Leucoanthocyanidin Test Leucoanthocyanidin and catechin Statistical Analysis

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2. Pew Test and Base Reaction Flavonoids

Flavonoids compounds were determined by two different methods, as follows: Pew Test One milliliter of extract was transferred to a test tube. Then, 0.5 g of Zn powder and 2 drops of 2 N HCl were added in the tube and the mixture was vortexed for 1 min. After that, 10 drops of 37 HCl were added in the mixture. The dark red color solution indicates the presence of flavanonol, and flavonol -3- glycoside, while the light color of solution indicates flavanone, and flavonol. Base Reaction One milliliter of extract was transferred to a test tube. Then, 5 drops of 9.5-10.5 ammonia solutions were added in the mixture. The yellow, orange-red, red-brown, or orange- brown color solutions indicated the presences of flavones, flavanol, and xanthone, flavanone, chalcone, ourone and flavanonol.

3. pH Test Anthocyanins

Briefly, one milliliter of the extract was transferred in a test tube then a drop of 2N HCl was added in the mixture. After that, 5 drops of 9.5-10.5 ammonia solutions were added in the mixture. The red color solution appeared after HCl addition and changed into blue color after ammonia addition indicated the presence of anthocyanin.

4. Leucoanthocyanidin Test Leucoanthocyanidin and catechin

One milliliter of the extract was transferred in the tube then 2 mL of HCl were added in the mixture. After that, the tube was heated in the water bath 50 o C. The red color solution and the yellow-brown color solution indicate the presence of leucoanthocyanidin and catechin, respectively.

5. Gelatin Test Tannin

Briefly, one milliliter of extract was transferred in the tube then 10 drops of 0.5-1 gelatin solution were added in tubes and adjusted the pH until 4.0 or added with NaCl for showing clear solution. Precipitation in the bottom of the tube indicates the presence of tannin.

1.2. Lignin Content Determination Aldaeus, 2010

The CGR 1 g was transferred into the beaker with volume at least 150 mL. In hydrolysis process, 5 mL of 72 H 2 SO 4 was added into beaker and stirred with glass rod until the sample begins to be dissolved. Then, the beaker was placed in the water bath at 30 ± 0.5 o C for 1 hr and stirred occasionally. After that, 85 mL of distilled water was added and beaker was closed by aluminum foil and heated in an autoclave at 120 ± 5 o C for 1 hr. After that, the solution was cooled down to 80 o C. Lignin content was determined by total amount of acid-insoluble residue AIR and acid- soluble lignin ASL in the sample. In determining AIR, sample which has been cooled down to 80 o C was filtered with glass filter. Then, the filtrate was transferred into beaker for ASL content determination. The residue on the glass filter was washed with hot water until the acidity of sample became neutral. Then, the glass filter containing the residue was dried in an oven at 105 o C overnight. After that, it was cooled down in a desiccator and weighted. To determine ASL, first, the absorbance of the obtained filtrate was determined at 205 nm. The filtrates must be diluted in order to obtain the absorbance at the range between 0.2-0.7. Then, the AIR, ASL, and total lignin content of the sample were calculated using the following equations; Acid Insoluble Residue AIR where: m = the dried residue weight g M = the sample dried matter g 11 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

First, the caffeine stock solution 1000 ppm was prepared by dissolving dried-caffeine 198.2 mg with dichloromethane in 200 mL volumetric flasks. Then, the working standards 5-50 ppm were prepared by diluting caffeine stock solution with dichloromethane in 50 mL volumetric flask. The absorbance was measured with spectrophotometer at 260 nm and dichloromethane was used as the blank. For caffeine extraction, 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 extract which dissolved in dichloromethane was measured at 260nm.

3.4. Anthocyanin Lee et al., 2005

Determination of anthocyanin in CGR was determined by pH differential method. Diluted extract 0.4mL was transffered into two 10mL volumetric flasks. The first volumetric flask was adjusted the volume with buffer pH 1.0 1.49 g of KCl in 100 ml of water and 0.2 M HCl, with a ratio of 25:67 and mixed. Then, the second volumetric flask was adjusted the volume with buffer pH 4.5 1.64 g of sodium acetate in 100 ml of water and mixed. The mixtures were measured the absorbance at 510 nm and 700 nm. The concentration of total anthocyanin was calculated using the following equation: Where: A = absorbance; A 510 nm - A 700 nm pH 1.0 - A 510 nm - A700 nm pH 4.5 MW = molecular weight of cyanidin-3-glucoside 433.2 DF = dilution factor e = extinction coefficient for cyanidin 3-glucoside of 31,600 L = cell path length 1 cm

4. Antioxidant Activity

The antioxidant activity was determined by using three extracts with the highest amounts of bioactive compounds. The activity was determined with two different methods, as follows:

4.1. FRAP Ferric Reducing Antioxidant Power Assay Molyneux, 2004

The ascorbic acid stock solution 10,000 µM was prepared by dissolving ascorbic acid 0.0176 g with distilled water in 10 mL volumetric flask. Then, the working standard 200-1000 µM was prepared by diluting ascorbic acid stock solution with distilled water in 10mL volumetric flasks. One milliliter of standard and diluted extract was transferred into tube. Then, 2.5 mL of 1 K 3 FeCN 6 was added into tube and the tube was placed in the water bath 50 o C for 30 min. After that, 2.5 mL of 10 trichloroacetic acid, 2.5 mL of distilled water and 0.5 mL of 1 ferric chloride were added into the tube, respectively. The mixture was measured the absorbance at 700 nm and distilled water was used as the blank. The antioxidant capacity of coffee ground residue extracts were expressed in mg ascorbic acid in 100 g dry basis. 13

4.2. DPPH 2,2-diphenyl-1-picrylhydrazyl Assay Molyneux, 2004

The trolox stock solution 10,000 µM was prepared by dissolving trolox 0.0250 g with methanol in 10 mL volumetric flasks. Then, the working standards 200-1000 µM were prepared by diluted trolox stock solution with methanol in 10 mL volumetric flasks. The DPPH solutions were prepared by dissolving DPPH 0.0024 g with methanol in 100 mL volumetric flask. Standard or diluted extracts 50 µL were transffered into the tube and followed by DPPH solutions 1950 µL. The mixture was kept in the room temperature for 30 min before analysis. The absorbance of solution was measured at 517 nm. Every step in DPPH assay must be avoided from the light. The antioxidant capacity of coffee ground residue extracts were expressed in mg trolox in 100 g dry basis.

5. Antimicrobial Activity

Three CGR extracts with highest bioactive compounds were measured the antimicrobial activity against several bacteria culture such as Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus as Gram-positive bacteria; Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa as Gram-negative bacteria. There were several preparations before starting antimicrobial activity as follows: a. Antibiotic Preparation Ampicillin and polymycin B sulphate were used in this work as positive control in agar well diffusion method. Ampicillin was used for all bacteria except P. aeruginosa, while Polymycin B sulphate was used for P. aeruginosa. The ampicillin stock solution 100mgmL was prepared by dissolving 1000 mg ampicillin powder with 10 mL sterile distilled water. Furthermore, the polymycin B sulphate solution 10mgmL was prepared by dissolving 100 mg polymycin B sulphate powder with 10mL sterile distilled water. The antibiotic solution was filtered into filter syringe before put in sterile sentrifuge tubes. The ampicillin stock solution 40 µL was diluted with distilled water in 10mL sterile volumetric flask to get 20µg50µL concentration of amphicilin. Then, the polymycin B sulphate stock solution 300µL was diluted with distilled water in 10mL sterile volumetric flask to get 300 unit50µL concentration of polymycin B sulphate. b. Mc Farland Preparation 0.5 Mc Farland solution was made by mixing 0.5 mL 1.175 wv BaCl 2 and 99.5 mL 1 vv H 2 SO 4 . The solution was checked the absorbance at 625 nm. The absorbance of 0.5 Mc Farland solutions must be between 0.08-0.13. Mc Farland solutions were kept in the room temperature at dark condition until used. c. Cultures Preparation The culture was activated in Nutrient Broth NB and incubated at 37 o C for 18-24 h. Then, the activated culture was subcultured before using in the antimicrobial assay. Muller Hinton Agar MHA was poured into petri dish and the culture was scratched on the agar. The petri dish was incubated at 37 o C for 18-24 h. After preparation, antimicrobial activity of CGR extracts was determined with two different methods as follows:

5.1. Agar-Well Diffusion Assay Lalitha, 2004

Agar well diffusion assay measured the effect of an antimicrobial agent against bacteria. The diameter of inhibition zone was measured by seeing the ability of the samples to inhibit the growth of the bacteria. The microorganism was cultured in Muller Hinton Broth MHB. Then, the turbidity of the culture was compared with 0.5 Mc Farland in black-white paper. If the cultures and Mc Farland had the same turbidity, the culture could be used in the analysis. The sample was prepared with different concentration 0.75 gmL, 1 gmL and 1.5 gmL. Before put into sterile vials, the sample was filtered into filter syringes. In agar well diffusion method, MHA was poured into petri disc. After 5-10 min, the fresh culture was swabed in the agar with cotton stick and kept until dry for 5 minutes. The well was made by using cork borer. After that, 50 µL of extract with different concentration, positive control, and negative control sterile water were added into the wells. The dish was placed in the 14 laminar for 2 h until the solution was absorbed into the agar. Then, the dish was incubated at 37oC for 18-24 h. Antimicrobial activity of the extract was determined by measuring clear zone diameter around the well. 5.2.Micro-Broth Dilution Assay Lalitha, 2004 Micro-broth dilution assay 96-wells plate method in the CGR is determined antimicrobial activity by measuring minimum inhibition concentration MIC and minimum bactericidal concentration MBC of the extracts. Extract was prepared in several concentrations. The initial concentration of each extract was 1500 mgmL. Further 1:2 serial dilution was performed by addition of culture broth to reach concentrations ranging from 500 to 0.98 mgmL. Extract 100 µL was transffered in 96-wells plates and 100 µL of bacterial culture in MHB was added in each well 2nd-11st well. Sterile control 200µL MHB and growth control 100 µL MHB and 100 µL bacterial cultures were also added in the well 1st well and 12th well, respectively. Positive control 0.01 BHT was also added in a well by added 100 µL BHT and 100 µL MHB. Then, 96-wells plates were incubated at 37oC for 18-24 h. The MIC value was evaluated after incubation, as the lowest concentration that completely inhibited the formation of visible growth have a clear turbidity compared with sterile controls and growth controls. The MBC determination was used to assess if the inhibitory effect observed in MIC determinations was through a lethal bactericidal action. Extract from well where the MIC results showed no bacterial growth was removed with a loop, inoculated into Muller-Hinton Agar MHA plate, and incubated at 37 o C for 18-24 h. MBC was considered to be the concentration at which microorganisms were totally unable to grow.

6. Statistical Analysis

Data analysis performed with the application of SPSS 16.0 software. Choosing three the best extracts from bioactive compounds results were determined by ANOVA Analysis of variance and comparing with Duncan’s Multiple Range Test. The other means were determined by ANOVA and comparing with Duncan’s Multiple Range Test. 15

IV. RESULTS AND DISCUSSIONS

A. PHYTOCHEMICAL SCREENING AND LIGNIN CONTENT OF CGR

SAMPLES Coffee ground residue Coffea arabica that were collected from 10 coffee shops in Chiang Rai Province, Thailand were labeled as A, B, C, D, E, F, G, H, I and J as shown in Appendix 1. The coffee shops were chose by several criterias such as scale of production, location and coffee bean distributor. The preliminary research included the phytochemical screening and determination of lignin content in dried-coffee ground residue less than 13 of moisture content Figure 5 Figure 5 Dried-CGR

1. Phytochemical screening

Phytochemical screening normally is used as preliminary research to know the chemical constituent contained in plant material. This screening could investigate qualitatively presence of some phythochemicals Kantamreddi et al., 2010. The presence of phytochemicals was shown by changing of color, turbidity, pH or other physical properties when small portions of extract was dissolved with tested chemical or reagents. Phytochemical screening in CGR revealed the presence of phenolic compounds, gallic acid, and chlorogenic acid in all samples and negative for the presence of anthocyanin, leucoanthocyanidin, flavonoids, tannin, and catechin Table 2. This result showed that CGR still had a large amount of those compounds. The other compounds that showed negative results in the test might be contained at the small amount in the dried-CGR. Therefore, the amount of phenolic compounds and chlorogenic acid were determined in the CGR extracts in the next part of this work. The amount of caffeine and anthocyanin were also determined.

2. Determination of Lignin Content

Lignin is complex non-soluble phenolic polymers and integral part of secondary cell walls of plants Poeteau et al., 2003. Lignin acts naturally as antioxidant against chemical, biological and mechanical stress in plants Kosikova, 2009. Total lignin in dried-CGR samples were shown in Figure 6. The result showed that total lignin content of dried-CGR ranged between 395 – 444 mgg. The result also showed that sample B had the highest lignin content 444.25 mgg. This result was slightly higher than that those reported by Caetano et al. 2012, which was 336 mgg. Lignin contained in a minor amount in coffee seeds than the other parts of coffee berry Murthy, 2011.