly found in ginseng plants (genus Panax), widely used as a herbal medicine around the world but primarily in Asian cultures. Most ginsenosides are members of the dammarane family and are triterpene saponins, a natural steroid glycoside. Although fGinsenosides are found throughout Panax, but isolation typically ginsenoside isolation typically focuses on the plant root. The root is typically dried and grounded into a fine powder to increase the surface area for extraction. Then the powder is extracted using solvents like ethanol, methanol, or water based on polarity by using soxhletSoxhlet extraction, maceration, or ultrasonication. The resultant filtrate is filtered, concentrated, and purified, and the extract is stored for analysis via HPLC or LC-MS.

Many ginsenosides have a dammarane skeleton (17 carbons in a four-ring structure) with various sugar moieties attached to the C-3 and C-20 positions. Over 30 ginsenosides have been identified and classified into two categories, 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT).

Separation of Ginsenosides from Panax Quinquefolius (American Ginseng)

Ginseng Root

Ginseng is one of the most well-known herbal medicines. It has been used in traditional Chinese medicine for centuries but is also popular in Western regions. Medicines derived from the ginseng root are used in the treatment or prevention of many health problems including anxiety, asthma, diabetes, headache, stress, depression and erectile dysfunction, among many others.

The unique compounds of panax are saponin triterpenoid glycosides, called ginsenosides. Their potential effects in humans are being studied intensively.

Figure 1. Chemical structures of the analyzed ginsenosides

Separating Ginsenosides by HPLC using a C18 Column

We achieved a good separation of seven different ginsenosides using an Ascentis^® Express C18 column. The application was first optimized using a ginsenoside standard mixture (Figure 2) and was then applied to a sample of American ginseng root (Figure 3). The seven targeted analytes are ginsenosides RB₁, RB₂, Re, Rc, RD, Rf and Rg₁ (Figure 1). The experimental conditions and chromatograms are shown in the table below.

Figure 2. Chromatogram of the standard mixture (50 µg/mL each component in 82:18, water:methanol)

Figure 3. Chromatogram of the American ginseng root extract

Table 1. Experimental Conditions

Column Conditions
column:	Ascentis® Express C18 15 cm x 4.6 mm, 2.7 µm particle size (53829-U)
mobile phase A:	water
mobile phase B:	acetonitrile
gradient:	min 0 1.5 13.5 14.5	%A 75 75 15 15	%B 25 25 85 85
flow rate:	1.5 mL/min
temp.:	60 °C
det.:	205 nm
injection:	10 µL
Extraction method used for ginseng root
NSF International ground plant material:	320 mg (or 36 mg for XRM material)
extraction solvent:	10 mL 50:50, water:ethanol
sonication:	30 min
temperature:	50 °C

Ginsenoside Standards

We offer a wide range of active components from ginseng, including the seven ginsenosides described in this article. Some of the ginsenosides are available as primary pharmaceutical reference standards manufactured by HWI Analytik using quantitative NMR for content assignment.