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2374 J. Med. Plants Res. Figure 1. Standard curve of rutin. The absorbance versus concentration is described by the equation: y = 0.0091 xs+0.0018 (R 2 = 0.9996), where, y is the absorbance and x is the content. (Shanghai, China). The AL204 electronic balance was from Mettler- Toledo Instruments Co., Ltd (Switzerland). Preparation of the rutin standard sample The present research used rutin as the contrast sample to determine total flavonoids. About 0.0100 g of rutin was accurately weighed and dissolved by appropriate amounts of 60% alcohol. Ethanol (30%) was then added until the solution volume was 10 mL, then the rutin standard sample (1 mg/ml) was obtained (Sun et al., 2004). Fabrication of the standard curve Seven 10 ml test tubes were prepared and numbered 0 to 6, to which 0, 0.1, 0.2, 0.3, 0.4, and 0.5 ml rutin standard samples were added. About 0.5 ml of 5% NaNO2 was added to all seven test tubes, which were shaken for 5 min. About 0.5 ml of 10% Al(NO3)3 was also added, and the tubes were shaken for 5 min. Water and 4 ml of 4% NaOH were then added, and the tubes were shaken for 15 min. Test tubes 0 was the contrast, and the absorbance was determined at 510 nm (Wu and Tan, 2006). The Microsoft Excel software was used to draw the standard curve (Figure 1). Extraction method and determination of total flavonoids from mulberry leaf Each extraction process was performed according to the following procedure. Mulberry leaf powder was accurately weighed, and then reflux-extracted with ethanol at 80°C. To our knowledge, 80°C is the optimal extraction temperature for flavonoids in mulberry (Cai et al., 2003; Gao et al., 2005; Li 2003; Chen and Chen, 2008). The volume of the extracting solution was concentrated to almost half using a rotary evaporator. The absorbance of 1 ml of the concentrated extracting solution was determined according to the standard curve. Calculation of the extraction rate The absorbance and volume of the extracting solution after concentration were determined. The extraction rate of total flavonoids was calculated via the following equation: Where A is the absorbance, V is the volume of the extracting solution after concentration, and M is the weight of the mulberry leaf powder. Precision test The absorbance of the 50 µg/mL rutin standard sample was determined for six times. Stability test for the extracts After extraction, the absorbance of the extracting solution made from 1.00 g of mulberry powder was determined nine times every hour. Reappearance test for the extraction of total flavonoids Six mulberry powder samples (1.00 g each) were prepared. The ,
Table 1. Precision test. extraction was performed at 80°C for 3 h using 80% alcohol and a solid-liquid ratio of 1:40. After the extracting solution was concentrated, the absorbance was determined at 510 nm. The extraction rate was then calculated. Recovery rate test About 6.00 g of mulberry leaf powder was weighed. The extraction conditions were the same as those in the reappearance test. After the extracting solution was concentrated, 10 mL of the extract and 1 mg/mL rutin standard sample were added into each of the 6 test tubes. The recovery rate was then calculated. Single factor experiments of hot reflux extractions with ethanol According to the extracting method, the alcohol concentration, solid-to-liquid ratio, number of extraction, and extracting period were chosen for single factors experiments. About 5.00 g of mulberry leaf powder was weighed before each extraction. Influence of the alcoholic concentration on the extraction Alcoholic concentrations of 40, 50, 60, 70 and 80% were used. The extraction was performed once every 3 h at 80°C using a solidliquid ratio of 1:30. The extraction rate was then calculated. Influence of the solid-liquid ratio on the extraction Solid-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50 were used. The extraction was performed once every 3 h at 80°C using 60% alcohol (v/v). The extraction rate was then calculated. Influence of the number of extraction on the extraction The numbers of extraction used were 1, 2, 3, 4, and 5. The extraction was performed for 3 h at 80°C using 60% alcohol and a solid-liquid ratio of 1:30. The extraction rate was then calculated. Influence of the extracting period on the extraction Extracting periods of 60, 90, 120, 150, and 180 min were used. The extraction was performed at 80°C using 60% alcohol and a solidliquid ratio of 1:30. The extraction rate was then calculated. Orthogonal design of total flavonoid extraction from mulberry leaf The alcohol concentration, solid-liquid ratio, number of extraction, Test No. Absorbance 1 0.455 2 0.453 3 0.450 4 0.455 5 0.454 6 0.452 Nie et al. 2375 and extracting period were chosen as the orthogonal factors. The orthogonal design L9 (3 4 ) was adopted to optimize the extraction conditions (Chen and Jiang, 2008). For each orthogonal test, 5.00 g of mulberry leaf powder was weighed. Determination of the optimal system using HPLC A standard solution of rutin was prepared as follow. About 0.0049 g of rutin was accurately weighed and dissolved in an ethanol solution. Ethanol was added until volume of 25 ml was reached in a 25 ml volumetric flask. About 1, 2, 4, 6, and 8 ml of rutin standard solution were diluted to 10 ml by ethanol. Rutin determination was performed as described above. Using the concentration of the rutin standard solution as the abscissa and the peak area as the ycoordinate, the linear chart was constructed. The regression equation was y = 507.15x+23.364, R 2 = 0.9998. The HPLC analysis was carried out on an Agilent Series 1100 liquid chromatograph. The UV detection system therein was connected to a reversedphase column (Kromasil C18; 5 μm, 250 mm × 4.6 mm). The HPLC mobile phase was a methanol-acetonitrile-1% acetic acid (13:18:69, v/v/v) solution. The absorbance was measured at 360 nm for the rutin detection. The flow rate was 0.5 ml/min, and the column temperature was maintained at 30°C. The sample acquired from the optimal system was injected at a volume of 10 μl after dilution. The peak area was used for quantification. The extraction rate was then measured and compared with the results from the UV spectrophotometer. RESULTS Precision test After the absorbance was determined, the results showed that the relative standard deviation (RSD) was 0.43% (RSD < 2.0%), as shown in Table 1. Stability test for the extracts Table 2 shows that the absorbance of flavonoids showed minor differences within 3 to 8 h. The RSD was 1.3% (RSD < 2.0%). Reappearance test for the extraction of total flavonoids Table 3 compares the results of the six numbers of
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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
Page 97 and 98: ACKNOWLEDGMENTS The authors would l
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Page 101 and 102: Figure 3. The most widely included
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Page 105 and 106: Table 1. List of plants included in
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Page 111 and 112: 14 7 1 15 1 35 17 16 Imported plant
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Page 117 and 118: DPPH inhibition (%) DPPH % inhibiti
Page 119 and 120: % Mortality Concentration (µg/ml)
Page 121 and 122: information on plants used for the
Page 123 and 124: Table 1. Levels of independent vari
Page 125 and 126: Arbutin (mg/g) Co-solvent Figure 1.
Page 129 and 130: Arbutin (mg/g) Extraction pressure
Page 131 and 132: Table 3. Arbuitn content of Asian p
Page 135 and 136: Arbutin (mg/g) Extraction pressure
Page 137 and 138: Arbutin (mg/g) Extraction temperatu
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Page 141 and 142: Table 1. Chemical composition of es
Page 143 and 144: Darjazi 2369 Table 2. Statistical a
Page 145 and 146: Table 3. Correlation matrix (number
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Page 153 and 154: Table 6. Results of ANOVA. Nie et a
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Page 157 and 158: Table 2. Plant data and MIC values
Page 159 and 160: exhibiting the polar extracts of P.
Page 161 and 162: Table 3. Contd. AcOEt ++ ++ ++ - Me
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Page 165 and 166: Warghat et al. 2391 Table 3. Summar
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Page 171 and 172: ice residue is an ideal raw materia
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2436 J. Med. Plants Res. budget dur
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2440 J. Med. Plants Res. Table 1. R
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2442 J. Med. Plants Res. Stability
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2444 J. Med. Plants Res. production
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2446 J. Med. Plants Res. Figure 2.
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2450 J. Med. Plants Res. Table 2. A
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2452 J. Med. Plants Res. Ghasemi Pi
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2456 J. Med. Plants Res. Table 3. M
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2460 J. Med. Plants Res. Department
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2466 J. Med. Plants Res. AOAC (2000
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2470 J. Med. Plants Res. Table 1. F
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2486 J. Med. Plants Res. components
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2490 J. Med. Plants Res. Inhibition
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2494 J. Med. Plants Res. can increa
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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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