Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-014-0206-9
ORIGINAL PAPER
Comparative pharmacokinetic interactions of Quercetin and Rutin in rats after oral administration of European patented formulation containing Hipphophae rhamnoides and Co-administration of Quercetin and Rutin Ananth Kumar Kammalla • Mohan Kumar Ramasamy Jyothi Chintala • Govind Prasad Dubey • Aruna Agrawal • Ilango Kaliappan
•
Received: 27 January 2014 / Accepted: 20 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract Quercetin and Rutin are most common flavone constituents of some herb extracts such as Hippophae rhamnoides L. Inter and intra herb pharmacokinetics interactions of Quercetin and Rutin were investigated in the present study. Pharmacokinetic study was investigated in the two groups of rats (n = 6) for pharmacokinetic interactions between the Quercetin and Rutin (2.5 mg/kg) mixture treated alone with European patented polyherbal formulation containing equivalent weight of the above. The total plasma concentrations of Quercetin and Rutin were determined by liquid chromatography mass spectrometry (LC–MS). A method was developed and validated according to the ICH guidelines. The results of the present study shows that there are great differences in the pharmacokinetics of Quercetin and Rutin when they are administered together and from the polyherbal formulation which will be interacted by many other constituents. The
A. K. Kammalla M. K. Ramasamy J. Chintala I. Kaliappan (&) Interdisciplinary School of Indian System of Medicine (ISISM), SRM University, Kattankulathur, Kancheepuram (Dt) 603203, Tamil Nadu, India e-mail: [email protected] G. P. Dubey National Facility for Tribal and Herbal Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India A. Agrawal Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India I. Kaliappan Department of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM University, Kattankulathur 603203, Tamil Nadu, India
bioavailability of Quercetin was lowered from the polyherbal formulation when compared with the co-administration, whereas the Rutin bioavailability has increased from the polyherbal formulation when compared with the co-administration. The maximum plasma concentration of Quercetin from coadministration and polyherbal formulation was 165.3 ± 31.9 and 90.8 ± 21.4 ng/mL, respectively, whereas in the case of Rutin it was 61.1 ± 29.3 and 121.7 ± 19.2 ng/mL. After polyherbal formulation administration to rats the AUC0–24, AUC0–? and AUMC0–? of both Quercetin and Rutin significantly increased when compared to co-administration. The above results proved that inter and intra herb pharmacokinetic interactions between Quercetin and Rutin. Possible interactions of the other constituents with hydrolyzing enzymes in the formulation enhances the oral bioavailability of Rutin. Accordingly besides the drug herb interactions, inter and intra herb interaction might be brought into view with the wide use of herbal remedies. Keywords Drug interaction Pharmacokinetics Quercetin Rutin Polyherbal formulation
1 Introduction Flavonoids are a group of polyphenolic compounds widely present in many plants and vegetables such as onions, apple, tea, etc., Many herbs containing flavonoids have been widely used in traditional medicines for various ailments. Their extracts are increasingly introduced into global market as complementary and alternative medicines such as Gingko flavones (Aruna and Naidu 2007) and total flavones of Hipphophae rhamnoides (Guowen et al. 2012), Soy isoflavones and St. John’s Wort extracts.
Eur J Drug Metab Pharmacokinet
Pharmacokinetics of active ingredients in natural products and traditional medicines are important to illustrate their mechanism of action. With the gradually increasing use of medicinal herbal extracts as an alternative medicine more and more clinical affairs derived from herb–drug interactions have emerged and are being investigated (Ilango et al. 2013). Most interactions are associated with pharmacokinetics (Brazier and Levine 2003). A polyherbal formulation for the management of cardiovascular and neurological disorders consist leaves of Bacopa monnieri (Scrophuliariaceae), fruits of Hipphophae rhamnoides (Elaganaceae) and bulbs of Dioscorea bulbifera (Dioscoraceae), developed by Dubey et al. (2005), was an European patented formulation. Quercetin and Rutin flavonoids are some of the major constituents present in the formulation. The chemical structures of Quercetin, Rutin and internal standard Kaempferol are depicted in Fig. 1a–c, respectively. Both these markers have related pharmacological actions like antioxidant activity such as modification of eicosanoid biosynthesis, prevention of low-density lipoprotein from oxidation, prevention of platelet aggregation and promotion of relaxation of cardiovascular muscle (Formica and Regelson 1995). Also it was proven
that Quercetin and its related bioflavonoids modulate the function and expression of P-glycoprotein and CYP3A4 which were primarily involved in the drug interactions (Ke et al. 2008). Previous investigations were carried out on the individual pure compounds or physical mixtures of two or more compounds. Studies have also shown that rate of elimination of Quercetin seems to be very low and rate of absorption of Rutin was slower than Quercetin. Guowen et al. studied the pharmacokinetic properties of Quercetin and other markers after oral gavage of total flavones of Hipphophae rhamnoides L. in rats and found the major biomarkers show the quite different pharmacokinetic behavior when compared with individual pure forms (Guowen et al. 2012). Ke et al. (2008) investigated on the intra herb pharmacokinetic interactions of Quercetin and Isorhamnetin. Interestingly, similar to the drug–herb interactions, Quercetin and Rutin with other constituents in this formulation would possibly cause potential intra-herb interaction. In the present study, we have investigated the pharmacokinetic behavior of Quercetin and Rutin from this formulation by in vivo pharmacokinetics in rats.
2 Materials and methods 2.1 Chemicals Quercetin (QU, 99 %) and Rutin (RU, 99 %), Internal standard Kaempferol (KM, 99.9 %) were purchased from Sigma Aldrich Co (MO, USA). Deionized water was produced by Milli Q system (Massachusetts). Acetonitrile (HPLC grade—Merck Co, India) and Formic acid (Rankem, India) were used for LCMS analysis. Formulation capsules with batch number SRM 001 were purchased from M/s Varanasi Bioresearch Pvt Ltd (Varanasi, India). 2.2 Animals Male Wistar Rats (250 ± 20 g) were supplied by the Kings Institute (Chennai, India). The experimental protocol number IAEC 152/2011 was approved by the Institutional Animal Ethics Committee of SRM College of pharmacy, SRM University for the use of experimental animals and all animal studies were carried out according to the CPCSEA guidelines. 2.3 Preparation of standard solution and quality control samples
Fig. 1 Chemical structures of a Quercetin b Rutin and c Kaempferol
The stock solution of Quercetin and Rutin and internal standard (Kaempferol) were prepared in the mixture of
Eur J Drug Metab Pharmacokinet
acetonitrile and water (50:50, v/v). The stock solution of the standard was further diluted in acetonitrile–water (50:50, v/v) to produce the combined standard mixture working solutions at concentrations of 50–1,600 ng/mL of Quercetin and Rutin. A quantity of Kaempferol was dissolved in mixture of acetonitrile and water (50:50, v/v) to prepare the IS solution with a concentration of 400 ng/mL. For the validation of the method three concentration levels of the standard solution containing Quercetin and Rutin (50, 200 and 800 ng/mL) were used for preparing QC plasma samples and all solutions were stored at 4 °C until needed. 2.4 Sample preparation Frozen plasma samples were thawed at room temperature and treated as follows: 100 lL of IS solution (400 ng/mL), 100 lL of the working solution for the calibration curve and QC samples and 200 lL of acetonitrile were added to 100 lL plasma samples. The mixture was vortexed for 5 min and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred into a clean eppendorf tube, and evaporated to dryness under the stream of nitrogen. The residue was dissolved in 200 lL of the Acetonitrile— Water (50:50, v/v), vortexed and centrifuged at 12,000 rpm for 15 min. The supernatant was passed through 0.2 l membrane filter and 20 lL of the filtrate solution was injected for the LC–MS analysis. 2.5 Liquid chromatography and mass spectrometry A Shimadzu LCMS 2020 (Shimadzu, Japan) equipped with binary solvent delivery system, column compartment and photo diode array detector (PDA) was used for all analysis. The chromatographic separation was performed on Phenomenex C18 column (i.d. 250 9 4.6 mm, 5 lm) and the column oven temperature was set at 30 °C. A linear gradient elution of trinary mobile consists of Methanol: Acetonitrile: Water (40:15:45 v/v/ v).Equilibration time was kept for 10 min prior to the injection of each sample and the flow rate was kept at 0.5 mL/min. The instrument was operated by switching electrospray ionization (ESI) source in positive and negative ionization modes in a single run. The ESI was performed using nitrogen gas to assist nebulization (the flow rate was set at 1.5 L/min), capillary voltage at 1.6 kV and temperatures of Curved Desolvation Line (CDL) and heat block at 250 and 300 °C were used. All instrumentation data were collected and synchronized by Lab solutions software (Version 7.1) from Shimadzu.
2.6 Pharmacokinetic study A single dose of Quercetin and Rutin (2.5 mg/kg) and test formulation containing equivalent dose of Quercetin and Rutin (2.5 mg/kg) were orally administered to two groups (n = 6). Both the preparations were administered as solutions by oral gavage at a volume of 10 mL/kg utilizing feeding needle. Blood samples (0.3–0.4 mL of venous blood) were collected from fossa orbitalis vein into heparinized centrifuge tubes at 15, 30, 60, 120, 180, 240, 360, 480, 600, 1,440, 2,160, 2,880 min after single oral administration. 30 min after blood withdrawal, the samples were centrifuged at 4,000 rpm for 10 min and the separated plasma samples were frozen in polypropylene tubes at -20 °C prior to analysis. 2.7 Validation of assay The current LC–MS assay was validated for specificity, linearity, precisions, accuracy and matrix effects according to ICH (International Conference on Harmonization 1996) guidelines. The specificity was determined by analyzing six blank plasma samples. There was no interference from endogenous or exogenous materials observed at the retention time in the ion channel of either the analytes or the IS. The calibration plasma samples were prepared by spiking 100 lL of working solution and 100 lL of IS solution to 100 lL of blank plasma and then the samples were treated according to the sample preparation. Quality control (QC) samples were prepared and assayed along with calibration curve samples. The calibration curve consisted of six concentration levels and each concentration was prepared and assayed on three separate days. The calibration curves were constructed by the plot of the peak area of the analyte versus concentrations of the calibration standards and described in the form of y = a ? bx. The concentrations of the analytes in unknown samples were assessed by interpolation from the calibration curve. The LOQ of the assay was defined as the lowest quantifiable concentration of the standard curve (LOQ, S/N = 10). The LOD was defined as the detectable amount (LOD, S/N = 3). The intra and inter-day precisions were defined as relative standard deviation (RSD) and the accuracy was assessed by comparing the measured concentration with its true value. The accuracy and precision were assessed by determining QC samples at three different validation batches. The intrabatch QC samples were prepared for six replicates. The acceptable intra and inter day precision and accuracy should be \15 %. The extraction recovery of analytes at three QC levels was evaluated by determining the peak area ratios of the
Eur J Drug Metab Pharmacokinet
analytes in the post-extraction spiked samples to that acquired from pre-extraction spiked samples.
3 Pharmacokinetic analysis 3.1 Non compartmental analysis All the pharmacokinetics parameters were determined by non-compartmental analysis. The peak plasma level (Cmax) and the time to reach peak plasma concentration (Tmax) were obtained directly from the concentration–time data. The elimination rate constant (Ke) was calculated from the slope of the logarithm of the plasma concentration versus time using the final four points. The apparent elimination half-life (T1/2) was calculated as 0.693/Ke. The area under the plasma concentration–time curve (AUC) and the area under the first moment curve (AUMC) were calculated by the trapezoidal rule. Total body clearance was established as Xo/AUC. The Mean Residence Time (MRT) after oral administration was calculated by dividing the AUMC by AUC. The values were calculated by Microsoft Excel (Microsoft, Seattle, Washington, USA) and each value was expressed as mean ± SD. 3.2 Wagner Nelson method This method involves mathematical calculation of the cumulative amount of drug absorbed at each time point Aa after drug administration and the total amount of the drug absorbed Aa?. The fraction Aa/Aa? represents the fraction of the absorbed dose that is already absorbed at each time point. The fraction remaining to be absorbed can be examined to determine the nature of the absorption process (Shargel et al. 2012). A plot of the fraction of the drug remaining to be absorbed versus time can be used to determine the order of the absorption process. Statistical significance was assessed by an unpaired Student’s t test and the significance level of P \ 0.05 was adopted for all statistical comparisons. All results were expressed as mean ± standard deviation (SD).
4 Results
suitably separated with in 20 min. As shown in Fig. 2 the retention time was 4.5 min for Rutin, 9.6 min for Quercetin and 15.6 min for Kaempferol (I.S). The selective ion monitoring chromatograms of Quercetin and Rutin from the standard mixture sample and spiked plasma are depicted in Figs. 2 and 3. Flavones in this study were examined in negative ion mode since it is known to yield better response for these compounds when compared to positive ion mode. Results of this study demonstrate that the mass signals of Rutin, Quercetin and Kaempferol (I.S) were high enough for detection in rat samples. The [MH]- ion of each standard was selected Selective Ion Monitoring (SIM) mode to obtain the selectivity and sensitivity for the determination of the two flavones. Kaempferol was selected as internal standard because of its similarity of chemical structure, chromatographic behavior, ionization efficiency, extraction efficiency and reproducibility at target ion of m/z 285.The content of Quercetin and Rutin in the capsule was estimated and found to be 0.09665 w/w and 0.0541 % w/w by HPLC, respectively. 4.2 Method validation 4.2.1 Specificity All the analytes and internal standard could be detected on their own selected ion chromatograms without any significant interference. 4.3 Linearity and lower limits of quantification The calibration curves of Quercetin and Rutin exhibited good linearity with correlation coefficients (R2) within the range from 50 to 1,600 ng/mL. The LLOQs were suitable for quantitative detection of analytes in the pharmacokinetics studies. Linear ranges, LOQ, LOD and correlation coefficients obtained from typical calibration curves were shown in Table 1. 4.4 Precision and accuracy The intraday, interday precision and accuracy date of Quercetin and Rutin at three concentration levels are summarized in Table 2. The RSD values of inter and intraday precision was \3.The accuracy ranged from 98 to 103 %.
4.1 Method development 4.5 Extraction recovery The LCMS conditions for the two flavonoids were optimized and shown in the materials and method section. The optimal condition allowed the two flavones (Quercetin and Rutin) and the internal standard (Kaempferol) to be
The extraction recoveries ranged from 82.6 to 103.2 % at three different concentration levels for the two analytes. The extraction recovery of the I.S. was more than 90 %.
Eur J Drug Metab Pharmacokinet Fig. 2 Selective ion chromatogram of Quercetin and Rutin from standard sample
Fig. 3 Selective ion chromatogram of Quercetin, Rutin and Kaempferol as internal standard from plasma spiked sample
Table 1 Method validation parameters Compound
Concentration range (lg/mL)
Rt (min)
SIM mode
Regression equation
R2
LOD (ng/mL)
LOQ (ng/mL)
Quercetin
0.05–1.6
9.8 ± 0.1
301
y = 1,642.4x-91,451
0.99
21.1
64.1
Rutin
0.05–1.6
4.8 ± 0.2
609
y = 471.72x-10,325
0.99
8.3
26.7
Eur J Drug Metab Pharmacokinet Table 2 Inter and intraday precision Inter-batch (n = 5) Nominal concentration (ng/mL)
Intra-batch(n = 10) Measured concentration (ng/mL)
Precision (%)
Accuracy (%)
Nominal concentration (ng/mL)
Measured concentration (ng/mL)
Precision (%)
Accuracy (%)
Quercetin 50
50.3
0.93
100.6
50
50.7
2.95
101.5
200
196.9
1.68
98.5
200
195.5
1.54
97.8
800
818.8
1.8
102.4
800
819.1
1.74
102.4
50
50.3
0.6
100.6
50
45.2
2.4
90.4
200
193.7
2.1
96.8
200
193.2
0.8
96.6
800
783.4
1.4
97.8
800
817.2
3.1
102.2
180
160
160
140
Concentration (ng/mL)
Concentration (ng/mL)
Rutin
140 Quercetin
120
Rutin
100 80 60 40 20
Quercetin Rutin
120 100 80 60 40 20 0
0
0 0
4
8
12
16
20
4
8
12
16
20
24
24
Time (hrs)
TIme (hrs)
Fig. 4 Area under curve of Quercetin and Rutin from the oral administration of mixture
4.6 Pharmacokinetics interaction of Quercetin and Rutin The developed and validated method was applied to the pharmacokinetic evaluation of polyherbal formulation in rats following oral administration. Plasma samples from single dose of 2.5 mg/kg equivalent weight of Quercetin were obtained after intragastric gavage. The mean plasma concentration of Quercetin and Rutin versus time curve is illustrated in Figs. 4 and 5. Corresponding to previous pharmacokinetics of individual flavonoids and in the form of Hipphophae extract in human and various animal species, Quercetin and Rutin showed two or more peaks of maximum plasma concentration in individual rats. This phenomenon was declining with coadministration. The main pharmacokinetics of Quercetin and Rutin are listed in Table 3. In Table 3, it can be seen that the AUC of Quercetin and Rutin after the co administration was significantly different
Fig. 5 Area under curve of Quercetin and Rutin from the polyherbal formulation
from the oral dose of polyherbal formulation (P \ 0.001 for both AUC and AUC, Students t test). After oral dose of polyherbal formulation, the oral bio availability of Rutin was significantly enhanced, whereas the Quercetin bioavailability shows insignificant enhancement when compared with Quercetin Rutin co-administration. The rate of absorption (Ka) calculated by the Wagner nelson method significantly differs from thepolyherbal formulation when compared with co-administration of quercetin and rutin by oral administration.
5 Discussion The results of the present study show that there is a great difference in the pharmacokinetics of the Quercetin and Rutin when they were administered together and from the polyherbal formulation which will be interacted by many other constituents. The bioavailability of Quercetin was
Eur J Drug Metab Pharmacokinet Table 3 Pharmacokinetic parameters Parameter Cmax (ng/mL Tmax (h) AUC (0–24) (ng/mL/h) AUC (0–?) (ng/mL/h) AUMC (ng/mL/h2) MRT (h) -1
Ka (h )
Quercetin (coadministration) 165.3 ± 31.9
Rutin (coadministration) 61.1 ± 29.3
Quercetin (polyherbal formulation) 90.8 ± 21.4*
Rutin (polyherbal formulation) 121.7 ± 19.2*
3.37 ± 0.51
4.17 ± 1.19
3.27 ± 0.86
4.43 ± 0.85
944.27 ± 70.57
389.45 ± 42.30
337.20 ± 14.75**
684.83 ± 15.52*
972.17 ± 85.77
456.67 ± 29.80
406.60 ± 24.78
6,729.27 ± 206.75
6,048.53 ± 151.78
5,135.37 ± 248.58
7.7 ± 1.1
11.9 ± 1.5
12.2 ± 1.5*
0.419 ± 0.006
0.242 ± 0.04
0.5537 ± 0.02**
731.40 ± 31.15** 6,295.57 ± 304.66 7.4 ± 1.1* 0.2753 ± 0.01
All values are expressed as Mean ± SD (n = 6). The statistical significance of differences was tested using the nonparametric Wilcoxon test. Significant values are expressed as *P \ 0.05, **P \ 0.01 and ***P \ 0.001 compared with corresponding co-administration Cmax maximum plasma concentration, Tmax Time to reach maximum concentration, AUC area under curve, AUMC area under first moment curve, MRT mean residence time, Ka rate of absorption
lowered from the polyherbal formulation when compared with the co-administration, whereas the Rutin bioavailability is increased from the polyherbal formulation when compared with the co-administration. Absorption of Quercetin and Rutin that occurred from the different parts of the gastrointestinal tract and inter individual variation in the absorption of Quercetin from Rutin, but not from Quercetin aglycone, was considerable. However, the mechanism of the absorption remained unclear. It has been reported that enzymes cleaving Quercetin glycoside are present in the small intestine and in the colon, and, therefore, it is likely that many of the compounds are hydrolyzed prior to the absorption of Quercetin aglycone (Erlund et al. 2000). Some studies indicate that the Quercetin glycosides containing glucose moieties could be absorbed intact (Hollman et al. 1999). Yang et al. proposed that rutin undergoes deglycosylation to quercetin-3-O-glucoside by an enzyme L-rhamnosidase which is being produced by human intestinal bacteria of isolated strains. Also they stated that lack of these enzymes could not metabolize further (Yang et al. 2012). The above results indicate that Rutin hydrolyzes before absorption; hence only very trace amount of Rutin was detected in plasma but the presence of other secondary metabolites in the polyherbal formulation interfaces with hydrolyzing enzymes in distal tubules of small intestine or colon, their by increasing in rutin bioavailability in polyherbal formulation. The absorption curve for the Quercetin was biphasic (Erlund et al. 2000). This bi-phasic phenomenon was observed in individual plasma concentration-time curve also. This phenomenon indicated the poor enterohepatic recirculation of the flavonoid compounds from the formulation. A possible explanation for this phenomenon is that Quercetin aglycone seems to be absorbed from the several parts of the gastrointestinal tract. When it was co administrated with Rutin alone it has been absorbed in the proximal parts of the gastrointestinal tract and traces
will be left by the time the distal parts of the small intestine or the colon are reached to allow another absorption peak to occur. Hollman et al. (1997) also stated that biphasic concentration profile in the elimination of quercetin from plasma indicates a rather fast distribution and elimination followed by a slow final elimination. When it was from the formulation due to many other constituent interactions the quantity of the Quercetin reaching the small intestine is increased and causes the increased bioavailability. The non compartmental model analysis which is based on the statistical moment theory was performed for the pharmacokinetic analysis. The Quercetin peak concentration in plasma occurred at 3.3 h from the co-administration and 3.2 h from the polyherbal formulation, while from the Rutin it was 4.1 and 4.7 h from co-administration and polyherbal formulation, respectively. These results indicate that Quercetin was absorbed more rapidly than Rutin likely due to quick metabolism of Quercetin after its absorption (Graefe et al. 2001). This finding is consistent with results reported by Lan et al. (2007). The mean residence time of the Quercetin was significantly higher from the polyherbal formulation, whereas Rutin residence time is significantly lowered from the polyherbal formulation. Vinson et al. reported that free Quercetin is one of the most potent antioxidants to inhibit lipid peroxidation (IC50 & 0.22 lmol/L) (Vinson et al. 1995). However, Quercetin is probably present mainly as conjugated forms, extensively bound to albumin (Manach et al. 1995) and the actual antioxidant potency of these forms is still uncertain and possibly lesser than that of the free forms. The method developed for the Quercetin and Rutin quantification from the plasma samples was highly sensitive and selective analytical method. The high recovery and precision values indicate that developed method is accurate for the extraction of the Quercetin and Rutin from the plasma.
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6 Conclusion In conclusion, the present study proved that the oral bioavailability of Quercetin and Rutin was enhanced from the polyherbal formulation when compared with this mixture administration. Therefore, it may be concluded that the oral dose of mixed compounds in herb extracts could gain a significant biopharmaceutical advantage when compared to the single compound. Consequently, inter and intra herb interaction of the Quercetin and Rutin was elucidated. Further elucidation of mechanism of interaction is under process. Acknowledgments The authors would like to thank the Department of Science and Technology, Government of India, for providing financial assistance to carry the work and Department of Pharmacology, SRM College of Pharmacy, SRM University, for providing facilities to carryout animal studies. Conflict of Interest
The authors declare no conflict of interest.
References Manach C et al (1995) Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin. J Nutr 125(7):1911–1922 Ilango K et al (2013) Emerging need of pharmacokinetics in Ayurvedic system of medicine. Int J Res Ayurveda Pharm 4(5):1–5 Aruna D, Naidu M (2007) Pharmacodynamic interaction studies of Gingiko biloba with cilostazol and clopidogrel in healthy human subjects. Br J Clin Pharmacol 63:333–338 Brazier N, Levine M (2003) Drug-herb interaction among commonly used conventional medicines: a compendium for health care professionals. Am J Ther 10:163–169
Dubey GP (2005) Herbal preparation for management of cardiovascular and neurologic disorders. European Union, Patent No. EP 1 569 666 B1 Erlund I et al (2000) Pharmacokinetics of quercetin from quercetin aglycone and rutin in healthy volunteers. Eur J Pharmacol 56:545–553 Formica J, Regelson W (1995) Review of the biology of Quercetin and related bioflavanoids. Food Chem Toxicol 33:1061–1080 Graefe E et al (2001) Pharmacokinetics and bioavailability of quercetin glycosides in humans. J Clin Pharmacol 41:492–499 Guowen L et al (2012) Pharmacokinetic properties of isorhamnetin, kaempferol and quercetin after oral gavage of total flavones of Hipphophae rhamnoides L. in rats using a UPLC-MS method. Fitoterapia 83:182–191 Hollman PC et al (1997) Relative bioavailability of the antioxidant flavanoids quercetin from various foods in man. FEBS Lett 418:152–156 Hollman P et al (1999) The sugar moiety is a major determininant of the absorption of dietary flavanoid glycosides in man. Free Radic Res 31:569–573 International Conference on Harmonization (1996) Guideline on validation of analytical procedure-methodology. IFPMA, Geneva Ke L et al (2008) Intra-herb pharacokinetics interaction between quercetina nd isorhamentin. Acta Pharmacologica Sinica 29(11):1376–1382 Lan K, Jiang X, He J (2007) Quantitative determiniation of isorhamnetin, quercetin and kaempferol in rat plasma by liquid chromatography with electron spray ionization tandem mass spectrometry and its application to the pharmacokinetic study of isorhamnetin. Rapid Commun Mass Spectrom 21:112–120 Shargel L, Susanna W-P, Andrew B (2012) Applied biopharmaceutics and pharmacokientics, 5th edn. McGraw-Hill, New York Vinson JA, Jang J, Dabbagh YA, Serry MM, Cai S (1995) Plant polyphenols exhibit lipoprotein-bond antioxidant activity using an in vitro oxidation model for heart disease. J Agric Food Chem 43:2798–2799 Yang J et al (2012) Identificaiton of rutin deglycosylated metabolites produced by human intestinal bacteria using UPLC-Q TOF/MS. J Chromatogr B 898(1):95–100
ORIGINAL PAPER
Comparative pharmacokinetic interactions of Quercetin and Rutin in rats after oral administration of European patented formulation containing Hipphophae rhamnoides and Co-administration of Quercetin and Rutin Ananth Kumar Kammalla • Mohan Kumar Ramasamy Jyothi Chintala • Govind Prasad Dubey • Aruna Agrawal • Ilango Kaliappan
•
Received: 27 January 2014 / Accepted: 20 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract Quercetin and Rutin are most common flavone constituents of some herb extracts such as Hippophae rhamnoides L. Inter and intra herb pharmacokinetics interactions of Quercetin and Rutin were investigated in the present study. Pharmacokinetic study was investigated in the two groups of rats (n = 6) for pharmacokinetic interactions between the Quercetin and Rutin (2.5 mg/kg) mixture treated alone with European patented polyherbal formulation containing equivalent weight of the above. The total plasma concentrations of Quercetin and Rutin were determined by liquid chromatography mass spectrometry (LC–MS). A method was developed and validated according to the ICH guidelines. The results of the present study shows that there are great differences in the pharmacokinetics of Quercetin and Rutin when they are administered together and from the polyherbal formulation which will be interacted by many other constituents. The
A. K. Kammalla M. K. Ramasamy J. Chintala I. Kaliappan (&) Interdisciplinary School of Indian System of Medicine (ISISM), SRM University, Kattankulathur, Kancheepuram (Dt) 603203, Tamil Nadu, India e-mail: [email protected] G. P. Dubey National Facility for Tribal and Herbal Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India A. Agrawal Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India I. Kaliappan Department of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM University, Kattankulathur 603203, Tamil Nadu, India
bioavailability of Quercetin was lowered from the polyherbal formulation when compared with the co-administration, whereas the Rutin bioavailability has increased from the polyherbal formulation when compared with the co-administration. The maximum plasma concentration of Quercetin from coadministration and polyherbal formulation was 165.3 ± 31.9 and 90.8 ± 21.4 ng/mL, respectively, whereas in the case of Rutin it was 61.1 ± 29.3 and 121.7 ± 19.2 ng/mL. After polyherbal formulation administration to rats the AUC0–24, AUC0–? and AUMC0–? of both Quercetin and Rutin significantly increased when compared to co-administration. The above results proved that inter and intra herb pharmacokinetic interactions between Quercetin and Rutin. Possible interactions of the other constituents with hydrolyzing enzymes in the formulation enhances the oral bioavailability of Rutin. Accordingly besides the drug herb interactions, inter and intra herb interaction might be brought into view with the wide use of herbal remedies. Keywords Drug interaction Pharmacokinetics Quercetin Rutin Polyherbal formulation
1 Introduction Flavonoids are a group of polyphenolic compounds widely present in many plants and vegetables such as onions, apple, tea, etc., Many herbs containing flavonoids have been widely used in traditional medicines for various ailments. Their extracts are increasingly introduced into global market as complementary and alternative medicines such as Gingko flavones (Aruna and Naidu 2007) and total flavones of Hipphophae rhamnoides (Guowen et al. 2012), Soy isoflavones and St. John’s Wort extracts.
Eur J Drug Metab Pharmacokinet
Pharmacokinetics of active ingredients in natural products and traditional medicines are important to illustrate their mechanism of action. With the gradually increasing use of medicinal herbal extracts as an alternative medicine more and more clinical affairs derived from herb–drug interactions have emerged and are being investigated (Ilango et al. 2013). Most interactions are associated with pharmacokinetics (Brazier and Levine 2003). A polyherbal formulation for the management of cardiovascular and neurological disorders consist leaves of Bacopa monnieri (Scrophuliariaceae), fruits of Hipphophae rhamnoides (Elaganaceae) and bulbs of Dioscorea bulbifera (Dioscoraceae), developed by Dubey et al. (2005), was an European patented formulation. Quercetin and Rutin flavonoids are some of the major constituents present in the formulation. The chemical structures of Quercetin, Rutin and internal standard Kaempferol are depicted in Fig. 1a–c, respectively. Both these markers have related pharmacological actions like antioxidant activity such as modification of eicosanoid biosynthesis, prevention of low-density lipoprotein from oxidation, prevention of platelet aggregation and promotion of relaxation of cardiovascular muscle (Formica and Regelson 1995). Also it was proven
that Quercetin and its related bioflavonoids modulate the function and expression of P-glycoprotein and CYP3A4 which were primarily involved in the drug interactions (Ke et al. 2008). Previous investigations were carried out on the individual pure compounds or physical mixtures of two or more compounds. Studies have also shown that rate of elimination of Quercetin seems to be very low and rate of absorption of Rutin was slower than Quercetin. Guowen et al. studied the pharmacokinetic properties of Quercetin and other markers after oral gavage of total flavones of Hipphophae rhamnoides L. in rats and found the major biomarkers show the quite different pharmacokinetic behavior when compared with individual pure forms (Guowen et al. 2012). Ke et al. (2008) investigated on the intra herb pharmacokinetic interactions of Quercetin and Isorhamnetin. Interestingly, similar to the drug–herb interactions, Quercetin and Rutin with other constituents in this formulation would possibly cause potential intra-herb interaction. In the present study, we have investigated the pharmacokinetic behavior of Quercetin and Rutin from this formulation by in vivo pharmacokinetics in rats.
2 Materials and methods 2.1 Chemicals Quercetin (QU, 99 %) and Rutin (RU, 99 %), Internal standard Kaempferol (KM, 99.9 %) were purchased from Sigma Aldrich Co (MO, USA). Deionized water was produced by Milli Q system (Massachusetts). Acetonitrile (HPLC grade—Merck Co, India) and Formic acid (Rankem, India) were used for LCMS analysis. Formulation capsules with batch number SRM 001 were purchased from M/s Varanasi Bioresearch Pvt Ltd (Varanasi, India). 2.2 Animals Male Wistar Rats (250 ± 20 g) were supplied by the Kings Institute (Chennai, India). The experimental protocol number IAEC 152/2011 was approved by the Institutional Animal Ethics Committee of SRM College of pharmacy, SRM University for the use of experimental animals and all animal studies were carried out according to the CPCSEA guidelines. 2.3 Preparation of standard solution and quality control samples
Fig. 1 Chemical structures of a Quercetin b Rutin and c Kaempferol
The stock solution of Quercetin and Rutin and internal standard (Kaempferol) were prepared in the mixture of
Eur J Drug Metab Pharmacokinet
acetonitrile and water (50:50, v/v). The stock solution of the standard was further diluted in acetonitrile–water (50:50, v/v) to produce the combined standard mixture working solutions at concentrations of 50–1,600 ng/mL of Quercetin and Rutin. A quantity of Kaempferol was dissolved in mixture of acetonitrile and water (50:50, v/v) to prepare the IS solution with a concentration of 400 ng/mL. For the validation of the method three concentration levels of the standard solution containing Quercetin and Rutin (50, 200 and 800 ng/mL) were used for preparing QC plasma samples and all solutions were stored at 4 °C until needed. 2.4 Sample preparation Frozen plasma samples were thawed at room temperature and treated as follows: 100 lL of IS solution (400 ng/mL), 100 lL of the working solution for the calibration curve and QC samples and 200 lL of acetonitrile were added to 100 lL plasma samples. The mixture was vortexed for 5 min and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred into a clean eppendorf tube, and evaporated to dryness under the stream of nitrogen. The residue was dissolved in 200 lL of the Acetonitrile— Water (50:50, v/v), vortexed and centrifuged at 12,000 rpm for 15 min. The supernatant was passed through 0.2 l membrane filter and 20 lL of the filtrate solution was injected for the LC–MS analysis. 2.5 Liquid chromatography and mass spectrometry A Shimadzu LCMS 2020 (Shimadzu, Japan) equipped with binary solvent delivery system, column compartment and photo diode array detector (PDA) was used for all analysis. The chromatographic separation was performed on Phenomenex C18 column (i.d. 250 9 4.6 mm, 5 lm) and the column oven temperature was set at 30 °C. A linear gradient elution of trinary mobile consists of Methanol: Acetonitrile: Water (40:15:45 v/v/ v).Equilibration time was kept for 10 min prior to the injection of each sample and the flow rate was kept at 0.5 mL/min. The instrument was operated by switching electrospray ionization (ESI) source in positive and negative ionization modes in a single run. The ESI was performed using nitrogen gas to assist nebulization (the flow rate was set at 1.5 L/min), capillary voltage at 1.6 kV and temperatures of Curved Desolvation Line (CDL) and heat block at 250 and 300 °C were used. All instrumentation data were collected and synchronized by Lab solutions software (Version 7.1) from Shimadzu.
2.6 Pharmacokinetic study A single dose of Quercetin and Rutin (2.5 mg/kg) and test formulation containing equivalent dose of Quercetin and Rutin (2.5 mg/kg) were orally administered to two groups (n = 6). Both the preparations were administered as solutions by oral gavage at a volume of 10 mL/kg utilizing feeding needle. Blood samples (0.3–0.4 mL of venous blood) were collected from fossa orbitalis vein into heparinized centrifuge tubes at 15, 30, 60, 120, 180, 240, 360, 480, 600, 1,440, 2,160, 2,880 min after single oral administration. 30 min after blood withdrawal, the samples were centrifuged at 4,000 rpm for 10 min and the separated plasma samples were frozen in polypropylene tubes at -20 °C prior to analysis. 2.7 Validation of assay The current LC–MS assay was validated for specificity, linearity, precisions, accuracy and matrix effects according to ICH (International Conference on Harmonization 1996) guidelines. The specificity was determined by analyzing six blank plasma samples. There was no interference from endogenous or exogenous materials observed at the retention time in the ion channel of either the analytes or the IS. The calibration plasma samples were prepared by spiking 100 lL of working solution and 100 lL of IS solution to 100 lL of blank plasma and then the samples were treated according to the sample preparation. Quality control (QC) samples were prepared and assayed along with calibration curve samples. The calibration curve consisted of six concentration levels and each concentration was prepared and assayed on three separate days. The calibration curves were constructed by the plot of the peak area of the analyte versus concentrations of the calibration standards and described in the form of y = a ? bx. The concentrations of the analytes in unknown samples were assessed by interpolation from the calibration curve. The LOQ of the assay was defined as the lowest quantifiable concentration of the standard curve (LOQ, S/N = 10). The LOD was defined as the detectable amount (LOD, S/N = 3). The intra and inter-day precisions were defined as relative standard deviation (RSD) and the accuracy was assessed by comparing the measured concentration with its true value. The accuracy and precision were assessed by determining QC samples at three different validation batches. The intrabatch QC samples were prepared for six replicates. The acceptable intra and inter day precision and accuracy should be \15 %. The extraction recovery of analytes at three QC levels was evaluated by determining the peak area ratios of the
Eur J Drug Metab Pharmacokinet
analytes in the post-extraction spiked samples to that acquired from pre-extraction spiked samples.
3 Pharmacokinetic analysis 3.1 Non compartmental analysis All the pharmacokinetics parameters were determined by non-compartmental analysis. The peak plasma level (Cmax) and the time to reach peak plasma concentration (Tmax) were obtained directly from the concentration–time data. The elimination rate constant (Ke) was calculated from the slope of the logarithm of the plasma concentration versus time using the final four points. The apparent elimination half-life (T1/2) was calculated as 0.693/Ke. The area under the plasma concentration–time curve (AUC) and the area under the first moment curve (AUMC) were calculated by the trapezoidal rule. Total body clearance was established as Xo/AUC. The Mean Residence Time (MRT) after oral administration was calculated by dividing the AUMC by AUC. The values were calculated by Microsoft Excel (Microsoft, Seattle, Washington, USA) and each value was expressed as mean ± SD. 3.2 Wagner Nelson method This method involves mathematical calculation of the cumulative amount of drug absorbed at each time point Aa after drug administration and the total amount of the drug absorbed Aa?. The fraction Aa/Aa? represents the fraction of the absorbed dose that is already absorbed at each time point. The fraction remaining to be absorbed can be examined to determine the nature of the absorption process (Shargel et al. 2012). A plot of the fraction of the drug remaining to be absorbed versus time can be used to determine the order of the absorption process. Statistical significance was assessed by an unpaired Student’s t test and the significance level of P \ 0.05 was adopted for all statistical comparisons. All results were expressed as mean ± standard deviation (SD).
4 Results
suitably separated with in 20 min. As shown in Fig. 2 the retention time was 4.5 min for Rutin, 9.6 min for Quercetin and 15.6 min for Kaempferol (I.S). The selective ion monitoring chromatograms of Quercetin and Rutin from the standard mixture sample and spiked plasma are depicted in Figs. 2 and 3. Flavones in this study were examined in negative ion mode since it is known to yield better response for these compounds when compared to positive ion mode. Results of this study demonstrate that the mass signals of Rutin, Quercetin and Kaempferol (I.S) were high enough for detection in rat samples. The [MH]- ion of each standard was selected Selective Ion Monitoring (SIM) mode to obtain the selectivity and sensitivity for the determination of the two flavones. Kaempferol was selected as internal standard because of its similarity of chemical structure, chromatographic behavior, ionization efficiency, extraction efficiency and reproducibility at target ion of m/z 285.The content of Quercetin and Rutin in the capsule was estimated and found to be 0.09665 w/w and 0.0541 % w/w by HPLC, respectively. 4.2 Method validation 4.2.1 Specificity All the analytes and internal standard could be detected on their own selected ion chromatograms without any significant interference. 4.3 Linearity and lower limits of quantification The calibration curves of Quercetin and Rutin exhibited good linearity with correlation coefficients (R2) within the range from 50 to 1,600 ng/mL. The LLOQs were suitable for quantitative detection of analytes in the pharmacokinetics studies. Linear ranges, LOQ, LOD and correlation coefficients obtained from typical calibration curves were shown in Table 1. 4.4 Precision and accuracy The intraday, interday precision and accuracy date of Quercetin and Rutin at three concentration levels are summarized in Table 2. The RSD values of inter and intraday precision was \3.The accuracy ranged from 98 to 103 %.
4.1 Method development 4.5 Extraction recovery The LCMS conditions for the two flavonoids were optimized and shown in the materials and method section. The optimal condition allowed the two flavones (Quercetin and Rutin) and the internal standard (Kaempferol) to be
The extraction recoveries ranged from 82.6 to 103.2 % at three different concentration levels for the two analytes. The extraction recovery of the I.S. was more than 90 %.
Eur J Drug Metab Pharmacokinet Fig. 2 Selective ion chromatogram of Quercetin and Rutin from standard sample
Fig. 3 Selective ion chromatogram of Quercetin, Rutin and Kaempferol as internal standard from plasma spiked sample
Table 1 Method validation parameters Compound
Concentration range (lg/mL)
Rt (min)
SIM mode
Regression equation
R2
LOD (ng/mL)
LOQ (ng/mL)
Quercetin
0.05–1.6
9.8 ± 0.1
301
y = 1,642.4x-91,451
0.99
21.1
64.1
Rutin
0.05–1.6
4.8 ± 0.2
609
y = 471.72x-10,325
0.99
8.3
26.7
Eur J Drug Metab Pharmacokinet Table 2 Inter and intraday precision Inter-batch (n = 5) Nominal concentration (ng/mL)
Intra-batch(n = 10) Measured concentration (ng/mL)
Precision (%)
Accuracy (%)
Nominal concentration (ng/mL)
Measured concentration (ng/mL)
Precision (%)
Accuracy (%)
Quercetin 50
50.3
0.93
100.6
50
50.7
2.95
101.5
200
196.9
1.68
98.5
200
195.5
1.54
97.8
800
818.8
1.8
102.4
800
819.1
1.74
102.4
50
50.3
0.6
100.6
50
45.2
2.4
90.4
200
193.7
2.1
96.8
200
193.2
0.8
96.6
800
783.4
1.4
97.8
800
817.2
3.1
102.2
180
160
160
140
Concentration (ng/mL)
Concentration (ng/mL)
Rutin
140 Quercetin
120
Rutin
100 80 60 40 20
Quercetin Rutin
120 100 80 60 40 20 0
0
0 0
4
8
12
16
20
4
8
12
16
20
24
24
Time (hrs)
TIme (hrs)
Fig. 4 Area under curve of Quercetin and Rutin from the oral administration of mixture
4.6 Pharmacokinetics interaction of Quercetin and Rutin The developed and validated method was applied to the pharmacokinetic evaluation of polyherbal formulation in rats following oral administration. Plasma samples from single dose of 2.5 mg/kg equivalent weight of Quercetin were obtained after intragastric gavage. The mean plasma concentration of Quercetin and Rutin versus time curve is illustrated in Figs. 4 and 5. Corresponding to previous pharmacokinetics of individual flavonoids and in the form of Hipphophae extract in human and various animal species, Quercetin and Rutin showed two or more peaks of maximum plasma concentration in individual rats. This phenomenon was declining with coadministration. The main pharmacokinetics of Quercetin and Rutin are listed in Table 3. In Table 3, it can be seen that the AUC of Quercetin and Rutin after the co administration was significantly different
Fig. 5 Area under curve of Quercetin and Rutin from the polyherbal formulation
from the oral dose of polyherbal formulation (P \ 0.001 for both AUC and AUC, Students t test). After oral dose of polyherbal formulation, the oral bio availability of Rutin was significantly enhanced, whereas the Quercetin bioavailability shows insignificant enhancement when compared with Quercetin Rutin co-administration. The rate of absorption (Ka) calculated by the Wagner nelson method significantly differs from thepolyherbal formulation when compared with co-administration of quercetin and rutin by oral administration.
5 Discussion The results of the present study show that there is a great difference in the pharmacokinetics of the Quercetin and Rutin when they were administered together and from the polyherbal formulation which will be interacted by many other constituents. The bioavailability of Quercetin was
Eur J Drug Metab Pharmacokinet Table 3 Pharmacokinetic parameters Parameter Cmax (ng/mL Tmax (h) AUC (0–24) (ng/mL/h) AUC (0–?) (ng/mL/h) AUMC (ng/mL/h2) MRT (h) -1
Ka (h )
Quercetin (coadministration) 165.3 ± 31.9
Rutin (coadministration) 61.1 ± 29.3
Quercetin (polyherbal formulation) 90.8 ± 21.4*
Rutin (polyherbal formulation) 121.7 ± 19.2*
3.37 ± 0.51
4.17 ± 1.19
3.27 ± 0.86
4.43 ± 0.85
944.27 ± 70.57
389.45 ± 42.30
337.20 ± 14.75**
684.83 ± 15.52*
972.17 ± 85.77
456.67 ± 29.80
406.60 ± 24.78
6,729.27 ± 206.75
6,048.53 ± 151.78
5,135.37 ± 248.58
7.7 ± 1.1
11.9 ± 1.5
12.2 ± 1.5*
0.419 ± 0.006
0.242 ± 0.04
0.5537 ± 0.02**
731.40 ± 31.15** 6,295.57 ± 304.66 7.4 ± 1.1* 0.2753 ± 0.01
All values are expressed as Mean ± SD (n = 6). The statistical significance of differences was tested using the nonparametric Wilcoxon test. Significant values are expressed as *P \ 0.05, **P \ 0.01 and ***P \ 0.001 compared with corresponding co-administration Cmax maximum plasma concentration, Tmax Time to reach maximum concentration, AUC area under curve, AUMC area under first moment curve, MRT mean residence time, Ka rate of absorption
lowered from the polyherbal formulation when compared with the co-administration, whereas the Rutin bioavailability is increased from the polyherbal formulation when compared with the co-administration. Absorption of Quercetin and Rutin that occurred from the different parts of the gastrointestinal tract and inter individual variation in the absorption of Quercetin from Rutin, but not from Quercetin aglycone, was considerable. However, the mechanism of the absorption remained unclear. It has been reported that enzymes cleaving Quercetin glycoside are present in the small intestine and in the colon, and, therefore, it is likely that many of the compounds are hydrolyzed prior to the absorption of Quercetin aglycone (Erlund et al. 2000). Some studies indicate that the Quercetin glycosides containing glucose moieties could be absorbed intact (Hollman et al. 1999). Yang et al. proposed that rutin undergoes deglycosylation to quercetin-3-O-glucoside by an enzyme L-rhamnosidase which is being produced by human intestinal bacteria of isolated strains. Also they stated that lack of these enzymes could not metabolize further (Yang et al. 2012). The above results indicate that Rutin hydrolyzes before absorption; hence only very trace amount of Rutin was detected in plasma but the presence of other secondary metabolites in the polyherbal formulation interfaces with hydrolyzing enzymes in distal tubules of small intestine or colon, their by increasing in rutin bioavailability in polyherbal formulation. The absorption curve for the Quercetin was biphasic (Erlund et al. 2000). This bi-phasic phenomenon was observed in individual plasma concentration-time curve also. This phenomenon indicated the poor enterohepatic recirculation of the flavonoid compounds from the formulation. A possible explanation for this phenomenon is that Quercetin aglycone seems to be absorbed from the several parts of the gastrointestinal tract. When it was co administrated with Rutin alone it has been absorbed in the proximal parts of the gastrointestinal tract and traces
will be left by the time the distal parts of the small intestine or the colon are reached to allow another absorption peak to occur. Hollman et al. (1997) also stated that biphasic concentration profile in the elimination of quercetin from plasma indicates a rather fast distribution and elimination followed by a slow final elimination. When it was from the formulation due to many other constituent interactions the quantity of the Quercetin reaching the small intestine is increased and causes the increased bioavailability. The non compartmental model analysis which is based on the statistical moment theory was performed for the pharmacokinetic analysis. The Quercetin peak concentration in plasma occurred at 3.3 h from the co-administration and 3.2 h from the polyherbal formulation, while from the Rutin it was 4.1 and 4.7 h from co-administration and polyherbal formulation, respectively. These results indicate that Quercetin was absorbed more rapidly than Rutin likely due to quick metabolism of Quercetin after its absorption (Graefe et al. 2001). This finding is consistent with results reported by Lan et al. (2007). The mean residence time of the Quercetin was significantly higher from the polyherbal formulation, whereas Rutin residence time is significantly lowered from the polyherbal formulation. Vinson et al. reported that free Quercetin is one of the most potent antioxidants to inhibit lipid peroxidation (IC50 & 0.22 lmol/L) (Vinson et al. 1995). However, Quercetin is probably present mainly as conjugated forms, extensively bound to albumin (Manach et al. 1995) and the actual antioxidant potency of these forms is still uncertain and possibly lesser than that of the free forms. The method developed for the Quercetin and Rutin quantification from the plasma samples was highly sensitive and selective analytical method. The high recovery and precision values indicate that developed method is accurate for the extraction of the Quercetin and Rutin from the plasma.
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6 Conclusion In conclusion, the present study proved that the oral bioavailability of Quercetin and Rutin was enhanced from the polyherbal formulation when compared with this mixture administration. Therefore, it may be concluded that the oral dose of mixed compounds in herb extracts could gain a significant biopharmaceutical advantage when compared to the single compound. Consequently, inter and intra herb interaction of the Quercetin and Rutin was elucidated. Further elucidation of mechanism of interaction is under process. Acknowledgments The authors would like to thank the Department of Science and Technology, Government of India, for providing financial assistance to carry the work and Department of Pharmacology, SRM College of Pharmacy, SRM University, for providing facilities to carryout animal studies. Conflict of Interest
The authors declare no conflict of interest.
References Manach C et al (1995) Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin. J Nutr 125(7):1911–1922 Ilango K et al (2013) Emerging need of pharmacokinetics in Ayurvedic system of medicine. Int J Res Ayurveda Pharm 4(5):1–5 Aruna D, Naidu M (2007) Pharmacodynamic interaction studies of Gingiko biloba with cilostazol and clopidogrel in healthy human subjects. Br J Clin Pharmacol 63:333–338 Brazier N, Levine M (2003) Drug-herb interaction among commonly used conventional medicines: a compendium for health care professionals. Am J Ther 10:163–169
Dubey GP (2005) Herbal preparation for management of cardiovascular and neurologic disorders. European Union, Patent No. EP 1 569 666 B1 Erlund I et al (2000) Pharmacokinetics of quercetin from quercetin aglycone and rutin in healthy volunteers. Eur J Pharmacol 56:545–553 Formica J, Regelson W (1995) Review of the biology of Quercetin and related bioflavanoids. Food Chem Toxicol 33:1061–1080 Graefe E et al (2001) Pharmacokinetics and bioavailability of quercetin glycosides in humans. J Clin Pharmacol 41:492–499 Guowen L et al (2012) Pharmacokinetic properties of isorhamnetin, kaempferol and quercetin after oral gavage of total flavones of Hipphophae rhamnoides L. in rats using a UPLC-MS method. Fitoterapia 83:182–191 Hollman PC et al (1997) Relative bioavailability of the antioxidant flavanoids quercetin from various foods in man. FEBS Lett 418:152–156 Hollman P et al (1999) The sugar moiety is a major determininant of the absorption of dietary flavanoid glycosides in man. Free Radic Res 31:569–573 International Conference on Harmonization (1996) Guideline on validation of analytical procedure-methodology. IFPMA, Geneva Ke L et al (2008) Intra-herb pharacokinetics interaction between quercetina nd isorhamentin. Acta Pharmacologica Sinica 29(11):1376–1382 Lan K, Jiang X, He J (2007) Quantitative determiniation of isorhamnetin, quercetin and kaempferol in rat plasma by liquid chromatography with electron spray ionization tandem mass spectrometry and its application to the pharmacokinetic study of isorhamnetin. Rapid Commun Mass Spectrom 21:112–120 Shargel L, Susanna W-P, Andrew B (2012) Applied biopharmaceutics and pharmacokientics, 5th edn. McGraw-Hill, New York Vinson JA, Jang J, Dabbagh YA, Serry MM, Cai S (1995) Plant polyphenols exhibit lipoprotein-bond antioxidant activity using an in vitro oxidation model for heart disease. J Agric Food Chem 43:2798–2799 Yang J et al (2012) Identificaiton of rutin deglycosylated metabolites produced by human intestinal bacteria using UPLC-Q TOF/MS. J Chromatogr B 898(1):95–100
