Take Home Test Chromatography Up 13

TAKE HOME TEST-CHROMATOGRAPHY

GRADUATE PROGRAM OF PHARMACY, UNIVERSITY OF PANCASILA,

JAKARTA

1. In an aphrodisiac drink, the powder formula consists of coffee, and Pasak Bumi roots.

However, the National Agency for Drug and Food Control suspected that this formula

contains Sidenafil and Tadalafil. Design an experiment using various

chromatographic methods that this formula is containing coffee, Pasak Bumi, and

contaminated by Sidenafil and Tadalafil.

2. There is an MLM honey product called Propulis. This Propulis contains flavonoids,

and aminoacids. Using various chromatographic method, you have to design an

experiments, what are the amino acids, and the flavonoids. Predict the bees have taken

the honey from flowers of which plants.

3. It was known that a various pheophorbides derived chlorophyll were active for anti

hepatitis virus. Design an experiment using various chromatographic methods,

determining the pheophorbides.

4. An expensive perfume was made of cananga, eagle wood and vetiver oil. Design an

experiment using chromatographic methods that this perfume containing these

essential oils. Determine how to quantify the major compound of each essential oil.

5. Various fruits are claimed containing rich of antioxidant, phenol, flavonoid, and

tannin. Design a combined chromatographic and spectroscopy experiments to

evaluate these claims, antioxidant capacity, total phenolic, flavonoids and tannin

content. Design equivalency for each constituent, namely, of antioxidant with

Ascorbic Acid, phenol with galic acid, flavonoid with quercetin and tannin with

cathecin.

Name : Nur Aji, S. Farm., AptNIM : 5413220025 (Kosmetika Bahan Alam SM. I)

ANSWER SHEETS

1. In an aphrodisiac drink, the powder formula consists of coffee, and Pasak Bumi roots.

However, the National Agency for Drug and Food Control suspected that this formula

contains Sidenafil and Tadalafil. Design an experiment using various

chromatographic methods that this formula is containing coffee, Pasak Bumi, and

contaminated by Sidenafil and Tadalafil.

Answer :1

Analysis of sildenafil and tadanafil can use the method of HPLC & LC- MS with a

column C18. While the tools and materials used are: Sildenafil citrate and tadalafil

use as standar, Methanol, acetonitrile (analytical grade) and formic acid. Method &

procedure is :

a. Recovery experiments were performed to evaluate the reliability and suitability of

the optimized conditions coffee. In this study, blank coffee powder was spiked

with 10 μg/g standard concentration. Two different types of extraction solvents

which were acetonitrile (A) and methanol (M) with two different extraction steps;

without nitrogen evaporation and with nitrogen evaporation step as used were

evaluated.

b. Extraction with evavoration : A 1 g sample was weighed into 50 mLcentrifuge

tube, added with 10 mLsolvent and being shake by overhead, sonicated and

centrifuged at 4000 rpm. The supernatant was then filtered with 0.2 μm PVDF

micro filter and injected into instruments. Result collected from this method were

labelled as A1 (using acetonitrile as extraction solvent) and M1 (using methanol

as extraction solvent).

c. Extraction with evaporation step : The first part of the extraction were the same as

above mentioned, however following the centrifugation step, the sample

supernatant collected was further evaporated using nitrogen evaporator until

complete dryness, reconstituted with 1 mL acetonitrile: water (1:1), filtered and

ready for instrumentation. Result collected from this method were labelled as A2

(using acetonitrile as extraction solvent) and M2 (using methanol as extraction

solvent).

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d. Optimization of sample extraction : Two different extraction techniques; without

the evaporation step and with evaporation step were applied in order to choose the

most efficient technique to analyze coffee sample. On both methods, two type of

extraction solvent were tested, acetonitrile (A) and methanol (M).. However,

coffee contains more matrix interferences such as fat, caffeine, sugar and we

presumed much lower concentration of adulterant added (if any) as compared to

pharmaceutical and herbal products. Thus, the methodology was optimised in

order to improve the efficiency for our samples. Modification aimed to obtain

more concentrated extract, thus the amount of sample weighed were increased to

one gram and the final extract was not diluted as recommended. The only

modification made on this method was the replacement of dichloromethane:

isopropanol (9:1) with either acetonitrile or methanol as extraction solvent, since

most studies reported the efficiency of these solvents in the analysis. The mean

recoveries and standard deviation for all four methods attempted is illustrated in

Tables 1 and 2. All analytes by HPLC-DAD displayed recovery of more than 80%

and 30% to 102% from LC-MS-TOF. The standard deviation was less than 10%

for all samples extracted and detected by LC-MS-TOF, but only sample extracted

with acetonitrile (Method A1 and A2) demonstrated standard deviation of less

than 10% by HPLC-DAD. Methanol was found not suitable to extract PDE5

inhibitors in coffee, as it generates very high interferences with DAD detection.

That acetonitrile proved a good recovery results as compared with the methanol

which generated higher background noise. The report also stated that acetonitrile

was necessary solvent for tadalafil due to its poor solubility in methanol.

e. Optimization of HPLC : attempted using two different combinations of mobile

phase: The combination of water and acetonitrile with modification on gradient

elution programme to obtain the best separation and the combination of water and

methanol with modification on gradient elution programme to obtain the best

separation. The selected combination of mobile phase was further optimized by

two different matrix modifier to find the most suitable matrix modifier in this

analysis: 10 mM ammonium format and 0.1% formic acid. Chromatogram data

were collected from 190 nm to 500 nm wavelength, within 20 min of run time.

The UV signal were monitored at 230 nm, 245 nm and 290 nm.

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f. Selection of mobile phase for HPLC analysis :The combination of water and

methanol as the mobile phase are able to separate well tadalafil from vardenafil

and sildenafil with high intensities. However, the background noise is noticibly

high and affect the identification of the analytes of interest. The peak of vardenafil

and sildenafil however are not separated well with the use of methanol as both

analytes are very similar in the molecular structure and its polarity. The peak of

vardenafil and sildenafil are not well separated when methanol is used as mobile

phase. The use of acetonitrile and water however provide better separation with

high intensities for all three compounds. All three peaks of vardenafil, sildenafil

and tadalafil were distinguishly separated. The peak of caffein is also separated

from the three interested compounds. At the same time, the background noise is

not too significant as to interfere with the peak of interest. The presence of several

Natoms in the molecular structure of target compounds would cause serious peak

tailing if no modifier was added into mobile phase in HPLC analysis. Ammonium

formate was used as matrix modifier to separate sildenafil, tadalafil and other

drugs in herbal products by LC-MS/MS. It was found in this study that the use of

ammonium format was not suitable to separate both sildenafil and vardenafil by

HPLC. Peaks of vardenafil and sildenafil found to be overlapping with each other

and these two compounds failed to be separated either by isocratic or gradient

elution. The use of ammonium formate was presumably suitable to analyze both

sildenafil and vardenfil simultaneously if using mass spectrometry because the

chromatogram of each target compound can be extracted individually, but not

with DAD detector. The separation problem was resolved by adding formic acid

in mobile phase is recommended. It was found that the use of 0.1% formic acid

was able to separate these two peaks of sildenafil and vardenfil. It was reported

that mobile phase in acidic condition help to improve the sensitivity of sildenafil

in liquid chromatography. As a conclusion, the use of acetonitrile and water with

0.1% formic acid as matrix modifier added to both solution was found to be the

most suitable mobile phase in this study.

g. Optimization of gradient elution: A linear solvent gradient using 0.1% formic acid

in water and 0.1% formic acid in acetonitrile with flow rate of 1 ml/min was the

most suitable combinations in terms of analysis time, resolution and sensitivity.

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Best separation obtained when composition of 0.1% formic acid in acetonitrile

was linearly ramped from 3% to 80% in 10 min and being maintained within 5

min before dropped instantly to 3% for another 5 min. Prominent peaks of

vardenafil, sildenafi l and tadalafi l were resolved at 8.459 min, 9.109 min and

11.157 min, respectively. The compounds were identified by comparing the

retention time with the individual compounds retention time.

h. Wavelength selection : The wavelengths of 290 nm and 230 nm were commonly

selected to quantitate. It was found that at 290 nm the peak intensities of all

analytes were highest as compared with 230 nm and 245 nm. However in coffee

sample, interferences signal was also higher and made quantification more

difficult as it is very close to the compound of interest. The intensities of

background matrix interference were less at 230 nm and yet peak intensities of

analytes of interest were very small. Thus, the identification and quantitation of

vardenafil, sildenafil and tadalafil in coffee was selected at 245 nm as it provided

prominent peak intensities with least matrix interferences and more stable

baseline.

i. Optimization of LC-MS-TOF : The developed HPLC method was then transferred

to LC-MS-TOF analysis but the gradient programme was optimized to get best

separation. The mass spectrometer was operated in the positive ion mode.

Nitrogen gas was utilized as the nebulizer gas and electrospray ionization (ESI)-

low concentration tuning mix solution used as the calibrations solution. All the

masses were corrected using internal reference ions of m/z 322.0481, 622.0289

and 922.0098 with mass error less than 5 ppm.

j. Statistical analysis : The results were expressed as mean value ± standard

deviation of three replicates. Analysis of sample was performed using SPSS

software version 11.5 2002 to determine the variation of recovery among four test

methods and individual analyte. The level of significance was p<0.05.

k. Evaluation of method performance : Statistical analysis showed that all four

extraction methods were significantly different (p<0.05) for both instruments,

HPLC-DAD and LC-MS-TOF. Further statistical analysis found that tadalafil was

significantly different (p<0.05) for all four methods detected with HPLC-DAD as

can be seen in Table 1. However, sildenafil and vardenafil were not differently

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significant (p>0.05) by method A1 and A2. Result of LC-MS-TOF showed that

tadalafil and sildenafil were not significantly different (p>0.05) for method M1

and A2, yet differ significantly (p<0.05) for method A1 and M2 as indicated in

Table 2. Vardenafil found to be significantly different (p<0.05) for all four

methods. According to the guideline on the acceptance of method performance by

European Union (2002/657/EC), the accuracy must be within 80% and 110% for

sample spiked at concentration of 10 μg/g. Therefore, methods A1 and M1

fulfilled the criteria for acceptance of performance using LC- MS-TOF whereas

method A1 and A2 by HPLC-DAD. Methods A1 and A2 were finally selected for

additional statistic analysis due to quantitation to be conducted by HPLC-DAD.

Based on statistical test between obtained result and target value (100%), it was

proven that there was no significant difference (p>0.05) for all analytes extracted

by method A1 by HPLC-DAD. However, method A2 showed there were no

significant different (p>0.05) only for vardenafil, whereas sildenafil and tadalafil

were significantly different (p< 0.05). Based on the presented results, method A1

was finally selected for future studies to determine the presence of tadalafil,

sildenafil and vardenafil simultaneously in coffee. Furthermore, the extraction

technique was faster as compared with method A2, which took 3 h longer. Figure

1(b) illustrates a HPLC chromatogram of spiked sample at concentration of 0.5

mg/kg which was extracted using method A1 and Figure 2(b) for LC-MS-TOF

extracted ion chromatogram.

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Figur (1). (a) Vardenafil, (b) Sidenafil, (c) Tadanafil

2. There is an MLM honey product called Propulis. This Propulis contains flavonoids,

and aminoacids. Using various chromatographic method, you have to design an

experiments, what are the amino acids, and the flavonoids. Predict the bees have taken

the honey from flowers of which plants.

Answer :2

a. Determining of Flavonoid in Propolis

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HPLC analysis. For the quantification of flavonoids, cinnamic acid and its derivatives

in propolis extracts and further for the evaluation of these bioactive compounds in

propolis, an Agilent 1100 HPLC apparatus was used; for the HPLC analysis a Zorbax

SB-C18 column, with 250 mm length, 4.6 mm i.d., and 5 m particle diameter was

used; for flavonoid detection the wavelenght of 337 nm was set; the mobile phase

was acetonitrile:water at 48:52 ratio; the temperature was 25ºC, with a flow of 0.3

ml/min; the injected sample volume was 20 µl. Diluted standard solutions of rutin,

quercetin, apigenin, kaempferol , acacetin, chrysin, pinocembrine, cinnamic acid, and

caffeic acid were analyzed in the same HPLC conditions and furthermore the

calibration of the detector response was done. The calibration curves were used for

the quantification of the main bioactive compounds from propolis.

On the basis of calibration curves from HPLC analysis for standard

flavonoids and cinnamic acid derivatives, the concentration of these bioactive

compounds in extracts and furthermore in propolis (raw material) was evaluated.

Thus, for the water “hot” propolis extracts, P(H)_0_a, only rutin, caffeic acid,

and quercetin were identified. For rutin, the retention time cannot be exactly

established (6.7-7.0 min) probably due to the partial hydrolytic degradation of this

compound under HPLC conditions (Fig.1). In the case of standard quercetin, two very

close peaks appear on the chromatogram (at ~13 min); this fact can be explained by

the possibility of enolization of quercetin (Fig. 2).

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The concentrations of these compounds extracted from propolis with water

were 0.3 mg/g propolis for rutin, 0.1 mg/g for caffeic acid and 0.13 mg/g for

quercetin. For these HPLC conditions (non-polar column), the more hydrophilic

compounds (like rutin and caffeic acid) are separed in the first part of chromatogram.

Not all separed compounds can be identified. For the similar “cold” extract the

concentration of rutin was lower (0.06 mg/g), but the concentrations of caffeic acid

and quercetin were higher

(2.1 mg/g and 0.7 mg/g, respectively) (Fig. 3). Concentrations under 0.2 mg/g were

obtained for all the rest of flavonoids identified in both water extracts. For the “hot”

propolis extracts conducted in 20% ethanol (in triplicate, P(H)_20_a/b/c), a great

number of compounds were separed by HPLC analysis, especially those more

hydrophilic, which are separed in the first part of chromatogram (Fig. 4). Among

rutin, caffeic acid, and quercetin, there were also identified apigenin, kaempferol, and

chrysin. The most concentrated was caffeic acid (3 mg/g), followed by rutin and

chrysin (1.6 and 2.2 mg/g, respectively; Table 1). For the “cold “extract (in duplicate,

P(C)_20_a/b, Fig. 4 and Table 1), rutin have been identified in similar concentration

(2.2 mg/g), but caffeic acid was identified in concentration of 5.6 mg/g, quercetin 1.4

mg/g, apigenin 1.5 mg/g, and chrysin 0.05 mg/g. No significative concentration of

kaempferol was identified in

“cold” extract.

b. Determining of Amino Acids in Propolis

The propolis presents a complex product, but its quality and its value conditions

depend on the chemical composition and of the ecological situate ion, the study of its

content and the dynamic of amino-acids in the compos ition has a great theoretical

and practice importance.

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Material and Methods

There were taken the samples to determine the amino-acids content in the propolis

composition during the active season from the collected propolis from the bees

families. There was determined the composition and the quantity of amino-acids in

the propolis using the special analysers (AAA-T 339) at the Centre of Metrology and

Automation of Scientific Researches at the Academy of Science of Moldova. The

received results have been worked using the statistical variation by the computer

programmes.

3. It was known that a various pheophorbides derived chlorophyll were active for anti

hepatitis virus. Design an experiment using various chromatographic methods,

determining the pheophorbides.

Answer : 3

In analogy with chlorophyll a and pheophytin a, pheophorbide a and

methylpheophorbide a in sediment samples are important compounds as indicators of

early diagenetic process. However, there is no remarkable difference in absorption

and/ or fluorescence spectral properties of chlorophyll or its degradation products.

The excitation and emission maximal wavelengths of these compounds in acetone

were as follows: chlorophyll a (430 nm/666 nm), pheophytin a (410 nm/ 670 nm),

pheophorbide a (410 nm/ 670 nm) and methylpheophorbide a (415 nm/ 670 nm),

respectively.'' 2 This indicates that coexistence of a large amount of the degradation

products causes a large error in the determination of early diagenetic products in

chlorophyll a. High performance liquid chromatography has been used for separation

and identification of chlorophylls in sediment.

Experimental

Apparatus

The high performance liquid chromatograph was a Hitachi model 655 equipped with a

Rheodyn 7125 syringe-loading sample injector (20 mm3 loop) and a Zorbax ODS

column (250 mmX4.6 mm I.D.). The fluorescence detector was a Hitachi model F-

1000 spectrofluorometer with a 12 mm3 flow cell. The excitation light source was a

150 W xenon arc lamp and the detector was a R-928F photomultiplier.

Materials and Reagents

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Chlorophyll a was isolated from confrey (Symphytum officinalle L.) with methanol

and acetone using the dioxane method4, and was purified by the use of cellulose

column chromatography.2 Pheophorbide a was prepared from chlorophyll a by

addition of 1 mol dm 3 hydrochloric acid. Pheophorbide a was prepared from

chlorophyll a according to the Hynninen method.5 Methylpheophorbide.

Chromatographic conditions

Separation of chlorophyll and chlorophyll derivatives was performed at 25°C by using

acetonitrileacetone (45 : 55 v/ v) as the mobile phase at a flow rate of 0.6 cm3 min-1.

The effluent from the column was monitored with a spectrofluorometer by using an

excitation wavelength of 410 nm and an emission wavelength of 670 nm.

Results and Discussion

Determination of chlorophylls by HPLC

A typical chromatogram obtained by the mixed standard solutions of chlorophyll a,

pheophytin a, pheophorbide a, and methylpheophorbide a is shown in Fig. 1. For a

reversed phase type column, Zorbax ODS, a variety of solvent conditions were

examined to obtain the complete separation of chlorophyll and chlorophyll

derivatives. The most suitable capacity factors (k') were obtained by using

acetonitrile-acetone (45 : 55 v/ v) for chlorophylls. The k' values at 25° C

were 0.26 for pheophorbide a, 0.33 for methylpheophorbide a, 1.78 for chlorophyll a,

and 4.23 for pheophytin a, respectively. The determination of chlorophyll a and

pheophytin a was carried out by means of the peak height method. And the relative

standard deviation inherent in the peak height method for chlorophyll a and

pheophytin a was less than 1.8% (for 9 determinations). However, the peak for

methylpheophorbide a was greatly influenced by the

tailing peak of pheophorbide a. This indicates that coexistence of even a small amount

of pheophorbide a causes a large error in the determination of methylpheophorbide a.

In order to eliminate the error from pheophorbide a, the narrow base line method was

used for the quantitative analysis of methylpheophorbide a.

Determination of methylpheophorbide a by the narrow baseline method

The narrow baseline method for the spectrophotometric determination was first

proposed by Commins.6 This method was applied to the fluorometric determination

of polynuclear aromatic hydrocarbons by Matsushita et al.' In these studies, the

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baseline was set by connection of two points each about 5 nm apart from the peak on

the spectrum. This baseline was called a narrow baseline (NBL) and the height to the

peak from the NBL was taken as analytical data. In this paper, the unit on abscissa

(wavelength/ nm) is replaced by "retention time/ min". The peak height of

methylpheophorbide a increased with increasing peak height of pheophorbide a, as

shown in Fig. 2. The observed value of methylpheophorbide a was 30% higher than

the theoretical value, when the peak height of pheophorbide a was equal to that of

methylpheophorbide a. Therefore, the narrow baseline method was applied for the

determination of methylpheophorbide a by HPLC. In order to set a narrow baseline,

we determined the two peaks (a and b) each 5 min apart from the peak of

methylpheophorbide a on the chromatogram, as shown in Fig. 3. The analytical signal

(I) was given in the next equation; I=1o-(Ia+Ib)/2, where Io, Ia and Ib were peak

height of the maximum peak, point a and point b, respectively. The relative standard

deviation inherent in this method for a sample containing 8.1X10.8 mol dm-3 was less

than 2.4% (for 6 determinations).

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Determination of chlorophylls in core samples

Core samples collected, were analyzed by this method. Two grams of the wet sample were

transferred to a centrifuge tube containing 20 cm3 of acetone and shaken for 5 min. The

suspension was centrifuged at 2000 r.p.m. for 10 min. The total extracts that were obtained

from 5 separate runs were collected through a separatory funnel. The yellow supernatant

solution thus obtained (acetone extract, ca. 100 cm3) was mixed with 10 cm3 of petroleum

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ether and then was added to an equial volume (approximately) of saturated sodium chloride

solution. In these procedures, chlorophylls were extracted to the petroleum ether layer. This

layer was evaporated in vacuo and was made up to 10 cm3 with acetone or mobile phase

{acetonitrile : acetone=45 : (v/ v)}. Five mm3 of the resulting solution was injected in a

sample loop of the HPLC with a microsyringe. The results are summarized in Table 1. These

results confirm that HPLC combined with the narrow baseline method will contribute, as a

convenient technique, to the determination of chlorophyll and its degradation products.

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4. Determination of suspected fragrance contains essential oils Cananga, wooden eagle

and vitiver. The method can be used is GC-MS. Essential oils were analyzed by

GC/MS using a split injection onto a capillary column with a stationary phase

containing derivatized cyclodextrin macromolecules, achieving optimum separations

of enantiomers.

Answer : 4

Methods

Before analyzing the various oils, the chromatography was optimized. The parameters

were selected based on the information provided in the “Restek Guide to the Analysis

of Chiral Compounds by GC”. The guide recommends higher linear velocities (80

cm/sec), slower temperature ramps (1 – 2 °C/min), an appropriate initial operating

temperature (40 to 60°C), and on-column concentrations of 50 ng or less. The

Trennzahl values increase with an increase in linear velocity, improving resolution.

As the oven temperature ramp decreases below 3°C/min, the Trennzahl values

increase with enantiomeric resolution factors.2 A split/splitless injector was

configured for a split injection at 200°C with a 3 mm id silanized glass split liner. A 1

μL injection was made with an AS 2000 fully programmable autosampler. The oils

were diluted to 1% in methylene chloride. The analytical column was a Restek® Rt-

βDEXse 0.32 mm x 30 meter, 0.25 micron film with a carrier gas flow of 5 cc/min

helium or linear velocity of 80 cm/sec and a split flow of 50 cc/min (Split ratio of

10/1). The oven was programmed at 2°C/min during the elution. The Finnigan™

TRACE™ DSQ quadrupole mass spectrometer from

Thermo Electron was autotuned for a classical tune, and default values from the

Autotune file were used. The mass spectrometer was able to handle the high carrier

gas flow by configuring with the 250 liter pump option. A Full Scan analysis was set

to scan from 35 to 300 m/z at a scan rate of 1,000 amu/sec. The source was set at

200°C.

To analyze the essential oil perfume containing cananga, eagle wood and vitiver, then

use the comparison of pure essential oil cananga , eagle wood and vitiver.

5. Various fruits are claimed containing rich of antioxidant, phenol, flavonoid, and

tannin. Design a combined chromatographic and spectroscopy experiments to

15


evaluate these claims, antioxidant capacity, total phenolic, flavonoids and tannin

content. Design equivalency for each constituent, namely, of antioxidant with

Ascorbic Acid, phenol with galic acid, flavonoid with quercetin and tannin with

cathecin.

Answer : 5, 6, 7

It has been proven that free radicals play an important role in many diseases, such as

cardiovascular diseases, cancer, neurodegenerative diseases, diabetes and ageing.

Vitamins and polyphenols in fruits and vegetables are considered to be responsible for

such antioxidant activity, with polyphenols being the most active. The natural

antioxidants are expected to be an alternative to synthetic ones because of their

potential health benefits. Antioxidant activities of many fruits, vegetables, spices,

medicinal plants and microalgae have been evaluated, and the results showed that

some of them could be rich sources of natural antioxidants, so the search for raw

materials containing potent natural antioxidants continues to attract the attention of

researchers.

a. Antioxidant capacities of fruits

The antioxidants in fruits are either water-soluble or fat-soluble. When determining

total antioxidant activities of fruits, both water-soluble and fat-soluble components

should be considered. In this study, the fat-soluble components of wild fruits were

extracted with tetrahydrofuran, and their water-soluble components were extracted

with a mixture of methanolacetate acid-water. The ferric reducing antioxidant power

(FRAP) assay was used to evaluate antioxidant capacities of the 56 wild fruits. The

FRAP assay, which is a simple, inexpensive and widely employed method for the

evaluation of antioxidant capacity, is based on the capacity of antioxidants to reduce

ferric(III) ions to ferrous(II) ions.

The antioxidant capacities of plant samples may be influenced by a lot of factors, such

as the extraction solvent and test systems used, and cannot be fully described by any

one single method. A reliable antioxidant evaluation protocol requires measurement

of more than one property because most natural antioxidants are multifunctional, so it

is essential to perform different antioxidant activity assessments to take into account

the various mechanisms of antioxidant action. Therefore, the Trolox equivalent

antioxidant capacity (TEAC) assay was used to evaluate the free radical scavenging

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capacities of the fruits. The TEAC assay is based on the ability of antioxidants to

scavenge the ABTS radical, and can measure antioxidant capacities of hydrophilic

and lipophilic compounds in the same sample. The TEAC assay is a simple, rapid and

commonly used method for the evaluation of antioxidant capacity, and could offer

reproducible results [21,22]. In the literature, natural and synthetic antioxidants, such

as ascorbic acid, vitamin E, butylated hydroxytoluene, butylated hydroxyacetone and

Trolox, were often used as positive controls.

Methods

The fresh, whole plant (3 kg) was collected and shade dried to obtain 500 g dry

sample which was later coarsely powdered in a Willy Mill to 60-mesh size and used

for solvent extraction. For sample preparation, 500 g of dried sample were extracted

twice (2000 ml for each) with 95% methanol at 25°C for 48 h and concentrated using

a rotary evaporator under reduced pressure at 40°C to yield the TLM (11.5%). The

residue was suspended in water (50 ml) and partitioned successively with n-hexane,

chloroform, ethyl acetate, n-butanol (a total of two aliquots of 100 ml each) and

soluble residual aqueous fraction yielding respectively the TLH (5.4%), TLC (4.3%),

TLE (6.1%), TLB (4.8%) and TLA (8.2%).

Chemicals

Ascorbic acid; aluminum chloride, 2, 20 - azino-bis-(3- ethylbenzothiazoline-6-

sulphonic acid) (ABTS); ferric chloride (FeCl3); Folin-Ciocalteu; bovine serum

albumin (BSA); potassium persulphate; 2,20-diphenyl-1-picrylhydrazyl (DPPH); nitro

blue tetrazolium (NBT); phenazine methosulphate (PMS); reduced glutathione

(GSH); 1,2- dithio-bis nitro benzoic acid (DTNB); sulphosalicylic acid;thiobarbituric

acid (TBA) and trichloroacetic acid (TCA). Sulphuric acid; 2-deoxyribose; riboflavin;

sodium carbonate (Na2CO3); sodium hydroxide (NaOH); sodium nitrite (NaNO2);

disodium hydrogen phosphate (Na2HPO4) and hydrogen peroxide (H2O2). Potassium

ferricyanide [K3Fe (CN)6]; triflouroacetic acid; sodium dihydrogen phosphate

(NaH2PO4) and all solvents n-hexane (99.8%); chloroform (99.8%); ethyl acetate

(99.8%) and n-butanol (99.8%) used were of analytical grade. Distilled deionized

water (dd.H2O) was prepared by Ultrapure TM water purification system.

In vitro studies

Antioxidant assays

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Each sample was dissolved in 95% methanol to make a concentration of 1 mg/ml and

then diluted to prepare the series concentrations for antioxidant assays. Reference

chemicals were used for comparison in all assays. DPPH radical scavenging activity

assay. The free radical scavenging activity of the fractions was measured in vitro by

2,20- diphenyl-1-picrylhydrazyl (DPPH) assay according to the method described

earlier.. The stock solution was prepared by dissolving 24 mg DPPH with 100 ml

methanol and stored at 20°C until required. The working solution was obtained by

diluting DPPH solution with methanol to attain an absorbance of about 0.98±0.02 at

517 nm using the spectrophotometer. A 3 ml aliquot of this solution was mixed with

100 μl of the sample at various concentrations (10 - 500 μg/ml). The reaction mixture

was shaken well and incubated in the dark for 15 min at room temperature. Then the

absorbance was taken at 517 nm. The control was prepared as above without any

sample.

Phosphomolybdate assay (total antioxidant capacity)

The total antioxidant capacity of the fractions was determined by phosphomolybdate

method using ascorbic acid as a standard. An aliquot of 0.1 ml of sample solution was

mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate

and 4 mM ammonium molybdate). The tubes were capped and incubated in a water

bath at 95°C for 90 min. After the samples had cooled to room temperature, the

absorbance of the mixture was measured at 765 nm against a blank. A typical blank

contained 1 ml of the reagent solution and the appropriate volume of the solvent and

incubated under the same conditions. Ascorbic acid was used as standard.

b. Total phenolic content of fruits

The total phenolic contents of fruits were estimated using the Folin–Ciocalteu

method, which relies on the transfer of electrons from phenolic compounds to the

Folin–Ciocalteu reagent in alkaline medium, and is a simple, rapid and reproducible

method. In addition, it should be pointed out that some non-phenolic reducing

compounds, such as organic acids and sugars, could interfere the determination of

total phenolic contents by the Folin-Ciocalteu method, which leads to an

overvaluation of the phenolic content. Furthermore, different phenolics might present

different responses with the Folin Ciocalteu reagent, such as gallic acid and rutin have

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similar behaviours, but several flavonoids present low absorption, which leads to an

underestimation of various compounds.

Estimation of total phenolic content

The total phenolic content was determined by the spectrophotometric method. In

brief, a 1 ml of sample (1 mg/ml) was mixed with 1 ml of Folin-Ciocalteu’s phenol

reagent. After 5 min, 10 ml of a 7% Na2CO3 solution was added to the mixture

followed by the addition of 13 ml of deionized distilled water and mixed thoroughly.

The mixture was kept in the dark for 90 min at 23°C, after which the absorbance was

read at 750 nm. The TPC was determined from extrapolation of calibration curve

which was made by preparing gallic acid solution. The estimation of the phenolic

compounds was carried out in triplicate. The TPC was expressed as milligrams of

gallic acid equivalents (GAE) per g of dried sample.

c. Total Flavonoid

Total flavonoid content was determined in quercetine. In a 10 ml test tube, 0.3 ml of

extracts quercetine, 3.4 ml of 30% methanol, 0.15 ml of NaNO2 (0.5 M) and 0.15 ml

of AlCl3.6H2O (0.3 M) were mixed. After 5 min, 1 ml of NaOH (1 M) was added.

The solution was mixed well and the absorbance was measured against the reagent

blank at 506 nm. The standard curve for total flavonoids was made using rutin

standard solution (0 to 100 mg/l) under the same procedure as earlier described. The

total flavonoids were expressed as milligrams of rutin equivalents per g of dried

fraction.

d. Test for tannins

i. 50 mg of catechine was boiled in 20 ml of distilled water and filtered. A few

drops of 0.1% FeCl3 was added in filtrate and observed for colour change;

brownish green or a blue-black colouration was taken as evidence for the

presence of tannins.

ii. The tannins were determined by Folin and Ciocalteu method. 0.1 ml of the

sample extract was added with 7.5 ml of distilled water and adds 0.5 ml of

Folin Phenol reagent, 1 ml of 35% sodium carbonate solution and dilute to 10

ml with distilled water. The mixture was shaken well, kept at room

temperature for 30 min and absorbance was measured at 725 nm. Blank was

prepared with water instead of the sample. A set of standard solutions of gallic

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acid is treated in the same manner as described earlier and read against a

blank. The results of tannins are expressed in terms of gallic acid mg/g of

extract

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REFERENCES

1. A.M. Zailina,AM, Aminah, A. & Ambar, YM, Optimization of Extraction Methods for Determination of Phosphodiesterase-5 (PDE5) Inhibitors in Premix Coffee. Sains Malaysiana 42(2)(2013): 135–142.

2. Coneac, G, et al. Flavonoid Contents of Propolis from the West Side of Romania and Correlation with the Antioxidant Activity. Chem. Bull. "POLITEHNICA" Univ. (Timisoara). Volume 53(67), 1-2, 2008.

3. Yoshitake, Y, et al.Determination of Pheophorbide a and Methylpheophorbide a by Fuorosence –HPLC : Availability of Narrow Baseline Method. Analyitical Science Vol 2. 1986.

4. Mariot, PJ, et al. Gas chromatographic technologies for the analysis of essential oils. Journal of Chromatography A, 936 (2001) 1–22.

5. Sacchetti, Gianni et al,. Comparative Evaluation of 11 Essential Oils of Different Origin as Functional Antioxidants, Antiradicals, and Antimicrobials in Foods.Universita Degli Studi di Ferrara. Italy. 2004.

6. Li Fu et al. Antioxidant Capacities and Total Phenolic Content of 56 Wild Fruits from South China. Sun Yat-Sen University. Guangzhou, China. 2010 (Artikel dari www.mdpi.com/journal/molecules.com diakses tanggal 2 Januari 2014.)

7. Sae et al. Antioxidant activity, Total Phenolic, and Total FlVONOID Contents of Whole Plant Extracts Torilis leptophylla L.BMC Complementary and Alternative Medicine. 2012., 12:221 (http://www.biomedcentral.com/1472-6882/12/221 diakses tanggal 2 Januari 2014)

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