Introduction

Berberine is a natural medicine derived from the flowering plant Coptis japonica and is reputed to have beneficial effects in the treatment of conjunctivitis and stomatitis as well as being a stomachic, intestinal remedy and antidiarrheic. It is suggested that berberine, one of alkaloids contained in Coptis japonica, which has yellow color and bitter taste may have antibacterial and anti-inflammatory effects. This article shows the separation of berberine obtained from powder extracts of Coptis japonica using analytical HPLC, the separation is scaled-up using semi-preparative HPLC for isolation and purification.

LC-4000 semi-preparative HPLC system

Experimental

Experimental conditions
<Conventional HPLC>
Column: YMC-PACK Pro C18 (4.6 mm ID x 250 mmL, 5 µm)
Eluent: 0.1% TFA in Acetonitrile / Water (30/70)
Eluent flow rate: 1.0 mL/min
Column temp.: 25 °C
Wavelemgth: 220 ~ 450 nm, 345 nm
Injection volume: 10 µL
Standard sample: Powdered Coptis japonica (0.5 g/50 mL in methanol / 10% hydrochloric acid (100/1))

<Semi-Preparative HPLC>
Column: YMC-PACK Pro C18 (20 mm ID x 250 mmL, 5 µm)
Eluent: 0.1% TFA in Acetonitrile / Water (30/70)
Eluent flow rate: 15 mL/min
Column temp.: 25 °C
Wavelemgth: 345 nm
Injection volume: 5 mL
Standard sample: Powdered Coptis japonica (0.5 g/50 mL in methanol / 10% hydrochloric acid (100/1))

Preparation (extraction)

1. Weigh 0.5 g of powdered Coptis japonica and place in a centrifuge tube.
2. Add 30 mL of methanol/10% hydrochloride mixture (100/1) and mix for 15 minutes.
3. Centrifuge (3,000 rpm, 10mim) and decant the supernatant into a 50 mL measuring flask.
4. Add 20 mL of methanol/10% HCl mixture (100/1) to the residue and repeat the procedure.
5. Add methanol/10% HCl mixture (100/1) to the collected supernatant in measuring flask and make up to 50 mL.

Figure 1. Structural formula of Berberine

Keywords

Analytical separation, Semi-preparative separation, Coptis japonica, Berberine, Neutraceutical, Natural medicine

Results

Figure 2 is Chromatogram and contour plot of the extracts from Coptis japonica powder using conventional HPLC separation. Using PDA detector and with spectral comparison, the separation of the target compound Berberine from the other components was optimized and good separation was achieved within 15 minutes.

Figure 2. Chromatogram of the extract from Coptis japonica powder

Figure 3 is Chromatogram of the extract from Coptis japonica powder using semi-preparative HPLC scaled up from the analytical scale method.

Figure 3. Semi-preparative chromatogram of the extract from Coptis japonica powder

To optimize recovery of Berberine the injection volume was increased. However, this caused a problem as the elution power of the extraction solvent was stronger than the mobile phase, the target compound was not retained on column. To resolve this, the sample was diluted five times in water and 5 mL of the diluted sample was injected. Berberine was retained as shown in Figure 3. the separation efficiency increased but with a small sacrifice of peak shape. Figure 4 shows the fraction collected using the ChromNAV chromatography data system. The peak fraction and sample rack position for the target are highlighted in green.

Figure 4. Collected fraction of the extract from Coptis japonica powder (ChromNAV screen)

Figure 5 shows the sample purity chromatogram of this fraction under the same conditions as in Figure 2. It is confirmed that Berberine was isolated as single component.

Figure 5. Chromatogram of the collected fraction (10 mL Injected)