Life Science Archives (LSA)

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1 Available online at Life Science Archives (LSA) ISSN: Volume 2; Issue - 2; Year 2016; Page: Research Article ORAL ADMINISTRATION OF A FLAVANOID QUERCETIN IN RAT PLASMA ON BIOAVAILABILITY STUDY ANALYSIS BY HPLC S. Kanimozhi*, Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar , Tamilnadu, India. Abstract The present study has been performed to assess the role of quercetin, a dietary flavonoid on bioavailability study in rat plasma. The bioavailability study was achieved by HPLC to predict the pharmacokinetic parameters of quercetin after oral administration at the dose of 50 mg/kg b.w to male Wistar rats, blood samples were collected and separated at different time intervals (0, 6, 12 and 24 hrs). Separation was achieved on Eclipse XDB-C18 column ( mm) using methanol: acetonitrile (30: 70, v/v) as a mobile phase at a flow rate of 1.0 ml/min and data were analyzed using one-way analysis of variance (ANOVA). From the calibration curve, the amount of quercetin was calculated. In plasma sample, the rentention time of quercetin was 2.34 and produced significantly different pharmacokinetic parameters in Tmax, Cmax, half life (t 1/2 ) and area under curve (AUC). Our results demonstrate that the quercetin in the pharmacokinetic study could improve the ability to illustrate their bioavailability, understand the mechanism of drug action and help to promote the drug development phase. Article History Received : Revised : Accepted : Introduction Phytochemicals are produced in plants and it protects the plant against insect invasion, infection, ultraviolet light damage and diseases due to its natural biological action. Plants also derive characteristics of colour, flavour, and odour from phytochemicals. Flavonoids constitute a class of phytochemical are divided into different subclasses and the contributions of flavonoids to health are based on their molecular structure (Nijveldt et al., 2001). Flavonoids are nonenergetic and are not considered as indispensable as are vitamins, yet the supply of some of them, or maybe of a complex mix, may have a positive effect on health. Two recent epidemiological *Corresponding author: S. Kanimozhi E.mail sivakani.14@gmail.com Key words: Quercetin, Bioavailability, HPLC and Plasma. studies support the view that flavones and flavonols protect against cardiovascular mortality (Hertog et al., 1995; Rimm et al., 1996). Also, they have been shown to inhibit the growth of various cancer cell lines in vitro, and to reduce tumor development in experimental animals (Manach et al., 1996). Quercetin is a flavonol and is generally originate in several plant based foods such as apples, onions, berries, broccoli, red wine, red grapes, bark roots, citrus fruits, flowers, and tea (Nutrient et al., 2001). Quercetin supplements are marketed to the people as on other therapy for treating allergies, arthritis, gout, eye disorders, asthma, hypertension, bacterial infections, and neurodegenerative disorders, quercetin as an antihypertensive agent and also decrease blood

2 S. Kanimozhli /Life Science Archives (LSA), Volume 2, Issue 2, Page 508 to 513, pressure. Quercetin is a versatile molecule among numerous pharmacological properties as well as antioxidant, antimicrobial, anti-viral, antiinflammatory, anticancer, antiobesity agent neurological, cardiovascular, hepatoprotective and protective of the reproductive system (Jan et al., 2010). The quercetin bioavailability is dependent on the form of quercetin is resulting from the glycoside forms in better absorption than quercetin aglycone (Manach et al., 2005) Other factors like intestinal flora and dietary mechanism could affect the bioavailability of various isoforms of quercetin. Such as rutin and pectin supplementation results that increased quercetin level in mice plasma concentrations (Tamura et al., 2007). Same results were observed in rats fed a diet elevated in pectin that was orally administered a single (50 mg/kg) dose of quercetin (Nishijima et al., 2009). In pigs, the quercetin aglycone and quercetin- 3-O-glycoside both are enhance absorption in the dietary fat (Lesser et al., 2004; Lesser et al., 2006). Some studies investigated the ability of previous polyphenolic compounds to influence quercetin absorption while, the co-supplementation of epigallocatechin gallate and quercetin could raise epigallocatechin gallate absorption in rats (Kale et al., 2010). The aim of the current study examined to analyse the bioavailability study of quercetin in plasma by HPLC. 2. Materials and Methods Chemicals All the chemicals used in the experiments were of HPLC grade. Quercetin was procured from Sigma Aldrich, (St. Louis, MO, USA) and the solvents were procured from Hi-media Laboratories Pvt, Ltd., Mumbai, India. Experimental animal Male Wistar rats ( g) from Central Animal House, Annamalai University, Tamil Nadu and India were maintained at 24 ± 2 o C on 12 hrs light/dark cycles. The rats were nourished with standard diet pellets (Hindustan Lever Ltd., Bangalore, India) and water ad libitium. Investigations were performed conferring to the ethical norms of the Institutional Animal Ethics Committee of Annamalai University (Approval No. 994, dated ). The rats were acclimatized for a week before starting the experiments Experimental Study The developed HPLC method was used in a pharmacokinetic disposition study after oral administration of quercetin (50 mg/kg body weight) to male Wistar rats ( g). Venous blood samples were collected at 0, 6, 12 and 24 hrs in heparinised tubes. Blood samples were immediately centrifuged at 3000 rpm for 5 min and harvested plasma samples stored at -20 C until analysis. The standard solution was prepared by stock solution of quercetin (1 mg/lml) in methanol. These solutions were spiked in to drug free rat plasma sample to determine the recover, precision, accuracy and detection limit of the HPLC method. Sampling procedure Plasma samples (0.1 ml) were shaken with 1.0 ml methanol for 2 min and centrifuged at 3000 rpm for 10 min. The methanol extract was transferred to dry tube. The procedure was repeated twice and the methanol extracts collected were dried at 40 ºC under a nitrogen stream. The residue was dissolved in 100 μl methanol and then injected into the chromatographic system. Quercetin content was analysed by reverse phase - HPLC with a UV detector and a vaccum degasser run by Auto chro software (Acme 9000 series; Young Lin,USA). Chromatographic separation was achieved by using a reverse phase C18 coloumn (150 mm and 4.6 mm, pore size 5 µm). The test was carried out by injecting 25 μl of mixture of standard solution at assay concentration of quercetin. The mobile phase consisted of 73 % methonal and water (99.5:0.5) (solvent A) and 27 % acetonitrile (solvent B), ph adjusted to 3.64 with glacial acetic acid. The separation was performed under an isocratic condition with a constant flow rate 1.5 ml/min, coloumn temperature 25 o C and the detector wavelength 347 nm. The experiment was performed three times and the mean was used for

3 S. Kanimozhli /Life Science Archives (LSA), Volume 2, Issue 2, Page 508 to 513, the calculations. The data was analyzed by linear regression. Statistical Analysis The results were presented as mean ± SD. One-way analysis of variance (ANOVA), followed by Duncan s multiple range test (DMRT) was done (SPSS software version 13.0) and a p- value of <05 was considered significant. 3. Results Bioavailibility of quercetin by HPLC HPLC chromatogram of quercetin standard and plasma from rats at different time intervals (0, 6, 12 and 24 hrs), after oral administration of quercetin at dose of 50 mg/kg body weight (Figure 1 and 2) The oral administration of free quercetin in rats, The retention time of quercetin was detected at 2.34 and the maximum plasma concentration (C max ) achieved was ± 0.08 µg/ml in 6 hours, T max was achieved at 6, t 1/2 was 6.02 ± 0.36 and AUC was calculated at 18 ± 0.84 (Table -1). Discussion The present study demonstrates noticeable differences in the rate of absorption and of appearance of quercetin in blood plasma, when provided as quercetin. When rats were fed semipurified diets containing either forms of this flavonol, quercetin was more rapidly absorbed than the aglycone moiety of rutin. Furthermore, quercetin appeared in detectable amounts in the blood circulation, long before the bolus had reached the cecum. By contrast, in rats fed diet, quercetin metabolites could be found in blood plasma. Thus, with a quercetin diet, the possibilities of absorption of this flavonol appear greater, since quercetin is available for digestive absorption both in the small intestine and in the large bowel. During the post absorptive period, whilst absorption in the small intestine was declining (due to transit of digesta and dilution of remaining digesta by endogenous materials), flavonol absorption took place in the large intestine. Figure -1a: HPLC Chromotogram of quercetin standard Figure -1b: HPLC Chromatogram of plasma sample 6h after an oral administration of quercetin at dose of 50 mg / kg bw Figure -1c: HPLC Chromatogram of plasma sample 12h after an oral administration of quercetin at dose of 50 mg / kg bw

4 Concentration µg/ml S. Kanimozhli /Life Science Archives (LSA), Volume 2, Issue 2, Page 508 to 513, Table -1: Pharmacokinetic parameters of quercetin (50 mg / kg bw) in male Wistar rats after oral administration PK Parameters Mean ± SD Cmax ± 0.08 Tmax 6 t 1/ ±0.36 AUC 18 ±0.84 Cmax = Maximum plasma concentration, tmax = Time for maximum plasma concentration, AUC= Area under plasma concentration time curve, t 1/2 = Elimination half life. Values that are not sharing common superscript in the same column differ significantly at P < 0.05 was performed by one-way analysis of variance Time (h) Figure -2: Plasma concentration-time profiles of Quercetin after oral administration in rats at dose The mean quercetin concentration time of 50 mg / kg profiles in the plasma after oral administration of quercetin bw in plasma of rat generated the 50 mg/kg of quercetin in different formulations. pharmacokinetic parameters after oral The peak area of quercetin was measured when administration using the absorption elimination sample working solution was injected. Plasma curve was tabulated. Cmax = Maximum plasma concentration declined rapidly after 6 hours in concentration, tmax = Time for maximum rats, which indicating that the drug was distributed plasma concentration, AUC= Area under and metabolized rapidly. Manach et al., (1999) plasma concentration time curve, t1/2= reported that quercetin is quickly metabolized by Elimination half life, Kel = Elimination rate the intestinal enzymes and strongly bound to constant. albumin, which might delay their emigration and Our present study showed that HPLC so as to preferentially excrete in bile (Jain et chromatogram of quercetin standard and plasma al.,2013). The various concentration of from rats at different time intervals (0, 6, 12 and 24), after oral administration of quercetin at dose of 50 mg/kg body weight. The oral administration

5 S. Kanimozhli /Life Science Archives (LSA), Volume 2, Issue 2, Page 508 to 513, of free quercetin in rats, The retention time of quercetin was detected at 2.34 and the maximum plasma concentration (Cmax) achieved was ± 0.08 µg/ml in 6 hours, Tmax was achived at 6, t 1/2 was 6.02 ± 0.36 and AUC was calculated at 18 ± 0.84 this is indicating that the quercetin was distributed and metabolized rapidly. Elsewhere, it is reported that quercetin is quickly metabolized by the intestinal enzymes (Manach et al., 1999). 4. Conclusion The bioavailability study of quercetin (50 mg /kg b.w) produced extensively efficient pharmacokinetic parameters in Tmax, Cmax, half life and AUC. This kind of pharmacokinetic studies of active compounds could improve the ability to illustrate their mechanisms of action and help to promote the drug development phase in to lead development. Acknowledgement I thank Professor Dr. P. Subramanian, Department of Biochemistry and Biotechnology, Annamalai University, India, for the support and guidance of my research work as my research supervisor. This research work is supported by the Indian Council of Medical Research ICMR 45/3/2013-Bio/BMS dated , New Delhi, India, in the form of Senior Research Fellowship to S. Kanimozhi. 5. References 1) Nijveldt, R. J., van Nood, E., van Hoorn, D. E., Boelens, P. G., van Norren, K., van Leeuwen, P. A. (2001). Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr., 74: ) Hertog, M. G. L., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F et al. (1995). Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study Arch. Intern. Med., 155: ) Rimm, E. B., Katan, M. B., Ascherio, A., Stamper, M. J., Wille W. C. (1996). Relation between intake of flavonoids and risk for coronary heart disease in male health professionalsann. Intern. Med., 125: ) Manach, C., Régérat, F., Texier, O., Agullo, G., Demigne, C., Rémésy, C. (1996). Nutr. Res., 16: ) Nutrient Data Laboratory, Food Composition Laboratory. USDA database for the flavonoid content of selected foods. Beltsville, MD: Beltsville Human Nutrition Research Center, Agriculture Research Service, USDA; January ) Jan, A. T., Kamli, M. R., Murtaza, I., Singh, J. B., Ali, A., Haq, Q. M. R. (2010). Dietary Flavonoid Quercetin and Associated Health Benefits An Overview. Food Rev Int., 26: ) Manach, C., Williamson, G., Morand, C., Scalbert, A., Remesy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr., 81:230S 42S. 8) Tamura, M., Nakagawa, H., Tsushida, T., Hirayama, K., Itoh, K. (2007). Effect of pectin enhancement on plasma quercetin and fecal flora in rutin-supplemented mice. J Food Sci., 72: ) Nishijima, T., Iwai, K., Saito, Y., Takida, Y., Matsue, H. (2009). Chronic ingestion of apple pectin can enhance the absorption of quercetin. J Agric Food Chem., 57: ) Lesser, S., Cermak, R., Wolffram, S. (2004). Bioavailability of quercetin in pigs is influenced by the dietary fat content. J Nutr., 134: ) Lesser, S., Cermak, R., Wolffram, S. (2006). The fatty acid pattern of dietary fat influences the oral bioavailability of the flavonol quercetin in pigs. Br J Nutr., 96: ) Kale, A., Gawande, S., Kotwal, S., Netke, S., Roomi, W., Ivanov, V., Niedzwiecki, A., Rath, M. (2010). Studies on the effects of oral administration of nutrient mixture, quercetin and red onions on the bioavailability of epigallocatechin gallate from green tea extract. Phytother Res., 24 : S48 55.

6 S. Kanimozhli /Life Science Archives (LSA), Volume 2, Issue 2, Page 508 to 513, ) Jain, A. K., Thanki, K., Jain, S. (2013). Coencapsulation of tamoxifen and quercetin in polymeric nanoparticles: implications on oral bioavailability, antitumor efficacy, and drug-induced toxicity. Mol Pharm., 10: ) Manach, C., Texier, O., Morand, C., et al (1999). Comparison of the bioavailability of quercetin and catechin in rats. Free Radic Biol Med., 27: