INTRODUCTION:
Hesperidine, a bioflavonoid, is an abundant and inexpensive by-product of Citrus cultivation. A deficiency of this substance in the diet has been linked with abnormal capillary leakiness as well as pain in the extremities causing aches, weakness and night leg cramps. No signs of toxicity have been observed with the normal intake of hesperidine or related compounds1. Hesperidine is chemically (S)-7-[[6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-gluco pyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, is a white to yellow crystalline powder melting at ca 2600 C 2. Flavonoids are a group of polyphenolic compounds, which are widely distributed throughout the plant kingdom. To date about 3000 varieties of flovonoids are known3.
Many have low toxicity in mammals and some of them are widely used in medicine for maintenance of capillary integrity4. Flavonoids exhibit several biological effects such as anti-inflammatory, anti hepatotoxic and anti-ulcer actions5, 6. They also inhibit enzymes such as aldose reductase and xanthine oxidase. They are potent antioxidants and have free radical scavenging abilities. Many have anti-allergic, antiviral actions and some of them provide protection against cardiovascular mortality7, 8. They have been shown to inhibit the growth of various cancer cell lines in vitro, and reduce tumor development in experimental animals9.
Fig No.1. Structure of Hesperidine
Few analytical methods have been reported for the estimation of Hesperidine; they are UV 10, HPLC 11, TLC 12, GC 13, HPTLC 14, LC-MS 15, GC-MS 16, Fourier transform infrared spectroscopy (FT-IR), Nuclear Magnetic Resonance Spectroscopy (NMR) 17 and X-Ray Diffraction17 methods. The present work is focused on isolation and characterization of hesperidine present in dried peel of Citrus limetta and ayurvedic formulation by TLC, IR Spectroscopy, UV Spectroscopy and UFLC methods.
MATERIALS AND METHODS:
Collection:
Oranges were collected during December to January 2012, from the local market of Anantapur and was identified and authentified by a botanist. Peel was collected, washed with running water, shade dried and powdered.
Chemicals:
The Standard marker Hesperidine was obtained from HIMEDIA Laboratories Pvt. Limited, Mumbai. All chemicals and reagents were of analytical grade such as ethyl acetate (Merck Pvt. limited), Dimethyl Formamide (Merck Pvt. limited), Silica gel G (Finar chemicals Pvt. Ltd) and some were of HPLC grade such as water, Methanol and Acetonitrile. Ayurvedic formulation Madiphal rasayana (Baidyanath) liquid formulation was purchased from ayurvedic drug store.
Experimental Part:
Extraction procedure: 18
Extraction of hesperidine was achieved by using 1kg shade dried powder peel of Citrus limetta by using Soxhlet extractor. Petroleum ether (40 – 60°C) is filled in a 3 liter round bottom flask with magnetic stir bar. Dried and powdered orange peel are placed in the extraction sleeve of a Soxhlet extractor and covered with a little glass wool. A reflux condenser is put on the Soxhlet extraction unit, and then the reaction mixture is stirred and heated for 4 hours under strong reflux. The petroleum ether extract is discarded.
Afterwards the substance is placed again in an extraction sleeve and, like before, but with methanol, extracted unless the solvent leaving the extraction sleeve is colorless (1 to 2 hours).The extract is evaporated. The residue is mixed with 6% acetic acid; the precipitated solid is the crude hesperidine. It is sucked off with a Buchner funnel, washed with 6% acetic acid and dried at 60 °C until it is constant in weight.
Recrystallization:
A 5% solution of the crude product in Dimethyl sulfoxide is produced under stirring and heating to 60–80 °C. Afterwards the same amount of water is added slowly whilst stirring. When cooling to room temperature the hesperidine precipitates. It is sucked off, first washed with little warm water and then with iso-propanol and dried in the desiccators until it is constant in weight.
Preparation of standards:
Preparation of Standard stock solution:
Accurately weighed 10 mg quantity of API and isolated powder of hesperidine was dissolved in 0.5 ml of Dimethyl formamide (DMF) and water was added to make up the volume up to 100 ml to give a concentration of 100µg/ml.
Preparation of ayurvedic formulation sample:
Accurately measured 1 ml quantity of Marketed formulation was dissolved in 2.0 ml of Dimethyl formamide (DMF) and water was added to make up the volume up to 100 ml to give a concentration of 100µg/ml.
Analysis of Hesperidine:
Identification by IR spectroscopy:
The hesperidine was characterized by using FT-IR where the sample of standard and isolated were subjected for IR radiation and the IR spectrum was compared for standard and isolated.
UV spectroscopy:
For the prepared stock solutions of standard hesperidine, isolated hesperidine and ayurvedic formulation Madiphal rasayana absorbance was measured between 200 to 400 nm by using UV-Visible double beam Spectrophotometer, (Elico SL-244) and reported.
Identification by Thin Layer Chromatography (TLC):
Silica gel G was taken and made into slurry using water and slides were prepared by pouring the slurry on glass slides to get a uniform distribution. These plates were kept in oven for drying before spotting the plates were activated at 1050C. Saturated hesperidine solution was prepared by using DMF for standard, isolated and marketed formulation. Developing chamber was kept ready with proposed solvent system Ethyl acetate: methanol: Water (8.3:1.0:0.7 v/v/v) for saturation. The spotting was done in such a way that approximately 1 mm-sized spot of the solution is seen. The plate was kept slightly tilt in solvent system to run the solvent system 3/4th of the plate. The spots made visible by using iodine chamber and Rf was calculated.
UFLC analysis:
An isocratic Chromatographic
separation was developed by using UFLC Shimadzu LC 20 AD , column used was Pre-packed Phenomenex Luna 5u
C18, 250 x 4.6mm, 5µm mobile phase system consist of Water: Acetonitrile: Methanol
(50:25:25v/v/v), with a flow rate of 1.0 ml/min and injection volume was 20 
l with a vacuum degasser and PDA detector.
LC solutions software was utilized for instrument control, data collection and
data processing.
RESULTS AND DISCUSSION:
IR spectroscopy:
The structure of the extracted Hesperidine was confirmed by FTIR analysis. In this study, the IR spectra of the three isolated sample of Hesperidine were analyzed and compared with the IR spectrum of standard Hesperidine from orange peel. The isolated fractions gave IR spectra similar to that of the standard Hesperidine. Thus the results indicated a strong similarity between both the standard and extracted compound.
The spectra of Hesperidine showed (Fig. 7 and Fig. 8) no broad absorption band in the range 3000cm -1- 3500cm -1 attributed to O–H stretching vibrations. The peaks around 2885 and 1080 cm -1 in the FT-IR spectrum of Hesperidine shown in Fig. 2 and Fig. 3 are due to the stretching vibrations of aliphatic C– H and C–O–C, bonds respectively.
UV spectroscopy:
The entire standard, isolated and ayurvedic formulation stock solutions were prepared and individually scanned from 200-400nm and their maximum absorbance was determined from the spectra’s were shown in the figures Fig. 4, Fig.5 & Fig.6.
TLC:
TLC studies confirm the presence of Hesperidine in both the isolated and the ayurvedic formulation using the solvent system Ethyl acetate: methanol: Water (8.3:1.0:0.7 v/v). Both isolated compound and ayurvedic formulation appeared as a single spots having the same Rf values as that of standards and their Rf values were found to be 0.710, 0.708 and 0.702 respectively. These were shown in the Fig. 7 & Fig. 8.
UFLC:
Chromatographic separation was achieved by using Phenomenex Luna 5u C18 column with the mobile phase consisting of a mixture of water-Acetonitrile-methanol (50:25:25 v/v/v). The system was injected with standard, isolated and Madiphal rasayana. The retention time of hesperidine in all the three stock solutions was found to be 2.108minutes. The overlay spectrum of UFLC study was shown in the Fig: 9.