Prosedur Pengukuran Berbasis Fluorimetri Untuk Mengukur Konsentrasi Kurkumin Di Dalam Obat-Obat Herbal Komersial
FLUORIMETRIC BASED PROCEDURE FOR MEASURING
CURCUMIN CONCENTRATION IN COMMERCIAL
HERBAL MEDICINES
SITI NURMA NUGRAHA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
DECLARATION OF THESIS AND INFORMATION SOURCES OF
INFORMATION AND PATENT
I hereby declare that the thesis of “Fluorimetric Based Procedure for
Measuring Curcumin Concentration in Commercial Herbal Medicines” is true of
my own work under the direction of thesis committee and has not been submitted
in any form to any university. The content of this thesis has been examined by the
thesis committee and external examiner. Sources of information which is derived
or cited either from published or unpublished scientific paper from other writers
have mentioned in the script and listed in the reference at the end of this thesis.
I hereby assign the copyright of my thesis to Bogor Agricultural
University.
Bogor, May 2016
Siti Nurma Nugraha
ID G751130151
RINGKASAN
SITI NURMA NUGRAHA. Prosedur Pengukuran Berbasis Fluorimetri
untuk Mengukur Konsentrasi Kurkumin di dalam Obat-obat Herbal Komersial.
Dibimbing oleh HUSIN ALATAS dan IRMANIDA BATUBARA.
Masyarakat Indonesia telah secara turun-temurun menggunakan obat herbal
tradisional, yang dikenal sebagai jamu, untuk mengobati dan melindungi tubuh
dari penyakit. Kebiasaan penggunaan jamu ini memberi implikasi akan
pentingnya kebutuhan untuk meningkatkan dan mengembangkan beberapa standar
kualitas jamu. Atas dasar ini, sistem standardisasi yang baik sangat diperlukan
untuk mendapatkan kualitas jamu yang memenuhi standar kelayakan, efektif, dan
berkualitas tinggi. Kunyit (Curcuma longa) dan temulawak (Curcuma
Zanthorrhiza) adalah tanaman obat yang banyak dimanfaatkan sebagai bahan
utama jamu oleh orang Indonesia. Kedua bahan tersebut mengandung kurkumin
yang merupakan komponen aktif utama pada rhizoma (akar rimpang).
Berbagai jenis metode telah dikembangkan dalam penentuan kadar kurkumin
di dalam tumbuhan obat, seperti (1) teknik spektrofotometri UV-Vis, (2) teknik
kromatografi lapisan tipis (Thin-Layer Chromatography), dan (3) teknik High
Performance Liquid Chromatography (HPLC). Meskipun metode-metode
tersebut tersedia dan dapat digunakan, suatu teknik analisis kurkumin yang
penerapannya sederhana, memberikan hasil yang cepat, peka, tepat, dan berbiaya
murah masih menjadi masalah utama di Indonesia. Dalam studi ini kami
mengajukan dan menggunakan sebuah metode penentuan kadar kurkumin
alternatif berbasis fluorimetri. Metode ini dapat diterapkan untuk analisis obat
herbal yang mengandung komponen zat yang dapat berfluoresensi, seperti
kurkumin, yang menunjukkan gejala fluoresensi kuat di dalam pelarut organik.
Metode fluorimetri ini memiliki kadar sensitivitas dan selektivitas yang lebih
tinggi dibandingkan dengan teknik spektrofotometri dan metode ini juga lebih
sederhana dan murah dibandingkan dengan teknik analisis kromatografi.
Tiga tahap utama dalam studi ini meliputi: persiapan kurva standar kalibrasi
kurkumin, penentuan konsentrasi kurkumin di dalam sampel, dan perbandingan
terhadap hasil yang diperoleh menggunakan teknik HPLC. Pengukuran
fluoresensi dilakukan pada fluorometer FLUOstar Omega yang menggunakan
spektrometer UV-Vis. Level fluoresensi yang muncul berbanding lurus dengan
intensitas cahaya yang datang dan konsentrasi dari bahan fluoresen yang diamati.
Kurva kalibrasi standar yang dihasilkan menunjukkan hubungan yang linear dan
positif antara konsentrasi dan intensitas fluoresensi. Tingginya nilai koefisien
determinasi juga menunjukkan bahwa hubungan tersebut sangat jelas. Evaluasi
kinerja dari metode fluorimetri ini dilakukan dengan membandingkan konsentrasikonsentrasi yang diperoleh dari pembacaan hasil fluorometer terhadap hasil yang
diperoleh dari HPLC.
Kata Kunci: fluoresensi, kurkumin, kunyit, temulawak, fluorometer, analisa
HPLC.
SUMMARY
SITI NURMA NUGRAHA. Fluorimetric Based Procedure for Measuring
Curcumin Concentration in Commercial Herbal Medicines. Supervised by HUSIN
ALATAS and IRMANIDA BATUBARA.
People in Indonesia have historically used the traditional herbal medicines,
known as jamu, for the treatment and protection of diseases. It implies a need to
improve and develop some quality standard of jamu. To develop a jamu, various
species of medicinal plants were selected and identified carefully prior extraction
and manufacturing into jamu components. Standardization in this area is
necessary to achieve the quality of jamu that are eligible, effective, and of high
quality. Turmeric (Curcuma longa) and temulawak (Curcuma zanthorrhiza) are
medicinal plants, widely used as the main ingredients in jamu by Indonesian
people. Both have curcumin as the major active components of the rhizomes.
Numerous methods have been developed on curcumin determination in
medicinal plants such as (1) UV-vis spectrophotometry, (2) Thin Layer
Chromatography, and (3) High Performance Liquid Chromatography (HPLC).
Despite the availability of these methods, the implementation of a simple, rapid,
sensitive, precise, and more economic technique for curcumin analysis is still
considered an important issue in Indonesia. An alternative fluorimetric method is
proposed and used in this study. It can be applied for the analysis of herbal
medicines containing a fluorescent component, such as curcumin, that exhibits
strong fluorescence in organic solvents. The fluorimetric method has higher
sensitivity and selectivity compared to the spectrophotometric technique and it is
also simpler and less expensive compared to the chromatographic analysis.
Three main parts of this study include: preparation of standard calibration
curve of curcumin, determination of curcumin concentration in samples, and
comparison against measurements from HPLC. Fluorescence measurements were
performed on a FLUOstar Omega fluorometer which uses a UV-vis spectrometer.
Fluorescence is directly proportional to the intensity of the exciting light and the
concentration of the fluorescent material being investigated. The standard
calibration curve shows linear and positive relationship between concentration and
fluorescence intensity. High coefficient of determination also indicates that the
relationship is strong. Performance evaluation of the fluorimetric method was
carried out by comparing concentrations derived from the instrument with that
from HPLC.
Keyword : fluorescence, curcumin, turmeric, temulawak, fluorometer, HPLC
analysis
© Copyright of IPB, the year 2016
Copyright reserved by the law
Forbidden to quote part or all of these writings without including or
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not harm the interests of fair Bogor Agricultural University.
Prohibited announced and reproduce part or the whole paper in any form
without permission from Bogor Agricultural University.
FLUORIMETRIC BASED PROCEDURE FOR MEASURING
CURCUMIN CONCENTRATION IN COMMERCIAL
HERBAL MEDICINES
SITI NURMA NUGRAHA
A Thesis submitted in partial fulfillment of the
requirement for Master Degree
in Biophysics Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
External examiner :
Dr. Kiagus Dahlan
Thesis title
Name
ID
: Fluorimetric Based Procedure for Measuring Curcumin
Concentration in Commercial Herbal Medicines
: Siti Nurma Nugraha
: G751130151
Approved by
The Commission of Supervisors
Dr. Husin Alatas, M.Si
Supervisor
Dr. Irmanida Batubara, M.Si
Co-Supervisor
Certified by:
Head of Biophysics
Graduate Program
Dean of the IPB Graduate School
Dr. MersiKurniati, M.Si
Dr.Ir. DahrulSyah, MSc.Agr
Examination Date : May 20th 2016
Graduation date :
ACKNOWLEDGEMENTS
Firstly, I am grateful to Allah SWT for this amazing life, abundant favors
and blessings during my graduate study until I finish this thesis.
I wish to thank Dr. Husin Alatas as my thesis supervisor for devoting his
time for discussion and giving me his continued support, motivation and
suggestions for my research work. I have learned and improved more than I ever
expected.
I also would like to thank my thesis co-supervisor, Dr. Irmanida Batubara
for her expertise, guidance, and comments. My thanks also go to Dr. Kiagus
Dahlan as my thesis external examiner for his valuable suggestion and motivation,
Dr. Akhiruddin Maddu, Tony Sumaryada, PhD and Dr. Mersi Kurniati for their
support and useful suggestion, and also Dr. Mamat Rahmat and Sugianto Arjo,
M.Si for their helpful contribution.
I would like to thank the people at Biofarmaka Research Center, in
particular, Ms. Nunuk Kurniati, Ms. Laela Wulan Sari, Ms. Wiwi Widiyanti and
Mr. Endi Suhendi for their general help and technical support. I thank my
classmates in Biophysics IPB who all have been great friends and helped me in
this study.
This master study opportunity was made possible in part by the support
from the Bogor City Government and Principal of SMPN 2 Bogor. I also thank
my colleagues in SMPN 2 Bogor for their support, especially Ibu Yani Herliani,
M.Pd for her profound encouragement.
I would like to give my special gratitude to my husband for his
tremendous love and patience, my parents for giving those powerful words of
advice, my loving sister and brother for always being truthful to me. Without their
love and companionship, I could not go on to achieve what I have today.
Lastly, I dedicate this thesis to my students who have become the sources
of my inspiration.
Bogor, May 2016
Siti Nurma Nugraha
TABLE OF CONTENS
TABLE LIST
ii
FIGURE LIST
iii
APPENDICES LIST
iv
1 INTRODUCTION
Background
Objective
Hypotheses
Benefit
1
2
2
3
3
2 MATERIALS AND METHODS
Instruments
Fluorimetric Method
Chemical and Reagents
Determination of Curcumin Concentration and Comparison
Detection Limit (LOD) and Quantitation Limit (LOQ)
4
4
4
5
5
6
3 RESULTS AND DISCUSSION
7
4 CONCLUSION AND RECOMMENDATION
14
REFERENCES
15
APPENDICES
17
TABLE LIST
1 Linear Regression Parameters of Calibration Curve for Curcumin
2 LOD and LOQ value
3 Curcumin Concentration in Temulawak
4 Curcumin Concentration in Turmeric
8
9
10
11
FIGURE LIST
1 Chemical Structure of Curcumin
2 Fluorometer FLUOstar Omega
3 Fluorescence Diagram
4 Standard Calibration Curve
5 Percentage Difference Diagram of Temulawak
6 Percentage Difference Diagram of Turmeric
1
4
7
9
12
13
APPENDIX LIST
1 Flow chart of research
2 Concentration of temulawak for every fluorescence intensity
3 Concentration of turmeric for every fluorescence intensity
4 Percentage difference table
5 HPLC data for temulawak extract
6 HPLC data for turmeric extract
18
19
20
21
21
21
1
1 INTRODUCTION
Background
Indonesia has the second biggest biodiversity in the world expressed by
30,000 plant species and is a home to about 80% of the world’s medicinal plants.
Based on this plenteous source of medicinal plants, it is estimated that 40 million
of Indonesian have historically used the traditional herbal medicines, known as
jamu, for the treatment and protection of diseases (Elfahmi et al., 2014). In the
country, jamu is popular and common practice on diet and general health. It
implies a need to improve and develop some quality standard of jamu. Various
researches utilizing advanced technology have been carried out to improve the
quality of jamu and, eventually, to further boost consumer confidence. To develop
a jamu, various species of medicinal plants were selected and identified carefully
prior extraction and manufacturing into jamu components. Standardization in this
area is necessary to achieve the quality of jamu that are eligible, effective, and of
high quality.
Turmeric (Curcuma longa) and temulawak (Curcuma zanthorrhiza) are
medicinal plants, widely used as the main ingredients in jamu by Indonesian
people.
Both
main
ingredients
have
curcumin
[1,7-bis
(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-Dione] (Figure 1) as the major
active components of the rhizomes (Tonnesen et al., 1992; Sutrisno et al., 2008).
Curcumin, having nearly a two centuries old scientific history, is still attracting
researchers from all over the world. Starting from 1815, when curcumin was first
isolated from turmeric, there were only a few reports till the 1970s on its chemical
structure, synthesis, biochemical and antioxidant activity (Sharma, 1976). It has
three chemical entities in its structure: two aromatic ring systems containing
o-methoxy phenolic groups, connected by a seven carbon linker consisting of an
α,β-unsaturated β-diketone moiety (Grykiewicz, 2012).
Recent pharmacological researches revealed that curcumin is functioned as
anti-oxidant (Namita et al., 2012), anti-inflammatory (Anand et al., 2008),
anti-carcinogenic (Basnet et al., 2011), anti-diabetes (Merrel et al., 2009),
antimicrobial effects (Maheshwari et al., 2006), anti-tumor (Kunnumakkara et al.,
2007), anti-bacterial and anti-coagulant (Maiti et al., 2007).
Figure 1. Chemical structure of curcumin
2
Numerous methods have been developed on curcumin determination in
medicinal plants such as (1) UV-vis spectrophotometry (Jasim et al., 1988;
Sharma et al., 2012; Liu et al., 2016), (2) Thin Layer Chromatography (TLC;
Zhang et al., 2007), and (3) High Performance Liquid Chromatography (HPLC;
Pak et al., 2003). Although spectrophotometry is widely used for the
quantification of curcumin, it lacks of reproducibility (Govindrajan, 1980) and
need some complicated sample-preparation steps (Liu et al., 2016). On the other
hand, the chromatographic methods (TLC and HPLC) give some good
performances in producing simultaneous separation and determination of various
components with high accuracy and good precision (Yang et al., 2012). The HPLC
analysis has also been employed to detect low quantities of curcumin in biofluids
(Kim et al., 2013). However, it has the disadvantages of being more
time-consuming and expensive (Bisht et al., 2007; Nam et al., 2007; Tiyaboonchai
et al., 2007; Sou et al., 2008).
Despite the availability of these methods, the implementation of a simple,
rapid, sensitive, precise, and more economic technique for curcumin analysis is
still considered an important issue in Indonesia. An alternative fluorimetric
method (Diaz et al., 1992; Mazzarino et al., 2010) is proposed and used in this
study. It can be applied for the analysis of herbal medicines containing a
fluorescent component, such as curcumin, that exhibits strong fluorescence in
organic solvents (Diaz et al., 1992). The fluorimetric method has higher sensitivity
and selectivity compared to the spectrophotometric technique and it is also
simpler and less expensive compared to the chromatographic analysis (Zhang et
al, 2007). However, a standard procedure based on fluorimetric method for
measuring the curcumin concentration in commercial herbal medicines has not yet
been addressed scientifically.
Objectives
The objectives of this study are as follows:
1. To develope the corresponding simple procedure based on the corresponding
fluorimetric method for measuring curcumin concentration in two commercial
herbal medicines, containing turmeric and temulawak
2. To evaluate the result by comparison with the HPLC analysis.
Hypotheses
The relationship between fluorescence intensity and curcumin standard
concentration is expected to be linear and positive, and to show an acceptable
accuracy when compared with the HPLC measurement.
3
Benefits
This study is expected to have new standard procedure for measuring
concentration of curcumin in Indonesian commercial herbal medicines with have
some beneficial effect in technical and economic.
4
2 MATERIALS AND METHODS
Instruments
Fluorescence measurements were performed on a FLUOstar Omega
fluorometer (BMG Labtech, USA) which uses a UV-vis spectrometer that
instantaneously captures wavelengths in a range of 220-850 nm at 1 nm resolution
(Figure 2). This device do not need scanning or calibration since there is no
installed monochromator, and it is equipped with a high-intensity xenon flash
lamp and selected PMTs, coupled with high transmission band pass filters, to
provide a stable, low-noise signal platform for ultra-sensitive fluorescence
reading. All the measurements were carried out in triplicate (three repetitions).
Figure 2. Fluorometer FLUOstar Omega
Fluorimetric Method
Since fluorescent materials are relatively rare (which limits the application of
the techniques), fluorimetry is also a more specific analytic technique than
absorption spectroscopy. A useful aspect of fluorescence specificity is that the
fluorescent molecule has two characteristic spectra: the excitation spectrum (the
relative efficiency of different incidence wavelengths that cause fluorescence),
and the emission spectrum (the relative spectral distribution of emitted light). The
wavelength of the emission is always longer than the wavelength of excitation.
Also, the fluorescence emission spectrum is specific to the material. Thus, it is
5
possible to avoid interfering radiation caused by direct scattering of the exciting
radiation or by fluorescence of other substances, by using spectrally selective
filters in the fluorescence detectors. For this study, the excitation and emission
wavelengths were 420 nm and 530 nm, respectively (Wang et al, 2006). The
fluorescence intensity of all curcumin standards was measured by four different
experiments: (1) Bottom optic with Gain Adjustment (GA) equal to 500; (2)
Bottom optic with GA = 1000, (3) Top optic with GA = 500, and (4) Top optic
with GA = 1000. Bottom and top optics are illumination optics inside fluorometer
which are used to measure fluorescence, luminescence, and absorbance. Their
difference is located at the optic position that determines the light inlet, which is at
the left (right) side of reagent box for bottom (top) reading. GA is a parameter for
light intensity that is projected into the sample. The two selected GA levels (500
and 1000) have been tested to give optimum fluorescence intensity. Next, the
fluorescence intensity was measured by the fluorometer for the above series of
standard solutions. The calibration graph was obtained by plotting the
fluorescence intensity of the standard solutions against the theoretical standard
concentrations. The linearity was evaluated by linear least-square regression
analysis.
Chemical and Reagents
Curcumin (ChromaDex: CDXA-09-1789, MW: 166.7) was prepared at
concentration of 0.4, 0.8, 1.6 and 2.4 g/mL using methanol as the solvent. For
sample preparation, 10 milligrams of each extract (temulawak and turmeric
extract) were dissolved in 10 ml methanol to get a concentration of 1000 g/mL
and diluted to appropriate concentration with methanol as needed. Curcumin has
extensive absorption around 420 nm and can emit the fluorescence around 530 nm
in aqueous solution, but their intensities is very low (Feng Wang, 2006).
Determination of Curcumin Concentration and Comparison
Fluorescence intensity of the samples, as read out from the instrument, was
put into the regression equation (obtained from previous step) to get a final
estimate of curcumin concentration contained in sample. The result was in g/mL
unit which was then converted to mg/g according to the following formula
y'
y
csample
1000
(1)
where y′ is the curcumin concentration in mg/g, y is the concentration in g/mL,
and csample is the sample concentration (100 g/mL for temulawak and 10 g/mL
for turmeric). The factor 1000 was used to convert milligram to gram.
The estimates from this method were evaluated through comparison with
results from HPLC analysis. Percent difference (Pd) was computed using Equation
2 when comparing the two quantities.
6
Pd
c fluorometer cHPLC
cHPLC
with c denoting curcumin concentration in mg/g.
100
(2)
Detection Limit (LOD) and Quantitation Limit (LOQ)
Another important parameter that should be examined is the limit of detection
(LOD) and limit of quantitation (LOQ). The LOQ and LOD value was calculated
directly from the calibration graph. LOQ is defined as the lowest concentration in
the standard curve that can be measured with suitable precision and accuracy
(Mazzarino et al., 2010). LOD is defined as the lowest concentration in the
standard curve that can be detected, but not necessarily quantified as an exact
value (Mazzarino et al., 2010). Approximate value of LOD and LOQ defined by
IUPAC (Long and Winefordner, 1983), are LOD = 3SB/m and LOQ = 10SB/m (SB
is the standard deviation of the blank signals and m is the slope of the calibration
curve).
7
3 RESULTS AND DISCUSSIONS
Fluorimetry, the measurement and use of fluorescence, is a technique of
quantitative chemical analysis ideally suited to field use (Smith et al, 1981).
Fluorimetry is chosen for its extraordinary sensitivity, high specificity, simplicity,
and low cost as compared to other analytical techniques. It is a widely accepted
and powerful technique that is used for variety of environmental, industrial, and
biotechnology applications.
Fluorescence is an absorption and instantaneous re-emission of radiant energy
from a molecule or atom accompanied by a change in wavelength as well as
direction. When a quantum of light is absorbed by a molecule, the molecule is
raised to an excited state. There are a variety of ways the excited molecule can
manifest or dissipate this energy. The simplified diagram below shows absorption
by molecules to produce excited state and then emitted light (Figure 3).
Figure 3. Jablonski Energy Diagram of Fluorescence
Fluorescence is directly proportional to the intensity of the exciting light and
the concentration of the fluorescent material being investigated. The level of
fluorescence is proportional to concentration, frequently over a range of
concentration of several orders of magnitude. The linearity breaks down at a very
high concentration but this can be overcome either by simple dilution or by
preparing a calibration curve of fluorescence versus concentration. The standard
calibration curve of curcumin from the first replicate whereas the regression
parameters and the coefficient determinations (R2) from all experiments and
replications are summarized in Table 1.
8
Table 1. Linear regression parameters for standard calibration curve of curcumin
(y = mx+b where m is the slope, b is the intercept, R2 is the coefficient of
determination, and y is in ppm). BO and TO refer to Bottom Optic and
Top Optic respectively.
1st Replicate
2nd Replicate
3rd Replicate
Setup
m
m
m
b
R2
b
R2
b
R2
(10-2)
(10-2)
(10-2)
BO,
0.9
-0.170 0.98
0.8
-0.150 0.99
0.8
-0.120 0.98
GA500
BO,
0.012 -0.043 0.98 0,011 -0.042 0.99 0.011 -0.030 0.99
GA1000
TO,
1.5
-0.476 0.98
0.7
-0.115 0.99
0.8
-0.065 0.98
GA500
TO,
0,012 -0.015 0.96 0.01
-0.04 0.99 0.011 -0.002 0.98
GA1000
Slope parameters from all cases range from 1.0x10-4 to 1.5x10-2 (Table 1).
Comparing the GA level, the slopes are consistently higher at GA500 (both
bottom and top optic reading) than those at GA1000 by 2 orders of magnitude.
This finding indicates that, at identical fluorescence intensity, curcumin
concentration read out from the instrument at GA500 is larger than that read out at
GA1000. In general, real-time molecule-based experiments, it is beneficial to read
from the bottom of the microplate and not from the top. Bottom reading offers
several advantages for molecule detection. The light collector can be placed closer
to the sample, the cell layer adherent to the bottom of the well, decreasing light
dissipation. This factor positively affects sensitivity.
A measurement from the top of the microplate without lid will always give
higher signal-to-blank ratios (S:B) than measurements from the bottom. This is
mainly due to the fact that the plastic of the bottom of the microplate negatively
affects the light transmission, both for excitation and emission, resulting in lower
overall signals. Light reflection caused by the plastic surface and the plastic type
also increase blank values. This factors affect the value of LOD and LOQ (Table
2).
As seen in Figure 4, relationship between fluorescence intensity and curcumin
standard concentration is linear and positive, with the coefficient of determination
(R2) being more than 0.9 demonstrating a good sensing characteristic.
9
Figure 4. Standard calibration curve of curcumin from the first replicate. X-axis
shows fluorescence intensity (counts) and Y-axis shows curcumin
concentration (g/mL).
The LOD and LOQ were found to be between 0.0165-0.1113 g/mL and
0.0549-0.371 g/mL, respectively for all experiments, indicating that the method
was sufficiently sensitive to be used for curcumin determination in herbal
medicine. Detailed information for LOD and LOQ are summarized in Table 2.
Table 2. LOD and LOQ for quantitative analysis of standard curcumin. BO and
TO refer to Bottom Optic and Top Optic respectively.
Setup
BO, GA500
BO, GA1000
TO, GA500
TO, GA1000
LOD (g/mL)
0.0316
0.0165
0.1113
0.0569
LOQ (g/mL)
0.1053
0.0549
0.3710
0.1896
Fluorescence intensity and curcumin content of samples are given in Table 3
for temulawak and Table 4 for turmeric. Three replications of fluorescence
intensity measurement (per experiment) will be hereinafter denoted as P1, P2 and
10
P3 while three regression equations (per experiment) will be hereinafter referred
to as T1, T2 and T3.
For temulawak, fluorescence intensity is in the range of 24-1734 counts
(Table 3). The intensity is higher at GA1000 than that at GA500 with ratio
between the two reaching up to 75. Comparing both optic positions, bottom
reading gives fluorescence intensity that is smaller than the top reading (at the
same GA level) and their difference ranges between 1-15 counts for GA500 and
154-236 counts for GA1000. Regarding the curcumin content, concentration
varies between 4.2-24.7 mg/g, being maximal in T3 and minimal in T2, as seen in
all replicates (P1-P3) and experiments. For experiment using top optic and
GA500, the curcumin content is relatively higher than the other three experiments.
Table 3. Fluorescence intensity (counts) and curcumin concentration (g/mL) in
temulawak sample. P1, P2, and P3 refer to the 1st, 2nd, and 3rd replicate
respectively of the fluorescence intensity measurement. T1, T2, and T3
refer to the 1st, 2nd, and 3rd regression equations from the standard
calibration curves. BO and TO refer to Bottom Optic and Top Optic
respectively.
Curcumin concentration (g/mL)
Fluorescence intensity
Setup
(counts)
T1
T2
T3
BO, GA500
BO, GA1000
TO, GA500
TO, GA1000
P1
P2
P3
P1
P2
P3
P1
P2
P3
P1
P2
P3
36
24
32
1340
1498
1350
39
38
37
1186
1734
1526
0.154
0.046
0.118
0.118
0.137
0.119
0.109
0.094
0.079
0.127
0.193
0.168
0.138
0.042
0.106
0.105
0.123
0.107
0.158
0.151
0.144
0.078
0.133
0.112
0.168
0.072
0.136
0.117
0.135
0.119
0.247
0.239
0.231
0.128
0.188
0.166
For turmeric, fluorescence intensity ranges from 23 to 634 counts (Table 4).
Same with findings for temulawak, the ratio on GA1000 to GA500 experiments
reaches around 15-30. Bottom optic experiments show lower fluorescence
intensity than top optic with their difference being up to 12 counts for GA500 and
around 75 counts for GA1000. Curcumin content in turmeric ranges between
0.10-2.07 mg/g, which is 12-42 times smaller than those in temulawak. The
concentration is higher in T3 and smaller in T2, hence similar as those obtained
for temulawak.
11
Table 4. Fluorescence intensity (counts) and curcumin concentration (g/mL) in
turmeric sample. P1, P2, and P3 refer to the 1st, 2nd, and 3rd replicate
respectively of the fluorescence intensity measurement. T1, T2, and T3
refer to the 1st, 2nd, and 3rd regression equations from the standard
calibration curves. BO and TO refer to Bottom Optic and Top Optic
respectively.
Curcumin concentration (g/mL)
Fluorescence intensity
Setup
(counts)
T1
T2
T3
BO, GA500
BO, GA1000
TO, GA500
TO, GA1000
P1
P2
P3
P1
P2
P3
P1
P2
P3
P1
P2
P3
23
28
26
559
562
591
34
35
33
500
634
544
0.037
0.082
0.064
0.024
0.022
0.028
0.034
0.049
0.019
0.045
0.061
0.050
0.034
0.074
0.058
0.019
0.019
0.023
0.123
0.130
0.116
0.010
0.023
0.014
0.064
0.104
0.088
0.031
0.031
0.035
0.207
0.215
0.199
0.053
0.068
0.058
The sample concentrations for temulawak and turmeric are different since
they are selected according to the range of fluorescence intensity found in linear
regression of curcumin standard. The determination of this concentration level
plays a main role in estimating the percentage of difference (Pd) against HPLC
measurement. The Figures 5 and 6 present bar charts of Pd for the evaluation of
fluorimetric analysis. For temulawak, curcumin concentration derived from the
fluorometer differs by -60% to 132% with the concentration measured by HPLC
(10.637 mg/g). The best estimates (indicated by smaller absolute Pd) are found in
bottom optic at GA1000 experiment, particularly in T2, P3 combination (Figure
5). This experiment also gives better results when compared with those using
bottom optic at GA500, as seen in all nine combinations. For top optic, curcumin
concentration tends to overestimate those from HPLC (Pd = 2-132%), especially
at GA500.
12
Figure 5. Percentage difference between curcumin concentration in temulawak
from fluorometer and that from HPLC (Y-axis). P1, P2, and P3 refer to
the 1st, 2nd, and 3rd replicate respectively of the fluorescence intensity
measurement. T1, T2, and T3 refer to the 1st, 2nd, and 3rd regression
equation of the curcumin calibration curve. Curcumin concentration
measured by HPLC is 10.636 mg/g.
With regards to turmeric, curcumin content analyzed by the fluorometer tends
to be higher than the one by HPLC (Figure 6). Pd ranges from -70% to 511%,
which is higher than the range found for temulawak. At GA500, better
performance is seen in T2, P1, for both bottom and top optic experiments. At
GA500, top optic yields curcumin content that is consistently larger than the
HPLC and its performance is inferior to the corresponding bottom optic
experiment.
13
Figure 6. Percentage difference between curcumin concentration in turmeric from
fluorometer and that from HPLC (Y-axis). P1, P2, and P3 refer to the
1st, 2nd, and 3rd replicate respectively of the fluorescence intensity
measurement. T1, T2, and T3 refer to the 1st, 2nd, and 3rd regression
equation of the curcumin calibration curve. Curcumin concentration
measured by HPLC is 0.339 mg/g.
14
4 CONCLUSION AND RECOMMENDATION
Conclusion
We have developed a standard procedure for measuring the concentration of
herbal medicines containing temulawak and turmeric, using fluorimetric method.
Three main parts of the study include: preparation of standard calibration curve of
curcumin, determination of curcumin concentration in samples, and comparison
against measurements from HPLC. The standard calibration curve shows linear
and positive relationship between concentration and fluorescence intensity. High
coefficient of determination also indicates that the relationship is strong. For both
samples, fluorometer yields higher fluorescence intensity on higher GA level.
Comparing optic position, top optic reading shows greater intensity than the
bottom optic counterpart. Using information the fluorescence intensity of sample
and regression equation fitting from the curve, the results imply that curcumin
concentration in temulawak is higher compared to that in turmeric. It is partially
due to different source where the samples were obtained. Performance evaluation
of the fluorimetric method was carried out by comparing concentrations derived
from the instrument with that from HPLC. It was found that the fluorometer
estimates are closer to HPLC only for bottom optic measurement at GA1000.
Hence, it can be concluded that this method has a potential to be used as an
alternative quality standard for curcumin content determination in temulawak and
turmeric as a sample.
Recommendation for Future Work
Further research on fluorescence intensity estimation for curcumin standard,
by adding the variation of concentration, is needed. Doing so would be useful to
get a better LOD value and based on the regression equations reported in this
study we could better estimate curcumin level in samples. In additional, this
fluorimetry method should be validated in order to determine whether the method
have been properly developed and are under control.
15
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APPENDICES
18
Appendix 1 Flow chart of the research
Materials and Equipment Preparation
Solution Preparation
Preparation of Standard
Calibration of Curcumin
Measurement of Fluorescence Intensity
for Temulawak and Turmeric Extracts
Determination of Curcumin
Concentration in Samples
Comparison Against
Measurements from
HPLC
Report Arrangement
Determination of
LOD and LOQ
19
Appendix 2 Concentration of temulawak for every fluorescence intensity
Bottom Optic (GA=500)
Temulawak
curcumin (1)
(Intensity)
36
0.154
24
0.046
32
0.118
curcumin (2)
curcumin (3)
0.138
0.042
0.106
0.168
0.072
0.136
curcumin (2)
curcumin (3)
0.1054
0.12278
0.1065
0.1174
0.13478
0.1185
curcumin (1)
curcumin (2)
curcumin (3)
0.109
0.094
0.079
0.158
0.151
0.144
0.247
0.239
0.231
curcumin (1)
curcumin (2)
curcumin (3)
0.12732
0.19308
0.16812
0.0786
0.1334
0.1126
0.12846
0.18874
0.16586
Bottom Optic (GA=1000)
Temulawak
curcumin (1)
(intensity)
1340
0.1178
1498
0.13676
1350
0.119
Top Optic
(GA=500)
Temulawak
(intensity)
39
38
37
Top Optic
(GA=1000)
Temulawak
(intensity)
1186
1734
1526
20
Appendix 3 Concentration of turmeric for every fluorescence intensity
Bottom Optic (GA=500)
turmeric (intensity) curcumin (1)
23
0.037
28
0.082
26
0.064
curcumin (2)
0.034
0.074
0.058
curcumin (3)
0.064
0.104
0.088
Bottom Optic (GA=1000)
turmeric (intensity) curcumin (1)
559
0.02408
562
0.02444
591
0.02792
curcumin (2)
0.01949
0.01982
0.02301
curcumin (3)
0.03149
0.03182
0.03501
Top Optic
(GA=500)
turmeric (intensity)
34
35
33
curcumin (1)
0.034
0.049
0.019
curcumin (2)
0.123
0.13
0.116
curcumin (3)
0.207
0.215
0.199
Top Optic
(GA=1000)
turmeric (intensity)
500
634
544
curcumin (1)
0.045
0.06108
0.05028
curcumin (2)
0.01
0.0234
0.0144
curcumin (3)
0.053
0.06774
0.05784
21
Appendix 4 Percentage difference table
Instrument
Setup
BO500
B01000
TO500
TO1000
Instrument
Setup
BO500
B01000
TO500
TO1000
T1,P1 T2,P1
44.78 29.74
10.94
1.28
2.48 48.55
19.40 25.73
Percentage Difference of temulawak
T3,P1 T1,P2 T2,P2 T3,P2 T1,P3 T2,P3 T3,P3
57.95 56.75 60.51
32.31 10.94
0.34 27.86
10.00 28.80 15.64
26.92 11.88
0.60 11.88
132.2 11.63 41.96 124.70 25.73 35.38 117.18
20.34 81.45 25.04
77.69 57.95
6.24 56.07
T1,P1 T2,P1
9.24
0.38
29.14 43.90
0.38 263.1
32.86 70.48
Percentage Difference of turmeric
T3,P1 T1,P2 T2,P2 T3,P2 T1,P3 T2,P3 T3,P3
88.96 142.10 118.48 69.29 88.96
71.24 159.8
8.47
29.14
40.95
5.52 17.33
32.09
3.34
511.1
44.67 283.82 534.7 43.90 242.49 487.5
56.48
80.10
32.09 100.7 47.62
58.67 71.24
Appendix 5 HPLC data for temulawak extract
Sample
Code
40IV15
Compound
BISDESMETOKSI KURKUMIN
DESMETOKSI KURKUMIN
KURKUMIN
Mass spl
(g)
0.0521
0.0521
0.0521
Diluted to
(mL)
50
50
50
sample
(mg/g)
0.2382
4.1203
10.6365
Sample
(%b/b)
0.0238
0.412
1.0636
Mass spl
(g)
0.0500
0.0500
0.0500
Diluted to
(mL)
50
50
50
sample
(mg/g)
0.1007
0.1301
0.3387
Sample
(%b/b)
0.01007
0.01301
0.03387
Appendix 6 HPLC data for turmeric extract
Sample
Code
050/XI
Compound
BISDESMETOKSI KURKUMIN
DESMETOKSI KURKUMIN
KURKUMIN
22
CURRICULUM VITAE
The author was born in Tangerang on June 20, 1982 as the first child of Eddy
Supriyadi and Ruchyati. The author passed her high school from SMAN 5
Tangerang in 1999. At the same year, pursued undergraduate program at Physics
IPB and then graduated in 2004. The author was accepted as a graduate student at
Biophysics SPS IPB in 2013 and was supported by the Bogor City Government.
Currently, the author works as a science teacher of SMPN 2 Bogor.
CURCUMIN CONCENTRATION IN COMMERCIAL
HERBAL MEDICINES
SITI NURMA NUGRAHA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
DECLARATION OF THESIS AND INFORMATION SOURCES OF
INFORMATION AND PATENT
I hereby declare that the thesis of “Fluorimetric Based Procedure for
Measuring Curcumin Concentration in Commercial Herbal Medicines” is true of
my own work under the direction of thesis committee and has not been submitted
in any form to any university. The content of this thesis has been examined by the
thesis committee and external examiner. Sources of information which is derived
or cited either from published or unpublished scientific paper from other writers
have mentioned in the script and listed in the reference at the end of this thesis.
I hereby assign the copyright of my thesis to Bogor Agricultural
University.
Bogor, May 2016
Siti Nurma Nugraha
ID G751130151
RINGKASAN
SITI NURMA NUGRAHA. Prosedur Pengukuran Berbasis Fluorimetri
untuk Mengukur Konsentrasi Kurkumin di dalam Obat-obat Herbal Komersial.
Dibimbing oleh HUSIN ALATAS dan IRMANIDA BATUBARA.
Masyarakat Indonesia telah secara turun-temurun menggunakan obat herbal
tradisional, yang dikenal sebagai jamu, untuk mengobati dan melindungi tubuh
dari penyakit. Kebiasaan penggunaan jamu ini memberi implikasi akan
pentingnya kebutuhan untuk meningkatkan dan mengembangkan beberapa standar
kualitas jamu. Atas dasar ini, sistem standardisasi yang baik sangat diperlukan
untuk mendapatkan kualitas jamu yang memenuhi standar kelayakan, efektif, dan
berkualitas tinggi. Kunyit (Curcuma longa) dan temulawak (Curcuma
Zanthorrhiza) adalah tanaman obat yang banyak dimanfaatkan sebagai bahan
utama jamu oleh orang Indonesia. Kedua bahan tersebut mengandung kurkumin
yang merupakan komponen aktif utama pada rhizoma (akar rimpang).
Berbagai jenis metode telah dikembangkan dalam penentuan kadar kurkumin
di dalam tumbuhan obat, seperti (1) teknik spektrofotometri UV-Vis, (2) teknik
kromatografi lapisan tipis (Thin-Layer Chromatography), dan (3) teknik High
Performance Liquid Chromatography (HPLC). Meskipun metode-metode
tersebut tersedia dan dapat digunakan, suatu teknik analisis kurkumin yang
penerapannya sederhana, memberikan hasil yang cepat, peka, tepat, dan berbiaya
murah masih menjadi masalah utama di Indonesia. Dalam studi ini kami
mengajukan dan menggunakan sebuah metode penentuan kadar kurkumin
alternatif berbasis fluorimetri. Metode ini dapat diterapkan untuk analisis obat
herbal yang mengandung komponen zat yang dapat berfluoresensi, seperti
kurkumin, yang menunjukkan gejala fluoresensi kuat di dalam pelarut organik.
Metode fluorimetri ini memiliki kadar sensitivitas dan selektivitas yang lebih
tinggi dibandingkan dengan teknik spektrofotometri dan metode ini juga lebih
sederhana dan murah dibandingkan dengan teknik analisis kromatografi.
Tiga tahap utama dalam studi ini meliputi: persiapan kurva standar kalibrasi
kurkumin, penentuan konsentrasi kurkumin di dalam sampel, dan perbandingan
terhadap hasil yang diperoleh menggunakan teknik HPLC. Pengukuran
fluoresensi dilakukan pada fluorometer FLUOstar Omega yang menggunakan
spektrometer UV-Vis. Level fluoresensi yang muncul berbanding lurus dengan
intensitas cahaya yang datang dan konsentrasi dari bahan fluoresen yang diamati.
Kurva kalibrasi standar yang dihasilkan menunjukkan hubungan yang linear dan
positif antara konsentrasi dan intensitas fluoresensi. Tingginya nilai koefisien
determinasi juga menunjukkan bahwa hubungan tersebut sangat jelas. Evaluasi
kinerja dari metode fluorimetri ini dilakukan dengan membandingkan konsentrasikonsentrasi yang diperoleh dari pembacaan hasil fluorometer terhadap hasil yang
diperoleh dari HPLC.
Kata Kunci: fluoresensi, kurkumin, kunyit, temulawak, fluorometer, analisa
HPLC.
SUMMARY
SITI NURMA NUGRAHA. Fluorimetric Based Procedure for Measuring
Curcumin Concentration in Commercial Herbal Medicines. Supervised by HUSIN
ALATAS and IRMANIDA BATUBARA.
People in Indonesia have historically used the traditional herbal medicines,
known as jamu, for the treatment and protection of diseases. It implies a need to
improve and develop some quality standard of jamu. To develop a jamu, various
species of medicinal plants were selected and identified carefully prior extraction
and manufacturing into jamu components. Standardization in this area is
necessary to achieve the quality of jamu that are eligible, effective, and of high
quality. Turmeric (Curcuma longa) and temulawak (Curcuma zanthorrhiza) are
medicinal plants, widely used as the main ingredients in jamu by Indonesian
people. Both have curcumin as the major active components of the rhizomes.
Numerous methods have been developed on curcumin determination in
medicinal plants such as (1) UV-vis spectrophotometry, (2) Thin Layer
Chromatography, and (3) High Performance Liquid Chromatography (HPLC).
Despite the availability of these methods, the implementation of a simple, rapid,
sensitive, precise, and more economic technique for curcumin analysis is still
considered an important issue in Indonesia. An alternative fluorimetric method is
proposed and used in this study. It can be applied for the analysis of herbal
medicines containing a fluorescent component, such as curcumin, that exhibits
strong fluorescence in organic solvents. The fluorimetric method has higher
sensitivity and selectivity compared to the spectrophotometric technique and it is
also simpler and less expensive compared to the chromatographic analysis.
Three main parts of this study include: preparation of standard calibration
curve of curcumin, determination of curcumin concentration in samples, and
comparison against measurements from HPLC. Fluorescence measurements were
performed on a FLUOstar Omega fluorometer which uses a UV-vis spectrometer.
Fluorescence is directly proportional to the intensity of the exciting light and the
concentration of the fluorescent material being investigated. The standard
calibration curve shows linear and positive relationship between concentration and
fluorescence intensity. High coefficient of determination also indicates that the
relationship is strong. Performance evaluation of the fluorimetric method was
carried out by comparing concentrations derived from the instrument with that
from HPLC.
Keyword : fluorescence, curcumin, turmeric, temulawak, fluorometer, HPLC
analysis
© Copyright of IPB, the year 2016
Copyright reserved by the law
Forbidden to quote part or all of these writings without including or
mentioning the source. Citing is only for educational purposes, research, writing
papers, drafting reports, writing criticism, or review an issue, and citing it does
not harm the interests of fair Bogor Agricultural University.
Prohibited announced and reproduce part or the whole paper in any form
without permission from Bogor Agricultural University.
FLUORIMETRIC BASED PROCEDURE FOR MEASURING
CURCUMIN CONCENTRATION IN COMMERCIAL
HERBAL MEDICINES
SITI NURMA NUGRAHA
A Thesis submitted in partial fulfillment of the
requirement for Master Degree
in Biophysics Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
External examiner :
Dr. Kiagus Dahlan
Thesis title
Name
ID
: Fluorimetric Based Procedure for Measuring Curcumin
Concentration in Commercial Herbal Medicines
: Siti Nurma Nugraha
: G751130151
Approved by
The Commission of Supervisors
Dr. Husin Alatas, M.Si
Supervisor
Dr. Irmanida Batubara, M.Si
Co-Supervisor
Certified by:
Head of Biophysics
Graduate Program
Dean of the IPB Graduate School
Dr. MersiKurniati, M.Si
Dr.Ir. DahrulSyah, MSc.Agr
Examination Date : May 20th 2016
Graduation date :
ACKNOWLEDGEMENTS
Firstly, I am grateful to Allah SWT for this amazing life, abundant favors
and blessings during my graduate study until I finish this thesis.
I wish to thank Dr. Husin Alatas as my thesis supervisor for devoting his
time for discussion and giving me his continued support, motivation and
suggestions for my research work. I have learned and improved more than I ever
expected.
I also would like to thank my thesis co-supervisor, Dr. Irmanida Batubara
for her expertise, guidance, and comments. My thanks also go to Dr. Kiagus
Dahlan as my thesis external examiner for his valuable suggestion and motivation,
Dr. Akhiruddin Maddu, Tony Sumaryada, PhD and Dr. Mersi Kurniati for their
support and useful suggestion, and also Dr. Mamat Rahmat and Sugianto Arjo,
M.Si for their helpful contribution.
I would like to thank the people at Biofarmaka Research Center, in
particular, Ms. Nunuk Kurniati, Ms. Laela Wulan Sari, Ms. Wiwi Widiyanti and
Mr. Endi Suhendi for their general help and technical support. I thank my
classmates in Biophysics IPB who all have been great friends and helped me in
this study.
This master study opportunity was made possible in part by the support
from the Bogor City Government and Principal of SMPN 2 Bogor. I also thank
my colleagues in SMPN 2 Bogor for their support, especially Ibu Yani Herliani,
M.Pd for her profound encouragement.
I would like to give my special gratitude to my husband for his
tremendous love and patience, my parents for giving those powerful words of
advice, my loving sister and brother for always being truthful to me. Without their
love and companionship, I could not go on to achieve what I have today.
Lastly, I dedicate this thesis to my students who have become the sources
of my inspiration.
Bogor, May 2016
Siti Nurma Nugraha
TABLE OF CONTENS
TABLE LIST
ii
FIGURE LIST
iii
APPENDICES LIST
iv
1 INTRODUCTION
Background
Objective
Hypotheses
Benefit
1
2
2
3
3
2 MATERIALS AND METHODS
Instruments
Fluorimetric Method
Chemical and Reagents
Determination of Curcumin Concentration and Comparison
Detection Limit (LOD) and Quantitation Limit (LOQ)
4
4
4
5
5
6
3 RESULTS AND DISCUSSION
7
4 CONCLUSION AND RECOMMENDATION
14
REFERENCES
15
APPENDICES
17
TABLE LIST
1 Linear Regression Parameters of Calibration Curve for Curcumin
2 LOD and LOQ value
3 Curcumin Concentration in Temulawak
4 Curcumin Concentration in Turmeric
8
9
10
11
FIGURE LIST
1 Chemical Structure of Curcumin
2 Fluorometer FLUOstar Omega
3 Fluorescence Diagram
4 Standard Calibration Curve
5 Percentage Difference Diagram of Temulawak
6 Percentage Difference Diagram of Turmeric
1
4
7
9
12
13
APPENDIX LIST
1 Flow chart of research
2 Concentration of temulawak for every fluorescence intensity
3 Concentration of turmeric for every fluorescence intensity
4 Percentage difference table
5 HPLC data for temulawak extract
6 HPLC data for turmeric extract
18
19
20
21
21
21
1
1 INTRODUCTION
Background
Indonesia has the second biggest biodiversity in the world expressed by
30,000 plant species and is a home to about 80% of the world’s medicinal plants.
Based on this plenteous source of medicinal plants, it is estimated that 40 million
of Indonesian have historically used the traditional herbal medicines, known as
jamu, for the treatment and protection of diseases (Elfahmi et al., 2014). In the
country, jamu is popular and common practice on diet and general health. It
implies a need to improve and develop some quality standard of jamu. Various
researches utilizing advanced technology have been carried out to improve the
quality of jamu and, eventually, to further boost consumer confidence. To develop
a jamu, various species of medicinal plants were selected and identified carefully
prior extraction and manufacturing into jamu components. Standardization in this
area is necessary to achieve the quality of jamu that are eligible, effective, and of
high quality.
Turmeric (Curcuma longa) and temulawak (Curcuma zanthorrhiza) are
medicinal plants, widely used as the main ingredients in jamu by Indonesian
people.
Both
main
ingredients
have
curcumin
[1,7-bis
(4-hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-Dione] (Figure 1) as the major
active components of the rhizomes (Tonnesen et al., 1992; Sutrisno et al., 2008).
Curcumin, having nearly a two centuries old scientific history, is still attracting
researchers from all over the world. Starting from 1815, when curcumin was first
isolated from turmeric, there were only a few reports till the 1970s on its chemical
structure, synthesis, biochemical and antioxidant activity (Sharma, 1976). It has
three chemical entities in its structure: two aromatic ring systems containing
o-methoxy phenolic groups, connected by a seven carbon linker consisting of an
α,β-unsaturated β-diketone moiety (Grykiewicz, 2012).
Recent pharmacological researches revealed that curcumin is functioned as
anti-oxidant (Namita et al., 2012), anti-inflammatory (Anand et al., 2008),
anti-carcinogenic (Basnet et al., 2011), anti-diabetes (Merrel et al., 2009),
antimicrobial effects (Maheshwari et al., 2006), anti-tumor (Kunnumakkara et al.,
2007), anti-bacterial and anti-coagulant (Maiti et al., 2007).
Figure 1. Chemical structure of curcumin
2
Numerous methods have been developed on curcumin determination in
medicinal plants such as (1) UV-vis spectrophotometry (Jasim et al., 1988;
Sharma et al., 2012; Liu et al., 2016), (2) Thin Layer Chromatography (TLC;
Zhang et al., 2007), and (3) High Performance Liquid Chromatography (HPLC;
Pak et al., 2003). Although spectrophotometry is widely used for the
quantification of curcumin, it lacks of reproducibility (Govindrajan, 1980) and
need some complicated sample-preparation steps (Liu et al., 2016). On the other
hand, the chromatographic methods (TLC and HPLC) give some good
performances in producing simultaneous separation and determination of various
components with high accuracy and good precision (Yang et al., 2012). The HPLC
analysis has also been employed to detect low quantities of curcumin in biofluids
(Kim et al., 2013). However, it has the disadvantages of being more
time-consuming and expensive (Bisht et al., 2007; Nam et al., 2007; Tiyaboonchai
et al., 2007; Sou et al., 2008).
Despite the availability of these methods, the implementation of a simple,
rapid, sensitive, precise, and more economic technique for curcumin analysis is
still considered an important issue in Indonesia. An alternative fluorimetric
method (Diaz et al., 1992; Mazzarino et al., 2010) is proposed and used in this
study. It can be applied for the analysis of herbal medicines containing a
fluorescent component, such as curcumin, that exhibits strong fluorescence in
organic solvents (Diaz et al., 1992). The fluorimetric method has higher sensitivity
and selectivity compared to the spectrophotometric technique and it is also
simpler and less expensive compared to the chromatographic analysis (Zhang et
al, 2007). However, a standard procedure based on fluorimetric method for
measuring the curcumin concentration in commercial herbal medicines has not yet
been addressed scientifically.
Objectives
The objectives of this study are as follows:
1. To develope the corresponding simple procedure based on the corresponding
fluorimetric method for measuring curcumin concentration in two commercial
herbal medicines, containing turmeric and temulawak
2. To evaluate the result by comparison with the HPLC analysis.
Hypotheses
The relationship between fluorescence intensity and curcumin standard
concentration is expected to be linear and positive, and to show an acceptable
accuracy when compared with the HPLC measurement.
3
Benefits
This study is expected to have new standard procedure for measuring
concentration of curcumin in Indonesian commercial herbal medicines with have
some beneficial effect in technical and economic.
4
2 MATERIALS AND METHODS
Instruments
Fluorescence measurements were performed on a FLUOstar Omega
fluorometer (BMG Labtech, USA) which uses a UV-vis spectrometer that
instantaneously captures wavelengths in a range of 220-850 nm at 1 nm resolution
(Figure 2). This device do not need scanning or calibration since there is no
installed monochromator, and it is equipped with a high-intensity xenon flash
lamp and selected PMTs, coupled with high transmission band pass filters, to
provide a stable, low-noise signal platform for ultra-sensitive fluorescence
reading. All the measurements were carried out in triplicate (three repetitions).
Figure 2. Fluorometer FLUOstar Omega
Fluorimetric Method
Since fluorescent materials are relatively rare (which limits the application of
the techniques), fluorimetry is also a more specific analytic technique than
absorption spectroscopy. A useful aspect of fluorescence specificity is that the
fluorescent molecule has two characteristic spectra: the excitation spectrum (the
relative efficiency of different incidence wavelengths that cause fluorescence),
and the emission spectrum (the relative spectral distribution of emitted light). The
wavelength of the emission is always longer than the wavelength of excitation.
Also, the fluorescence emission spectrum is specific to the material. Thus, it is
5
possible to avoid interfering radiation caused by direct scattering of the exciting
radiation or by fluorescence of other substances, by using spectrally selective
filters in the fluorescence detectors. For this study, the excitation and emission
wavelengths were 420 nm and 530 nm, respectively (Wang et al, 2006). The
fluorescence intensity of all curcumin standards was measured by four different
experiments: (1) Bottom optic with Gain Adjustment (GA) equal to 500; (2)
Bottom optic with GA = 1000, (3) Top optic with GA = 500, and (4) Top optic
with GA = 1000. Bottom and top optics are illumination optics inside fluorometer
which are used to measure fluorescence, luminescence, and absorbance. Their
difference is located at the optic position that determines the light inlet, which is at
the left (right) side of reagent box for bottom (top) reading. GA is a parameter for
light intensity that is projected into the sample. The two selected GA levels (500
and 1000) have been tested to give optimum fluorescence intensity. Next, the
fluorescence intensity was measured by the fluorometer for the above series of
standard solutions. The calibration graph was obtained by plotting the
fluorescence intensity of the standard solutions against the theoretical standard
concentrations. The linearity was evaluated by linear least-square regression
analysis.
Chemical and Reagents
Curcumin (ChromaDex: CDXA-09-1789, MW: 166.7) was prepared at
concentration of 0.4, 0.8, 1.6 and 2.4 g/mL using methanol as the solvent. For
sample preparation, 10 milligrams of each extract (temulawak and turmeric
extract) were dissolved in 10 ml methanol to get a concentration of 1000 g/mL
and diluted to appropriate concentration with methanol as needed. Curcumin has
extensive absorption around 420 nm and can emit the fluorescence around 530 nm
in aqueous solution, but their intensities is very low (Feng Wang, 2006).
Determination of Curcumin Concentration and Comparison
Fluorescence intensity of the samples, as read out from the instrument, was
put into the regression equation (obtained from previous step) to get a final
estimate of curcumin concentration contained in sample. The result was in g/mL
unit which was then converted to mg/g according to the following formula
y'
y
csample
1000
(1)
where y′ is the curcumin concentration in mg/g, y is the concentration in g/mL,
and csample is the sample concentration (100 g/mL for temulawak and 10 g/mL
for turmeric). The factor 1000 was used to convert milligram to gram.
The estimates from this method were evaluated through comparison with
results from HPLC analysis. Percent difference (Pd) was computed using Equation
2 when comparing the two quantities.
6
Pd
c fluorometer cHPLC
cHPLC
with c denoting curcumin concentration in mg/g.
100
(2)
Detection Limit (LOD) and Quantitation Limit (LOQ)
Another important parameter that should be examined is the limit of detection
(LOD) and limit of quantitation (LOQ). The LOQ and LOD value was calculated
directly from the calibration graph. LOQ is defined as the lowest concentration in
the standard curve that can be measured with suitable precision and accuracy
(Mazzarino et al., 2010). LOD is defined as the lowest concentration in the
standard curve that can be detected, but not necessarily quantified as an exact
value (Mazzarino et al., 2010). Approximate value of LOD and LOQ defined by
IUPAC (Long and Winefordner, 1983), are LOD = 3SB/m and LOQ = 10SB/m (SB
is the standard deviation of the blank signals and m is the slope of the calibration
curve).
7
3 RESULTS AND DISCUSSIONS
Fluorimetry, the measurement and use of fluorescence, is a technique of
quantitative chemical analysis ideally suited to field use (Smith et al, 1981).
Fluorimetry is chosen for its extraordinary sensitivity, high specificity, simplicity,
and low cost as compared to other analytical techniques. It is a widely accepted
and powerful technique that is used for variety of environmental, industrial, and
biotechnology applications.
Fluorescence is an absorption and instantaneous re-emission of radiant energy
from a molecule or atom accompanied by a change in wavelength as well as
direction. When a quantum of light is absorbed by a molecule, the molecule is
raised to an excited state. There are a variety of ways the excited molecule can
manifest or dissipate this energy. The simplified diagram below shows absorption
by molecules to produce excited state and then emitted light (Figure 3).
Figure 3. Jablonski Energy Diagram of Fluorescence
Fluorescence is directly proportional to the intensity of the exciting light and
the concentration of the fluorescent material being investigated. The level of
fluorescence is proportional to concentration, frequently over a range of
concentration of several orders of magnitude. The linearity breaks down at a very
high concentration but this can be overcome either by simple dilution or by
preparing a calibration curve of fluorescence versus concentration. The standard
calibration curve of curcumin from the first replicate whereas the regression
parameters and the coefficient determinations (R2) from all experiments and
replications are summarized in Table 1.
8
Table 1. Linear regression parameters for standard calibration curve of curcumin
(y = mx+b where m is the slope, b is the intercept, R2 is the coefficient of
determination, and y is in ppm). BO and TO refer to Bottom Optic and
Top Optic respectively.
1st Replicate
2nd Replicate
3rd Replicate
Setup
m
m
m
b
R2
b
R2
b
R2
(10-2)
(10-2)
(10-2)
BO,
0.9
-0.170 0.98
0.8
-0.150 0.99
0.8
-0.120 0.98
GA500
BO,
0.012 -0.043 0.98 0,011 -0.042 0.99 0.011 -0.030 0.99
GA1000
TO,
1.5
-0.476 0.98
0.7
-0.115 0.99
0.8
-0.065 0.98
GA500
TO,
0,012 -0.015 0.96 0.01
-0.04 0.99 0.011 -0.002 0.98
GA1000
Slope parameters from all cases range from 1.0x10-4 to 1.5x10-2 (Table 1).
Comparing the GA level, the slopes are consistently higher at GA500 (both
bottom and top optic reading) than those at GA1000 by 2 orders of magnitude.
This finding indicates that, at identical fluorescence intensity, curcumin
concentration read out from the instrument at GA500 is larger than that read out at
GA1000. In general, real-time molecule-based experiments, it is beneficial to read
from the bottom of the microplate and not from the top. Bottom reading offers
several advantages for molecule detection. The light collector can be placed closer
to the sample, the cell layer adherent to the bottom of the well, decreasing light
dissipation. This factor positively affects sensitivity.
A measurement from the top of the microplate without lid will always give
higher signal-to-blank ratios (S:B) than measurements from the bottom. This is
mainly due to the fact that the plastic of the bottom of the microplate negatively
affects the light transmission, both for excitation and emission, resulting in lower
overall signals. Light reflection caused by the plastic surface and the plastic type
also increase blank values. This factors affect the value of LOD and LOQ (Table
2).
As seen in Figure 4, relationship between fluorescence intensity and curcumin
standard concentration is linear and positive, with the coefficient of determination
(R2) being more than 0.9 demonstrating a good sensing characteristic.
9
Figure 4. Standard calibration curve of curcumin from the first replicate. X-axis
shows fluorescence intensity (counts) and Y-axis shows curcumin
concentration (g/mL).
The LOD and LOQ were found to be between 0.0165-0.1113 g/mL and
0.0549-0.371 g/mL, respectively for all experiments, indicating that the method
was sufficiently sensitive to be used for curcumin determination in herbal
medicine. Detailed information for LOD and LOQ are summarized in Table 2.
Table 2. LOD and LOQ for quantitative analysis of standard curcumin. BO and
TO refer to Bottom Optic and Top Optic respectively.
Setup
BO, GA500
BO, GA1000
TO, GA500
TO, GA1000
LOD (g/mL)
0.0316
0.0165
0.1113
0.0569
LOQ (g/mL)
0.1053
0.0549
0.3710
0.1896
Fluorescence intensity and curcumin content of samples are given in Table 3
for temulawak and Table 4 for turmeric. Three replications of fluorescence
intensity measurement (per experiment) will be hereinafter denoted as P1, P2 and
10
P3 while three regression equations (per experiment) will be hereinafter referred
to as T1, T2 and T3.
For temulawak, fluorescence intensity is in the range of 24-1734 counts
(Table 3). The intensity is higher at GA1000 than that at GA500 with ratio
between the two reaching up to 75. Comparing both optic positions, bottom
reading gives fluorescence intensity that is smaller than the top reading (at the
same GA level) and their difference ranges between 1-15 counts for GA500 and
154-236 counts for GA1000. Regarding the curcumin content, concentration
varies between 4.2-24.7 mg/g, being maximal in T3 and minimal in T2, as seen in
all replicates (P1-P3) and experiments. For experiment using top optic and
GA500, the curcumin content is relatively higher than the other three experiments.
Table 3. Fluorescence intensity (counts) and curcumin concentration (g/mL) in
temulawak sample. P1, P2, and P3 refer to the 1st, 2nd, and 3rd replicate
respectively of the fluorescence intensity measurement. T1, T2, and T3
refer to the 1st, 2nd, and 3rd regression equations from the standard
calibration curves. BO and TO refer to Bottom Optic and Top Optic
respectively.
Curcumin concentration (g/mL)
Fluorescence intensity
Setup
(counts)
T1
T2
T3
BO, GA500
BO, GA1000
TO, GA500
TO, GA1000
P1
P2
P3
P1
P2
P3
P1
P2
P3
P1
P2
P3
36
24
32
1340
1498
1350
39
38
37
1186
1734
1526
0.154
0.046
0.118
0.118
0.137
0.119
0.109
0.094
0.079
0.127
0.193
0.168
0.138
0.042
0.106
0.105
0.123
0.107
0.158
0.151
0.144
0.078
0.133
0.112
0.168
0.072
0.136
0.117
0.135
0.119
0.247
0.239
0.231
0.128
0.188
0.166
For turmeric, fluorescence intensity ranges from 23 to 634 counts (Table 4).
Same with findings for temulawak, the ratio on GA1000 to GA500 experiments
reaches around 15-30. Bottom optic experiments show lower fluorescence
intensity than top optic with their difference being up to 12 counts for GA500 and
around 75 counts for GA1000. Curcumin content in turmeric ranges between
0.10-2.07 mg/g, which is 12-42 times smaller than those in temulawak. The
concentration is higher in T3 and smaller in T2, hence similar as those obtained
for temulawak.
11
Table 4. Fluorescence intensity (counts) and curcumin concentration (g/mL) in
turmeric sample. P1, P2, and P3 refer to the 1st, 2nd, and 3rd replicate
respectively of the fluorescence intensity measurement. T1, T2, and T3
refer to the 1st, 2nd, and 3rd regression equations from the standard
calibration curves. BO and TO refer to Bottom Optic and Top Optic
respectively.
Curcumin concentration (g/mL)
Fluorescence intensity
Setup
(counts)
T1
T2
T3
BO, GA500
BO, GA1000
TO, GA500
TO, GA1000
P1
P2
P3
P1
P2
P3
P1
P2
P3
P1
P2
P3
23
28
26
559
562
591
34
35
33
500
634
544
0.037
0.082
0.064
0.024
0.022
0.028
0.034
0.049
0.019
0.045
0.061
0.050
0.034
0.074
0.058
0.019
0.019
0.023
0.123
0.130
0.116
0.010
0.023
0.014
0.064
0.104
0.088
0.031
0.031
0.035
0.207
0.215
0.199
0.053
0.068
0.058
The sample concentrations for temulawak and turmeric are different since
they are selected according to the range of fluorescence intensity found in linear
regression of curcumin standard. The determination of this concentration level
plays a main role in estimating the percentage of difference (Pd) against HPLC
measurement. The Figures 5 and 6 present bar charts of Pd for the evaluation of
fluorimetric analysis. For temulawak, curcumin concentration derived from the
fluorometer differs by -60% to 132% with the concentration measured by HPLC
(10.637 mg/g). The best estimates (indicated by smaller absolute Pd) are found in
bottom optic at GA1000 experiment, particularly in T2, P3 combination (Figure
5). This experiment also gives better results when compared with those using
bottom optic at GA500, as seen in all nine combinations. For top optic, curcumin
concentration tends to overestimate those from HPLC (Pd = 2-132%), especially
at GA500.
12
Figure 5. Percentage difference between curcumin concentration in temulawak
from fluorometer and that from HPLC (Y-axis). P1, P2, and P3 refer to
the 1st, 2nd, and 3rd replicate respectively of the fluorescence intensity
measurement. T1, T2, and T3 refer to the 1st, 2nd, and 3rd regression
equation of the curcumin calibration curve. Curcumin concentration
measured by HPLC is 10.636 mg/g.
With regards to turmeric, curcumin content analyzed by the fluorometer tends
to be higher than the one by HPLC (Figure 6). Pd ranges from -70% to 511%,
which is higher than the range found for temulawak. At GA500, better
performance is seen in T2, P1, for both bottom and top optic experiments. At
GA500, top optic yields curcumin content that is consistently larger than the
HPLC and its performance is inferior to the corresponding bottom optic
experiment.
13
Figure 6. Percentage difference between curcumin concentration in turmeric from
fluorometer and that from HPLC (Y-axis). P1, P2, and P3 refer to the
1st, 2nd, and 3rd replicate respectively of the fluorescence intensity
measurement. T1, T2, and T3 refer to the 1st, 2nd, and 3rd regression
equation of the curcumin calibration curve. Curcumin concentration
measured by HPLC is 0.339 mg/g.
14
4 CONCLUSION AND RECOMMENDATION
Conclusion
We have developed a standard procedure for measuring the concentration of
herbal medicines containing temulawak and turmeric, using fluorimetric method.
Three main parts of the study include: preparation of standard calibration curve of
curcumin, determination of curcumin concentration in samples, and comparison
against measurements from HPLC. The standard calibration curve shows linear
and positive relationship between concentration and fluorescence intensity. High
coefficient of determination also indicates that the relationship is strong. For both
samples, fluorometer yields higher fluorescence intensity on higher GA level.
Comparing optic position, top optic reading shows greater intensity than the
bottom optic counterpart. Using information the fluorescence intensity of sample
and regression equation fitting from the curve, the results imply that curcumin
concentration in temulawak is higher compared to that in turmeric. It is partially
due to different source where the samples were obtained. Performance evaluation
of the fluorimetric method was carried out by comparing concentrations derived
from the instrument with that from HPLC. It was found that the fluorometer
estimates are closer to HPLC only for bottom optic measurement at GA1000.
Hence, it can be concluded that this method has a potential to be used as an
alternative quality standard for curcumin content determination in temulawak and
turmeric as a sample.
Recommendation for Future Work
Further research on fluorescence intensity estimation for curcumin standard,
by adding the variation of concentration, is needed. Doing so would be useful to
get a better LOD value and based on the regression equations reported in this
study we could better estimate curcumin level in samples. In additional, this
fluorimetry method should be validated in order to determine whether the method
have been properly developed and are under control.
15
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APPENDICES
18
Appendix 1 Flow chart of the research
Materials and Equipment Preparation
Solution Preparation
Preparation of Standard
Calibration of Curcumin
Measurement of Fluorescence Intensity
for Temulawak and Turmeric Extracts
Determination of Curcumin
Concentration in Samples
Comparison Against
Measurements from
HPLC
Report Arrangement
Determination of
LOD and LOQ
19
Appendix 2 Concentration of temulawak for every fluorescence intensity
Bottom Optic (GA=500)
Temulawak
curcumin (1)
(Intensity)
36
0.154
24
0.046
32
0.118
curcumin (2)
curcumin (3)
0.138
0.042
0.106
0.168
0.072
0.136
curcumin (2)
curcumin (3)
0.1054
0.12278
0.1065
0.1174
0.13478
0.1185
curcumin (1)
curcumin (2)
curcumin (3)
0.109
0.094
0.079
0.158
0.151
0.144
0.247
0.239
0.231
curcumin (1)
curcumin (2)
curcumin (3)
0.12732
0.19308
0.16812
0.0786
0.1334
0.1126
0.12846
0.18874
0.16586
Bottom Optic (GA=1000)
Temulawak
curcumin (1)
(intensity)
1340
0.1178
1498
0.13676
1350
0.119
Top Optic
(GA=500)
Temulawak
(intensity)
39
38
37
Top Optic
(GA=1000)
Temulawak
(intensity)
1186
1734
1526
20
Appendix 3 Concentration of turmeric for every fluorescence intensity
Bottom Optic (GA=500)
turmeric (intensity) curcumin (1)
23
0.037
28
0.082
26
0.064
curcumin (2)
0.034
0.074
0.058
curcumin (3)
0.064
0.104
0.088
Bottom Optic (GA=1000)
turmeric (intensity) curcumin (1)
559
0.02408
562
0.02444
591
0.02792
curcumin (2)
0.01949
0.01982
0.02301
curcumin (3)
0.03149
0.03182
0.03501
Top Optic
(GA=500)
turmeric (intensity)
34
35
33
curcumin (1)
0.034
0.049
0.019
curcumin (2)
0.123
0.13
0.116
curcumin (3)
0.207
0.215
0.199
Top Optic
(GA=1000)
turmeric (intensity)
500
634
544
curcumin (1)
0.045
0.06108
0.05028
curcumin (2)
0.01
0.0234
0.0144
curcumin (3)
0.053
0.06774
0.05784
21
Appendix 4 Percentage difference table
Instrument
Setup
BO500
B01000
TO500
TO1000
Instrument
Setup
BO500
B01000
TO500
TO1000
T1,P1 T2,P1
44.78 29.74
10.94
1.28
2.48 48.55
19.40 25.73
Percentage Difference of temulawak
T3,P1 T1,P2 T2,P2 T3,P2 T1,P3 T2,P3 T3,P3
57.95 56.75 60.51
32.31 10.94
0.34 27.86
10.00 28.80 15.64
26.92 11.88
0.60 11.88
132.2 11.63 41.96 124.70 25.73 35.38 117.18
20.34 81.45 25.04
77.69 57.95
6.24 56.07
T1,P1 T2,P1
9.24
0.38
29.14 43.90
0.38 263.1
32.86 70.48
Percentage Difference of turmeric
T3,P1 T1,P2 T2,P2 T3,P2 T1,P3 T2,P3 T3,P3
88.96 142.10 118.48 69.29 88.96
71.24 159.8
8.47
29.14
40.95
5.52 17.33
32.09
3.34
511.1
44.67 283.82 534.7 43.90 242.49 487.5
56.48
80.10
32.09 100.7 47.62
58.67 71.24
Appendix 5 HPLC data for temulawak extract
Sample
Code
40IV15
Compound
BISDESMETOKSI KURKUMIN
DESMETOKSI KURKUMIN
KURKUMIN
Mass spl
(g)
0.0521
0.0521
0.0521
Diluted to
(mL)
50
50
50
sample
(mg/g)
0.2382
4.1203
10.6365
Sample
(%b/b)
0.0238
0.412
1.0636
Mass spl
(g)
0.0500
0.0500
0.0500
Diluted to
(mL)
50
50
50
sample
(mg/g)
0.1007
0.1301
0.3387
Sample
(%b/b)
0.01007
0.01301
0.03387
Appendix 6 HPLC data for turmeric extract
Sample
Code
050/XI
Compound
BISDESMETOKSI KURKUMIN
DESMETOKSI KURKUMIN
KURKUMIN
22
CURRICULUM VITAE
The author was born in Tangerang on June 20, 1982 as the first child of Eddy
Supriyadi and Ruchyati. The author passed her high school from SMAN 5
Tangerang in 1999. At the same year, pursued undergraduate program at Physics
IPB and then graduated in 2004. The author was accepted as a graduate student at
Biophysics SPS IPB in 2013 and was supported by the Bogor City Government.
Currently, the author works as a science teacher of SMPN 2 Bogor.