Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2011 , Vol 15 , Num 1
Quantitative determination of α-tocopherol and quality control studies in Sarcopoterium spinosum L.
Buket Bozkurt Sarıkaya, Hüsniye Kayalar
Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey DOI : 10.12991/201115438


Sarcopoterium spinosum L. ekstrelerinde α-tokoferol miktar tayini TLC-dansitometri ve HPLC-UV yöntemleriyle araştırılmıştır. S. spinosum'un α-tokoferol içeriği 0.017210-0.023744 % (TLC-dansitometri) ve 0.025966-0.037212 % (HPLC-UV) aralığında tespit edilmiştir. En yüksek α-tokoferol miktarı, her iki yöntemde de meyveli dönem toprak üstü kısımlarından elde edilmiştir. Bunlara ek olarak, bitki örneklerinin nem içeriği, total kül ve sülfat külü miktarı DAB 10'a göre tayin edilmiştir.


Sarcopoterium spinosum L. (Rosaceae) is low, mound-forming spiny common shrub in the Eastern Mediterranean countries[1]. In folk medicine, S. spinosum is reported to have antidiabetic effect[2,3]. In the literature there are several reports about the antidiabetic activity of S. spinosum[4-8].

Vitamine E has eight naturally occuring stereoisomers, four tocopherols (α, β, γ, δ) and four tocotrienols (α, β, γ, δ)[9]. Among these compounds, most studies on vitamine E are carried out with α-tocopherol. Vitamine E has been shown to possess several biological properties, such as antioxidant, cancer-preventive effect[10]. In addition, it could decrease the risk of the heart damage to the oxidative stress[11].

For the quantitative determination of α-tocopherol; spectrophotometric[12], HPLC[13], TLC[14] and GC-MS[15] methods have been suggested. In this study, aerial and underground parts of S. spinosum in two vegetation periods have been used to determine α-tocopherol content by performing modified TLC and HPLCUV methods. The results of two different analytical methods have been compared in this text. Furthermore, contents of humidity, ash and sulphated ash of drug specimens were performed in accordance with DAB 10[16] for the purpose of obtaining data for expectative monographs.


Plant Material
The aerial and underground parts of S. spinosum were collected from Seferihisar, Izmir during both flower ing and fruiting periods. The plant was identified by Prof. M. Ali Onur from the Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Izmir (Turkey). A voucher specimen (No. 1323) is deposited in the Herbarium of the Faculty of Pharmacy, Department of Pharmacognosy, Ege University.

Humidity, Total Ash, Sulphated Ash Determinations
Quality control determinations on plant materials (humidity, total ash and sulphated ash) were conducted according to methods in DAB 10. Aerial and underground parts of S. spinosum in flowering and fruiting periods were individually studied to the determine the quality control of plant.


Four different extracts were prepared from the specimens of S. spinosum: A (aerial parts, flowering), B (aerial parts, fruiting), C (underground parts, flowering) and D (underground parts, fruiting). 100 g of each samples of S. spinosum were extracted with n-hexane (1 x 600 ml first for 5 h and than 2 x 600 ml for 8h) under stirring and filtered. The extraction solvent was evaporated in vacuo at 40ºC.

dl-α-Tocopherol (Roche) was used as a standard. The n-hexane used for the extraction was obtained from Merck, whereas the methanol used as eluent in the high-performance liquid chromatography (HPLC) system was purchased from Lab- Scan. Other solvents and reagents were obtained from Merck.

Sample Solutions
10 mg of each extract was dissolved in 2.5 ml methanol for HPLC-UV method and 20-60 mg extract in 2 ml chloroform for TLC densitometric assay.

TLC-densitometric assay
A Shimadzu high-speed TLC scanner CS-920 was used with the following settings: beam size of 0.4 X 0.4 mm, X=24, Y=10, L=3; AZS off, wavelength of 350 nm. Silica gel 60F254 (20 X 20 cm, 0.25 mm thick, Merck) plates were used. Cyclohexane/diethylether (4:1) was used as mobile phase. The samples were implemented with Hamilton syringes (15 mm from the bottom line of the plate). The mobile phase was allowed to run a distance of 100 mm in the saturated tank.

Silica plates were prewashed in chloroform/methanol (1:1), dried and activated at 1000C for 10 min. α-Tocopherol solutions (2, 4, 6 and 8 μl) were applied on a TLC plate and developed under the same conditions. The developed plates were initially air-dried and then oven-dried for 15 min at 1000C, and sprayed with CuSO4-phosphoric acid reagent (10% CuSO4 / 8% phosphoric acid, 1:1) followed by charring at 1900C for 10 min. The α-tocopherol quantity of the samples were measured by Thin Layer Scanner at 350 nm using a D2 lamp. The calibration curve exhibited a linear relationship between the quantities and areas on TLC plates (Fig. 1). 20 μl of sample solutions were applied on TLC plate and after the development the areas of the spots were integrated by TLC-densitometry. Each analysis was carried out in triplicate.

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FIGURE 1: Calibration Curve for the Determination of α-Tocopherol by TLCDensitometry

High-Pressure Liquid Chromatography-UV Method

The HPLC system (Hewlett Packard 1100 series) equipped with a UV variable-wavelength detector (HP 1100) set at 292 nm. A hichrom 5 C18 column (25 cm x 4.6 mm i.d.) was eluted with methanol at a flow rate of 2 ml/min. A manual injector with 20 μl loop (HP 1100 G1328A Rheodyne 7725i) and the column temperature was adjusted to 40ºC.

For the preparation of the calibration curve of α-tocopherol, 2 mg of the standard was dissolved in 1 ml methanol. 0.5, 1, 2, 4, 5, 10, 15, 20 and 25 μg/20μl concentrations were prepared from stock solution. Twenty microliters of the standard solutions were injected on the HPLC column. Then, the calibration curve of α-tocopherol was drawn (Fig. 2). 10 mg of extracts were dissolved in methanol (2.5 ml). Each aliquot was injected into the HPLC column with a volume of 10 μl. For each sample, the procedure was repeated three times.

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FIGURE 2: The Calibration Curve for the HPLC-UV Determination of α-Tocopherol


Quality Control Studies
Quality control determinations (humidity, total ash and sulphated ash) on drug specimens prepared separately from plants in flowering and fruiting periods were conducted according to methods in DAB 10.

The results of the assays (Table 1) provide knowledge for the prospective monographs of Herba and Radix Poterii spinosi.

TABLE 1: Quality Control Determinations on Herba and Radix Poterii spinosi prepared from Sarcopoterium spinosum

TLC-Densitometric Assay
α-Tocopherol content of Sarcopoterium spinosum extracts was quantitatively determined by TLC-densitometric method. Each extracts and standard compound were tested for three times. The results of our studies revealed that α-tocopherol is not present in underground parts of S. spinosum collected during both flowering and fruiting seasons (Sample C and D). For the calculation of the α-tocopherol content of extracts, TLCdensitometric curve and following linear equation were used.

y = 1576.5 x + 1177.9; R2 = 0.9995

where x is the α-tocopherol concentration (μg/ml) and y is the area (integration unit). The results of the assay are shown in Table 2.

TABLE 2: Contents of α-Tocopherol (% on dried wt.) in S. spinosum as determined by TLC-Densitometry and HPLC-UV Method

HPLC-UV Method
The identification and quantitative determination of α-tocopherol in the extracts were carried out by a comparison of retention times and areas with that of standard α-tocopherol. The results showed that α-tocopherol is not present in sample C and D. The α-tocopherol content in the n-hexane extracts of the aerial parts of the S. spinosum was calculated from the following regression equation of the calibration curve:

y = 198.13 x + 3.4285; R2 = 0.9992

where x is the α-tocopherol concentration (μg/ml) and y is the peak area. The results of the assay are shown in Table 2.


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