Open Access proceedings Journal of Physics: Conference series
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BÀI TẬP THAM KHẢO KIỂM TRA CUỐI HỌC KÌ 1 SINH 8
| 2. Materials and Methods
The research was started by collecting the guava leaves from Conservation and Cultivation Station Unit of Tropical Biopharmaca Research Center at Darmaga Campus, Bogor. The leaves were powdered prior to extraction. The toxicity by BSLT toxicity test method, the total tannin content, total phenolic content, the total flavonoid content, and thin layer chromatography profile of extracts were determined. The kaempferol and quercetin content of the selected extract, then determined by high performance liquid chromatography to select the best extraction method. 2.1. Extractions Guava leaves are divided into two parts; the first part, directly extracted with 4 kinds of techniques, namely maceration (M1), maceration by sonication (SN1), reflux (R1), and Soxhlet (S1). The second part was extracted with n-hexane (Soxhlet method), and the resulting residue is further divided into two parts. The first part, directly extracted with 4 kinds of techniques, namely maceration (M2), maceration by sonication (Sn2), reflux (R2), and Soxhlet (S2). The second part was extracted further with ethyl acetate (Soxhlet method); the residue was then extracted with 4 kinds of techniques, namely maceration (M3), maceration by sonication (SN3), reflux (R3), and Soxhlet (S3). The whole extract is obtained (M1, M2, M3, SN1, Sn2, SN3, R1, R2, R3, S1, S2, and S3) after concentrated by rotary evaporator 2.1.1. Maceration. The extraction process is based on Erosa-Rejon et al [13]. Leaf samples were extracted with ethanol at room temperature for 1 week. Extraction is done 3 times. 2.1.2. Maceration by sonication. The extraction process is based on Tang et al [12]. Leaf samples were extracted with methanol-water (85:15) with the aid of sonication for 3 hours. The extraction was repeated 3 times. 2.1.3. Reflux. The extraction process is based on Zang et al [6]. Leaf samples were added with 70% ethanol and then refluxing at a temperature of 60-70ºC for 3 hours. 2.1.4. Soxhlet. The extraction process is based on Loizzo et al [14]. Leaf samples were added with methanol70% and the Soxhlet process was performed. 2.2. Total Phenolic Content Determination Each about 25 mg diluted with methanol: water (1: 1) into a 25 ml flask. About 0.9 mL of standard solution (gallic acid) and extract was added by 4.5 mL reagent Folin-Ciocalteau, and shaken with a vortex. After 3 minutes, each solution was added to 3.6 mL of Na 2 CO 3 7.5%, shaken and incubated for 1 hour. The absorbance of the standard solution and the sample was measured by UV-Vis 2 ISS IOP Publishing IOP Conf. Series: Earth and Environmental Science 58 (2017) 012060 doi:10.1088/1755-1315/58/1/012060 spectrophotometer at a wavelength of 765 nm. The total phenol content of samples is determined using a regression equation of standard gallic acid. 2.3. Tannin Content Determination Extract of 0.5 g was dissolved in DMSO, added to 10 ml of distilled water and heated at a temperature of 40-60 ° C for 30 minutes. The solution was filtered and diluted with distilled water until 50 mL and 5 mL of the solution was then added by 5 mL indigo carmine. The solution was subsequently titrated with 0.1 N KMnO 4 . 2.4. Total Flavonoid Content Determination The extracts about 200 mg was added with 1 mL hexamethylenetetramine (HMT) 0.5%, 20 mL acetone, and 2 ml of HCl. The mixture was hydrolyzed by refluxed for 30 minutes. The result was filtered and added with acetone to 100 ml. About 20 mL of solution was added to 20 ml of water and 15 ml ethyl acetate. Ethyl acetate fraction was collected in a 50 mL volumetric flask. Extraction was repeated by adding 10 mL ethyl acetate. The total ethyl acetate fraction was added with ethyl acetate until 50 ml. Furthermore, 10 ml of the mixture was added to 1 mL of 2% AlCl 3 and 5% glacial acetic acid in methanol. The mixture was homogenized and allowed to stand 15-30 minutes. The absorbance value at a wavelength of 425 nm was measured with a UV-VIS spectrophotometer 2.5. Brine Shrimp Lethality Test Ten larvae of Artemia salina were added to 1 mL extract with varying concentrations of 200-14000 g/ml. Control without the addition of extract was used as negative control. After 1 day (24 hours) the number of dead shrimp larvae was determined and the LC 50 value was determined by probit analysis. 2.6. Thin Layer Chromatography Profile Each extract was analyzed by Thin Layer Chromatography on silica gel G 60 F 254 and n-hexane:ethyl acetate (2:98) as eluent. The detection was observed in the UV lamp 254 and 366 nm. An extract containing a fluorescent and Rf value the same with kaempferol and quercetin standards will be analyzed further. 2.7. Kaempferol and Quercetin Content Determination About 30 mg of each extract was added to 2 ml of HCl 4 M, shaken and heated for 30 minutes and extracted by 2 mL ethyl acetate. Ethyl acetate fractions were separated and rinsed with 1 mL ethyl acetate 2 times. The collected ethyl acetate fraction evaporated and diluted with methanol till 10 ml. Standard solutions and the sample were then filtered with 0:45 micro μm membrane and 20 l was injected in high performance liquid chromatography (HPLC) for analysis. HPLC Conditions used is C18 column, UV detector at λ 370 nm, oven temperature 30 ° C, mobile phase acetonitrile 30% in KH 2 PO 4 0.025 M buffer pH 2.5 with isocratic elution, and flow rate of 1 ml / min 14 . The kaempferol and quercetin content were determined by comparing the peak area with the peak area of standards. 2.8. Statistical analysis All data obtained were analyzed using the analysis of variance at 95% confidence level (p<0.05) using the Duncan’s test with SPSS 16. tải về 0.65 Mb. Chia sẻ với bạn bè của bạn:
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