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.

ANSWER SHEETS

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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 g/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 m 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).
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
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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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.
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

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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.
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

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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
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 g/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.
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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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.

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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
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
6

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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 25C, with a flow of 0.3
ml/min; the injected sample volume was 20 l. 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
flavonoids and cinnamic acid

for standard

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).

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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.
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
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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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 F1000 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
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.
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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

Chromatographic conditions
Separation of chlorophyll and chlorophyll derivatives was performed at 25C 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
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
11

Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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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Name : Nur Aji, S. Farm., Apt

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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
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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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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
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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 60C), 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 3C/min, the Trennzahl values increase
with enantiomeric resolution factors.2 A split/splitless injector was configured for a
split injection at 200C with a 3 mm id silanized glass split liner. A 1 L 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-DEXse 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 2C/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
200C.
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
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

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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
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

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for solvent extraction. For sample preparation, 500 g of dried sample were extracted
twice (2000 ml for each) with 95% methanol at 25C for 48 h and concentrated using
a rotary evaporator under reduced pressure at 40C 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-6sulphonic 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
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 20C until required. The working solution was obtained by
diluting DPPH solution with methanol to attain an absorbance of about 0.980.02 at
517 nm using the spectrophotometer. A 3 ml aliquot of this solution was mixed with
100 l of the sample at various concentrations (10 - 500 g/ml). The reaction mixture
was shaken well and incubated in the dark for 15 min at room temperature. Then the

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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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 95C 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 FolinCiocalteu
method, which relies on the transfer of electrons from phenolic compounds to the
FolinCiocalteu 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
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-Ciocalteus 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 23C, 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

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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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
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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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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): 135142.

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) 122.
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.

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Name : Nur Aji, S. Farm., Apt

NIM : 5413220025 (Kosmetika Bahan Alam SM. I)

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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