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Acid-soluble lignin ASL
where: A = absorption at 205 nm D = dilution factor
V = volume of the filtrate L A = extinction coefficient of lignin 110 gl cm
b = cuvette path length cm
Total Lignin Content Total lignin content = AIR + ASL
2. CGR Extraction
CGR was extracted with two methods as follows:
2.1. Hot Water Extraction
Hot water extraction was done in order to be the control treatment that could be compared with microwave assisted extraction. Briefly, 200 mL of distilled water 90
o
C was mixed with 20 g of the CGR. The temperature of the mixture was maintained at 90
o
C for 5 min with occasionally stirred. The mixture was then filtered through Whatman filter paper No.1 with vacuum pump
suction. Then, the pH of the filtrate was determined. Then, the filtrate was evaporated using a evaporator 50
o
C, 120 rpm to obtain the crude extract. The crude extract was transferred to the vial and closed with perforated-paraffin paper and kept in a desiccator until the weight was stable.
The vials was tightly closed with aluminum foil and kept in a refrigerator 4
o
C until used for analysis.
2.2 Microwave Assisted Extraction Upadhyay, 2011
The CGR was transferred into a beaker. Then, distilled water 200mL was added into the beaker and the mixture was homogenized for 2 min and pH of mixture was determined. After that,
the beaker was placed in a microwave with the power of 800 watt for various extraction time 3, 4 and 5 min. The mixture was then filtered through Whatman filter paper No.1 with vacuum pump
suction. Then, the filtrate was evaporated using a evaporator 50
o
C, 120 rpm to obtain the crude extract. The crude extract was transferred to the vial and closed with perforated-paraffin paper and
kept in a desiccator until the weight was stable. The vials was tightly closed with aluminum foil and kept in a refrigerator 4
o
C until used for analysis.
3. Bioactive Compound Determination
The bioactive compounds of the CGR extract were determined quantitatively, as follows:
3.1. Phenolic Compounds Singhelton and Rossi, 1965
The total phenolic content was determined by Folin-Ciocalceteu method. Gallic acid was used as standard. Gallic acid stock solutions 1000 µgmL were prepared by dissolving 10 mg of
dry gallic acid in 10 mL distilled water. Working standards 10-100 µgmL were prepared by diluting the stock solution with distilled water in 10mL volumetric flasks.
Diluted extract 0.5mL and standard 0.5mL were transferred in the tube. Then, 2.5 mL of 10 vv Folin and 2 mL of 7.5 wv Na
2
CO
3
were added in the mixture. The mixture was kept for an hour in the room temperature. After that, the absorbance was measured at 765nm.
Distilled water was needed as the blank. The standard calibration curves of gallic acid 10-100 µgmL were plotted and the total phenolic content was expressed as gallic acid equivalent
µgmL.
3.2. Chlorogenic Acid Belay and Gholap, 2009
The chlorogenic acid in the extract was determined by uv-spectrophotometer method. First, chlorogenic acid stock solution 80µgmL was prepared by dissolving dried-chlorogenic
12
acids 40 mg with distilled water in 500mL volumetric flask. Working standards 4-16 µgmL were prepared by diluting chlorogenic acid stock solutions with distilled water in 100 mL
volumetric flask. After that, the mixture was measured the absorbance at 324nm and distilled water was used as the blank.
In determination of chlorogenic acid in the extract, the diluted extract was mixed with dichloromethane at ratio 25:25 and the mixture was stirred for 10 minutes. After that, the mixture
was separated using separatory funnel. The caffeine extraction process was repeated 4 times by adding 25 mL dichloromethane in the mixture. The caffeine which dissolved in dichloromethane
was kept for caffeine analysis and the chlorogenic acid which dissolved in distilled water was measured the absorbance at 324nm. Every step in this procedure must be avoided from light.
3.3. Caffeine Belay et al., 2007
