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Journal of Medicinal Plants Research Vol. 6(12), pp. 2402-2409, 30 March, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1328 ISSN 1996-0875 ©2012 <strong>Academic</strong> <strong>Journals</strong> Full Length Research Paper Determination of scavenging effect of Chinese medicinal herbs on hydroxyl radical using a new chemiluminescence system Cai Zhuo*, Qiu Xia-lin, Liang Xin-yuan, Mo Li-shu and Jiang Cai-ying College of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, P. R. China. Accepted 17 February, 2012 A novel flow injection chemiluminescence (FI-CL) method was developed for determination of scavenging effect of Chinese medicinal herbs on hydroxyl radical (·OH). It was shown that a strong chemiluminescence (CL) signal was observed when methylene bule (MB) was mixed with Fenton reagent in an acidic medium. The CL intensity was decreased significantly when the extract of Chinese medicinal herb was added to the reaction system and partially scavenged the hydroxyl radicals in the solution. The extent of decrease in the CL intensity had a good stoichiometrical relationship with herb concentration. Based on this, we developed a new method for the determination of scavenging effect of Chinese medicinal herbs on hydroxyl radical using a flow injection chemiluminescence (FI-CL) technique. The proposed method was successfully applied to the evaluations of hydroxyl radical scavenging capacity of 18 kinds of Chinese medicinal herbs. The results showed that Rhus chinensis Mill has the highest antioxidant activity. Key words: Flow injection, chemiluminescence, hydroxyl radical, Chinese medicinal herbs. INTRODUCTION Hydroxyl radical (•OH) is by far the most damaging free radical; its action can have devastating effects within the body. Ageing, cancer, radiation damage and phagocytosis are associated with it (Cheng et al., 2002; Knight, 1995). Therefore, the investigation of natural antioxidants and their capacity of scavenging hydroxyl radicals have always attracted attentions. Many Chinese medicinal herbs contain chemical active composition such as polyphenols, flavonoids, saponins and polysaccharides (Chen et al., 2006), therefore, they are the potential resource of natural antioxidants which have many significant advantages over the synthetic antioxidants. It had been found that the effectiveness of Chinese herbal medicines is closely associated with their antioxidant capacity (Pan et al., 2004; Yang et al., 2006). In fact, some anti-free radical active substances have been screened out from the Chinese medicinal herbs (Cai et al., 2004). Thus, it is important for medicine and *Corresponding author. E-mail: zcai01@yahoo.com food industries to develop a simple and sensitive method for quantitative evaluation of hydroxyl radical scavenging ability of plants. Several analytical methods such as electron spin trap (Espinoza et al., 2009), highperformance liquid chromatography (HPLC) (Pezo et al., 2008), capillary electrophoresis (CE) (Kati et al., 2009), ultraviolet spectrophotometry (UV) (Chen et al., 2008), spectrophotometry (Yang et al., 2006) and fluorescence (Qu et al., 2004) have been utilized for this purpose. However, both HPLC, CE and fluorescence methods involve laborious processes or time-consuming procedures; while the UV and spectrophotometry methods suffer from lack of high sensitivity. The chemiluminescence (CL) method was widely employed in the analysis of pharmaceutical compounds due to its simplicity, ease of manipulation, and high sensitivity (Kricka, 2003); it therefore could be an effective tool in determination of the scavenging effect of Chinese medicinal herbs on hydroxyl radical. Methylene blue, a flourescent dye usually adopted in spectroscopic analysis (Sheng et al., 2008; Zhang et al., 2005) was chosen as the chemiluminescent reagent in
our new CL system. Fenton reaction is a typical reaction of generating hydroxyl radical and often used to evaluate the hydroxyl radical-scavenging capacity of plant active constituents (Julio et al., 2009; Ivan et al., 2009). It was observed that a strong CL emission signal occurred when methylene blue reacted with the hydroxyl radicals generated from Fenton reagent in an acidic medium. The water extract of Chinese medicinal herb was found to be able to inhibit the CL intensity by partially scavenging the hydroxyl radicals in the solution. The extent of CL intensity reduction exhibited a good stoichiometric relationship with the herbs concentration. Based on this, we established a new flow injection chemiluminescence (FI-CL) method for the evaluation of hydroxyl radical-scavenging capacity of Chinese medicinal herbs. EXPERIMENTAL Reagents and solutions All the chemicals used were of analytical grade and deionized water was used throughout the experiments. A stock solution of 2.0 × 10 -2 mol/L methylene blue (Shenyang Reagent Plant) was prepared by dissolving 0.7478 g methylene blue in 5 ml of deionized water and diluting to 100 ml. Hydrogen peroxide (Guang Zhou Xinjian Fine Chemical Plant) was stored in a fridge at 4°C, and diluted to a suitable concentration just before usage. A stock solution of 1.5 × 10 -3 mol/L ferrous iron (Fe 2+ ) was prepared daily by dissolving 0.1471 g ferrous ammonium sulfate 6-Hydrate (Guangdong Shantou Xilong Chemical Co., Ltd.) in 50 ml of sulfuric acid solution (0.1 mol/L) and diluting to 250 ml with deionized water. The sulfuric acid stock solution (0.1 mol/L) was prepared by diluting 2.72 ml of 98% concentrated sulfuric acid (Shanghai Shiyi Chemicals Reagent Co.,Ltd.) to 500 ml with deionized water. Sample preparation 18 commonly used Chinese medicinal herbs, with major active components varying from phenolic acids to fatty acids, were carefully selected and purchased from local traditional Chinese medicine shop for this study. The scientific names, family, used parts, and representative components of the herbs are listed in Table 1. Considering that Chinese medicinal herbs are traditionally boiled in water to produce a soup for patients to drink, therefore herb samples were prepared using boiling water extraction method. 1.0 g smashed plant was soaked with 80 ml of deionized water in a conical flask for 20 min and then extracted at 100°C for 40 min, the extract was filtered and the residue was washed with 5 ml hot water twice; the washings and the filtrate were mixed together and diluted to 100 ml with deionized water. Apparatus The CL measurements were performed using a FI-CL analyzer (IFFL-E, Xi’an Remex Analysis Instrument Co., Ltd. China). This analyzer consisted of two peristaltic pumps and a six-way injection valve equipped with a 75 μl sample loop (Figure 1). A PTFE tubing (0.8 mm i.d.) was used to connect all components in the flow system. The flow-cell was a coil of glass tubing (1 mm i.d., total length 100 mm), positioned in front of the detection window of a Zhuo et al. 2403 sensitive photomultiplier tube (PMT) which was operated at a negative high pressure of 750 V. The CL signal collected by the PMT was recorded with a computer employing CL analysis software. All ultrasonic oscillations of reagents were performed using an ultrasonic cleaner (KQ-2200E, Kunshan Ultrasonic Instrument Co., Ltd.). Procedure As shown in the schematic diagram of experimental manifold (Figure 1), the solutions of Fe 2+ and sample (or deionized water) were mixed by a 3-way mixing valve and propelled by the peristaltic pump (P1) into the flow-cell as carrier stream at the flow rate of 4.7 ml/min; H2O2 and methylene blue solutions were merged together by another 3-way mixing valve and propelled by the peristaltic pump (P2) at the flow rate of 7.0 ml/min through a 10 cm tubing into a sixway valve which was then injected 75 μl of the mixed solution into the carrier stream. The light output from the flow-cell was detected by a photomultiplier tube (PMT). The scavenging rate of a Chinese medicinal herb was calculated based on the decrement of the CL emission intensity (ΔI) according to the following equation: Scavenging rate (s) = [(I0-Is)/ I0]×100% (1) Here, I0 and Is are the CL emission intensities in the absence and presence of Chinese medicinal herb, respectively. RESULTS AND DISCUSSION Kinetic curve of CL reaction The kinetic characteristic curve of the reactive system is shown in Figure 2. The CL signal generated from the mixed solution of methylene blue and Fenton reagent in an acidic medium reached its maximum intensity at 3.5 s and then extinguished immediately within 3 s thereafter (Figure 2a), indicating that the luminescence reaction was a rapid reaction. In the presence of Rubia cordifolia L. extract, a relatively weaker CL signal with the same CL emission time and similar peak shape was obtained (Figure 2b). Selection of the experimental manifold and apparatus parameter Various types of the flow injection (FI) manifolds were investigated; the results showed that the maximal CL emission signal was obtained when using the manifold depicted in Figure 1; therefore, this manifold was employed in this study. The flow rate is important to FI-CL detection. If the flow rate is too high or too low, a suitable CL emission signal can not be obtained. The effects of flow rate on the CL intensity were examined in the range of 3.5 to 7.5 ml/min. It was shown that the CL intensity was kept at a constant with the increase in the flow rate of the carrier stream delivered by P1, while it was significantly enhanced with
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Journal of Medicinal Plants Researc
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Editors Prof. Akah Peter Achunike E
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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Figure 1. Chuanxiong Rhizoma (http:
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Table 1. Identification of main pea
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Table 3. Pharmacokinetic parameters
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active component study, many invest
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and 1185 kg ha -1 in the first site
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Table 1. Recommendation for use fer
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Sadanandan AK, Hamza S (1996c). Stu
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and it has been traced to South Ame
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Table 3. Results of phytochemical a
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(a) (b) (c) Figure 1. (a) Normal ce
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emedies in the south of Brazil. J.
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known fatty acids and terpenoids we
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Table 1. Renal function tests of ra
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Al-Radahe et al. 2271 Figure 3.nnnn
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namely disruption of the vascular e
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Zayachkivska OS, Konturek SJ, Drozd
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β-carotene were purchased from Sig
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Figure 1. Effect of ethanol concent
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Table 3. Climatic and soil factors
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content of polysaccharides. Phospha
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Serge et al. 2285 Table 1. Relative
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Percentage inhibition % of inhibiti
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Table 1. Result of phytochemical sc
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Table 3. Result of thin layer chrom
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Table 2. MIC of a compound 1 from c
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Zirihi et al. 2301 Figure 2. Termin
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Figure 5. T. catappa Linn. (Combret
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Figure 8. A. fumigatus: Aspect of c
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Figure 12. Sensitivity of A. fumiga
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Noormi et al. 2311 Table 1. Callus
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Table 2. Contd. NoC=No callus. 4.0
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Figure 2. Contd. at 2.0 mg/L yellow
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Table 1. Biochemical parameters in
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Figure 3. Bcl-xL reactions in inter
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ACKNOWLEDGMENTS The authors would l
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Figure 1. The first page of the boo
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Figure 3. The most widely included
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Figure 4. Lithographic print “St.
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Table 1. List of plants included in
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Table 1. Contd. Nedelcheva 2333 Cin
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Table 1. Contd. Nedelcheva 2335 Rhe
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14 7 1 15 1 35 17 16 Imported plant
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Academy of Science Publishing House
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et al., 1996; van Wyk and Gericke,
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DPPH inhibition (%) DPPH % inhibiti
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% Mortality Concentration (µg/ml)
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information on plants used for the
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Table 1. Levels of independent vari
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2466 J. Med. Plants Res. AOAC (2000
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2486 J. Med. Plants Res. components
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2500 J. Med. Plants Res. Flowering
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2524 J. Med. Plants Res. ACKNOWLEDG
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UPCOMING CONFERENCES 15th European
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