Identification and estimation of stevioside in the commercial samples of stevia leaf and powder by HPTLC and HPLC

Stevioside, a major sweet component of leaves of Stevia rebaudiana (Bertoni), is about 300 times sweeter than sucrose. Highly refined stevia leaf extracts and stevioside are officially used as low calorie sweeteners in many countries. The present study describes a method for rapid identification and estimation of stevioside in commercial samples using HPTLC and HPLC. Identification of stevioside in samples was done by HPTLC. The separation was achieved on a pre-coated silica gel 60F254 plate with mobile phase; ethyl acetate: methanol: water (75:15:10 v/v/v). Densitometric scanning was perfomed at 510 nm after visualization with a solution of acetic anhydride: sulphuric acid: ethanol (1:1:10 v/v/v). The identity of the peak corresponding to stevioside was further confirmed by spectral analysis. HPLC was used for the estimation of stevioside content. The calibration curve was linear in the concentration range from 0.1 to 1 mg/ml. The detection limit for stevioside was 0.05 mg/ml (1 μg per injection). The percentage stevioside content of the samples i.e. stevia powder and stevia leaf was found to be; 8.859 % and 3.703 % respectively. The method allows rapid identification and quantification of stevioside in different samples and can be used for routine analysis of stevioside in commercial samples. Key-Words: Stevioside; Stevia rebaudiana (Bertoni); HPTLC; Stevia powder; HPLC. Introduction Stevia rebaudiana (Bertoni), often referred to as ‘‘the sweet herb of Paraguay’’, is a perennial shrub belonging to Asteraceae (Compositae) family. It is native to certain regions of South America like Paraguay and Brazil. The major sweet component present in the leaves of Stevia rebaudiana (Bertoni), Stevioside, tastes about 300 times sweeter than sucrose (0.4% solution). The plant as well as its extract has been used since long time as a sweetener in various regions viz. South America, Asia, Japan and China. The leaves are found to contain a complex mixture of eight sweet diterpene glycosides, including stevioside, steviolbioside, rebaudiosides (A,B,C,D,E) and dulcoside A. Stevia leaves, stevioside and its highly refined extracts are officially used as a low calorie Sweetener in countries such as Brazil, Korea and Japan. Several studies have reported the antihyperglycaemic, insulinotropic, glucagonostatic and antihypertensive effects of stevia glycosides. * Corresponding Author: E-mail: [email protected] Tel.: +91-820-2571-998/201 ext. 2248. Many studies have also been conducted to determine the toxicological effects of stevia extract and stevioside, however no significant toxicity has been reported with either stevioside or stevia extract. JECFA (Joint FAO/WHO Expert Committee on Food Additives) in 2004, established an ‘‘Acceptable Daily Intake” or ADI of 0–2 mg/kg body weight/day of total steviol glycosides in a finished product on a temporary basis. Various methods have been described for the estimation of stevioside content in stevia leaves including enzymatic determination, near infrared reflectance spectroscopy (NIRS) and HPLC. All these methods are either time consuming or require highly sophisticated and costly instruments. The present study describes a method for rapid identification and estimation of stevioside content in commercial samples using HPTLC and HPLC. Material and Methods Stevioside standard was obtained from Sigma Aldrich, USA. Commercial stevia leaf powder and stevia powder were purchased from Maruti chemicals company, Ahmedabad, India. Acetonitrile (ACN) (HPLC grade) was from Merck, Germany, water (for Research Article [Khan et al., 2(9): Sep., 2011]