Asian Pac J Trop Biomed 2014; 4(4): 262-266 262 Asian Pacific Journal of Tropical Biomedicine journal homepage: www.apjtb.com Document heading doi:10.12980/APJTB.4.2014C1007 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. 襃 Quantitative estimation of hesperidin by HPTLC in different varieties of citrus peels 1 1 2 3 Prawez Alam *, Aftab Alam , Md. Khalid Anwer , Saleh I Alqasoumi 1Department of Pharmacognosy, College of Pharmacy, Salman Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Kingdom of Saudi Arabia 2Department of Pharmaceutics, College of Pharmacy, Salman Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Kingdom of Saudi Arabia 3Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Kingdom of Saudi Arabia PEER REVIEW ABSTRACT Peer reviewer Objective: To develop a simple, selective, sensitive and accurate high-performance thin layer P r o f . M a g e d S A b d e l - K a d e r , chromatography (HPTLC) method to determine the quantity of hesperidin in different varieties of Department of Pharmacognosy, College citrusMethods: fruits. of Pharmacy, Alexandria University, The method was carried out in aluminum-backed silica gel 60 F254 plates with ethyl 21215 15 3 2 ( ) Alexandria , Egypt. acetate-methanol-waterResults: : : %, v/v as mobile phase. R E-mail: [email protected] A compact band was obtained for hesperidin at f value of (0.40依0.04). The calibration Comments plot was linear in the range of 100-800 ng/spot of hesperidin and the correlation coefficient of 0.998 6 was indicative of good linear dependence of peak area on concentration. Limit of detection (8.87 This is a good study in which the ng/spot), limit of quantification (23.21 ng/spot), accuracy (less than 2%) and recovery (ranging from ) authors work to estimate the amount 98Conclusions:.55-99.38 were found satisfactory. of hesperidin in different varieties of The method developed can be used for routine analysis of hesperidin in crude drug citrus peels. Statically data proved that as well as in herbal and pharmaceutical dosage form containing citrus fruits as an ingredient. proposed method can be used in wide range for detection and estimation of hesperidin in different varieties of citrus peels. KEYWORDS Details on Page 266 HPTLC, Citrus, Hesperidin 1. Introduction anticancer activity against the selected cell lines[8]. In literature, several analytical methods for the determination Hesperidin is a flavanone glycoside found abundantly in of hesperidin have been reported. Several high performance citrus fruits and possess antioxidant activity[1,2]. Hesperidin liquid chromatography (HPLC) methods were developed alone, or in combination with other citrus bioflavonoids for the estimation of hesperidin either alone or in mixture is used for blood vessel conditions such as hemorrhoids, with other flavonoids in plant juices and pharmaceutical ( ) I [9-15] A varicose veins, and poor circulation venous stasis . t formulations .- liquid chromatography tandem mass is also used to treat lymphedema, a condition involving spectrometry (LC MS/MS) method was developed for the fluid retention that can be a complication of breast simultaneous determination of naringin, hesperidin, cancer surgery[3]. The antiallergic activity of hesperidin is neohesperidin, naringenin and hesperetin in rat plasma, activated by intestinal microflora[4]. Hesperidin has anti- using liquiritin as the internal standard[16,17]. No single inflammatory and analgesic effects[5-7]. In addition, the method has been reported for the quantitative estimation of results revealed that hesperidin exhibited pronounced hesperidin by high performance thin layer chromatography *Corresponding author: Dr. Prawez Alam, Department of Pharmacognosy, College Article history: of Pharmacy, Salman Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Received 11 Jan 2013 Arabia. Received in revised form 19 Jan, 2nd revised form 23 Jan, 3rd revised form 5 Feb 2014 Tel: +966-532771723 Accepted 22 Feb 2014 E-mail: [email protected] Available online 28 Apr 2014 Foundation Project: Supported by Deanship of Scientific Research, Salman Bin Abdulaziz University, Al-Kharj, KSA (Grant No.33/S/54). Prawez Alam et al./Asian Pac J Trop Biomed 2014; 4(4): 262-266 263 (HPTLC) in different varieties of citrus fruits. Therefore the volumetric flask. aim of present investigation was to develop a simple, precise 2.4. Chromatographic conditions and accurate HPTLC densitometric method for the estimation of hesperidin (Figure 1). OH HPTLC densitometric analysis was performed on 10 cm伊20 HO cm aluminium-backed plates coated with 0.2 mm layers of HO silica gel 60 F254 (E-Merck, Germany). Samples were applied H3C O TLC 6 C A OH to the plates as mm bands using a amag utomatic O TLC Sampler 4 (ATS4) sample applicator (Switzerland) fitted OCH3 C A HO O with a amag microlitre syringe. constant application O O 150 L L HO rate of n /s was used. inear ascending development of OH the plates to a distance of 80 mm were performed with ethyl acetate-methanol-water 15:3:2 (%, v/v) hesperidin as mobile C A D C (ADC ) OH O phase in a amag utomatic eveloping hamber 2 2 Figure 1. 30 Chemical structure of hesperidin. previously° saturated with mobile phase vapour for min at 22 C. 2. Materials and methods 2.5. Method validation 2.1. Plant material The proposed HPTLC method was validated according to Citrus limetta Citrus sinensis the guidelines of international conference on harmonization M ( ) ( ) L (ICH)[18] T Citrus osambi lemon , orange Citrus paradise, emon . he linearity of the method for hesperidin was ( ) and grape fruits ( ) were checked between 100 and 800 ng/spot and concentration was purchased from local market of Al Kharj, Kingdom of plotted against peak area. Saudi Arabia. The plant was collected and identified by Accuracy, as recovery, was determined by the standard Dr. Mohammad Atiqur Rahman; taxonomist of Medicinal, addition method. Pre-analyzed samples of hesperidin (300 Aromatic and Poisonous Plants Research Center (MAPPRC), ng/spot) were spiked with extra hesperidin standard (0%, College of Pharmacy, King Saud University, Saudi Arabia. 50%, 100%, and 150%) and the mixtures were reanalyzed. A voucher specimen 13998 was deposited in the Herbarium Percentage recovery and relative standard deviation (RSD, %) of the College of Pharmacy, King Saud University, Saudi were calculated for each concentration level. Arabia. Precision was assessed by determination of repeatability R 2.2. Chemicals and intermediate precision. epeatability of sample was determined as intra-day variation whereas intermediate precision was determined by assessment of inter-day Standard hesperidin was purchased from Sigma-Aldrich, variation for analysis of hesperidin at three different St. Louis, MO, USA. All the solvents were of HPLC grade and amounts (300, 400, and 500 ng/spot) in six replicates. other chemicals used were of analytical reagent grade. Robustness of the proposed TLC densitometric method was Accurately weighed 1 mg of standard hesperidin (purity determined to evaluate the influence of small deliberate 99%) was dissolved in 5 mL methanol and heated on a water changes in the chromatographic conditions during bath to dissolve completely and then make up the volume determination of hesperidin. Robustness was determined by 10 up to mL with methanolµ in a volumetric flask to give changing the polarity of the mobile phase. 100 L D L (LOD) (LOQ) concentration i.e. of g/m . ifferent µvolumes of working imit of detection and limit of quantification standard, 1, 2, 3, 4, 5, 6, 7 and 8 L were applied on were determined by standard deviation (SD) method. They thin-layer chromatography (TLC). The calibration curve were determined from the slope of the calibration (S) curve 100 800 SD was plotted in the range of - ng/spot, using data of and of the伊 blank sample using following equations: T LOD 3 3 SD S peak areas against the corresponding amount per spot. his = . 伊 / solution was used as a reference solution (stock solution) for LOQ=10 SD/S hesperidin. Specificity of the proposed TLC densitometric was Rf 2.3. Sample preparation for analysis of hesperidin in confirmed by analyzing and comparing the values and methanolic extract of citrus fruits spectra of the spot for hesperidin in the samples with that of the standards. 2.6. Quantification of hesperidin in different varieties of T (5 ) he air-dried peel g of citrus fruits were coarsely methanolic extract of citrus peels powdered, defatted with petroleum ether and then exhaustively extracted in a Soxhlet apparatus with methanol for 72 h. The solvent was evaporated to dryness under The test samples were applied and chromatograms were reduced pressure by use of a rotary vacuum evaporator and obtained under the same conditions as for analysis of the residue was separately dissolved in methanol in 50 mL standard hesperidin. The area of the peak corresponding to Prawez Alam et al./Asian Pac J Trop Biomed 2014; 4(4): 262-266 264 R 3.3. Method validation the f value of hesperidin standard was recorded and the amount present was calculated from the regression equation 3.3.1. Precision obtained from the calibration plot. The accuracy of the method, as recovery, was 98.55-99.38%, RSD 0 68 1 12 3. Results with values in the range . - . These results indicated the accuracy of the method (Table 2). Results 3.1. Method development from determination of repeatability and intermediate SD (%) T 3 RSD precision, expressed as are shown in able- . was in the range 0.59-1.06 for repeatability and 0.76 1.20 for The mobile phase composition was optimized to establish intermediate precision. These low values indicated that the a suitable and accurate densitometric HPTLC method for method is precise[19]. analysis of hesperidin.