Total Flavonoid Content of Lemongrass Leaf  
(Cymbogoncitratus (DC.) Stapf) Extract and 
Antioxidant Activity with Frap 
 

Ahda Maulida Ulufan Nurinnafi’a*, Kusumaningtyas Siwi Artini, Desy Ayu Irma 
Permatasari 
Department of Pharmacy, Faculty of Health Science, Universitas Duta Bangsa Surakarta, Jl. Bhayangkara No.55, Types, Serengan, Surakarta, 
Central Java 57154, Indonesia 

Abstract 
Lemongrass (Cymbogoncitratus (DC.) Stapf) leaves contain 
alkaloids, saponins, tannins, anthraquinones, steroids, phenols and 
flavonoids. Flavonoids act as antioxidants as they can reduce free 
radicals. This study aims to determine the total flavonoid content, 
antioxidant activity, and IC50 value of lemongrass leaf extract 
(Cymbogoncitratus (DC.) Stapf). Extracts were made by maceration 
using 96% ethanol as solvent. Testing of total flavonoid content 
with the AlCl3 method using UV-Vis spectrophotometry was carried 
out three times. The antioxidant activity test used the FRAP (ferric 
reducing antioxidant power) method on extracts containing 100, 
200, 300, 400, and 500 ppm. The test results showed that the leaf 
extract of citronella (Cymbogoncitratus (DC.) Stapf) had a total 
flavonoid content of 22.60 mg QE/g extract. Furthermore, there was 
antioxidant activity in the leaf extract of lemongrass 
(Cymbogoncitratus (DC.) Stapf indicated by the formation of a blue 
color purplish when reacted with FRAP solution, and IC50 extract 
value was 71.59 ppm and included in the category of strong 
antioxidants. 
 
Keywords: Antioxidant Activity; Lemongrass Leaf Extract 
(Cymbogoncitratus (DC.) Stapf); Total Flavonoid Content 

 
 
 
 
 
 
 
 
 

 

 
Data of article 

Received 
Reviewed 
Accepted 

: 
: 
: 

19 Jul 2022 
05 Aug 2022 
29 Aug 2022 

 
DOI 
10.18196/jfaps.v3i1.15556 
 
Type of article: 
Research 
 

INTRODUCTION 
 
Indonesia is a country with a tropical 
climate where there are various kinds of 
plants that have therapeutic activity.1 The 
use of herbal medicines from plants 
increases disease prevention efforts as the 
side effects tend to be harmless and can be 
minimized.2 One of the wild plants widely 
used by the community is lemongrass 
(Cymbogon citratus (Dc.) Stapf). The 
extract of lemongrass leaf (Cymbopogon 
citratus (DC.) Stapf) contains several 

 
* Corresponding author, e-mail: ahdamaulida@gmail.com 

vegetable constituents, namely essential 
oils.3 Lemongrass (Cymbopogon citratus 
(DC.) Stapf) can empirically be used as a 
medicine for headaches, coughs, stomach 
pains, diarrhea, body warmers, fever 
reducers, and mosquito repellents.4 It 
contains saponins, tannins, alkaloids, and 
flavonoids. Flavonoids are secondary 
metabolites often found in various green 
plants so they can be found in every plant 
extract. Flavonoids are found in many 
green plants, so they can be found in every 
plant extract. Flavonoids contribute to 



Ahda Maulida Ulufan Nurinnafi’a, Kusumaningtyas Siwi Artini, & Desy Ayu Irma Permatasari | Total Flavonoid Content 
of Lemongrass Leaf (Cymbogoncitratus (DC.) Stapf) Extract and Antioxidant Activity with Frap 

31 

pigment production and belong to the 
water-soluble polyphenol family.5 

Flavonoids can counteract free radicals by 
liberating hydrogen atoms from their 
hydroxyl groups; thus, flavonoids are 
called antioxidants.6 

 
Antioxidants are compounds that can 
scavenge free radicals. Free radicals are 
generated due to several factors, such as 
smoke, dust, pollution, and consuming 
fast food that is not balanced between 
carbohydrates, proteins, and fats.7 When 
free radical compounds meet other 
molecules, they can form new free 
radicals, resulting in chain reactions. This 
kind of reaction will continue and will stop 
if antioxidants reduce the reactivity.8 
Antioxidant compounds will donate one 
electron to unstable free radicals so that 
these free radicals can be neutralized and 
no longer interfere with body metabolism.  
Total flavonoid content can be measured 
by identifying the absorbance value at a 
certain wavelength based on the Lambert-
Beer principle of each herbal plant using a 
UV-Vis spectrophotometer.9  
 
The method used to determine total 
flavonoids is the AlCl3 method, using UV-
Vis spectrophotometry as flavonoids 
contain a conjugated aromatic system so 
that they show strong absorption bands in 
the ultraviolet and visible spectrum 
regions. The antioxidant activity test uses 
the FRAP (Ferric Reducing Antioxidant 
Power), which is used to determine the 
antioxidant activity of a material based on 
the ability of antioxidant compounds to 
reduce Fe3+ ions (ferri-tripyridyl-trazine) to 
Fe2+ (ferro-tripyridyl-trazine).3 In this study, 
the total flavonoid content of the 
lemongrass leaf extract (Cymbogon 
citratus (DC.) Stapf) will be tested using 
the UV-Vis spectrophotometry method, 
and the antioxidant activity will be tested 
by using the FRAP method. 

METHOD 
 
Plastic bag, knife, scissors, label paper, 
oven (Memmert), herb grinder, drop 
pipette, micro pipette, analytical balance 
(Ohaus), test tube, test tube clamp, test 
tube rack, maceration jar, stirrer, spatula, 
60 mesh sieve, filter paper, funnel, 
measuring cup, beaker, handscoon gloves, 
aluminum foil, rotary vacuum evaporator, 
tissue, cuvette, spectrophotometry UV-
Vis (EMCLab), TLC chamber, Silica Gel 60 
F TLC plate (Merck), UV lamp 366 nm, 
citronella leaf (Cymbopogon citratus), 
ethanol96%, potassium acetate 120mM 
(Merck), aquadest, quercetin (Sigma), 
AlCl3(Merck, 98%), concentrated 
H2SO4(Merck, 95-97%), sodium acetate 
trihydrate (Merck 99-101%), acetic acid 
(Merck, 99.8%), TPTZ powder (Sigma), 
concentrated HCl (Merck), FeCl3.6H2O 
(Merck), ascorbic acid (Merck 99-
100.05%), oxalic acid1% (Merck), 
chloroform, concentrated ammonia 
(Merck), Dragendorf reagent, Mayer, 
Wagner, Lieberman Burchard, FeCl31%, 
ether, n-hexane, and ethyl acetate. 
 
Lemongrass leaf samples were obtained 
from Sribit Village, Delanggu, Klaten, and 
then determined at the Biology 
Laboratory of Universitas Ahmad Dahlan. 
Lemongrass leaves were washed with 
running water, drained, dried in an oven, 
and ground by a herb grinder to become 
Simplicia powder before being extracted. 
Extraction was done by maceration 
method with 96% ethanol solvent for 3x24 
hours. The filtrate was filtered and 
evaporated to obtain a thick extract. 
Furthermore, phytochemical analysis was 
carried out on the lemongrass leaf extract, 
including the test for alkaloids, flavonoids, 
saponins, tannins, and steroids/ 
triterpenoids by TLC (Thin Layer 
Chromatography). The total flavonoid 
content test in the extract was started by 



Journal of Fundamental and Applied Pharmaceutical Science, 3(1), August 2022 
 

32 

determining the maximum wavelength of 
the quercetin standard solution, carried 
out by running the quercetin solution in 
the UV-Vis wavelength range of 370 - 450 
nm. The determined wavelength was 400 
nm. Furthermore, the standard curve of 
quercetin was made by making several 
concentrations, namely 10 ppm, 20 ppm, 
25 ppm, 30 ppm, and 35 ppm. For each 
standard, 1 mL of quercetin was added 
with 1 mL of 2% AlCl3 and 1 mL of 120 mM 
potassium acetate and incubated for 30 
minutes. The absorbance was measured 
using UV-Vis spectrophotometry with a 
determined wavelength (400 nm). The 
total flavonoid content of lemongrass leaf 
extract was determined using the AlCl3 
method by dissolving 10 mg of extract in 
10 mL of 96% ethanol. 1 mL of extract was 
added with 1 mL of 2% AlCl3 solution and 1 
mL of 120mM potassium acetate and 
incubated for 30 minutes at room 
temperature. The absorbance was 
determined by UV-Vis 
spectrophotometry. Three replications 
were made for each analysis, and the 
average absorbance was then identified 
Determination of the antioxidant activity 
of lemongrass leaf extract started with 
making a FRAP solution by mixing 25 mL 
of acetate buffer solution, 2.5 mL of TPTZ 
solution, and 2.5 mL of FeCl3.6H2O 
solution, then added with aquadest to 100 
mL in a volumetric flask. Determination of 
the maximum wavelength of the FRAP 
solution was carried out by mixing 3 mL of 
FRAP reagent and 1 mL of distilled water 
and then measuring the absorption at a 

wavelength of 400-600 nm using UV-Vis 
spectrophotometry. Based on the result, 
the maximum wavelength was 
determined at 595 nm. Ascorbic acid was 
used as a standard antioxidant solution 
also as a positive control. A standard curve 
solution of 100 ppm was prepared by 
dissolving 10 mg of ascorbic acid dissolved 
in 1% oxalic acid to the 100 mL volumetric 
flask limit. Furthermore, the stock solution 
of 100 ppm was diluted into several 
concentrations, namely 10, 15, 20, 25, and 
30 ppm. 1 mL of FRAP reagent was put and 
then added with 3 mL of ascorbic acid from 
each concentration, then homogenized 
and allowed to stand for 10 minutes at 
37ºC. The absorbance was then observed 
at a determined wavelength of 595 nm. 
The IC50 value of lemongrass leaf extract 
was determined by taking 50 mg extract, 
dissolved in 50 mL of 96% ethanol, 
obtained a concentration of 1000 ppm, 
and then diluted into several 
concentrations, namely 500, 400, 300, 
200, and 100 ppm. 1 mL of FRAP reagent 
was put in, and 3 mL of sample were added 
from each concentration. It was 
homogenized and allowed to stand for 10 
minutes at 37ºC, then observed the 
absorbance at a determined wavelength 
of 595 nm. 
 
RESULTS AND DISCUSSION 
 
The results of the extraction of lemongrass 
leaves (Cymbopogon citratus (DC.) Stapf) 
can be seen in table 1 below. 
 

 
Table 1.The results of the extraction of lemongrass leaves (Cymbopogon citratus (DC.) 

Stapf) 

Powder weight 
(g) 

Extract 
weight (g) 

Yield (%) (w/w) 

426 57.37 13.46 

  



Ahda Maulida Ulufan Nurinnafi’a, Kusumaningtyas Siwi Artini, & Desy Ayu Irma Permatasari | Total Flavonoid Content 
of Lemongrass Leaf (Cymbogoncitratus (DC.) Stapf) Extract and Antioxidant Activity with Frap 

33 

The yield of lemongrass leaf extract was 
13.46% w/w. A good extract yield is 
indicated by a yield >10%.10 The higher the 
yield of the extract is, the higher the 
content of substances will be attracted to 
the raw material.11 It was also presumably 
due to the maceration time that has been 
done for three days. The remaceration was 
done two times so that the chemical 
compound extraction process could be 
maximized. 
 
Phytochemicals 
Based on the phytochemical 
identification, lemongrass leaf extract 
contained alkaloids, flavonoids, saponins, 
tannins, and steroids. The results of the 
flavonoid test using TLC showed the spots 
between the extract and quercetin were 
nearly by using eluents n-Hexane: ethyl 
acetate: ethanol: water in a ratio of 
2.5:1:0.5:6.5. The Rf value of the extract 
was 0.51, and the Rf value of quercetin was 

0.56. The difference in the Rf value was 
0.05. The results would be declared 
positive containing quercetin if the Rf 
value had a difference of 0.05.12 
 
Results of Total Flavonoid Content Test 
of Lemongrass Leaf Extract 
(Cymbopogon citratus (DC.) Stapf 
Quantitative tests were carried out to 
determine the total flavonoid content of 
the lemongrass leaf extract, using 
quercetin as a standard solution. The 
maximum wavelength of quercetin was 
400 nm. Furthermore, the standard curve 
of quercetin was made by making several 
concentrations, namely, 10 ppm, 20 ppm, 
25 ppm, 30 ppm, and 35 ppm. Each 
concentration was measured at a 
wavelength of 400 nm. Based on these 
measurements, the linear regression 
equation for the standard solution of 
quercetin was y = 0.025x - 0.072, which can 
be seen in figure 1 below. 

 

 
Figure 1.The Standard Curve of Quercetin 

 

y = 0.025x - 0.072
R² = 0.969

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0 5 10 15 20 25 30 35 40

A
b

so
rb

a
n

ce

Quercetion concentration (ppm)

The Standard Curve of Quercetin

Y-Values

Linear (Y-Values)





Ahda Maulida Ulufan Nurinnafi’a, Kusumaningtyas Siwi Artini, & Desy Ayu Irma Permatasari | Total Flavonoid Content 
of Lemongrass Leaf (Cymbogoncitratus (DC.) Stapf) Extract and Antioxidant Activity with Frap 

33 

Furthermore, the absorbance 
measurement of the lemongrass leaf 
extract was made for 3 replications. The 
sample solution was added with 1 mL of 
aluminum chloride (AlCl3), which could 
form a complex, resulting in a shift in 
wavelength towards the visible indicated 

by the solution producing a more yellow 
color. 1 mL of potassium acetate was then 
added to maintain the wavelength in the 
visible region 13. It was measured by using 
UV-Vis spectrophotometry to identify the 
extract absorbance (Table 2). 
 

 
Table 2. Results of Total Flavonoid Content of Lemongrass Leaf Extract (Cymbopogon 

citratus (DC.) Stapf) 

Replication Sample Weight 
(g) 

Absorbance mg QE/g extract 

I 0.01 0.437 20.36 
II 0.01 0.427 19.96 
III 0.01 0.650 28.88 

Average 23.06 

The results of this study showed that the 
total flavonoid content of lemongrass leaf 
extract (Cymbopogon citratus (DC.) Stapf 
was 23.06 mg QE/g extract, which can be 
seen in table 2. The higher the flavonoid 
content is, the higher the benefits of 
flavonoids as antioxidants will be.14 

Sample III has a higher absorbance than 
the other two samples, probably due to 
the less precise determination of 
operating time. The three replications 
were analyzed in minutes of 10. In previous 
studies, it was shown that starting in the 
minutes of 30, the flavonoid compounds 
had completely reacted with the AlCl3 
reagent, and the absorbance reading was 
stable in the minutes of 30.15 
 
Results of Antioxidant Test of 
Lemongrass Leaf Extract (Cymbopogon 
citratus (DC.) Stapf) 
An antioxidant activity test was carried out 
to determine the IC50 value of the 

lemongrass leaf extract. The maximum 
wavelength of the FRAP solution was 595 
nm. Qualitative analysis was carried out by 
taking a sample solution of lemongrass 
leaf extract. The FRAP reagent was added, 
and then color changes were observed. As 
a result, the sample turned purplish blue, 
indicating that the sample had antioxidant 
activity.3 Furthermore, vitamin C was used 
to compare as it functions as a secondary 
antioxidant. It can reduce free radicals and 
prevent chain reactions.16 The IC50 value of 
vitamin C was 23.60 ppm. Quantitative 
tests were conducted by making leaf 
extract of lemongrass (Cymbopogon 
citratus (DC.) Stapf) in five concentration 
series, namely, 100, 200, 300, 400, and 500 
ppm. It was added with FRAP reagent at 
each concentration series and read the 
absorbance at the maximum wavelength 
of 595 nm. 
 

 

  



Journal of Fundamental and Applied Pharmaceutical Science, 3(1), August 2022 
 

34 

Table 3. Results of Antioxidant Activity Test of Lemongrass Leaf Extract (Cymbopogon 
citratus (DC.) Stapf) 

Extract Concentration 
(ppm) 

Absorbance Average % inhibition 

100 0.355 50.89 
200 0.306 57.67 
300 0.218 69.84 
400 0.199 72.47 
500 0.159 78.00 

The results of the absorbance 
measurement were used to obtain the % 
inhibition value. The value of % inhibition 
was used to find the IC50 value to 
determine the strength of antioxidant 

activity in the sample. Data in the form of 
concentration and % inhibition of 
lemongrass leaf extract showed a linear 
regression equation y = 0.069x + 45.06 
(Figure 2) to calculate the IC50 value. 

 

 
The calculation of the IC50 value of 
lemongrass leaf extract was as follows:  
y  = 0.069 x +45.06 
50 = 0.069 x +45.06 
50 – 45.06 = 0.069x 

𝑥 =
4.94

0.069
 

𝑥= 71.59 ppm 

 
The IC50 value of lemongrass leaf extract 
(Cymbopogon citratus (DC.) Stapf) was 
71.59 ppm. Specifically, phytochemicals 
are categorized as highly strong 
antioxidants when the IC50 value is <50 
ppm, strong when the IC50 value is 50-
100ppm, moderate when the IC50 value is 

y = 0.069x + 45.06
R² = 0.956

0

10

20

30

40

50

60

70

80

90

0 100 200 300 400 500 600

%
 i

n
h

ib
it

o
n

Extract concentration

The Standard Curve of Extract



Ahda Maulida Ulufan Nurinnafi’a, Kusumaningtyas Siwi Artini, & Desy Ayu Irma Permatasari | Total Flavonoid Content 
of Lemongrass Leaf (Cymbogoncitratus (DC.) Stapf) Extract and Antioxidant Activity with Frap 

35 

100-150ppm, and weak when the IC50 
value is 151-200ppm.17 
 
CONCLUSION 
 
Based on the result of this research, it can 
be concluded that the total flavonoid 
content of the lemongrass leaf extract 
(Cymbogon citratus (DC.) Stapf) was 23.06 
mg QE/g. There was antioxidant activity in 
the lemongrass leaf extract indicated by 
the formation of a purplish blue color on 
the test with FRAP solution. Furthermore, 
the IC50 value of lemongrass leaf extract 
was 71.59 ppm, classified as a strong 
antioxidant. 
 
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