HPLC Method Development for Naringenin and its Glucoside in Rat Serum and their Bioavailibilty Studies

Variety of botanicals and dietary products contains number of non-steroidal phytoestrogens. Several preclinical studies have confirmed positive effect of flavanones on bone functions. After isoflavones, the flavanones are the subgroup of polyphenols with most in vivo preclinical evidence responsible for improved bone health [1]. When young male rats were fed with a semi-purified diet to which oranges were added at 1 g.day-1 dose, significant inhibition of bone resorption was observed [2]. Hesperidin, a citrus flavanone glycoside, slows down femoral bone loss when administrated in Ovariectomized (OVx) mice [3]. Naringenin (N) (Figure 1), a major flavanone present in grapefruit, and its analogs, having disaccharide grouping attached to position 7 of N and which is hydrolyzed to N by gut flora prior to being absorbed, have been implicated in bone health for their oestrogen-‘like’ effects but low bioavailability impedes clinical potential. Earlier we reported that young male rats receiving N rich nutrients were found to enhance bone formation during alveolar bone development [4]. N has been shown to have positive skeletal effects in OVx mice. The doses of naringin (7-(2-O-(6-deoxy-alpha-L-mannopyranosyl)-betaD-glucopyranosyloxy)-2, 3-dihydro-4’, 5, 7-trihydroxyflavone) used were 0.2 and 0.4 mg.g-1.day-1 (200-400 mg.kg-1.day-1) that would yield very high doses when translated to humans [5]. However, these reports importantly point to N being a promising pharmacophore model for exploration of more potent osteogenic derivatives.


Introduction
Variety of botanicals and dietary products contains number of non-steroidal phytoestrogens. Several preclinical studies have confirmed positive effect of flavanones on bone functions. After isoflavones, the flavanones are the subgroup of polyphenols with most in vivo preclinical evidence responsible for improved bone health [1]. When young male rats were fed with a semi-purified diet to which oranges were added at 1 g.day -1 dose, significant inhibition of bone resorption was observed [2]. Hesperidin, a citrus flavanone glycoside, slows down femoral bone loss when administrated in Ovariectomized (OVx) mice [3]. Naringenin (N) (Figure 1), a major flavanone present in grapefruit, and its analogs, having disaccharide grouping attached to position 7 of N and which is hydrolyzed to N by gut flora prior to being absorbed, have been implicated in bone health for their oestrogen-'like' effects but low bioavailability impedes clinical potential. Earlier we reported that young male rats receiving N rich nutrients were found to enhance bone formation during alveolar bone development [4]. N has been shown to have positive skeletal effects in OVx mice. The doses of naringin (7-(2-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranosyloxy)-2, 3-dihydro-4', 5, 7-trihydroxyflavone) used were 0.2 and 0.4 mg.g -1 .day -1 (200-400 mg.kg -1 .day -1 ) that would yield very high doses when translated to humans [5]. However, these reports importantly point to N being a promising pharmacophore model for exploration of more potent osteogenic derivatives.
In the course of our search for more potent analogs of N with positive skeletal effects, we first screened various naturally occurring analogs of N in an assay for osteoblast differentiation. Naringenin-6-C-Glucoside (NCG), isolated from an Indian medicinal plant, Ulmus wallichiana (Himalayan Elm), [6] was identified as the most potent member of the N analogs in inducing osteoblast differentiation. Because the bioavailability is vital for any compound to exert biological effects, so we determined oral bioavailability of NCG and N. Subsequently, the effects of NCG on bone properties of OVx mice and in primary osteoblast cultures were studied and compared with N [4]. In this paper we are reporting a new validated method for simultaneous analysis of N and NCG in serum.

Materials and reagents
The sample of Naringenin (N) and its glucoside (NCG) were provided by Dr. Naibaidhya Chattopadhya, Endocrinology Division of our Institute. HPLC grade methanol and acetonitrile were purchased from M/S Merck Limited (Mumbai, India). Triple distilled water, from an all glass apparatus, was used. Phosphoric acid and other reagents used were of analytical grade.

HPLC analysis
For analysis, 20 μl of each sample was injected into a HPLC column. Elution was performed using a mixture of 0.5% phosphoric acid in triple distilled water and acetonitrile (75:25). Both the solutions were filtered and degassed before use. Chromatography was performed at 25 ± 3°C at a flow rate of 1.5 ml/minute. The flavanones (N and NCG) were quantified with isocratic conditions with absorbance monitored at 290 nm and 325 nm.

Preparation of standard stock solution
Stock solution of N & NCG was prepared by dissolving, in methanol, about 5 mg of the compounds in 25 ml volumetric flask to give a final concentration of 208 µg/ml & 206 µg/ml respectively. A series of standard solutions with concentrations in the range of 1.04-10.40 µg/ml for N and 1.03-10.30 µg/ml for NCG were obtained by serial dilution method with methanol. All the solutions were stored at -20°C and were brought to room temperature before use.

Sample processing
To each 0.2 ml of standard calibration samples and serum samples for analysis, 0.4 ml of methanol was added. The resulting mixtures were vortexed for 5 minutes and centrifuged for 10 min at 3,000 rpm at room temperature. Supernatants (500 µl) were taken in other clean tubes and solvents were evaporated under reduced pressure (using HETO system) and reconstituted in 50 μl methanol.

Extraction recovery
The absolute recovery was measured as the response of a processed spiked matrix standard expressed as a percentage of the response of a pure standard, which has not been treated and indicates the response for the entire amount of analyte that is present in the sample.

Analyte Stability
The stability of N and NCG in serum was investigated under a variety of storage conditions: performing three cycles of freeze (-20°C)-thaw (room temperature), 24 h storage at room temperature and under -20°C freezer at least one month.

Method development and Optimization
Several stationary and mobile phases were tried. Columns like C-18, C-8 and CN were tried with mobile phase consisting of ACN and water (1:1) at a flow rate of 1.5 ml/min. and detection at 290 and 325 nm. As NCG was a highly polar compound it is poorly retained on reverse phase HPLC columns. Its peak gets merged with the peaks of solvent and other serum impurities (Figure 2a-2c). Peak of N also came at about 4 minutes and it is also contaminated with the other impurities. Both compounds N and NCG were phenolic and, therefore, acidic in nature and to make them more lipophilic, we had to reduce their ionization. This could be achieved by optimization of the pH by adding an acid. Method parameters such as mobile phase composition, its pH, flow rate, etc, (Figure 2a and 2d) were optimized on the basis of peak characteristics and run time. Finally, we used a Lichrocart Lichrosphere C18 column (250 mm, 4 mm, 5 µm) (Merck) using a mixture of 0.5% phosphoric acid in triple distilled water and acetonitrile (75:25) with a flow rate of 1.5 ml/minute as mobile phase The column effluents were monitored at 290 nm and 325 nm (Figure 2d).

Assay specificity
HPLC analysis of standard solutions of N and NCG indicated that, in given chromatographic conditions, NCG had retention time about 2.5 min, whereas N eluted at about 14.5 min. There was no interference of other impurities at these retention times.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
Based on the signal to noise ratio (S/N) of three and ten the LOD and LOQ were found to be 104 ng/ml and 208 ng/ml (N) and 103 ng/ ml and 206 ng/ml (NCG) respectively.

Linearity and range
Under the above optimized chromatographic conditions, the linearity of the method was evaluated over concentration range of 208-1040 ng/ml (N) and 206-1030 ng/ml (NCG).

Accuracy and precision
Accuracy and precision of the analytical method was determined by analyzing Quality Control samples (QC) at three different concentrations within the calibration range in triplicate (n = 3). The precision (% RSD) of the analytical procedure was evaluated by determining intra-and interday coefficient of variation. Percentage relative standard deviation (% RSD) was found to be less than 15-20 % for intra and inter-day precision of QC standards in serum, which were under acceptable limits [7][8][9][10] and proved that method is precise. The results are given in Table 1.

Stability
Stability was assessed by comparing against the freshly thawed samples. The % mean stability for N and NCG were within the acceptance limits of 85 to 115%. The samples were found to be stable for all the parameters studied as per the experimental protocol ( Table  2).

Robustness
The method was found to be robust as minor changes in the method parameters did not alter method performance.

Recovery
The % mean recoveries for N and NCG at three concentration levels in serum were 84-89% (Table 3).

Comparative Bioavailability Studies of N and NCG
This study was designed to assess the influence of per oral N and NCG administration on its distribution profiles in rat serum. The concentration of N and NCG in extracted serum samples was checked after storage over 5 days. Both were found to be stable under the storage conditions. In rats fed a single dose of 5mg.kg -1 , the rate of appearance of NCG and N in plasma differed. Figure 3     was recorded in serum after 2 h. It showed three peaks in the serum due to hepatic recirculation. The highest serum concentration of N (C max ) was recorded after 4 h of dosing and reached 1584 ± 439 ng/ml, followed by a marked decrease up to 24 h ( Figure 3). In case of NCG (5 mg/kg dose) highest concentration 738 ± 300 ng/ml was obtained in 3 hours (C max ) (Figure 4). At 8 h, serum concentrations of NCG was again significantly increased and reached to 728 ng/ml. Twenty-four hours after dosing, high levels of NCG (570 ± 97 ng) were present in serum. The bioavailability results of N and NCG are summarized in Table 4. These results suggested that absorption of N and NCG took place early in the digestive tract (stomach or small intestine), as reported before for other flavonoids [11]. The AUC 0→∞ of N and NCG were 46247ng/ ml and 71557ng/ml respectively. This indicated that NCG had more bioavailability than N. N has low oral bioavailability might be due to its hydrophobic rings structure [12]. Several reports on N and naringin bioavailability in rat model served as the reference point [12,13].