Hepatoprotective Activity Of Some Quercetin Derivatives
C Roy, A Das
Keywords
carbon tetrachloride, hepatoprotection, paracetamol, psidium guajava linn., quercetin derivatives.
Citation
C Roy, A Das. Hepatoprotective Activity Of Some Quercetin Derivatives. The Internet Journal of Alternative Medicine. 2009 Volume 8 Number 2.
Abstract
The study was designed to evaluate the hepatoprotective activity of some quercetin derivatives 6-Bromoquercetin (Comp 1), 6,8-Dibromoquercetin (Comp 2), 2',5',6,8-Tetrabromoquercetin (Comp 3) and ,5',6,6',8-Pentabromoquercetin (Comp 4) in acute experimental liver injury induced by carbon tetrachloride and paracetamol. The effects observed were compared with a known hepatoprotective agent, silymarin. Four quercetin derivative was prepared by bromination, 6-Bromoquercetin (Comp 1), 6,8-Dibromoquercetin (Comp 2), 2',5',6,8-Tetrabromoquercetin (Comp 3) and ,5',6,6',8-Pentabromoquercetin (Comp 4) respectively. In the acute liver damage induced by different hepatotoxins, quercetin derivatives 6-Bromoquercetin (Comp 1) and 6,8-Dibromoquercetin (Comp 2)
Introduction
Natural flavonoids are distinguished by the breadth of their therapeutic action and their low toxicity. They exert an influence on the enzyme and immune systems of the organism [Kubo et al.,1992, Carte BK et al., 1991] and on metabolic processes [Petrushkova AM et al. 1992], and exhibit antiarrhythmic [Babaskin VS et al. 1992], antimicrobial [Ankhwinala MD et al., 1991, Joshi V et al., 1991, Aumente Rubio AD et al. 1988], antiviral [Dombi G, 1989, Shier W et al. 1992], antioxidant [Faur M et al., 1990, Beck G et al., 1990, Budzianowski J et al. 1991], and antitumoral and radioprotective [Kabiev OK et al. 1975] properties. With the aim of expanding the arsenal of drugs with a physiological action we have carried out a directed synthesis of bromine derivatives of a natural hydroxyflavone - quercetin, 3,5,7,3',4'-pentahydroxyflavone (1).The bromination of aromatic compounds has been studied and provides a logical theoretical basis for analyzing the reaction mechanism and the structures of the compounds obtained [P. Sykes, 1970, Mabry TJ et al. 1970].
We studied bromination in organic solvents. To fred the optimum conditions for obtaining mono- and dibromo derivatives of quercetin, we varied the molar ratio of quercetin and bromine from 1:1 to 1:4, the reaction temperature from 20 to 85°C, and the reaction time from 1 to 72 h. 6-Monobromoquercetin (2) was obtained in 2 h at 20-25°C by the action of an equimolar amount of bromine in dioxane, while 6,8-dibromoquercetin (3) was obtained in acetic acid at 35-40°C in 1 h. Compound (2) can be used both as the final product and as an intermediate for the introduction of new prognosticated groups. It must be mentioned that in the dynamics of the bromination of quercetin a decisive role is played by the temperature regime; in particular, elevated temperatures lead to an intensification of electrophilic substitution with the formation of a mixture of bromine derivatives. The main objective of the study is to find out the hepatoprotective potency of the synthetized derivatives.
Materials and Methods
Experimental
Acute hepatitis models
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Group 1: distilled water (1 ml/kg, p.o)
Group 2: distilled water for 9 days + CCl4 (1ml/kg, p.o) on ninth day
Group 3: silymarin (100mg/kg /day, p.o) for 9 days + CCl4 (1ml/kg, p.o) on ninth day
Group 4: derivative 1 for 9 days + CCl4 (1ml/kg, p.o) on ninth day
Group 5: derivative 2 for 9 days + CCl4 (1ml/kg, p.o) on ninth day
Group 6: derivative 3 for 9 days + CCl4 (1ml/kg, p.o) on ninth day
Group 7: derivative 4 for 9 days + CCl4 (1ml/kg, p.o) on ninth day
Food was withdrawn 12 hr before carbon tetrachloride administration to enhance the acute liver damage in animals of groups 2, 3, 4, 5, 6 and 7. The animals were sacrificed 24 hr after the administration of CCl4. Blood samples were collected and the serum was used for assay of marker enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and serum bilirubin. The liver was immediately isolated and washed with normal saline, blotted with filter paper and weighed. The liver was then subjected to histopathological examination (Matsuda H et al. 1970).
Statistical analysis
The statistical significance was assessed using one way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. The values are expressed as mean
Results
Synthesis of 6-Bromoquercetin (Derivative 1):
Yeild: 1.08 gms
Colour: yellow brownish.
State: solid.
Odour: Slight pungent.
Melting Point: 270-2720 C
Rf value: 0.78.
IR spectra (KBr,
NMR Spectra: (100 MHz, DMSO, ppm): 6.47 (s, H-8), 6.77 (d, H-5'), 7.59 (d, H-2'), 7.77
(d, H-6').
Mass Spectra: C 47.89, 47.75; H 2.94, 2.63; Br 21.85, 21.02; C15H9O7Br.
Synthesis of 6,8-Dibromoquercetin (Derivative 2):
Yeild: 1.2 gms
Colour: yellow green.
State: solid.
Odour: Slight odour.
Melting Point: 254-2560 C
Rf value: 0.79.
IR spectra (KBr,
NMR Spectra: (100 MHz, DMSO, ppm) 6.71(d, H-5'), 7.56(d, H-2'), 7.64(d, H-6').
Mass Spectra: C 39.3, 38.9; H 1.8, 1.53; Br 33.50, 33.92; C15H807Br 2.
.
Synthesis of 2',5',6,8-Tetrabromoquercetin (Derivative 3):
Yeild: 1.00 gms
Colour: nearly colourless.
State: solid.
Odour: Slight odour.
Melting Point: 248-2500 C
Rf value: 0.87.
IR spectra (KBr,
NMR Spectra: (100 MHz, DMSO, ppm):8.09 (s, H-6').
Mass Spectra: C 28.07; H 1.24; Br 51.01; C15H607Br 4.
Synthesis of 2',5',6,6',8-Pentabromoquercetin (Derivative 4)
Yeild: 0.9 gms
Colour: nearly colourless.
State: solid.
Odour: Slight odour.
Melting Point: 135-1370 C
Rf value: 0.90.
IR spectra (KBr,
NMR Spectra: No signals reported.
Mass Spectra: C 24.1; H 0.82; Br 56.9; C15H5O7Br 5.
Pharmacological Studies:
Carbon Tetrachloride (CCl) Induced Acute Hepatic Injury
A significant difference in biochemical markers was observed between normal and CCl4 control groups. Comparative analysis on the effect of ALT, AST and ALP and between the quercetin derivatives revealed that quercetin derivatives (Comp 1 and Comp 2) have almost similar activity compared to the standard silymarin (100mg/Kg body weight). Comparative analysis revealed that Comp 3 and Comp 4 prevented the increase in biomarker levels but were not significant when compared to standard silymarin, a known hepatoprotective agent. Significant reduction of liver weight was also seen in animals treated with Comp 1 and Comp 2, whereas other quercetin derivatives (Comp 3 and Comp 4) did not show significant reduction in the liver weight (Figure 1 and 2). Significant reduction in bilirubin level was seen in in silymarin, Comp 1 and Comp 2 whereas the other two compounds did not show much decrease in bilirubin level. (Figure 3).
Paracetamol (PCM) Induced Acute Hepatic Injury:
Serum Biochemical markers and liver weight:
Forty eight hours after treatment with paracetamol, the parameters ALT, AST, ALP and bilirubin levels in the serum increased remarkably. A significant difference in biochemical markers was observed between normal and PCM control groups. Comparative analysis on the effect of ALT, AST and ALP and between the extracts revealed that quercetin derivative Comp 1 and Comp 2 have almost similar activity compared to the standard silymarin (100mg/Kg body weight). The other derivatives Comp 3 and Comp 4 did not show significant activity when compared to standard silymarin. Significant reduction of the liver weight was observed in groups administered with standard silymarin, Comp 1 and Comp 2 whereas the other extracts did not show any reduction of the liver weights (Figure 4 and 5). Significant reduction in bilirubin levels were seen in silymarin, Comp 1, Comp 2 whereas Comp 3 and Comp 4 did not show much reduction in bilirubin levels. (Figure 6).
Figure 1
Values are mean S.E.M, n = 6, a p<0.001 vs. vehicle control, b p<0.01, c p>0.05 vs. vehicle control, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. CCl4 treated control.
Figure 2
Values are mean S.E.M, n = 6, a p<0.001 vs. vehicle control, b p<0.01, c p>0.05 vs. vehicle control, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. CCl4 treated control.
Figure 3
Values are mean S.E.M, n = 6, a p<0.001 vs. vehicle control, b p<0.01, c p>0.05 vs. vehicle control, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. CCl4 treated control.
Figure 4
Values are mean S.E.M, n = 6, a p<0.001 vs. vehicle control, b p<0.01, c p>0.05 vs. vehicle control, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. PCM treated control.
Figure 5
Values are mean S.E.M, n = 6, a p<0.001 vs. vehicle control, b p<0.01, c p>0.05 vs. vehicle control, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. PCM treated control.
Figure 6
Values are mean S.E.M, n = 6, a p<0.001 vs. vehicle control, b p<0.01, c p>0.05 vs. vehicle control, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 vs. CCl4 treated control.
Histopathology
Histological examination of the liver tissue from CCl4 treated animals revealed that CCl4 had produced profound inflammation and congestion especially in the sinusoids. Hydropic degeneration and steatosis in the periportal region was also observed (fig 7a). In animals pretreated with silymarin, and derivative 1 found to reduce the inflammation, degenerative changes and steatosis (fig 7b).
Figure 7
Histopathological examinations of the liver tissues showed severe congestion of blood vessels, mild hydropic degeneration, pyknosis of nucleus and occasional necrosis in PCM treated animals (fig 8a). Silymarin reduced the pyknosis of hepatocytes when compared to PCM treated control. Animals treated with derivative 1
Figure 8
Discussions
Derivatives were prepared by bromination of quercetin, four compounds were synthesized Derivative 1 (6-Bromoquercetin), Derivative 2 (6,8-Dibromoquercetin), Derivative 3 (2',5',6,8-Tetrabromoquercetin) and Derivative 4 (2',5',6,6',8-Pentabromoquercetin). 6 bromoquercetin was prepared by a solution of 0.2 ml (0.0039 mole) of bromine in 10 ml of dioxane which was slowly added dropwise to a solution of 1.0 g (0.0033 mole) of quercetin (1) in 80 ml of dioxane. The reaction was continued at 20-22°C for 2 h. The resulting precipitate was washed with water and dried. 6,8-Dibromoquercetin was prepared at 35-40°C where 0.4 ml (0.0078 mole) of bromine was slowly added dropwise to a solution of 1.0 g (0.0033 mole) of 6-Bromoquercetin in 100 ml of glacial acetic acid. After an hour's vigorous stirring, a yellow-green precipitate deposited. The reaction mixture was concentrated, and the precipitate was washed with water and dried. 2',5',6,8-Tetrabromoquercetin was prepared at 75-80°C where 0.8 ml (0.0154 mole) of bromine in 40 ml of dioxane was slowly added dropwise to a solution of 1.0 g (0.0033 mole) of 6-Bromoquercetin in 100 ml of dioxane. After eight hours' vigorous stirring, the reaction mixture was evaporated to dryness. The residue was dissolved in water and the solution was brought to a boil and filtered. On cooling, it deposited a precipitate of 2',5',6,8-Tetrabromoquercetin. 2',5',6,6',8-Pentabromoquercetin was prepared by separating it with paper chromatography on FN-11 paper in system 1 to get 2',5',6,6',8-Pentabromoquercetin.
Pharmacological activity of the synthesized derivatives (Comp 1, Comp 2, Comp 3 and Comp 4) revealed that Comp 1 (Derivative 1) and Comp 2 (Derivative 2) has significant hepatoprotection when compared to silymarin (100mg/kg body weight) which is a standard hepatoprotective agent.
In carbon tetrachloride induced hepatoxic model significant difference in biochemical markers was observed between normal and CCl4 control groups. Comparative analysis on the effect of ALT, AST and ALP and between the quercetin derivatives revealed that quercetin derivatives (Comp 1 and Comp 2) have almost similar activity compared to the standard silymarin (100mg/Kg body weight). Comparative analysis revealed that Comp 3 and Comp 4 prevented the increase in biomarker levels but were not significant when compared to standard silymarin, a known hepatoprotective agent. Significant reduction of liver weight was also seen in animals treated with Comp 1 and Comp 2, whereas other quercetin derivatives (Comp 3 and Comp 4) did not show significant reduction in the liver weight. Significant reduction in bilirubin level was seen in in silymarin, Comp 1 and Comp 2 whereas the other two compounds did not show much decrease in bilirubin level.
In paracetamol (PCM) treated acute hepatic injury a significant difference in biochemical markers was observed between normal and PCM control groups. Comparative analysis on the effect of ALT, AST and ALP and between the extracts revealed that quercetin derivative Comp 1 and Comp 2 have almost similar activity compared to the standard silymarin (100mg/Kg body weight). The other derivatives Comp 3 and Comp 4 did not show significant activity when compared to standard silymarin. Significant reduction of the liver weight was observed in groups administered with standard silymarin, Comp 1 and Comp 2 whereas the other extracts did not show any reduction of the liver weights. Significant reduction in bilirubin levels were seen in silymarin, Comp 1, Comp 2 whereas Comp 3 and Comp 4 did not show much reduction in bilirubin levels.
Acknowledgement
The authors are thankful to Prof. Suresh Nagpal, Chairman, Krupanidhi Educational Trust, Bangalore, India, Prof. Sunil Dhamanigi, Secretary, Krupanidhi Educational Trust, Bangalore, India for their help.
