Quantitative Determination of α-Tocopherol in Globularia alypum Using High-Performance Liquid Chromatography with UV Detection

Abstract

A quantitative determination of α-tocopherol in Globularia alypum L. was established by high-performance liquid chromatography (HPLC)-UV method. The investigations were carried out to evaluate the α -tocopherol content of the extracts from the air-dried aerial parts and the leaves of the plant. The calibration curve was used to calculate the content of the α-tocopherol, which was estimated as 0.002% and 0.0007% on the dried weight of the leaves and the aerial parts of G. alypum.

Keywords:

• Globularia alypum
• Globulariaceae
• α-tocopherol
• HPLC-UV

Introduction

Globularia alypum L. (GA) is a wild plant belonging to the Globulariaceae family. Its leaves are traditionally used as hypoglycemic agent and laxative (Bellakhdar et al., 1991). It is also used as cholagogue, stomachic, purgative, and sudorific (Sijelmassi, 1993). Investigations were carried out to evaluate the hypoglycemic activity of G. alypum (Skim et al., 1999). A recent investigation has demonstrated that G. alypum had produced significant decrease of blood glucose levels (Jouad et al., 2002).

In our current work, we had studied the aerial parts and the leaves of G. alypum for the content of α-tocopherol, previously shown to have antioxidant and anti-atherosclerotic activities (Yusoff, 2002).

Materials and Methods

Plant material

Globularia alypum was collected in March 2003 from Içmeler, Urla, in Izmir. The plant material was identified by Esat Çetin, and the voucher specimen (no. 1317) was deposited at the Herbarium of the Department Pharmacognosy, Faculty of Pharmacy, Ege University (Izmir, Turkey).

Extraction

The air-dried and ground aerial parts and the leaves of G. alypum (20 g) were extracted with n-hexane under stirring. The organic phases were filtered and distilled in vacuo to yield the n-hexane extracts.

Reagents and solvents

α-Tocopherol (Sigma) was used as a standard. Then-hexane used for the extraction was purchased from Riedel, whereas the methanol used as eluent in the High Pressure Liquid Chromatography (HPLC) system was purchased from Lab-Scan.

Standard and sample solutions

The standard solutions were prepared by dissolving α-tocopherol in methanol to yield concentrations of 0.2, 0.04, 0.008, and 0.0016 g/100 ml. Twenty microliters of the standard solutions were injected on the HPLC column. Then, the calibration curve was drawn (Fig. 1).

Figure 1 The calibration curve for the HPLC-UV determination of α-tocopherol.

Sample solutions were prepared by dissolving the n-hexane extracts of the leaves and the aerial parts of the plant in methanol (10 mg/2.5 ml). Ten microliters of each aliquot was injected on the HPLC column. Each analysis was carried out in triplicate.

HPLC-UV method

The HPLC system consisted of a QuatPump (Hewlett Packard Series 1100), an injector fitted with a 20-µl loop, and a UV detector (HP 1100) set at 292 nm. A Hichrom 5 C18 column (25 cm × 4.6 mm i.d.) was eluted with methanol at a flow rate of 2 ml/min. The column temperature was adjusted to 40°C.

Results and Discussion

α-Tocopherol in G. alypum was quantitatively determined by the HPLC-UV method. The calibration curve (Fig. 1) was used for the calculation. The α-tocopherol content in the n-hexane extracts of the leaves and the aerial parts of the plant was calculated from the following regression equation of the calibration curve:

y:	=	is the peak area
x:	=	is the α-tocopherol concentration (µg/µl)

The results of the assay are shown in Table 1.

Table 1 HPLC-UV determination of the α-tocopherol content of Globularia alypum.

Sample	α-Tocopherol (% on dried weight)	α-Tocopherol (% on the n-hexane extract)
Leaves	0.002149	0.101154
Aerial parts	0.000766	0.079130

The method, found to be specific and suitable for the routine analysis because of its simplicity and sensitivity, can be used for the analysis of α-tocopherol in other plant extracts.