Optimization of Solvent Extractions

3553 words (14 pages) Essay

29th Jan 2018 Chemistry Reference this

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CHAPTER 3

METHODOLOGY

3.1 Introduction

In this study, the leaves of A. Malanccensis, formulated gaharu tea bags from Gaharu Tea Valley, Gopeng and Kebun Rimau, Tawau were extracted by using solvent extraction method. The optimization of solvent extractions were determined in this study as well. Chemical composition of the crude extracts were analyzed by using GC-MS and HPLC. Besides, the obtained crude extracts were evaluated for their antioxidant property.

3.2 Chemicals and Apparatus

Throughout this study, several of chemical and apparatus were used as listed in table 3.1 and table 3.2.

Table 3.1 Chemical and reagent used

Chemical and reagents

Brand

Manufacturer

1,1-diphenyl-2-pycrylhydrazyl (DPPH)

Sigma

USA

2,4,6-tripyridyl-striazine

Sigma

USA

Sodium acetate

   

Acetic acid

M&B

Germany

Aluminum chloride hexahydrate

   

Ascorbic acid

Systerm

Malaysia

Ethanol

Systerm

Malaysia

Folin-Ciocalteu’s reagent

Sigma

USA

Gallic acid

   

Hydrochloric acid

   

Iron (II) sulphate

Sigma

USA

Iron (III) Chloride

Systerm

Malaysia

Methanol

Merck

Germany

Potassium bromide

   

Quercetin

   

Sodium carbonate

   

Sodium carbonate anhydrous

   

Sodium hydroxide

Merck

Germany

Sodium nitrate

   

Helium gas

Novaland

Malaysia

Nitrogen gas

Novaland

Malaysia

Table 3.2 Apparatus used

Apparatus

Brand

Manufacturer

Aluminium foil

Diamond brand

Malaysia

Analytical balance

Mettler-Toledo

Switerland

Beaker 100 mL

Schoot Duran

Germany

Beaker 250 mL

Schoot Duran

Germany

Beaker 500 mL

Iwaki

Japan

Conical flask 250 mL

Isolab

Germany

Filter funnel

   

Frontier Transform Infra-red spectrometer

Perkin Elmer

Germany

Gas Chromatography-Mass spectrometer

Perkin-Elmer

Germany

Measuring cylinder

Iwaki

Japan

Micropipette

Eppendrof

England

Oven

Memmet

USA

Parafilm “M”

Pechiney

USA

Rotatory evaporator

Buchi Labortechinc

Switzerland

Round bottle flask 250 mL

Favorit

Malaysia

Test tube 15 mL

   

UV-Vis spectrometer

Perkin Elmer

Germany

Volumetric flask

Exelo

Malaysia

Water bath

Cifton NE 5-289

England

Whatman No. 1 Filter paper

Buchi Reiii

Switzerland

Grinder

Kika

Germany

3.3 Extraction of the Gaharu Leaves and Formulated Gaharu Tea

3.3.1 Raw Material

The samples of gaharu leaves and formulated gaharu tea bag were used in this research were obtained from the Kebun Rimau Sdn Bhd, Tawau and Gaharu Tea Valley, Gopeng. Gaharu leaves were dried in the shade for 7 days at roomtemperature (28 ±2°C). After drying, leaves were cut into small pieces. The pieces of dried gaharu leaves and the formulated gaharu tea bag are used for the aqueous extraction and ethanolic extraction.

3.3.2Aqueous Extraction of Gaharu Leaves and Formulated Gaharu Tea

50g of dried leaves powder and contents of formulated gaharu tea bag were weighed and put into 500mL beaker, respectively. 250 mL of distilled water was added into both beaker and boil for 30 minutes. Beakers were wrapped with aluminium foil (Diamond brand) to prevent spilling of mixture and light exposure.The pooled extracts were filtered by using Whatman No. 1 filterpaper (Buchi Reiii) with 0.45µm membrane. The remains were re-extracted under same condition and filtered. The obtained crude extracts were concentrated at 50°C using a rotary evaporator under low pressure. The concentrated crude extracts from gaharu leaves and formulated gaharu tea bag were collected in air-tight containers and covered by the aluminum foil to prevent the active components in the extracts from decomposing by light. They were stored in refrigerator at 4 °C to prevent fungal attacking. All the extractions were carried out in replicates.

3.3.3 Ethanolic Extraction of the Gaharu Leaves and FormulatedGaharu Tea

10g of dried leaves powder and 6g of the formulated gaharu tea bag were weighed and put into 100 mL beaker, respectively. 50mL ethanol was poured into both beaker. Beakers were sealed with parafilm (Pechiney) and wrapped with aluminum foil (Diamond brand) to prevent spilling of mixture and light exposure, respectively. The pooled extracts were filtered by using Whatman No. 1 filter paper (Whatman International, England). The remains were re-extracted under same condition and filtered. The obtained crude extracts were concentrated at 40°C using a rotary evaporator under low pressure. The concentrated crude extracts from gaharu leaves and formulated gaharu tea bag were collected in air-tight containers and covered by the aluminum foil to prevent the light decomposition of bioactive components in the crude extracts. They were stored in refrigerator at 4 °C to avoid fungal attacking. All the extractions were carried out in replicates.

3.4 Optimization of the Gaharu Leaves and Formulated Gaharu Tea Extraction

3.4.1Ethanol Concentration

With the constants – 180 minutes and temperature 25OC, samples were extracted with six different concentrations of ethanol starting from 0% to 100% (v/v). The extraction procedures were described in section 3.2.3. The best ethanol concentration which related to the values of total phenolic contain (TPC) (mg gallic acid equivalent, GAE/g dry weight, DW) and total flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) were selected.

3.4.2Extraction time

The samples were extracted with the optimal ethanol concentration which was determined in the section 3.3.1 for 60, 120, 180, 240, 300 minutes by fixing the extraction temperature constant at 25oC. The best extraction time which related to the values of total phenolic contain (TPC) (mg gallic acid equivalent, GAE/g dry weight, DW) and total flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) eres selected.

3.4.3Temperature

The samples were extracted at different temperatures, which were 25, 35, 45, 55, and 65oC with the optimal temperature and extraction time as determined in section 3.3.1 and section 3.3.2. The best extraction temperature which related to the values of total phenolic contain (TPC) (mg gallic acid equivalent, GAE/g dry weight, DW) and total flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) weres selected.

3.5Chemical Characterization of Gaharu Leaves and Formulated Gaharu Tea

3.5.1Characterization using FT-IR

3.5.1.1 Sample Preparation

The method of FT-IR analysis by Khalil et al. (2013) was used with minor modifications (Khalil et al., 2013). The formation of transparent pellet (Thin disc) was done by mixing 2mg of crude extract powder with 40mg of potassium bromide (1:20), a mold was used to compress the mixture under a pressure of 7 tons. The analysis was carried out with the wavelength starting from 4000 to 400 cm-1. About 3 minutes were taken by the spectrum recording. FT-IR software Spectrum version 6.35 (Perkin Elmer) was used to perform the acquisition of the spectra and peak assignment.

3.5.2Characterization Using Gas Liquid Chromatography- Mass Spectroscopy (GC-MS)

3.5.2.1 Sample Preparation

The method of GC-MS analysis by Khalil et al. (2013) and Soetardjo et al., (2007) were used with the modification of experimental conditions. The crude extract powder was dissolved in 60% (v/v) methanol solution and filtered by using membrane filter with 0.45 µm pore size. The 0.5 µL of samples were injected into a gas chromatograph equipped with Perkin Elmer Clarus 500 mass spectrometer and Elite-5MS column [30m (length) x 0.25 mm (diameter), film thickness 0.25 µm]. The column temperature was programmed to 50oC for 6 min, with 5oC increase per min to 250oC. The temperature of detector and injector were both maintained at 250 oC. Hellium was used as the carrier gas with a linear velocity of 1 ml/min and the splitting ratio was 10:1. The mass spectrometer was operated in the electron impact ionization (EI) mode at 70 eV. The constituents of gaharu crude extract powder were identified by comparing their mass spectra with those of NIST02 library data of the GC-MS system. The condition of GC-MS are summarized in Table 3.4.

Table 3.4 Condition used for GC-MS analysis.

Parameters

Conditions

Gas chromatography

Perkin Elmer Clarus 500 gas chromatography

Mass selective detector

Perkin Elmer Clarus 500 mass spectrometer

Capillary columns

Elite-5MS (30 m (length) x 0.25 mm (diameter) ; film thickness 0.25 µm)

Injection volume

0.5 µL

Carrier gas

Helium

Initial oven temperature

50 oC

Final oven temperature

260-300 oC

Inlet temperature

250 oC

Injection

Split

Injector pressure

8.00-9.43 psi

Injector temperature

80 oC

Flow rate

1 mL min -1

Electron energy

70 eV

Source temperature

180 oC

Solvent delay

2 minutes

Data library

NIST 02

3.5.2.2 Kovats Retention Index

Kovats retention index is an idea used in the gas chromatography to convert retention times into system-independent constant. The collected data of GC-MS were processed by using the Kovats retention index (Equation 3.1). The calculated Kovats index was compared with the retention time of n-alkane which were given by the GC-MS manufacture company (Perkin Elmer) based on the same type of column with higher ramp temperature (10oC instead of 5 oC, Appendix A).

I = 100 times left [ n + ( N - n ) frac{t_{r (unknown)} - t_{r (n)} }{t_{r (N)} - t_{r (n)} } right ] (Equation 3.1)

Where,

I = Kovats retention index,

n = the number of carbon atoms in the smaller n-alkane,

N = the number of carbon atoms in the larger n-alkane,

tr = the retention time.

3.6Determination of Total Phenolic Compounds and Total Flavonoid Compounds in Crude Extracts

3.6.1Evaluation of Total Phenolic Compounds in Gaharu Leaves and

Formulated Tea Crude Extracts

Total phenolic content (TPC) of concentrated rude extract of three samples with the different extraction condition were determined by using the Folin-Ciocalten (F-C) assay according to the method described by Tay et al. with a modification (Tay et al., 2014). Concentrated crude extractwas diluted 30 times before use. 15 mL test tube was wrapped with aluminum foil and 1 mL of diluted sample was placed into it. Then, 5 mL of Folin-Ciocalten reagent was added into the test tube. After 5 minutes, 4 mL of 7.5% (w/v) sodium carbonate was added. The test tube was shaken to mix the diluted sample and reagents for 5 seconds. Thus, the test tube was allowed to stand in the dark room at room temperature for 30 minutes. The blank solution was prepared by replacing 1 mL of samples with 1 mL of deionized water. Absorbance was measured against the blank at 765 nm by using UV-Vis spectrometer (Perkin Elmer).Besides, 1 mM gallic acid stock solution was prepared. 17.012 mg of gallic acid was weighed ad transferred into 100 mL conical flask. Thus, deionized water was added in until the volume of 100 mL was achieved. The 1 mM gallic acid stock solution was further diluted into 0.1, 0.2, 0.4, 0.6, 0.8 mM gallic acid standard solutions. Total phenolic content was determined and calculated based on gallic acid calibration curve and expressed as gallic acid equivalents (GAEs) in milligram per g dry weight (mg GAE/ g DW). The TPC test was repeated with the concentrated crude extract with different solvent extraction conditions.

3.6.2Evaluation of Total Flavonoid Compounds in Gaharu Leaves and Formulated Tea Crude Extracts

The total flavonoid content (TFC) of the concentrated crude extract of three samples with different extraction conditions were determined according to the procedures described by Thoo et al. with slight modifications (Thoo et al., 2010). Concentrated crude extract was diluted for 15 times. Thus, 1 mL of diluted crude extract was mixed with 5 mL of deionized water and 0.3 mL 5% sodium nitrite in a 15 mL aluminum foil-wrapped test tube. After 12 minutes, 0.6 mL 10% aluminum chloride hexahydrate was added into the test tube. In the next 10 minutes, 2 mL of 1M sodium hydroxide solution and 1.1 mL were added to the mixture. Thus, test tube was shaken to mix the diluted sample with the reagents for 10 seconds. A blank was prepared by replacing diluted crude extract with 1 mL of deionized water. The absorbance was measured immediately at 510 nm by using UV-Vis light spectrometer (Perkin Elmer). Besides, the 1 mM quercetin stock solution was prepared as well. 30.22 mg of quercetin was accurately weighed and transferred into 100 mL conical flask. Thus, the deionized water was added into the conical flask until the volume of 100 mL was reached. The 1 mM quercetin stock solution was further diluted to 0.1, 0.2, 0.4, 0.6, 0.8 mM quercetin standard solutions. The total flavonoid content of diluted crude extract was determined and calculated based on quercetin calibration curve and expressed as quecertin equivalents (QEs) in milligram per g dry weight (mg QE/ g DW).). The TFC test was repeated with the concentrated crude extract with different solvent extraction conditions.

3.7 Determination of Antioxidant Activity

3.7.12,2-diphenyl-1-picrylhydrazyl (DPPH) Free RadicalScavenging Assay

The antioxidant capacity of crude extracts were measured by the DPPH assay based on the method which were carried out by Asadujjamanet al. and Tay et al. with slight modification (Asadujjamanet al., 2013; Tay et al., 2014). The 0.1 mL concentrated crude extract was added with 3.9 mL 0.004% ethanolic DPPH solution in an aluminium-wrapped test tube. A parafilm was immediately used to cover the opening of test tube. Thus, the test tube was allowed to stand in dark room at room temperature for 30 minutes. The absorbance of DPPH solution was determined against a deionized water blank a 517 nm by using the UV-Vis spectrometer (Perkin Elmer Lambda 25). 0.1 mL of the crude extract was replaced by extract solvent in the preparing of negative control. Absorbance measurements of the crude extracts and negative control was carried out in triplicate. The result was expressed as a percentage of DPPH radical scavenging activity.The following formula (Equation 3.2) was used to calculate the DPPH radical scavenging activity of crude extract.

1 − × 100%= DPPH radical scavenging activityEquation 3.2

Where,

As= absorbance of crude extract

Ac = Absorbance of control

3.7.2Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was carried out according to procedures described by Kamonwannasitet al. with slight modification (Kamonwannasitet al., 2013).The FRAP reagent was made of 0.01 M TPTZ (2,4,6-tripyridyl-striazine) in 0.04 M HCl, 0.02 M FeCl3, and 0.03M acetate buffer (pH 3.6) in a ratio of 1:1:10(v/v/v). 0.03M acetate buffer (pH3.6) was prepared by mixing 46.3 mL of 0.2M acetic acid and 3.7 mL 0.2 M sodium acetate 0.5 mL together. Thus, the deionized water was added into mixture to make up the volume to 100mL. Then, 0.5g of the sample was added to 15 ml of FRAP reagent which was prepared in situ and warmed until 37°C before it was being used. The absorbance was measured at 593 nm by using an UV-Vis spectrophotometer (Perkin Elmer) after the solution was incubated for 5 minutes. Besides, 1 mM FeSO4stock solution was prepared as well. 0.278g of FeSO4 were dissolved into 1 L of deionized water. Thus, 1 mM FeSO4stock solution was diluted into 0.1, 0.2, 0.4, 0.6, 0.8 mM FeSO4 standard solution. A standard calibration curve was constructed by using different concentration of FeSO4 solution. The results were expressed as μmol Fe2+/mg dry weight of plant material. All measurements were carried out in triplicate and the mean values were calculated.

3.8 Statistical analysis

The experimental results in this study were calculated and analyzed by using the IBM software (SPSS Statistics version 21). All values were expressed as the mean ± standard deviation (SD) of triplicate measurements of replicate extraction. Simple correlation was used to determine the relationship between total amount of flavonoids in the crude extracts and their antioxidant capacities.

CHAPTER 3

METHODOLOGY

3.1 Introduction

In this study, the leaves of A. Malanccensis, formulated gaharu tea bags from Gaharu Tea Valley, Gopeng and Kebun Rimau, Tawau were extracted by using solvent extraction method. The optimization of solvent extractions were determined in this study as well. Chemical composition of the crude extracts were analyzed by using GC-MS and HPLC. Besides, the obtained crude extracts were evaluated for their antioxidant property.

3.2 Chemicals and Apparatus

Throughout this study, several of chemical and apparatus were used as listed in table 3.1 and table 3.2.

Table 3.1 Chemical and reagent used

Chemical and reagents

Brand

Manufacturer

1,1-diphenyl-2-pycrylhydrazyl (DPPH)

Sigma

USA

2,4,6-tripyridyl-striazine

Sigma

USA

Sodium acetate

   

Acetic acid

M&B

Germany

Aluminum chloride hexahydrate

   

Ascorbic acid

Systerm

Malaysia

Ethanol

Systerm

Malaysia

Folin-Ciocalteu’s reagent

Sigma

USA

Gallic acid

   

Hydrochloric acid

   

Iron (II) sulphate

Sigma

USA

Iron (III) Chloride

Systerm

Malaysia

Methanol

Merck

Germany

Potassium bromide

   

Quercetin

   

Sodium carbonate

   

Sodium carbonate anhydrous

   

Sodium hydroxide

Merck

Germany

Sodium nitrate

   

Helium gas

Novaland

Malaysia

Nitrogen gas

Novaland

Malaysia

Table 3.2 Apparatus used

Apparatus

Brand

Manufacturer

Aluminium foil

Diamond brand

Malaysia

Analytical balance

Mettler-Toledo

Switerland

Beaker 100 mL

Schoot Duran

Germany

Beaker 250 mL

Schoot Duran

Germany

Beaker 500 mL

Iwaki

Japan

Conical flask 250 mL

Isolab

Germany

Filter funnel

   

Frontier Transform Infra-red spectrometer

Perkin Elmer

Germany

Gas Chromatography-Mass spectrometer

Perkin-Elmer

Germany

Measuring cylinder

Iwaki

Japan

Micropipette

Eppendrof

England

Oven

Memmet

USA

Parafilm “M”

Pechiney

USA

Rotatory evaporator

Buchi Labortechinc

Switzerland

Round bottle flask 250 mL

Favorit

Malaysia

Test tube 15 mL

   

UV-Vis spectrometer

Perkin Elmer

Germany

Volumetric flask

Exelo

Malaysia

Water bath

Cifton NE 5-289

England

Whatman No. 1 Filter paper

Buchi Reiii

Switzerland

Grinder

Kika

Germany

3.3 Extraction of the Gaharu Leaves and Formulated Gaharu Tea

3.3.1 Raw Material

The samples of gaharu leaves and formulated gaharu tea bag were used in this research were obtained from the Kebun Rimau Sdn Bhd, Tawau and Gaharu Tea Valley, Gopeng. Gaharu leaves were dried in the shade for 7 days at roomtemperature (28 ±2°C). After drying, leaves were cut into small pieces. The pieces of dried gaharu leaves and the formulated gaharu tea bag are used for the aqueous extraction and ethanolic extraction.

3.3.2Aqueous Extraction of Gaharu Leaves and Formulated Gaharu Tea

50g of dried leaves powder and contents of formulated gaharu tea bag were weighed and put into 500mL beaker, respectively. 250 mL of distilled water was added into both beaker and boil for 30 minutes. Beakers were wrapped with aluminium foil (Diamond brand) to prevent spilling of mixture and light exposure.The pooled extracts were filtered by using Whatman No. 1 filterpaper (Buchi Reiii) with 0.45µm membrane. The remains were re-extracted under same condition and filtered. The obtained crude extracts were concentrated at 50°C using a rotary evaporator under low pressure. The concentrated crude extracts from gaharu leaves and formulated gaharu tea bag were collected in air-tight containers and covered by the aluminum foil to prevent the active components in the extracts from decomposing by light. They were stored in refrigerator at 4 °C to prevent fungal attacking. All the extractions were carried out in replicates.

3.3.3 Ethanolic Extraction of the Gaharu Leaves and FormulatedGaharu Tea

10g of dried leaves powder and 6g of the formulated gaharu tea bag were weighed and put into 100 mL beaker, respectively. 50mL ethanol was poured into both beaker. Beakers were sealed with parafilm (Pechiney) and wrapped with aluminum foil (Diamond brand) to prevent spilling of mixture and light exposure, respectively. The pooled extracts were filtered by using Whatman No. 1 filter paper (Whatman International, England). The remains were re-extracted under same condition and filtered. The obtained crude extracts were concentrated at 40°C using a rotary evaporator under low pressure. The concentrated crude extracts from gaharu leaves and formulated gaharu tea bag were collected in air-tight containers and covered by the aluminum foil to prevent the light decomposition of bioactive components in the crude extracts. They were stored in refrigerator at 4 °C to avoid fungal attacking. All the extractions were carried out in replicates.

3.4 Optimization of the Gaharu Leaves and Formulated Gaharu Tea Extraction

3.4.1Ethanol Concentration

With the constants – 180 minutes and temperature 25OC, samples were extracted with six different concentrations of ethanol starting from 0% to 100% (v/v). The extraction procedures were described in section 3.2.3. The best ethanol concentration which related to the values of total phenolic contain (TPC) (mg gallic acid equivalent, GAE/g dry weight, DW) and total flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) were selected.

3.4.2Extraction time

The samples were extracted with the optimal ethanol concentration which was determined in the section 3.3.1 for 60, 120, 180, 240, 300 minutes by fixing the extraction temperature constant at 25oC. The best extraction time which related to the values of total phenolic contain (TPC) (mg gallic acid equivalent, GAE/g dry weight, DW) and total flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) eres selected.

3.4.3Temperature

The samples were extracted at different temperatures, which were 25, 35, 45, 55, and 65oC with the optimal temperature and extraction time as determined in section 3.3.1 and section 3.3.2. The best extraction temperature which related to the values of total phenolic contain (TPC) (mg gallic acid equivalent, GAE/g dry weight, DW) and total flavonoid contain (TFC) (mg quercetin equivalent, QE/g DW) weres selected.

3.5Chemical Characterization of Gaharu Leaves and Formulated Gaharu Tea

3.5.1Characterization using FT-IR

3.5.1.1 Sample Preparation

The method of FT-IR analysis by Khalil et al. (2013) was used with minor modifications (Khalil et al., 2013). The formation of transparent pellet (Thin disc) was done by mixing 2mg of crude extract powder with 40mg of potassium bromide (1:20), a mold was used to compress the mixture under a pressure of 7 tons. The analysis was carried out with the wavelength starting from 4000 to 400 cm-1. About 3 minutes were taken by the spectrum recording. FT-IR software Spectrum version 6.35 (Perkin Elmer) was used to perform the acquisition of the spectra and peak assignment.

3.5.2Characterization Using Gas Liquid Chromatography- Mass Spectroscopy (GC-MS)

3.5.2.1 Sample Preparation

The method of GC-MS analysis by Khalil et al. (2013) and Soetardjo et al., (2007) were used with the modification of experimental conditions. The crude extract powder was dissolved in 60% (v/v) methanol solution and filtered by using membrane filter with 0.45 µm pore size. The 0.5 µL of samples were injected into a gas chromatograph equipped with Perkin Elmer Clarus 500 mass spectrometer and Elite-5MS column [30m (length) x 0.25 mm (diameter), film thickness 0.25 µm]. The column temperature was programmed to 50oC for 6 min, with 5oC increase per min to 250oC. The temperature of detector and injector were both maintained at 250 oC. Hellium was used as the carrier gas with a linear velocity of 1 ml/min and the splitting ratio was 10:1. The mass spectrometer was operated in the electron impact ionization (EI) mode at 70 eV. The constituents of gaharu crude extract powder were identified by comparing their mass spectra with those of NIST02 library data of the GC-MS system. The condition of GC-MS are summarized in Table 3.4.

Table 3.4 Condition used for GC-MS analysis.

Parameters

Conditions

Gas chromatography

Perkin Elmer Clarus 500 gas chromatography

Mass selective detector

Perkin Elmer Clarus 500 mass spectrometer

Capillary columns

Elite-5MS (30 m (length) x 0.25 mm (diameter) ; film thickness 0.25 µm)

Injection volume

0.5 µL

Carrier gas

Helium

Initial oven temperature

50 oC

Final oven temperature

260-300 oC

Inlet temperature

250 oC

Injection

Split

Injector pressure

8.00-9.43 psi

Injector temperature

80 oC

Flow rate

1 mL min -1

Electron energy

70 eV

Source temperature

180 oC

Solvent delay

2 minutes

Data library

NIST 02

3.5.2.2 Kovats Retention Index

Kovats retention index is an idea used in the gas chromatography to convert retention times into system-independent constant. The collected data of GC-MS were processed by using the Kovats retention index (Equation 3.1). The calculated Kovats index was compared with the retention time of n-alkane which were given by the GC-MS manufacture company (Perkin Elmer) based on the same type of column with higher ramp temperature (10oC instead of 5 oC, Appendix A).

I = 100 times left [ n + ( N - n ) frac{t_{r (unknown)} - t_{r (n)} }{t_{r (N)} - t_{r (n)} } right ] (Equation 3.1)

Where,

I = Kovats retention index,

n = the number of carbon atoms in the smaller n-alkane,

N = the number of carbon atoms in the larger n-alkane,

tr = the retention time.

3.6Determination of Total Phenolic Compounds and Total Flavonoid Compounds in Crude Extracts

3.6.1Evaluation of Total Phenolic Compounds in Gaharu Leaves and

Formulated Tea Crude Extracts

Total phenolic content (TPC) of concentrated rude extract of three samples with the different extraction condition were determined by using the Folin-Ciocalten (F-C) assay according to the method described by Tay et al. with a modification (Tay et al., 2014). Concentrated crude extractwas diluted 30 times before use. 15 mL test tube was wrapped with aluminum foil and 1 mL of diluted sample was placed into it. Then, 5 mL of Folin-Ciocalten reagent was added into the test tube. After 5 minutes, 4 mL of 7.5% (w/v) sodium carbonate was added. The test tube was shaken to mix the diluted sample and reagents for 5 seconds. Thus, the test tube was allowed to stand in the dark room at room temperature for 30 minutes. The blank solution was prepared by replacing 1 mL of samples with 1 mL of deionized water. Absorbance was measured against the blank at 765 nm by using UV-Vis spectrometer (Perkin Elmer).Besides, 1 mM gallic acid stock solution was prepared. 17.012 mg of gallic acid was weighed ad transferred into 100 mL conical flask. Thus, deionized water was added in until the volume of 100 mL was achieved. The 1 mM gallic acid stock solution was further diluted into 0.1, 0.2, 0.4, 0.6, 0.8 mM gallic acid standard solutions. Total phenolic content was determined and calculated based on gallic acid calibration curve and expressed as gallic acid equivalents (GAEs) in milligram per g dry weight (mg GAE/ g DW). The TPC test was repeated with the concentrated crude extract with different solvent extraction conditions.

3.6.2Evaluation of Total Flavonoid Compounds in Gaharu Leaves and Formulated Tea Crude Extracts

The total flavonoid content (TFC) of the concentrated crude extract of three samples with different extraction conditions were determined according to the procedures described by Thoo et al. with slight modifications (Thoo et al., 2010). Concentrated crude extract was diluted for 15 times. Thus, 1 mL of diluted crude extract was mixed with 5 mL of deionized water and 0.3 mL 5% sodium nitrite in a 15 mL aluminum foil-wrapped test tube. After 12 minutes, 0.6 mL 10% aluminum chloride hexahydrate was added into the test tube. In the next 10 minutes, 2 mL of 1M sodium hydroxide solution and 1.1 mL were added to the mixture. Thus, test tube was shaken to mix the diluted sample with the reagents for 10 seconds. A blank was prepared by replacing diluted crude extract with 1 mL of deionized water. The absorbance was measured immediately at 510 nm by using UV-Vis light spectrometer (Perkin Elmer). Besides, the 1 mM quercetin stock solution was prepared as well. 30.22 mg of quercetin was accurately weighed and transferred into 100 mL conical flask. Thus, the deionized water was added into the conical flask until the volume of 100 mL was reached. The 1 mM quercetin stock solution was further diluted to 0.1, 0.2, 0.4, 0.6, 0.8 mM quercetin standard solutions. The total flavonoid content of diluted crude extract was determined and calculated based on quercetin calibration curve and expressed as quecertin equivalents (QEs) in milligram per g dry weight (mg QE/ g DW).). The TFC test was repeated with the concentrated crude extract with different solvent extraction conditions.

3.7 Determination of Antioxidant Activity

3.7.12,2-diphenyl-1-picrylhydrazyl (DPPH) Free RadicalScavenging Assay

The antioxidant capacity of crude extracts were measured by the DPPH assay based on the method which were carried out by Asadujjamanet al. and Tay et al. with slight modification (Asadujjamanet al., 2013; Tay et al., 2014). The 0.1 mL concentrated crude extract was added with 3.9 mL 0.004% ethanolic DPPH solution in an aluminium-wrapped test tube. A parafilm was immediately used to cover the opening of test tube. Thus, the test tube was allowed to stand in dark room at room temperature for 30 minutes. The absorbance of DPPH solution was determined against a deionized water blank a 517 nm by using the UV-Vis spectrometer (Perkin Elmer Lambda 25). 0.1 mL of the crude extract was replaced by extract solvent in the preparing of negative control. Absorbance measurements of the crude extracts and negative control was carried out in triplicate. The result was expressed as a percentage of DPPH radical scavenging activity.The following formula (Equation 3.2) was used to calculate the DPPH radical scavenging activity of crude extract.

1 − × 100%= DPPH radical scavenging activityEquation 3.2

Where,

As= absorbance of crude extract

Ac = Absorbance of control

3.7.2Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was carried out according to procedures described by Kamonwannasitet al. with slight modification (Kamonwannasitet al., 2013).The FRAP reagent was made of 0.01 M TPTZ (2,4,6-tripyridyl-striazine) in 0.04 M HCl, 0.02 M FeCl3, and 0.03M acetate buffer (pH 3.6) in a ratio of 1:1:10(v/v/v). 0.03M acetate buffer (pH3.6) was prepared by mixing 46.3 mL of 0.2M acetic acid and 3.7 mL 0.2 M sodium acetate 0.5 mL together. Thus, the deionized water was added into mixture to make up the volume to 100mL. Then, 0.5g of the sample was added to 15 ml of FRAP reagent which was prepared in situ and warmed until 37°C before it was being used. The absorbance was measured at 593 nm by using an UV-Vis spectrophotometer (Perkin Elmer) after the solution was incubated for 5 minutes. Besides, 1 mM FeSO4stock solution was prepared as well. 0.278g of FeSO4 were dissolved into 1 L of deionized water. Thus, 1 mM FeSO4stock solution was diluted into 0.1, 0.2, 0.4, 0.6, 0.8 mM FeSO4 standard solution. A standard calibration curve was constructed by using different concentration of FeSO4 solution. The results were expressed as μmol Fe2+/mg dry weight of plant material. All measurements were carried out in triplicate and the mean values were calculated.

3.8 Statistical analysis

The experimental results in this study were calculated and analyzed by using the IBM software (SPSS Statistics version 21). All values were expressed as the mean ± standard deviation (SD) of triplicate measurements of replicate extraction. Simple correlation was used to determine the relationship between total amount of flavonoids in the crude extracts and their antioxidant capacities.

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