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Article

Synthesis and Bio-Activity Evaluation of Scutellarein as a Potent Agent for the Therapy of Ischemic Cerebrovascular Disease

1
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, China
2
Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2011, 12(11), 8208-8216; https://doi.org/10.3390/ijms12118208
Submission received: 28 June 2011 / Revised: 6 October 2011 / Accepted: 1 November 2011 / Published: 18 November 2011
(This article belongs to the Section Biochemistry)

Abstract

:
Scutellarein, the main metabolite of scutellarin in vivo, has relatively better solubility, bioavailability and bio-activity than scutellarin. However, it is very difficult to obtain scutellarein in nature compared with scutellarin. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. The in vitro antioxidant activities of scutellarein were evaluated by measuring its scavenging capacities toward DPPH, ABTS+•, OH free radicals and its protective effect on H2O2-induced cytotoxicity in PC12 cells using MTT assay method. The results showed that essential point to the synthesis was the implementation of H2SO4 in 90% ethanol in N2 atmosphere; scutellarein had stronger antioxidant activity than scutellarin. The results have laid the foundation for further research and the development of scutellarein as a promising candidate for ischemic cerebrovascular disease.

1. Introduction

Cerebrovascular disease is a common and frequently-occurring disease that seriously endangers human health. It is one of the leading causes of death and disability worldwide, especially ischemic cerebrovascular disease, which is the most frequently prevalent [1]. Increasing evidence suggests a critical role of oxidative stress in ischemic cerebrovascular disease [2,3], and natural flavonoid antioxidants are well known as free radical scavengers [4]. Scutellarin (4′,5,6-trihydroxyflavone-7-glucuronide), the major anti-oxidant constituent in breviscapine extracted from Chinese herb of Erigeron breviscapus (vant.) Hand.Mazz., showed the effectiveness on dilating blood vessels, improving microcirculation, increasing cerebral blood flow, and inhibiting platelet aggregation since the 1970s [57]. In addition, it has been clinically used to treat acute cerebral infarction and paralysis induced by cerebrovascular diseases such as hypertension, cerebral thrombosis, cerebral haemorrhage in China since 1984 [8].
Although scutellarin has been clinically used for a long time, scutellarin has low water-solubility (just 0.16 mg/mL [9]) and lipid-solubility (log P = −2.56 in PBS at pH 4.2 [10]). Moreover, the bioavailability of scutellarin was very low; the absolute bioavailability in Beagle dog administered orally was rarely 0.4% [11]. Furthermore, intravenous elimination half-life in dogs was as short as 52 min [12]. Thus, various new formulations of scutellarin have been studied to overcome the above disadvantages, but its poor solubility, poor absorption and low bioavailability have not been completely solved until now. Interestingly, some researchers found that scutellarin was mainly absorbed in the form of its hydrolyzed product scutellarein by intestinal [13], and scutellarein was much more easily absorbed with the triple bioavailability, after oral administration of scutellarin and scutellarein in equal amounts [14]. Furthermore, in the clinical trials [15], a large amount of scutellarein was found in urine and plasma after oral administration of breviscapine in subjects, indicating that breviscapine was firstly hydrolyzed into aglycone when reaching colon and was then absorbed as scutellarein as the real bioactive components in the body. Pharmacodynamics confirmed that scutellarein had better protective effect than scutellarin in rat cerebral ischemia [16].
There is little scutellarein in E. breviscapus compared with large amount of scutellarin [16]. Frakas [17] and Cui [18] had already completed the total synthesis of scutellarein, but the route was long and the yield was low. As a result, scutellarein cannot be purchased in the market like scutellarin, and it is also very difficult to obtain it through other methods. Thus, we tried the synthesis of scutellarein by hydrolyzing scutellarin in water according to a patent [19]. Unfortunately, it was found that the reactants did not react. As a result, this study was intended to establish an efficient route to the synthesis of scutellarein by hydrolyzing scutellarin, and to preliminarily investigate its antioxidant activity in comparison with scutellarin, which will guide the search for more potent protective agents for ischemic cerebrovascular disease.

2. Materials and Methods

2.1. Materials

Scutellarin was purchased from Mianning Jiexiang Co. Ltd. (Chengdu, China). 1,1-Diphenyl-2- picrylhydrazyl (DPPH), 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt (ABTS) were purchased from Sigma Chemical Co. (Shanghai, China). Commercial kits used for determination of hydroxyl radical (OH) scavenging activity was purchased from Jiancheng Institute of Biotechnology (Nanjing, China). Dulbecoo’s Modified Eagles Medium (DMEM) was the product of Gibco. Heat-inactivated fetal calf serum was purchased from Sijiqing Institute of Biotechnology (Hangzhou, China). 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Fluka. PC12 cell line was obtained from Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China).

2.2. General Procedure for the Synthesis of Scutellarein

All the reagents were commercially available and used directly. Air- and moisture-sensitive liquids and solutions were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation below 45 °C at approximately 20 mm Hg. All non-aqueous reactions were carried out under anhydrous conditions using flame-dried glassware within an argon atmosphere in dry and freshly distilled solvents, unless otherwise noted. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.15~0.20 mm Yantai silica gel plates (RSGF 254) using UV light as the visualizing agent. Chromatography was performed on Qingdao silica gel (160~200 mesh) using petroleum ether (60~90) and ethyl acetate as the eluting solvent.
Scutellarin (0.5 g) was added to 10 mL solution of H2SO4 in 0~90% ethanol (0.5~3 mol/L). Then the reaction mixture was refluxed at 90~120 °C in N2 atmosphere for 6~48 h. After cooling to 25 °C, the reaction mixture was added to ice water; the solid was filtered and then recrystallized with 90% ethanol to give the target compound scutellarein. The synthesis route of scutellarein was outlined in Figure 1. Detailed synthesis conditions were designed in Table 1.

2.3. DPPH Radical-Scavenging Activity Assay

The DPPH radical-scavenging activity assay was measured according to the method [20] with a few modifications. 100 μL of the sample (4~250 μmol/L, dissolved in ethanol) was added to 100 μL of ethanol solution containing DPPH radicals (80 μmol/L) in 96 well plates. The mixture was shaken and left for 30 min at room temperature in the dark, and the absorption was measured at 517 nm. Ascorbic acid was used as the positive control. A lower absorbance represents a higher DPPH scavenging activity. The percentage scavenging effect was calculated as scavenging rate (%) = [1 − (A1A2)/A0] × 100%, where A0 was the absorbance of the control (without sample), A1 was the absorbance in the presence of the sample and A2 was the absorbance without DPPH.

2.4. ABTS+• Radical-Scavenging Activity Assay

The ABTS+ radical-scavenging activity assay was measured according to a literature procedure [21]. ABTS (7 mmol/L) and K2S2O8 (2 mmol/L) were dissolved and mixed in deionized water, reacting for 12~16 h at room temperature. The mixture (ABTS+ stock solution) was then diluted by phosphate buffer solution (PBS) to give an absorbance near 0.7 at 734 nm, defined as the reference absorbance (A0). A0 decreased to a stable value (A1) when 100 μL of ABTS+ was mixed with 100 μL the sample (1~62.5 μmol/L) for 6 min. The percentage scavenging effect was calculated as scavenging rate (%) = [1 − (A1A2)/A0] × 100%, where A0 was the absorbance of the control (without sample), A1 was the absorbance in the presence of the sample and A2 was the absorbance without ABTS+•.

2.5. OH Radical-Scavenging Activity Assay

The Hydroxyl radical-scavenging activity was measured according to the instruction of the assay kit (Nanjing Jiancheng Co., China). The percentage scavenging effect was calculated as scavenging rate (%) = [1 − (A1A2)/A0] × 100%, where A0 was the absorbance of the control (without sample), A1 was the absorbance in the presence of the sample and A2 was the absorbance without OH.

2.6. MTT Assay for PC12 Cell Survival

Protection against oxidative stress in PC12 cells was determined using the method [22] with minor modifications. PC12 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2. PC12 cells were grown on 96-well plates at a density of 5 × 104 cells/mL (100 μL/well) for 24 h. The protective effect against oxidative stress were tested at different concentrations in three types of experiments: (a) coincubation of PC12 cells with sample and H2O2; (b) preincubation of cells for 30 min with sample before exposing them to H2O2; (c) preincubation of cells for 8 h with sample before exposing them to H2O2. After incubation for 3 h with H2O2 (400 μmol/L) at 37 °C in CO2 incubator, the cells were incubated with the 0.5 mg/mL MTT for 4 h. Then, all culture media were removed and 150 μL of DMSO was added to each well, vigorously shaking for 10 min. Finally, the absorbance was assessed at 517 nm. The inhibiting rate of H2O2-induced cytotoxicity in PC12 cells was calculated as inhibiting rate (%) = [(A1A2)/(A0A2)] × 100%, where A1 was the absorbance in the presence of sample and H2O2, A2 was the absorbance in the presence of H2O2 (model), A0 was the absorbance without sample and H2O2 (normal).

3. Results and Discussion

3.1. Optimization of Reaction Conditions for the Synthesis of Scutellarein

The optimization of reaction conditions were shown in Table 1. The concentrated sulfuric acid was selected as a catalyst. Firstly, 0.5 g of scutellarin was added to 10 mL of 1 mol/L H2SO4 in water, and the reaction was taken at 90 °C for 6~24 h in N2 atmosphere. However, there was no product; even the concentration of H2SO4 increased from 1 mol/L (Table 1, run 1) to 3 mol/L (Table 1, run 3), due to the poor water solubility of scutellarein. Then, the solvent of water was changed into ethanol, where scutellarein had good solubility. After several attempts, we found that increasing the concentration of ethanol (Table 1, run 46) accelerated its hydrolysis and the yield increased to 2.1% in 90% ethanol (Table 1, run 6). Subsequently, the concentration of H2SO4 was optimized, when the concentration of H2SO4 increased from 1.0 mol/L (Table 1, run 7) to 3.0 mol/L (Table 1, run 9), the yield of scutellarein was improved from 5.3% to 10.0%, and the yield could increase to 12.1% when the reaction time extended to 48 h (Table 1, run 10). Lastly, the exogenous reaction temperature was studied in this reaction. When the reaction temperature was raised from 90 °C (Table 1, Run 10) to 120 °C (Table 1, Run 12), the yield of scutellarein increased from 12.1% to 17.3%. As a result, 3.0 mol/L H2SO4 in 90% ethanol and in N2 atmosphere at 120 °C for 48 h was implemented in the synthesis of scutellarein.

3.2. DPPH, ABTS+•, OH Radical-Scavenging Activity

DPPH, ABTS+• (both stable radicals) and OH (generated by Fenton reaction), are widely used to evaluate the antioxidant capacity of complex mixtures and individual compounds [21]. The in vitro antioxidant activity of scutellarein in comparison with scutellarin was evaluated by DPPH, ABTS+•, OH radical-scavenging activity assays. The IC50 value is defined as the concentration of sample that causes 50% loss of the radical. As illustrated in Figure 2 and Table 2, scutellarein (IC50 16.84 μmol/L for DPPH, 3.00 μmol/L for ABTS+•, 0.31 mmol/L for OH) had stronger radical-scavenging capacities than scutellarin (IC50 17.56 μmol/L for DPPH, 3.53 μmol/L for ABTS+•, 3.19 mmol/L for OH), which indicated that scutellarein is an effective natural antioxidant that might be a promising candidate for ischemic cerebrovascular disease.

3.3. Protective Effect on H2O2-Induced Cytotoxicity in PC12 Cells

PC12 cells can adopt a neuronal phenotype and have been used extensively as a model for catecholamine-secreting neuronal cells [23]. Active mitochondria of living cells can cleave MTT to produce formazan, the amount of which is directly related to the number of living cells. As shown in Table 3, cell viability markedly decreased after PC12 cells were exposed to H2O2. However, when the cells were incubated with scutellarein or scutellarin, H2O2-induced cell toxicity was significantly attenuated, and the protective effect of scutellarein was dose-dependent. However, coincubation of cells showed better protective effect than preincubation for some time against H2O2 cytotoxicity, and the protective effect decreased as preincubation time was prolonged, which may be due to the unstability of scutellarein. Therefore, coincubation was considered to more effectively evaluate cytoprotection of scutellarein; and scutellarein showed a significantly better protective effect than scutellarin. The cellular protective effect of scutellarein might be resulted from its antioxidant action mainly including preventing lipid peroxidation by eliminating radicals [24,25].

4. Conclusion

In summary, an efficient route was reported for the synthesis of scutellarein by hydrolyzing scutellarin. Essential to the synthesis was the implementation of H2SO4 in 90% ethanol in N2 atmosphere. The in vitro antioxidant assays clearly demonstrated that scutellarein had stronger scavenging capacities toward DPPH, ABTS+•, OH free radicals than scutellarin, and had better protective effect on H2O2-induced cytotoxicity in PC12 cells. The results suggested that it would be a promising potent agent for the therapy of ischemic cerebrovascular disease. In addition, these findings concluded the need for further study on the pharmacokinetics and its bioactivities other than antioxidant activities.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 30873235, 81001382), the Program for New Century Excellent Talents by the Ministry of Education (NCET-09-0163), Research Fund for the Doctoral Program of Higher Education of China (20093237120012), 2009’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, National Key Technology R&D Program (2008BAI51B01), and Main Training Fund of Nanjing University of Chinese Medicine (10XPY02). This research was also financially supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010), and Construction Project for Jiangsu Engineering Center of Innovative Drug from Blood-conditioning TCM Formulae.

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Figure 1. Synthesis of scutellarein.
Figure 1. Synthesis of scutellarein.
Ijms 12 08208f1
Figure 2. (A) The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity assay; (B) 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonicacid]- diammonium salt (ABTS+•) radical-scavenging activity assay; (C) hydroxyl radical (OH) radical-scavenging activity assay.
Figure 2. (A) The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity assay; (B) 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonicacid]- diammonium salt (ABTS+•) radical-scavenging activity assay; (C) hydroxyl radical (OH) radical-scavenging activity assay.
Ijms 12 08208f2
Table 1. Optimization of reaction conditions in the synthesis of scutellarein by hydrolyzing scutellarin.
Table 1. Optimization of reaction conditions in the synthesis of scutellarein by hydrolyzing scutellarin.
RunReaction conditionsYield (%)
11.0 mol/L H2SO4 in water, 90 °C, 6~24 hNo product
22.0 mol/L H2SO4 in water, 90 °C, 6~24 hNo product
33.0 mol/L H2SO4 in water, 90 °C, 6~24 hNo product
40.5 mol/L H2SO4 in 70% ethanol, 90 °C, 6~24 hNo product
50.5 mol/L H2SO4 in 80% ethanol, 90 °C, 6~24 hNo product
60.5 mol/L H2SO4 in 90% ethanol, 90 °C, 24 h2.1
71.0 mol/L H2SO4 in 90% ethanol, 90 °C, 24 h5.3
82.0 mol/L H2SO4 in 90% ethanol, 90 °C, 24 h8.5
93.0 mol/L H2SO4 in 90% ethanol, 90 °C, 24 h10.0
103.0 mol/L H2SO4 in 90% ethanol, 90 °C, 48 h12.1
113.0 mol/L H2SO4 in 90% ethanol, 100 °C, 48 h15.2
123.0 mol/L H2SO4 in 90% ethanol, 120 °C, 48 h17.3
Table 2. In vitro antioxidant activity of scutellarein in comparison with scutellarin in DPPH assay (IC50 in μmol/L), ABTS+• assay (IC50 in μmol/L), OH assay (IC50 in mmol/L).
Table 2. In vitro antioxidant activity of scutellarein in comparison with scutellarin in DPPH assay (IC50 in μmol/L), ABTS+• assay (IC50 in μmol/L), OH assay (IC50 in mmol/L).
CompoundsIC50

DPPH (μmol/L)ABTS+• (μmol/L)OH (mmol/L)
Scutellarein16.843.000.31
Scutellarin17.563.533.19
Vitamin C24.8112.561.12
Table 3. Attenuation of H2O2-induced PC12 cell damage by scutellarein (x ± s, n = 5).
Table 3. Attenuation of H2O2-induced PC12 cell damage by scutellarein (x ± s, n = 5).
Drug (μmol/L)CoincubationPreincubation for 30 minPreincubation for 8 h

A517Inhibiting Rate (%)A517Inhibiting Rate (%)A517Inhibiting Rate (%)
Normal0.550 ± 0.0040.589 ± 0.0030.624 ± 0.004
H2O20.353 ± 0.006 ##0.319 ± 0.0123 ##0.375 ± 0.015 ##
Scutellarin/1000.433 ± 0.009 **40.780.440 ± 0.009 **44.810.482 ± 0.002 **42.97
Scutellarein/1000.540 ± 0.038 **94.920.500 ± 0.040 **67.040.458 ± 0.013 **33.33
Scutellarein/100.422 ± 0.007 **35.320.395 ± 0.019 **28.120.414 ± 0.002 **15.96
Scutellarein/10.362 ± 0.0024.570.336 ± 0.007 *6.100.383 ± 0.010
**p < 0.01,
*p < 0.05 vs. H2O2 group;
##p < 0.01 vs. Normal group.

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Qian, L.-H.; Li, N.-G.; Tang, Y.-P.; Zhang, L.; Tang, H.; Wang, Z.-J.; Liu, L.; Song, S.-L.; Guo, J.-M.; Ding, A.-W. Synthesis and Bio-Activity Evaluation of Scutellarein as a Potent Agent for the Therapy of Ischemic Cerebrovascular Disease. Int. J. Mol. Sci. 2011, 12, 8208-8216. https://doi.org/10.3390/ijms12118208

AMA Style

Qian L-H, Li N-G, Tang Y-P, Zhang L, Tang H, Wang Z-J, Liu L, Song S-L, Guo J-M, Ding A-W. Synthesis and Bio-Activity Evaluation of Scutellarein as a Potent Agent for the Therapy of Ischemic Cerebrovascular Disease. International Journal of Molecular Sciences. 2011; 12(11):8208-8216. https://doi.org/10.3390/ijms12118208

Chicago/Turabian Style

Qian, Li-Hua, Nian-Guang Li, Yu-Ping Tang, Li Zhang, Hao Tang, Zhen-Jiang Wang, Li Liu, Shu-Lin Song, Jian-Ming Guo, and An-Wei Ding. 2011. "Synthesis and Bio-Activity Evaluation of Scutellarein as a Potent Agent for the Therapy of Ischemic Cerebrovascular Disease" International Journal of Molecular Sciences 12, no. 11: 8208-8216. https://doi.org/10.3390/ijms12118208

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