Validation of UV-Vis Spectrophotometric Method of

Quercetin in Ethanol Extract of Tamarind Leaf

Erma Yunita1, Deni Yulianto, Siti Fatimah, Tirsa Firanita
Akademi Farmasi Indonesia, Jl. Veteran Gg. Jambu, Pandeyan, Umbulharjo, Kota Yogyakarta, Daerah Istimewa Yogyakarta 55161.

Abstract
Tamarindus indica L is a medicinal plant that has many benefits. Data of article
One of the chemical compounds contained therein is the flavonoid Received : 24 Feb 2020
quercetin type. The number of herbal products on the market Reviewed : 12 May 2020 makes
the quality assurance of herbal products need to be done by Accepted : 4 Jul 2020
perf
orming the assay of the active compounds using validated methods. This
study aims to validate the assay method quercetin DOI
in the extract of tamarind leaves. Tamarind leaf extract was 10.18196/jfaps.010102
macerated with hexane; then, it was re-macerated with 70% ethanol. The extract was
concentrated using a rotary evaporator. Type of article: The assay was performed
using the UV-Vis Spectrophotometry Research method, and parameter validation
specified in this study, including linearity, LOD, LOQ, precision, and accuracy.
Quercetin level obtained in extracts of tamarind leaves was at 21.52 mg/g. Based on
the test method validation, the correlation coefficient (r) was 0.9999, the regression
function coefficients (Vx0) was 0.59545%, LOD 0.1515 ppm, LOQ 0.4592 ppm,
coefficient of variation precision was < 2%, and recoveries range was in 97-103%.

Keywords: Tamarind; Tamarindus indica L.; UV-Vis

Spectrophotometric; Validation

INTRODUCTION deactivating enzymes and disrupting the

cell wall; thus, it has good bactericidal
Tamarindus indica L. can be used for many properties.3 Quercetin in plants is in
purposes, such as herbs, medicinal various forms of glycosides.4
materials, and cosmetics. Furthermore,
tamarind fruit pulp can be used as a mixture There are a considerable amount of
of traditional medicine and herbs.1,2 benefits of tamarind leaves, which can
Flavonoid compounds contained in increase the development of product
tamarind leaves are a kind of flavonoid innovation.5 Quality assurance of herbal
quercetin. Quercetin has potential as products can be done by performing the
antiviral, antibacterial, and assay of the active compounds using
antiinflammatory.1 Quercetin has a good methods that have been validated.6 The
antibacterial activity for the phenol group current widely used analytical method is
with protein coagulation mechanism by the UV-Vis spectrophotometry method.
1 Corresponding author, e-mail: ermayunita@afi.ac.id
 12 Erma Yunita , Deni Yulianto, Siti Fatimah, Tirsa Firanita | Validation of UV-Vis Spectrophotometric Method of
Quercetin in Ethanol Extract of Tamarind Leaf
The method provides a simple way to Preparation of stock solutions
determine the quantity of a little substance. The stock solution of quercetin and
The method used in the assay should be tamarind leaf extract was made at a
validated. Validation of the methods of concentration of 10,000 ppm. Stock
analysis is an assessment of the actions of solutions were created using Ethanol pa.
certain parameters based on laboratory
experiments. This activity is carried out to Determination of maximum wavelength
prove the parameters used to meet the The stock concentration of 100 ppm as
requirements for its use. Validation can also much as ± 3 ml was measured with a
be used to identify the spectrophotometer at a wavelength of
source of the unwanted variability.7 200-800 nm.

METHODS Preparation of quercetin standard curve

The standard curve was created by
The material used in this study was connecting the standard solution
tamarind leaves (Tamarindus indica L.) from concentration in the series concentration
Mantrijeron, Yogyakarta. Other materials of 4 ppm, 6 ppm, 8 ppm, 10 ppm, and 12
used included ethanol pro analysis (Merck), ppm with the absorption results obtained
70% ethanol (CV. from the measurement using UV-Vis
General Labora), filter paper, Quercetin spectrophotometry at maximum
(Sigma Aldrich), aquadest (CV. General wavelength.8
Labora), and aluminum foil. The analysis Determination of my quercetin levels in
instrument used in this study was the the tamarind leaf extract
Genesys 10S UV-Vis Spectrophotometer. Tamarind leaf extract of a concentration of
500 ppm measured its absorbance using
Preparation of Tamarind Leaves Extract UV-Vis spectrophotometry at a maximum
Tamarind leaf was macerated with wavelength. Sample solutions were made
nhexane then stirred using an electric in 3 trials.
stirrer for 1 hour. It was then soaked for 24
hours. The results of the maceration were Validation of Analysis Methods
filtered. The residue obtained was then Linearity was conducted by measuring the
dried and macerated by 70% ethanol and absorbance of the comparison solution.
then stirred using an electric stirrer for 1 Furthermore, the curve of the relationship
hour. The soaking process was conducted between concentration vs. absorption and
for 24 hours to protect it from light, while the linear regression equation and the
it was occasionally stirred. The results of correlation coefficient (x) and the
maceration were filtered to separate the correlation coefficient of the function (V xo)
pulp and filtrate. The macerating process were determined. The regression function
was concentrated by a rotary evaporator coefficient (Vxo) can be calculated using the
and thickened above the water bath until a formula below.6
thick extract was formed. The ethanol
extract could be calculated for its yield.

Rendemen calculation = Sy/x

Sxo
Vxo x 100%
Information:
Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 13

yr : Absorbance
Sy/x : Residual standard deviation Percent recovery = x 100%
Information:
The correlation coefficient is considered to CF : Total sample concentration obtained
be good if r has a value of ≥ 0.998. The from measurements CA : The actual
regression function coefficient (Vxo) is sample concentration C*A : The
deemed to be good if it has a value of ≤ 5%. concentration of the analyte added

Limit of detection and limit of quantitation RESULTS AND DISCUSSION

can be calculated statistically through linear
regression lines from the calibration curve. Tamarind leaf extract was carried out
The measurement value will be the same as using the maceration method. Maceration
the value of b in the linear line equation y = is the most widely used simple method.
a + bx, while the standard deviation of the This method is carried out by inserting the
blank is the same as the residual standard appropriate plant powder and solvent into
deviation an inert container, which is tightly closed
(Sy/x) or by the formula below.6 at room temperature.9 Maceration was
conducted using n-hexane solvent and
then macerated again using 70% ethanol.
LOD = Extraction with n-hexane solvent aimed to
LOQ = dissolve chlorofil contained in tamarind
Information: leaves as chlorophyll would interfere at
absorbance readings. N-hexane solvents
Sy/x : Residual standard deviation b :
were selected as chlorophyll is insoluble in
Response from slope (slope in
water. However, it can dissolve in various
line equation y = bx + a)
types of organic solvents10; thus, it can be
seen that chlorophyll is non-polar, while
Precision is carried out by measuring the
solvents are chosen to have the same
absorbance of the test solution, and then
properties as chlorophyll. The residue from
the percentage of correlation coefficients
maceration was then added with 70%
was calculated to identify the
ethanol until the entire powder was
reproducibility of the system used CV
submerged. The filtrate was then
requirements <2%6. Testing was
evaporated using a rotary evaporator at
conducted 7 times.
60˚C with a speed of 80 rpm to separate the
extract from the solvent. The extract
Accuracy in this study was carried out by
obtained was then evaporated again using
the standard addition method. The
a water bath at a temperature of 60˚C until
comparison between the extract and the
a thick extract was obtained. The thick
standard is 70:30 with a range of 80%,
extract obtained was 11.98 grams. The
100%, and 120%. The absorbance of the
extract yield was calculated by comparing
solution was measured using a UV
the weight of the thick extract with the
spectrophotometer at a maximum
weight of dry simplicia in percent. 11 The
wavelength, and the experiment was
calculation of extract yield aimed to
carried out 3 times. Accuracy testing was
estimate the amount of material to be used
calculated as percent recovery using the
to obtain the desired amount of extract.
formula:
The yield value of tamarind leaf extract
obtained was 9.74%.
 14 Erma Yunita , Deni Yulianto, Siti Fatimah, Tirsa Firanita | Validation of UV-Vis Spectrophotometric Method of
Quercetin in Ethanol Extract of Tamarind Leaf
0,8
Determination of the maximum wavelength 0,7
y = 0.0616x - 0.018
is needed to increase sensitivity and 0,6

Absorbance (A)
r = 0.9999
minimize errors when it repeated 0,5
measurements.12 In this study, the 0,4
determination of maximum wavelength 0,3
was carried out using a standard solution of 0,2
a quercetin concentration of 100 ppm, 0,1
which was measured in the wavelength 0
0 5 10 15
range of
200-800 nm using UV-Vis Concentration (ppm)
spectrophotometry. The measurements
were carried out at that range as sibling Figure 2. Quercetin standard curve
UV, and visible rays had wavelengths
ranging from 200-800 nm. Based on The results of the quercetin standard curve
optimization screening results, the obtained the intercept axis value 0.0616
maximum wavelength found two-peak and the slop value of -0.018; thus, the linear
spectra at 210 nm and 361.8 nm (Figure 1). regression line equation derived was y =
Ethanol, as a solvent, is known to have 0.0616 x –0.018.
maximum absorption at a wavelength of
210 nm.13 Therefore, it can be concluded Determination of quercetin levels was
that the maximum wavelength of carried out using a solution of tamarind
quercetin was 361.8 nm. Meanwhile, other leaves extract with a concentration of 500
studies showed that quercetin has a ppm. The test was carried out three times,
maximum wavelength of 380 nm.14 and it then calculated the average level of
quercetin in the tamarind leaf extract. The
results of level measurements are shown in
A standard curve was carried out by using
Table 1. Quercetin levels can be calculated
a standard solution of quercetin with series
by processing data from the results of
concentrations of 4 ppm, 6 ppm, 8 ppm, 10
absorption measurements with the linear
ppm, and 12 ppm. The absorbance results
regression line equation y = 0.0616x –
obtained from each series of
0.018. The measurement results showed
concentrations were then made linear
that quercetin levels were 10.76 ppm in 500
regression line equations, which can be
ppm tamarind leaf extract; thus, it can be
used to calculate the quaternary levels of
seen that in 1 gram of tamarind leaf extract,
quercetin in the tamarind leaf extract. The
there was 21.52 mg quercetin.
results of the quercetin standard curve are
Measurement of this level can be useful to
shown in Figure 2.
determine the amount of tamarind leaf
extract needed in producing drugs with
natural ingredients so that the desired
pharmacological effect can be achieved.

λmax of Ethanol 210nm

λmax of Quersetin 361,8

nm

Figure 1. Determination of quercetin maximum wavelength

Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 15

Validation of the assay method is Intercept axis (a) 0.0616

conducted to ensure that the analytical Correlation coefficient (r) 0.9999
methods are accurate, specific,
Table 1. Quercetin levels in tamarind Limit of Detection is the smallest number of
extracts analytes in the sample, which still gives a
Replication Absorbance Quercetin significant response compared to others.
(A) Levels (ppm) Limit of Detection is a limit test parameter.
1 0.645 10,763 Meanwhile, the limit of Quantification can
2 0.644 19,747 be interpreted as the smallest quantity of
3 0.645 10,763 analytes in a sample that had fulfilled the
𝒙 10.758 criteria of precision and accuracy 6. The LOD
and LOQ can be calculated statistically
reproducible, and resistant to the analyte based on the standard deviation response
range to be analyzed15. The validation and the standard slope (S) curve. LOD
parameters of the method observed in this results obtained in this test are 0.1515 ppm.
study are as follows: The LOQ results obtained were 0.4592
ppm. The result showed that the standard
Linearity is conveyed in terms of variance curve and the determination of quercetin
around the direction of the regression line, content in tamarind leaf extract had fulfilled
which is calculated based on mathematical the LOD and LOQ parameters as the
equations of data obtained from the test standard curve used the lowest
results of analytes in samples with various concentration of 4 ppm and quercetin levels
series of concentration analytes.6 The above the LOD and LOQ values of 9.34 ppm
parameter used in linearity testing is the in 500 ppm tamarind leaf extract.
correlation coefficient (r) in linear
regression analysis y = a + bx. The linear A precision method was conducted
test results are shown in Table 2. The repeatedly by the same analyst in a shorter
linear regression line equation obtained is time interval; thus, the precision can also be
y = 0.06160.018x with a correlation described as repeatability or
coefficient (r) 0.9999. Linearity can also be reproducibility. Precision was measured as
determined by calculating the value of Vxo, a standard deviation or coefficient of
which is 0.59545%. Based on the results of variation6. Precision parameters were
the correlation coefficient and the
calculation of Vxo, it can be seen that the
spectrophotometric method has good Table 3. Precision of quercetin
linearity for Vxo value is ≤ 5% with a carried out using various series of
correlation coefficient of 0.9999.16 concentrations in making quercetin raw
curves, namely 4 ppm, 6 ppm, 8 ppm, 10
Table 2. Linearity of quercetin ppm, and 12 ppm. The precision results in
this study are shown in Table 3. It shows
Concentration (ppm) Absorbance (A)
that the variation of coefficient values of
4 0.231 each concentration has a value of less than
6 0.349 2%.
8 0.474
10 0,600 Accuracy is a measure that shows the
12 0.722 degree of closeness of the analyst's results
Slop (b) -0,018 to the actual levels of analyte. Accuracy is
 16 Erma Yunita , Deni Yulianto, Siti Fatimah, Tirsa Firanita | Validation of UV-Vis Spectrophotometric Method of
Quercetin in Ethanol Extract of Tamarind Leaf
described as a percentage of the recovery. received for analyte levels of more than 1%
Accuracy testing performed in this study is is 97-103%6. The accuracy results in Table 4
a standard addition method for the tested show the value of percent recovery that
compounds suk terms of secondary has met the requirements. It indicates that
metabolite classes. Tests were carried out the determination of levels using the
in the range of 80%, 100%, and 120%, with spectrophotometric method fulfills the
differences in extract concentrations and parameters of accuracy so that it can
standard used. The range of % recovery provide accurate results.
Absorbance (A)
Replication
4 ppm 6 ppm 8 ppm 10 ppm 12 ppm
1 0.232 0.343 0.470 0.601 0.731
2 0.226 0.343 0.476 0.594 0.716
3 0.226 0.348 0.476 0.598 0.725
4 0.229 0.349 0.474 0.607 0.716
5 0.231 0.356 0.472 0.596 0.721
6 0.235 0.353 0.471 0.604 0.724
7 0.235 0.353 0.480 0.597 0.722
𝒙 0.231 0.349 0.474 0,600 0.722
CV (%) 1.629 1.452 0.736 0.779 0.731

Table 4 . Percent recov ery of quercetin

Range (%) Absorbance (A) CA C* A CF % recovery
0.474 5.6 2.4 7.987 99.45
80 0.479 5.6 2.4 8.068 102.83
0.472 5.6 2.4 7.954 98.08
𝒙 100.12
0.601 7.0 3.0 10.048 101.60
100 0.595 7.0 3.0 9.951 98.36
0.597 7.0 3.0 9.983 99.43
𝒙 99.80
0.720 8.4 3.6 11.980 99.44
120 0.719 8.4 3.6 11.964 99.00
0.725 8.4 3.6 12.061 101.69
𝒙 100.04
CA : The actual sample concentration (ppm)
C*A : The concentration of the analyte added (ppm)
CF : Total sample concentration obtained from measurements (ppm)
CONCLUSION
Based on the results of the study, it can be
identified that the UV-Vis Based on the results of the study, it can be
spectrophotometry method has fulfilled concluded that the UV-Vis
several parameters for the determination of spectrophotometry method has fulfilled
the content of quercetin from tamarind leaf several parameters for the determination of
extract. These parameters are linearity, the concentration of quercetin from
LOD, LOQ, precision, and accuracy. tamarind leaf extract. The quercetin level in
the extract in tamarind was 21.52 mg in 1 g
Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 17

extract. The correlation coefficient is Dandang Gendis (Clinacanthus nutans)

0.9999, and the regression function Berpotensi sebagai
coefficient (Vxo) ≤ 5% is 0.59545%. The LOD Antioksidan. [Skripsi]. Bandung: IPB.
is 0.1515 ppm, and LOQ is 0.4592 ppm. The 6. Harmita, H. (2012). Petunjuk
variation coefficient values for precision pelaksanaan validasi metode dan Cara
parameters have been less than 2%. Perhitungannya. Pharmaceutical
Meawnhile, the accuracy values have a Sciences and Research (PSR), 1(3), pp.
range of 97-103%. 117-135.
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ACKNOWLEDGMENT Experimental Design. Journal of
Validation Technology, 16(3), pp. 9-11.
The authors would like to thank the 8. Alwi, H. (2017). Validasi Metode
Ministry of Research, Technology and Analisis Flavonoid dari Ekstrak Etanol
Higher Education Republic of Indonesia Kasumba Turate (Carthamus tinctorius
(RISTEKDIKTI) for funding this research L.) secara
through the Beginner Lecturer Research Spektrofotometri. [Doctoral
grant in 2019. dissertation]. Makasar: Universitas
Islam Negeri Alauddin Makasar.
CONFLICT OF INTEREST 9. Tetti, M. (2014). Ekstraksi, pemisahan
senyawa, dan identifikasi senyawa
There is no potential for conflict of interest. aktif. Jurnal Kesehatan, 7(2), pp.
361367.
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Quercetin in Ethanol Extract of Tamarind Leaf
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