Page6175 Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876 Journal home page: http:///index.php/en/ INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH ESTIMATION OF NARINGIN AND HESPERIDIN FROM CITRUS FRUITS BY HPTLC Sameer H. More, Shruti B. Sathe, Archana A. Sonawane, Aruna P. Jadhav, and Dr. Vilasrao J. Kadam Department of Quality Assurance, Bharati Vidyapeeth s College of Pharmacy, C.B.D. Belapur, Navi Mumbai-400614, Maharashtra, India. ARTICLE INFO Article history Received 29/07/2013 Available online 30/08/2013 Keywords Citrus fruits, Hesperidin, HPTLC, Naringin, Estimation. ABSTRACT A simple, precise and accurate High Performance Thin Layer Chromatographic method has been developed for the estimation of Naringin and Hesperidin in Citrus fruits. The separation was carried out on Merck TLC aluminium sheets of silica gel 60F 254, using Ethyl acetate: Chloroform: Acetonitrile: Glacial acetic acid: Triethylamine (3:4:2:1:2, v/v/v/v/v) as mobile phase and densitometric analysis of compound was carried out in absorbance mode at 285 nm. The linear regression analysis data for the calibration plots for Naringin and Hesperidin showed good linear relationship with regression coefficient (r 2 ) 0.994 and 0.998 in the concentration range of 150 500 ng/spot and 200-550 ng/spot, respectively. The method was validated for linearity, specificity, recovery, precision, robustness, limit of detection (LOD) and limit of quantification (LOQ). The statistical analysis of the data showed that the method is reproducible and selective for estimation of both flavonoids. In conclusion, the proposed method was successfully applied for identification and quantitation of these flavonoids in Citrus fruits and marketed formulation. Corresponding author Dr. Aruna P. Jadhav, Department of Quality Assurance, Bharati Vidyapeeth s College of Pharmacy, C.B.D. Belapur, Navi Mumbai-400614, Maharashtra, India. E-mail: drarunajadhav@gmail.com Tel: +91-22-27571122/2131; Fax: +91-22-27574515 Please cite this article in press as Dr. Aruna Jadhav et.al. Estimation of naringin and hesperidin from citrus fruits by HPTLC. Indo American Journal of Pharm Research.2013:3(8). Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page6176 INTRODUCTION Citrus fruits are edible fruits of plants belonging to genus Citrus of the family Rutaceae including orange, grapefruit, pomelo, sweet lime, lemon etc[1]. These fruit contain hundreds of phytochemicals and there is an increasing interest in the possibility that these substances contribute to optimal health and have a range of health related effects including antioxidant, anti viral, anti allergenic, anti inflammatory and anti cancer[2]. Some of these activities are due to presence of flavonoids such as Naringin and Hesperidin present in it (figure 1). Literature survey reveals that Naringin is estimated individually and in combination with other drugs by HPLC from fruit juice[3-7], HPLC in human urine sample[8], LC-MS in rat plasma[9], LC-MS/MS in rat serum[10], UV spectrophotometry[11], TLC- calorimetry in grapefruits[12], UPLC-MS/MS in rat plasma[13], Chiral separation HPLC in Launeae arborescens extract[14], and HPTLC in Drynaria quercifolia extract[15] Similarly, Hesperidin is estimated individually and in combination with other drugs by RP-HPLC from fruit juice[3,5,7], LC-MS in rat plasma[9], LC-MS/MS in rat serum[10], Chiral separation HPLC in Launeae arborescens extract[14], UHPLC in red wine extact[16], UV spectrophotometry from Orange juice and peel extract[17] and HPTLC from bulk and pharmaceutical dosage forms[18]. Now-a-days HPTLC is becoming a routine analytical technique due to its distinct advantage that it offers over other analytical techniques, that is several samples can be separated in parallel on same plate resulting in a high throughput and rapid low cost analysis. The aim of present work is to develop an accurate, specific, repeatable and robust HPTLC method for the determination of Naringin and Hesperidin in Citrus fruits and pharmaceutical dosage form. The proposed method was validated as per ICH guidelines[19]. Figure 1: Structure of Naringin (A) and Hesperidin (B) MATERIAL AND METHODS Materials Standard Naringin and Hesperidin were procured from Nans Products Ltd. and Otto Chemie, Mumbai, India, respectively. All the chemicals used in the experiment are of analytical grade procured from SD fine Chemicals, India. Plant material Grapefruit, Pomelo and Orange fruits were collected from local market and authenticated for their correct botanical identity by Dr. H. M. Pandit, Botanist, Guru Nanak Khalsa College, Mumbai 400 019.
Page6177 Preparation of extracts Grapefruit, Pomelo and Orange fruits were peeled manually and dried in tray dryer at 40 C. 25 g of powdered peel was extracted with methanol using soxhlet extractor till complete exhaustion. Extract was concentrated using rotary vacuum evaporator at 40 C and stored in the vacuum desiccator. Preparation of standard stock solution Naringin and Hesperidin stock solutions (1000 μg/ml) were prepared by dissolving accurately weighed 100 mg of each standard in 100 ml methanol. Preparation of sample solution Extract solution: Accurately weighed 100 mg of each dried extract was dissolved in 80 ml of methanol and final volume was made up to 100 ml with methanol to get stock solution containing 1 μg/ ml. Tablet: 20 tablets were weighed and average weight was calculated. All 20 tablets were triturated and powder equivalent to 10 mg of Hesperidin was dissolved in methanol and further dilutions were made to obtain concentration 40 μg/ml. The sample solution was filtered through Whatman filter paper. Instrumentation and chromatographic conditions Spotting was performed on TLC aluminium plates (10 10 cm) pre-coated with silica gel 60F 254. Standard solutions of markers and sample were applied to the plates as bands 6.0 mm wide, 10.0 mm from the bottom edge of the same chromatographic plate by use of a Camag (Muttenz, Switzerland) Linomat 5 sample applicator equipped with a 100 μl Hamilton syringe. Ascending development to a distance of 80 mm was performed at room temperature (24 ± 2 C), with mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase vapor for 60 min. After development, the plates were dried and then scanned at 285 nm with a Camag TLC Scanner 3 using the deuterium lamp with wincats software. Calibration curve for Naringin and Hesperidin Serial dilutions were made in the range of 15-50 μg/ml and 20-55 μg/ml for Naringin and Hesperidin, respectively. Aliquot of above each solutions (10 μl) were applied with the band width of 6 mm, in triplicate on TLC plate (10 10 cm) to obtain in a concentration range of 150-500 ng/spot for Naringin and 200-550 ng/spot for Hesperidin. Peak area for each band was recorded. Calibration curve was obtained by plotting peak area vs concentration of Naringin and Hesperidin. Analysis of sample Each sample and the standard solution were spotted in triplicates on a plate, developed using above chromatographic conditions. The analyte was found to be completely separated from other components so the linear and compact zones were scanned and their peak areas were recorded. METHOD VALIDATION The optimized HPTLC method was validated with respect to the following parameters. The validation was performed as per the ICH guidelines.
Page6178 Linearity and specificity Linearity was determined by spotting series of concentrations of standard and finding regression. The specificity of the method was ascertained by comparing the R f value and the peak purity was assessed by comparing the spectrum of standard Naringin and Hesperidin with sample. Precision The precision was examined by performing the intra-day and inter-day assay of the standard solutions at three concentration levels (200, 300, 400 ng/spot) in triplicate. The intra-day assay precision was performed three times on same day, while the inter-day assay precision was performed over three different days. Limit of detection and limit of quantification LOD and LOQ were determined by k x SD/s where k is a constant (3 for LOD and 10 for LOQ), SD is the standard deviation of the analytical signal and s is the slope of the calibration curve. The recovery of the drug at different levels in the fruits and formulation was checked by spotting the test samples of known concentration of Naringin and Hesperidin on the plates. The spots were then spiked in three different concentrations (80%, 100% and 120% w/w) by further adding known amount of standard mixture of Naringin and Hesperidin. These samples were then analyzed and the results obtained were compared with expected results. Robustness The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. Following the introduction of small changes in the mobile phase composition (±0.2 ml for major component), the effect on the results was examined. The amount of mobile phase was varied over the range of ± 5 %. The saturation time of development chamber was varied by ± 5 min. The robustness of the method was determined at two concentration levels (300 and 400 ng/spot). RESULT AND DISCUSSION Selection of analytical wavelength In situ HPTLC spectral overlain of Naringin and Hesperidin were taken. Isoabsorptive point was found at 285 nm and was selected as scanning wavelength. Optimization of chromatographic conditions The experimental conditions for HPTLC such as wavelength of detection and mobile phase composition were optimized to provide accurate, precise and reproducible results for the determination of Naringin and Hesperidin. The mixed standard stock solution containing 100 µg/ml of Naringin and Hesperidin was spotted on to TLC plate and developed in different solvent systems. Good resolution and sharp peaks were obtained with minimum tailing by using mobile phase consisting of ethyl acetate: chloroform: acetonitrile: glacial acetic acid: triethylamine, 3:4:2:1:2 (v/v/v/v/v). Naringin and Hesperidin were satisfactorily resolved with R f values at 0.41 ± 0.02 and 0.51 ± 0.03, respectively (figure 2).
Page6179 Figure 2: Chromatogram of standard Naringin [R f : 0.41 ± 0.02] and Hesperidin [R f : 0.51 ± 0.03] Linearity Linear relationship was observed by plotting drug concentration against peak area for each compound. Naringin and Hesperidin showed linear response in the concentration range of 150-500 ng/spot and 200-550 ng/spot, respectively (figure 3). The linearity was validated by the high value of the correlation coefficients. The results are tabulated in Table 1. Figure 3: Calibration curve of Naringin (a) and Hesperidin (b)
Page6180 Table 1: Linear regression data for calibration plot for Naringin and Hesperidin (n=3). Parameters Naringin Hesperidin Linearity range (ng) 150-500 200-550 Correlation coefficient (r 2 ± S.D) 0.994 ±0.00476 0.998 ±0.00098 Slope (mean ± S.D) 13.43 ±0.44 13.36 ±0.08 Intercept (mean ± S.D) 782.06 ±143.57 757.88 ±23.70 P value < 0.0001 < 0.0001 SD= Standard Deviation. P value is < 0.0001, considered extremely significant Precision The % values depicted in Table 2 show that the proposed method provides acceptable intra-day and interday variation of Naringin and Hesperidin, which indicate good precision for the developed method. Table 2: Intra-day and inter-day precision results for Naringin and Hesperidin Compound Intraday Interday Concentration (ng/spot) Conc. Conc (ng/spot) (ng/spot) 200 198.746 1.603 205.187 2.265 Naringin 300 298.932 0.733 301.712 1.336 400 397.666 0.961 398.548 1.607 200 188.987 1.624 185.257 1.237 Hesperidin 300 295.336 1.944 290.746 0.925 400 402.622 0.975 396.958 2.098 = Relative Standard Deviation, Each result is an average of nine measurements Limit of detection and limit of quantification The LOD and LOQ were found to be 33.54, 18.86 ng/spot and 100.75, 57.18 ng/spot for Naringin and Hesperidin, respectively. As shown from the data in Table 3 and 4, the recovery of Naringin from Grapefruit and Pomelo peel was found to be 99.82 and 99.78 %, respectively while that of Hesperidin from the orange peel and tablet was 99.33 and 99.58 %, respectively. Table 3: data for Naringin Compound Grapefruit Extract Level of % recovery Amount added (ng)) amount (ng) 80 365.065 371.308 101.710 1.569 100 405.065 401.067 99.012 1.947 120 445.065 440.481 98.970 1.931 Mean 99.82 80 403.534 399.801 99.074 2.576 Pomelo 100 443.534 445.544 100.453 0.873 99.78 Extract 120 493.534 492.752 99.841 1.224 = Relative Standard Deviation, Each result is an average of three measurements
Page6181 Compound Orange extract Level of % recovery Table 4: data for Hesperidin Amount added (ng) amount (ng) 80 385.108 384.221 99.769 3.090 100 435.108 436.292 100.272 2.019 120 475.108 465.384 97.953 1.639 Mean 99.33 Marketed 80 360.660 359.221 99.601 2.074 formulation 100 400.660 398.218 99.390 1.251 99.58 extract 120 440.660 439.560 99.750 1.255 = Relative Standard Deviation, Each result is an average of three measurements Specificity It was observed that other constituents present in the extract did not interfere with the peak of Naringin and Hesperidin. Therefore the method is specific. The spectrum of standard Naringin and Hesperidin corresponds with sample. Robustness The % of the peak area was calculated in triplicate for changes in mobile phase composition, duration of saturation time and volume of mobile phase for 300 and 400 ng/spot. The values of % as shown in table 5 were less than 2% which indicated that the developed method is robust. Parameters 3.2:3.8:2:1:2 [v/v/v/v/v] 2.8:4.2:2:1:2 [v/v/v/v/v] Table 5: Robustness results for Naringin and Hesperidin. Naringin Hesperidin 300 ng/spot 400 ng/spot 300 ng/spot 400 ng/spot Conc (ng) Conc (ng) Conc (ng) Mobile phase composition Conc (ng) 303.723 0.307 413.874 1.874 314.635 2.284 412.739 0.915 303.887 1.224 391.883 1.586 304.905 1.984 402.060 1.246 Saturation Time +5 Min 311.541 3.901 412.335 1.050 301.412 0.911 399.241 1.512-5 Min 304.939 1.907 390.568 1.532 290.481 1.175 410.768 1.439 Mobile phase volume +5% 298.684 1.457 392.653 1.193 320.873 1.486 395.798 2.175-5 % 306.652 1.296 401.514 0.474 323.567 1.490 395.648 1.934 = Relative Standard Deviation, Each result is an average of three measurement
Page6182 Analysis of Herbal extract and marketed formulation The developed method was applied for the detection and quantification of Naringin in Grapefruit and Pomelo peel whereas Hesperidin in Orange peel and formulation. The peaks for Naringin and Hesperidin were observed at R f 0.41 and 0.51, respectively in the densitogram of the herbal extract and tablet. There was no interference from other compounds present in the fruit peel and tablet. The total Naringin content in Grapefruit and Pomelo peel was found to be 2.15 and 7.24 % w/w, respectively, whereas Hesperidin content in Orange peel and Formulation was found to be 1.61 and 7.54 % w/w, respectively, shown in Table 6 and 7. Statistical evaluation of the results was performed with regard to accuracy and precision using Student s t-test and the F-ratio at 95% confidence level. Table 6: Naringin Content in Grapefruit and Pomelo Naringin Content (% w/w) Developed Method Reported method[15] F value t value Grapefruit peel 2.149 ± 0.012 2.139 ± 0.0125 1.037 0.940 Pomelo peel 7.262 ± 0.071 7.253 ± 0.064 0.817 0.164 Each result is an average of three measurements, standard F-value at 95% confidence level = 19; standard t- value at 95% confidence level = 2.776. Table 7: Hesperidin Content in Orange and Tablet Hesperidin Content (% w/w) Developed Method Reported method[18] F value t value Orange peel 1.613 ± 0.011 1.599 ± 0.029 6.511 0.739 Marketed formulation 7.600 ± 0.175 7.443 ± 0.157 0.803 1.152 Each result is an average of three measurements, standard F-value at 95% confidence level = 19; standard t- value at 95% confidence level = 2.776. CONCLUSION The developed HPTLC method has been shown to be selective, linear, precise and accurate. Statistical analysis proves that the method is suitable for routine analysis of Naringin and Hesperidin in raw material, extracts and pharmaceutical preparations. This developed method can be used to study the degradation of Naringin and Hesperidin under different stress conditions as per the recommendations of ICH guidelines. REFERENCES 1. Mabberley D. A classification for edible Citrus (Rutaceae). Telopea. 1997;7:167-72. 2. Baghurst K. The Health Benefits of Citrus Fruits. Horticultural Australia Ltd, 2003. 3. Gorinstein S, Huang D, Leontowicz H, Leontowicz M, Yamamoto K, Soliva-Fortuny R, Belloso OM, Martinez Ayala AL et al. Determination of naringin and hesperidin in Citrus fruit by high-performance liquid chromatography. The antioxidant potential of Citrus fruit. Acta Chromatographica. 2006;17:108-24. 4. Widmer W. Determination of naringin and neohesperidin in orange juice by liquid chromatography with UV detection to detect the presence of grapefruit juice: collaborative study. J AOAC Int. 2000;83:1155-65. 5. Levaj B, Dragović-Uzelac V, Bursać-Kovačević D, Krasnići N. Determination of flavonoids in pulp and peel of mandarin fruits. Agriculturae Conspectus Scientificus. 2009;74:221-25.
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