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 by HPTLC in different varieties of 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 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 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 , hesperidin, cancer surgery[3]. The antiallergic activity of hesperidin is , and in rat plasma, activated by intestinal microflora[4]. Hesperidin has anti- using 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 , 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. The mobile phase ethyl acetate- Table 2 15 3 2 (% ) n methanol-water : : , v/v resulted R in a sharp, Accuracy of the proposed method ( =6). f (0 40依 symmetrical, and well resolved peak at value of . Excess drug added Theoretical Conc. found 0 04) ( 2) % Recovery % RSD . Figure . UV spectra measured for the bands showed to analyte (%) content (ng) (ng)依SD maximum absorbance at approximately 286 nm. 0 300 298.16依2.04 99.38 0.68 Track 6 ID: Standard 6 50 450 443 50 4 96 98 55 1 12 600 . 依 . . . AU 100 600 595.00依4.47 99.16 0.75 500 150 750 745 16 3 93 99 35 0 75 Hesperidine . 依 . . . 400 Conc.: Concentration.

300 Table 3 n ( 6) 200 Precision of the proposed method = . Repeatability (Intraday precision) Intermediate precision (Interday) Conc. 100 Standard % Standard % (ng/spot) Area依SD Area依SD RSD RSD 0 R error error 0 00 0 20 0 40 0 60 0 80 1 00 f Figure 2...... 300 5 794.16依61.50 25.15 1.06 5 794.00依69.69 28.45 1.20 HPTLC chromatogram of standard hesperidin. 3.2. Calibration curve 400 7 352.50依71.15 29.05 0.96 7 331.66依56.41 23.06 0.76 500 8 661.16依51.41 20.99 0.59 8 700.05依87.80 35.85 1.00 Conc.: Concentration. T he calibration plot of peak area against amount of 3.3.2. Robustness of the method hesperidin was linear in the range 100-800 ng/spot. Linear regression data for the plot confirmed the good linear Results of robustness are shown in Table 4. Low values R2 (T 1) T ( ) % RSD (0 46 1 29) relationship able . he correlation Pcoefficient was of . - . were obtained after introducing small 0 998 6 ( 0 05) T TLC . which was highly significantY x < . . he linearY deliberate change into the densitometric procedure 13 47 +1 657 5 HPTLC [20] regression equationX was = . . , where is proved the robustness of the proposed method . (F 3) Table 4 responseTable 1 and is amount of hesperidin igure . n n Robustness of the proposed HPTLC method ( =6). Linear regression data for the calibration curve of hesperidin ( =6). Mobile phase composition L D Conc. Results inear regression ata (Ethyl acetate: methanol: water) ( ) Linearity range (ng/spot) 100-800 ng/spot Rf Y x Original Used Area依SD % RSD R 13 47 +1 657 50 egression equation = . . 15:3.2.1 +0.1 7 310依75 1.02 0.41 C 0 998 6 orrelation coefficient . 400 15:3:2 15:3:2 0.0 7 290依59 0.81 0.40 S SD 13 470 0 031 lope依 . 依 . 15:3.1.9 -0.1 7 284依51 0.69 0.39 Intercept依SD 1 657.50依15.68 Conc.: Concentration. Standard error of slope 0.076 Standard error of intercept 38.41 3.3.3. Limit of detection and quantification - 95% confidence interval of slope 13.31 13.62 LOD and LOQ of the proposed method was found to be 8.87 95% confidence interval of intercept 1 580.1-1 734.9 and 23.21 ng/spot, for hesperidin, which indicated that the 14000 y x proposed method can be used in wide range for detection =13.47 +1657.50 12000 R2 [21] =0.998 and quantification of hesperidin effectively . 10000 3.3.4. Specificity 8000

6000 The peak purity of , hesperid in and ascorbic

4000 acid were assessed by comparing the overlaid spectra at peak start, peak apex and peak end position of the spot. 2000 The overlaid spectra of hesperidin standards and different 0 varieties ofmethanolic extracts of citrus peels was given in 0 100 200 300 400 500 600 700 800 900 Figure 3. F 4 Regression curve of standard hesperidin. igure . Prawez Alam et al./Asian Pac J Trop Biomed 2014; 4(4): 262-266 265

Spectra comparsion Track 10 ID: Orange 100.0 100.0 500 90.0 90.0 AU450 80.0 80.0 400 7 AU

AU 70.0 70.0 350 60.0 60.0 300 50.0 50.0 250 40.0 40.0 200 30.0 30.0 150 Hesperidin 5 6 20.0 20.0 100 3 4 1 10.0 10.0 50 0.0 0.0 0 R 200.0 250.0 300.0 350.0 400.0 0.00 0.20 0.40 0.60 0.80 1.00 f Figure 8. Figure 4. nm hesperidin HPTLC Overlay UV absorption spectra of the standard and chromatogram of methanolic extract of orange peels. extracts of four different varieties of citrus peels. Table 5 3.3.4. Quantification of hesperidin in different varieties of Contents of hesperidin in different varieties of methanolic extracts of methanolic extracts of citrus peels citrus peels. Samples Contents (mean依SD, % w/w) % RSD H Citrus paradisi esperidin peaks from different varieties methanolic Grape fruits ( ) 11.15依2.34 4.11 Citrus limetta extract of citrusR peels were identified by comparing their Mosambi ( ) 3.79依2.56 5.56 f 0 40依0 04 (F 5 8) Citrus lemon single spot at = . . igure - values with those Lemon ( ) 1.72依2.53 3.67 Citrus sinensis obtained by chromatography of the standard under the same Orange ( ) 1.29依1.78 5.87 conditions. The hesperidin content in methanolic extracts of four different varieties of citrus peels was quantified by use of the linear regression equation and concentrations are 4. Discussion presented in Table 5. Track 16 ID: Grapes 500 A validated HPTLC method has been developed to AU450 Hesperidin 400 6 determine the quantity of hesperidin in methanolic extracts 350 of four different varieties of citrus peels. The mobile phase 15 3 2 (% ) 300 ethyl acetate-methanol-water : : , v/v resultedR in 250 a sharp, symmetrical, and well resolved peak at f value 200 of 0.40. Linear regression data for the plot confirmed the 150 100 1 3 4 5 good linear relationship and the resulting equation was 50 operational in the concentration range of 100-800 ng/spot. RSD 0 Rf The method was accurate 98.55%-99.38%, with values in 0.00 0.20 0.40 0.60 0.80 1.00 Figure 5. 0 75 1 12 HPTLC chromatogram of methanolic extract of grapes peels. the range . - . after spiking the hesperidin with different concentrations of standard.The HPTLC method developed for Track 14 ID: Mosammi 600 quantitation of hesperidin was found to be simple, accurate, AU reproducible and sensitive and is applicable to the analysis 500 7 of methanolic extracts of four different varieties of citrus 400 peels. Also it will find wide applications in standardization Hesperidin 300 and quality control of herbal raw materials as well as formulations. Statistical data proves that the method is 200 1 2 6 reproducible and selective for the analysis of hesperidin 3 4 100 with added advantages of short time, minimal sample preparation, in addition to the low cost. 0 R 0.00 0.20 0.40 0.60 0.80 1.00 f Figure 6. HPTLC chromatogram of methanolic extract of mosambi peels. Conflict of interest statement

Track 10 ID: Lemon 600 AU We declare that we have no conflict of interest. 500 11 400 Acknowledgements

300 Hesperidin 200 7 33 54 8 This project was supported by Grant No. /S/ , Deanship 1 4 5 3 9 10 S R S B A U 100 2 of cientific esearch, alman in bdulaziz niversity, Al-Kharj, KSA. The authors are grateful to Department of 0 R 0.00 0.20 0.40 0.60 0.80 1.00 f Pharmacognosy, King Saud University, Riyadh, Kingdom of Figure 7. HPTLC chromatogram of methanolic extract of lemon peels. Saudia Arabia for providing facilities to carry out this study. Prawez Alam et al./Asian Pac J Trop Biomed 2014; 4(4): 262-266 266 Comments [6] G alati EM, Monforte MT, Kirjavainen S, Forestieri AM, Trovato A, Tripodo MM. Biological effects of hesperidin, a citrus . Background Farmaco 40 (Note I): antiinflammatory and analgesic activity. 1994; : HPTLC is an important technique for standardization and 709-712. identification of different chemical constituents from medicinal [7] E mim JA, Oliveira AB, Lapa AJ. Pharmacological evaluation of the anti-inflammatory activity of a citrus bioflavonoid, hesperidin, and plants. Hesperidin is flavanone glycoside found abundantly J the , duartin and claussequinone, in rats and mice. in citrus peels and possess various pharmacological activity. Pharm Pharmacol 46 Therefore the aim of present investigation was to develop a 1994; : 118-122. simple, precise and accurate HPTLC densitometric method [8] A l-Ashaal HA, El-Sheltawy ST. Antioxidant capacity of hesperidin from citrus peel using electron spin resonance and cytotoxic activity for the estimation hesperidin. The studies were carried out Pharm Biol 49 to quantitative estimation of hesperidin by HPTLC in different against human carcinoma cell lines. 2011; : 276-282. varieties of citrus peels. [9] X ia J, Kotani A, Hakamata H, Kusu F. Determination of Hesperidin in pericarpium citri reticulatae by semi-micro HPLC with J Pharm Biomed Anal 41 Research frontiers 2006 1401 1405 electrochemical’ detection. ; : - . The present aim of this research work is to develop and [10] C ornara L, D Arrigo C, Pioli F, Borghesi B, Bottino C, Patrone E, et al. Micromorphological investigation on the leaves of the rock validate a simple, accurate HPTLC method for the quantitative Crithmum maritimu HPTLC samphire ( L.): occurrence of hesperidin and estimation of hesperidin by in different varieties of citrus Plant Biosyst 143 peels. diosmincrystals. 2009; : 283. [11] K anaze FI, Gabrieli C, Kokkalou E, Georgarakis M, Niopas Related reports I. Simultaneous reversed-phase high-performance liquid No single method has been reported for the quantitative chromatographic method for the determination of diosmin, hesperidin HPTLC and naringin in different citrus fruit juices and pharmaceutical estimation of hesperidin by in different varieties of citrus J Pharm Biomed Anal 33 - peels as per literature review available in our resources. formulations. 2003; : 243 249. [12] H e JX, Zhang Q, Lin SH. Quantitative analysis of narigin and Innovations and breakthroughs Citrus aurantium hesperidin contents in juice by reverse-phase Modern Food Sci Technol The proposed HPTLC method can be used for quantitative high-performance liquid chromatography. estimation of hesperidin by HPTLC in different varieties of citrus 2008; doi: 10.3969/j.issn.1673-9078.2008.01.026. peels without interference. [13] E l-Shafae AM, El-Domiaty MM. Improved LC methods for the determination of diosmin and/or hesperidin in plant extracts and Applications J Pharm Biomed Anal 26 - pharmaceutical formulations. 2001; : 539 545. [14] S un Y, Wang J, Gu S, Liu Z, Zhang Y, Zhang X. Simultaneous The proposed method for estimation of hesperidin by HPTLC Citrus reticulata in different varieties of citrus peels is first validated HPTLC ‘determination’ of flavonoids in different parts of Chachi fruit by high performance liquid chromatography Photodiode method and its statistical data proves that the method is Molecules 15 reproducible and selective for the analysis of hesperidin with Array Detection. 2010; : 5378-5388. added advantages of, speed and minimal, low cost of reagents [15] L i K, Yuan J, Su W. Determination of liquiritin, naringin, hesperidin, satisfactory precision and accuracy. This method may be used thymol, imperatorin, honokiol, isoimperatorin, and magnolol in the traditional Chinese medicinal preparation Huoxiang-zhengqi liquid for quality control and standardization of different varieties of Yakugaku Zasshi using high-performance liquid chromatography. citrus peels and marketed herbal formulations. 126 2006; : 1185-1190. Peer review - [16] L i X, Xiao H, Liang X, Shi D, Liu J. LC MS/MS determination of naringin, hesperidin and neohesperidin in rat serum after orally This is a good study in which the authors work to estimate Bulpleurum falcatum Fractus administrating the decoction of L. and the amount of hesperidin in different varieties of citrus peels. aurantii J Pharm Biomed Anal 34 Statically data proved that proposed method can be used . 2004; : 159-166. in wide range for detection and estimation of hesperidin in [17] T ong L, Zhou D, Gao J, Zhu Y, Sun H, Bi K. 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