Journal of Medicinal Materials, 2015, Vol. 20, No. 3 (pp. 156 -160)
IN VIVO ANTI-HYPERURICEMIC ACTIVITIES OF CINNAMALDEHYDE DERIVATIVES ISOLATED FROM
TWIGS OF CINNAMOMUM CASSIA BLUME Tran Minh Ngoc''*, Nguyen Minh Khoi,' Vu Binh Duong'
' Vietnam National Institute of Medicinal Materials
^ Vietnam Military Medical University
•Corresponding author: [email protected] (Received May l l ' \ 2015)
Summary
In vivo Anti-Hyperuricemic Activities of Cinnamaldehyde Derivatives Isolated from Twigs of Cinnamomum cassia Blume
Xanthine oxidase (XOD) and xanthine dehydrogenase (XDH) are mammalian xanthine ox t do reductases involved in the catalysis of the oxidation of hypo xanthine and xanthine to uric acid. It may be a reasonable approach to hamper XOD/XDH activity for the treatment of hyperuricemia-associaled syndromes considering that excessive formation of uric acid can cause such syndromes. C cassia Blume (Lauraceae) is traditionally employed in the treatment of gastritis, blood circulation and gout. Cinnamaldehyde derivatives from C cassia exhibit potent in vivo XOD inhibitory activity and the mode of inhibition is investigated employing Linewcaver-Burk plot analysis. The active compounds are evaluated for their inhibitory potential lo reduce serum and hepatic uric acid levels in male ICR mice, murine hyperuricemia model induced by pretreatmenl with the uncase inhibitor potassium oxonate. The tested compounds exhibit remarkable reductions in hepatic XDH/XOD activity in a time-dependent manner, suggesting that the observed in vivo anti-uricemic activity is partly originating from their inhibitory action on the xanthine oxidoreductases.
Keywords: Hyperuricemia, Cinnamaldehyde derivatives, Cinnamomum cassia. Xanthine oxidoreductases 1. Introduction is widely employed in foods, beverages and the Xanthine oxidase (XOD) (EC 1.2.3.2) and cosmetic industry [7]. C. cassia is traditionally xanthine dehydrogenase (XDH) (EC 1.17.1.4) are utilized in the treatment of diseases including mammalian xanthine oxidoreductases that catalyze gastritis, blood circulation and gout [6],[8], the oxidation of hypoxanthine and xanthine to Indeed, Kong et al., (2000) validated that the uric acid. Excessive production of uric acid can methanol extract of twigs of C. cassia exhibits culminate in hyperuricemia-associated diseases powerful inhibition of XOD among 122 such as gout, in which the deposition of uric acid traditional Chinese medicinal plants frequently in joints leads to severe inflammation [1-3]. prescribed for the treatment of hyperuricemia- Therefore, it may be a promising therapeutic associated disorders. In addition, our previous approach to diminish serum levels of uric acid by study demonstrates that cinnamaldehyde derivatives inhibiting XOD/XDH activity for the treatment in the methanol extract exhibit potent in vitro of hyperuricemia-associated disorders. Currently, XOD inhibitory activity [9].
allopurinol is being clinically used as the only In our follow-up study, the active cinnamaldehyde XOD/XDH inhibitor [4]. Considering the side derivatives, cinnamaldehyde (1), 2-methoxy effects of the current agent including hepatitis, cinnamaldehyde (2), and 2-hydroxy cinnamaldehyde nephropathy, and allergic reactions [5],[6], there (3), are purified from the methanol extract from is a growing need to explore new lead compounds twigs of C. cassia and evaluated for their potential that can be developed into XOD/XDH inhibitors. to hamper XOD activity in vivo. The mode of
Cinnamon {Cirmamonium cassia Blume) (Lauraceae) inhibition of the cinnamaldehyde derivatives is 156 Journal of Medicinal Materials, 2015, Vol. 20 No. 3
investigated using a kinetic study based on Lineweaver-Burk plot analysis. The in vivo anti- uricemic ability of the compounds is evaluated employing a murine hyperuricemia model induced by the uricase inhibitor potassium oxonate.
2. Materials and methods 2.1. Reagents
Uric acid, allopurinol, xanthine, XOD, XDH, sodium carbonate, nicotinamide adenine dinucleotide (NAD+), xanthine oxidase (X1875) and potassium oxonate were purchased from Sigma (St. Louis, MO, USA).
2.2. Plant material
Twigs of C. cassia were collected from Yen Bai province, Vietnam in November 2010.
Extraction and isolation. Sliced twigs of C cassia {20.0 kg) were extracted with methanol (3 X 40 L), filtered and concentrated to yield the methanol extract (1260 g). The extract was suspended m H2O (3 L) and consecutively extracted with «-hexane ( 3 x 3 L), CH2CI2 ( 3 x 3 L), EtOAc (3 X 3 L) and BuOH ( 3 x 3 L).
Compounds 1, 2 and 3 were purified from the EtOAc layer (90 g) based on previous isolation protocols [9].
In vitro XOD inhibition assay. XOD inhibitory activity of the cinnamaldehyde derivatives was evaluated measuring the generation of uric acid from xanthine by a spectrophotometer [10]. The assay mixture consisted of 50 inM sodium carbonate buffer (pH 7.8), 50 ^iM xanthine, 0.1 mM EDTA, and with the presence or absence of tested compound in total volume of 495 |il. The reaction was initiated by adding of 5 pi xanthine oxidase (20 nM) and the increasing absorbance at 295 nm was measured after 5 min. The inhibitory activity was obtained after friplicate measurements and was expressed as IC50 from the generated amount of uric acid of the control. To determine the mode of inhibition of the cinnamaldehyde derivatives, an enzymatic kinetic study was performed using Lineweaver-Burk plot analysis.
The study was carried out with or without a test compound in the presence of various concentrations of xanthine as the substrate. An initial rate was determined on the basis of a rate of increase in
absorbance at 295 nm indicating the generation of uric acid (e = 9.5 mM'cm"'). The Michaelis constant {K^) and maximal velocity (K,n„) of XOD were determined by Lineweaver-Burk plot analysis.
Animals and experimental protocols. Male ICR mice (26-30 g) were purchased from Central Lab. Animal Inc. (Seoul, Korea) and were housed in plastic cages. They were allowed one week to adapt to a controlled environment before experiments. All animals were maintained at 25
°C with a 12 h light/12 h dark cycle. They were given standard chow and water ad libitum during experiments and fasted one hour before the administration. Various concentrations of compounds 1, 2 and 3 and allopurinol were dissolved or dispersed in 0.9 % NaCl. The administered volume of the solution or suspension was determined based on body weights measured prior to each administration. All compounds were given orally once a day at 14:00 p.m.-15:00 p.m.
With reference to a previous study [6], a murine hyperuricemia model was generated by the administration of potassium oxonate. Briefly, mice were injected intraperitoneal ly with potassium oxonate (250 mg/kg) one hour before the final administration. As shown in Figs. 3 and 4, four groups of mice (two negative control groups and two hyperuricemia model groups; n = 6) were orally administered 0.9 % NaCl for one and five days. As four positive control groups, allopurinol at a dose of 10 mg/kg was administered for one and five days. The other twelve groups of mice orally received compounds 1 - 3 at 50 mg/kg for 1 and 5 days, respectively. All procedures were in strict accordance with the Vietnam legislation on the use and care of laboratory animals and with the guidelines for Experimental Animals of National Institute of Medicinal Materials.
Uric acid assay. One hour after the final administration, blood samples were collected from mice by tail vein bleeding. The blood was allowed to clot for one hour at room temperature and centrifuged at 3000 rpm for 5 min. Tlie acquired serum was stored at -20 °C until tested in the uric acid assay. A mouse liver was harvested, frozen immediately and stored at -80 °C.
Journal of Medicinal Materials, 2015, Vol. 20, No. 3 157
Liver rissue samples were homogenized in five times volume of 80 mM sodium pyrophosphate buffer (pH 7.4). The homogenate was centrifuged at 3000 rpm for 10 min, the lipid layer was carefully removed and the supernatant was further centrifuged at 10,000 rpm for 60 min at 4
°C and used in the uric acid assay with reference to the phosphotungstic acid method [11].
Assays of hepatic XDH/XOD activity. The above-mentioned supernatant was used to assess XDH/XOD activity following the procedure described elsewhere [12,13]. Briefly, 100 fiL of the supernatant was added to a reaction mixture
containing 50 pM xanthine and 5 mM EDTA.
For the measurement of XDH activity, 200 \iM NAD* was added to the reaction mixture. The mixture (total 5mL) was incubated at 37 °C for 30 min and terminated by the addition of 0.5 mL HCl (0.58 M). The UV absorbance at 295 nm was measured to determine uric acid production.
The XDH/XOD activity was expressed as nmole uric acid produced per minute per milligram protein. Protein concentration was determined by the Bradford method using bovine serum albumin [14].
3. Results and discussion
Compounds R cinnamaldehyde (1) H 2-methoxy cinnamaldehyde (2) OCH3 2-hydroxy cinnamaldehyde (3) OH Figure 1. Structures of isolated cinnamaldehyde derivatives 1 - 3 from twigs of C cassia Repeated column chromatography of the n-
hexane layer of the methanol extract led to the isolation of three cinnamaldehyde derivatives 1,2 and 3 (Fig. 1). Based on our previous study that the cinnamaldehyde derivatives are potent in vitro XOD inhibitors [9], an enzyme kinetic study employing Lineweaver-Burk plot analysis was carried out for the investigation of a mode of inhibition. Various concentrations of xanthine as the substrate (10, 25, 50, 75, and 100 jiM) were used in the presence of compounds 1,2 and 3 and XOD as the en^me (Fig. 2). As shown in Fig. 2, the reciprocal plots of the tested compounds are intersected to the left of the 1/V axis, suggesting that the tested compounds inhibit XOD in a mixed-type manner. A kinetic model of XOD inhibition via the tested compounds appears to follow Michaelis-Menten kinetics (Fig. 2).
According to Table 1 obtained from analysis of
the Lineweaver-Burk plots, the ICjo's of the tested cinnamaldehyde were 7.8 ± 0.65, 13.8 ± 0.88 and 14.6 ± 1.18 ^ig/mL, respectively, indicating that compound 1 is tlie most powerful XOD inhibitor even though compound 3 possesses the greatest affinity {K,„) to the enzyme.
0.15 -0 05 -lE-l^/ixan(9ifift](nM) »
Figure 2. The Lineweaver-Burk plots of XOD inhibitory activity in the presence 10 fig/mL of I, 2, and 3 1 (•), 2 (•),
3 (•), and DMSO (o) as a control. The data represent the mean ± S.E of triplicate independent experiments Table 1. Inhibition etTects of isolated compounds 1-3 against XOD
1 ' ^ '
' f^n,., (fM/min) 1 7 8 ± 0 65
1 2 i
1 3 i
"Values present
I3 8±0.S8 14.6±1,18 mean ± S.E of triplicate
6.1 ±0.35 7.1 ±0.29 7.6 ±0.46 , ndependent experiments.
X O D ' /f„(mM) 94.9 ± 1.21 86.2 ± 1.39 84.8 ±1.04
Type of inhibition Mixed Mixed Mixed
158 Journal of Medicinal Materials, 2015, Vol. 20, No. 3
The uric acid levels in liver tissue and serum from hyperuricemic mice treated with the cinnamaldehyde derivatives were measured to evaluate in vivo anti-uricemic activity of the compounds. The potassium oxonate-induced
hyperuricemic mice were treated with the tested compounds at a dose of 50 mg/kg and the uric acid levels of serum and liver tissue were measured employing the phosphotungstic acid method [11] (Fig. 3).
Figure3. Activity of compounds 1 - 3 and allopurinol on the formation of serum and liver uric acid levels in hyperuricemic mice pretreated with potassium oxonate. Data represent mean values ± S.D of uric acid levels in scrum (mg/dL) and liver
(mg/g tissue) in 10 groups of mice for I day and other 10 groups ofmice for 5 days, '/*< 0.001, " P < 0 , 0 5 vs normal control group," /*< 0.001, *"/'< 0.01, "**/'< 0.05 vs. hyperuricemic control group (n = 6) The serum uric acid levels were diminished by
the treatment of compounds 1-3 (3.41, 3.14 and 2.95 mg/dL, respectively) compared to that of the hyperuricemic group (4.64 mg/dL). The five-day treadnent of the cinnamaldehyde derivatives further reduced serum levels (2.94, 2.72 and 2.40 mg/dL) to nearly that of the negative control ^oup (2.22 mg/dL). A similar inhibition pattem was observed when the uric acid levels of liver tissue were
I '
assessed. The administration of three compounds in one-day at a dose of 50 mg/kg hampered the formation of hepatic uric acid (0.61, 0.57 and 0.55 mg/g tissue, respectively) and five-day treatment with 1-3 reduced die levels of liver uric acid to 0.51, 0.49 and 0.44 mg/g tissue, respectively. The five-day treatment with, compound 1 significantly decreased the uric acid level, which was as low as that of the negative control.
5 _, ^
I
Figure 4. Effects of compounds I - 3 and allopurinol on the hepatic XHD and XOD activity in hjperuncemic mice pretreated with potassium oxonate. Data represent mean values ± S.D of the hepatic XDH and XOD activity (nmol uric acid/mim/mg protein) in 10 groups of mice for I day and other 10 groups of mice for 5 days.' P< 0.001, " P<0.05 vs.
normai control group." P < 0.001," P < 0 01, vs, hyperuricemic control group (n = 6)
Journal of Medicinal Materials, 2015, Vol. 20, No. 3 159
In VIVO hepatic XDH and XOD activity was monitored to address that the observed reduction of uric acid levels induced by the compounds (1- 3) was associated with their inhibitory action on XDH/XOD activity. As shown in Fig. 4, the hepatic levels of XDH/XOD activity induced by potassium oxonate elevated to 9.75/4.19 nmol uric acid/min per mg protein. The XDH activity was inhibited by 31.4, 35.9 and 38.0 % upon one- day administration of 1-3 at a dose 50 mg/kg and 33.9, 37.2 and 41.6 %, respectively, upon five- day treatment. Similarly, the hepatic XOD activity was hindered by 35.2, 42.1 and 48.4 % upon one-day treatment of the three tested compounds at the same dose and 41.9, 45.1 and 50.8 % after five-day treatment, respectively. In light of these findings, it is plausible that the decreased uric acid in the hepatic tissue by the treatment with the cinnamaldehyde derivatives is at least partially due to the compounds' ability to inhibit XOD/XDH activity.
4. Conclusion
In spite of numerous attempts to validate the biological activity of C. cassia, only a few studies have been performed to address its usage as an anti-gout agent. In the present study, three cinnamaldehyde derivatives, including cinnamaldehyde (1), 2 methoxycinnamaldehyde (2) and 2 - hydroxycinnamaldehyde (3), were purifed from
the active methanol extract and their anti- hyperuricemic effect was examined utilizing a murine hyperuricemia model. A kinetic study revealed that the cinnamaldehyde derivatives inhibited the oxidation activity of XOD in a mixed-type inhibition, which is similar to that of allopurinol [6]. The administration of 1-3 at 50 mg/kg significantly reduced serum and hepatic uric acid levels in the examined hyperuricemic mice in a time-dependent manner. The inhibitory action of the compounds on XDH and XOD was further investigated to elucidate the association between the inhibitory potency of the formation of uric acid and the potential to hamper the oxidative enzymatic activity. The tested compounds reduced the heparic XDH/XOD activity in a time- dependent manner, implying that the observed anti-hyperuricemic effect is at least partly derived from their inhibition of the hepatic XDH/XOD activity. Considering tlie potent anti-hyperuricemic effect originating from the simple moiety of the active compounds and their ease of acquisition in large quantities from C. cassia, the scaffold could be a reasonable starting structure that can be developed into a promising anti-gout agent with improved pharmacological properties.
Acknowledgements: This work was supported by a g-ant from the National Foundation for Science and Technology
Development. Vietnam (NAFOSTED 2010 - 2012).
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