Extraction of sennosides from senna tea leaves using ISOLUTE® C18 SPE cartridges

Figure 1. Structural formula of Sennoside.

Introduction

Some health foods marketed as having dietary benefits may contain senna stems as raw materials. However, the stems as well as the fruits, leaves, and leaf shafts of senna contain sennosides, which are pharmacological constituents and designated as pharmaceuticals. Sennosides are used to treat constipation, but have certain side effects such as digestive disorders when used excessively, and must be taken with caution.

In this application note, we examined the content of sennosides in senna tea leaves, which are marketed as health foods, using Biotage® SPE products.

The sample pre-treatment method is shown in the flowchart below.

When 100 mg of senna tea leaf was treated as described, ~15 mg of dried extract was obtained. This was dissolved in water, and the extract was fractionated using an ISOLUTE C18 1g/6mL SPE cartridge.

A Biotage® V-10 Touch high-speed evaporator system was used to evaporate extracts and solid-phase eluates.
Figure 2. Biotage® V10 Touch Evaporation System

LC/MS measurements were performed on both eluates fractionated using the ISOLUTE® C18 cartridge, and sennosides were observed in both Elute 1 and Elute 2. The content of sennosides calculated from the peak area values was similar to the nominal values.

Sample pre-treatment and C18 SPE fractionation method

UHPLC conditions

Instrument

Nexera LC-30AD (Shimadzu Corporation)

Column

ACQUITY UPLC® BEH C18 1.7 μm (2.1 mm × 50 mm column; Waters)

Mobile phase

A:0.1 % (v/v) aqueous formic acid
B: acetonitrile

Gradient conditions

Flow rate

0.4 mL/min

Column temperature

40 °C

Injection volume

1 µL

Mass spectrometry conditions

Instrument

LCMS-8060 (Shimadzu)

Ionization mode

ESI positive

Nebulizer gas flow rate

3.00 L/min

Drying gas flow rate

10.00 L/min

Heating gas flow rate

10.00 L/min

Interface temperature

350 ⁰C

DL temperature

200 ⁰C

Heat block temperature

350 ⁰C

CID gas

270 kPa

SRM transitions

Sennoside: m/z 861.25 > 386.25; Collision Energy: 40

Figure 3. SRM chromatogram (blue) of Sennoside A reference standard and SRM chromatogram (black) of sennosides in senna tea leaf extract.

Table 1. Dry weight of each elution section of senna tea leaf extract and sennoside A content in senna tea leaves.

*Elute 1: Methanol:Water (1:1, v/v)
**Elute 2: Methanol

Conclusion

Fractionation of senna leaf extracts using ISOLUTE® C18 solid phase extraction cartridges provided a useful tool in determina- tion of the content of the pharmacologically active sennoside A in the product.

Ordering information

Literature number: AN935