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 INTERNATIONAL CONFERENCE ON PHARMACEUTICAL RESEARCH AND PRACTICE
ISBN: 978-979-98417-5-9 | Universitas Islam Indonesia

Formulation and Antioxidant Test of Chromolaena odorata Leaf

Extract in Gel with DPPH Method (1,1-Diphenyl-2-Picril Hydrazil)
Desty Restia Rahmawati*, Isharyanto, Zafna Anggraeni Harianto, Nur Afifah Afianty, Latifa Amalia, Citra Ariani
Edityaningrum

Faculty of Pharmacy, University of Ahmad Dahlan, Jln. Prof. Dr. Soepomo, Janturan, Umbulharjo, Yogyakarta, Indonesia

Abstract. Chromolaena odorata leaf extract contains phenolic compounds that have activity as
powerful antioxidants that can counteract free radicals. Free radicals are ions that are triggered
by UV rays that can cause aging. The Antioxidant can stabilize free radicals so that overcome
aging of the skin. The purpose of this study was to find out the most optimal formula of gel and to
know its antioxidant activity. Chromolaena odorata leaf extract was obtained by maceration
method by using ethanol 70%. This gel is formulated using CMC-Na as gelling agent and with
dose variation of extract. The evaluation of gel were physical characteristic (spreadability and
adhesivity) and tested for antioxidant activity using DPPH method. The results showed that
percent inhibition of FI, FII, and FIII were 8.77±0.39, 9.33±0.09, and 56.6±0.13 respectively. The
antioxidant activity of FIII exceeded 1.5 times the vitamin C (p<0.05) which is a powerful
antioxidant. This is supported by testing the physical properties of the preparation according to
the pH of the skin which is 5.93±0.17 does not irritate the skin. Gel preparations also have good
homogeneity, dispersion, and adhesion. The formulation of Chromolaena odorata leaf extract in
gel contains powerful antioxidants that have capability as antiaging.

Keywords: Chromolaena odorata, leaf extract, gel, antioxidant

1. Introduction
butylated hydroxytoluene (BHT) and butylated
The skin has the main function as a protector, including hydroxyanisole (BHA) [19].
the skin from the rays of radiation and toxic substances.
In carrying out its functions, there are several problems
that can inhibit skin sustainability. The main and Thus, alternative solutions for antioxidant compounds
severe factors that occur in the body and can cause from natural ingredients which are not harmful to the
oxidative damage, commonly known as "Reactive skin are needed. Phenolic antioxidants play a major
Oxygen Stress" (ROS). The problem that can be role in fighting free radical species which are the main
ensnared by skin aging is the sun (photoaging), cause of various negative skin changes. Although
especially ultraviolet (UV) light which will increase isolated of leaf compounds have high potential for skin
ROS in cells. Skin exposed to sunlight will be at risk protection, herbal extracts show better potential
for aging skin aging, characterized by wrinkled skin, because of the complex composition of herbal sources.
dry, rough, and round-line [6]. Use antioxidants as a Many studies have shown that green plants can
preventative or best skin care effect with aging photos. improve skin damage due to UV exposure [14,20].
Antioxidants used in synthetic or natural ingredients. Phenolic is an effective source of antioxidants, which
Some anti-aging products use synthetic antioxidants, can be found in the leaves of the Chomolaena odorata
where the chemicals contained in antioxidant synthetic plant, which is a very abundant plant population on
materials will provide a long-term adverse risk such as Indonesian soil. This plant is just a weed plant that has
not been utilized properly in increasing its use value. In

*Corresponding author: [email protected]

42
 INTERNATIONAL CONFERENCE ON PHARMACEUTICAL RESEARCH AND PRACTICE
ISBN: 978-979-98417-5-9 | Universitas Islam Indonesia

the ethanolic extract of Chomolaena odorata leaves was smoothed using a blender and stratified with 50
has a higher total phenolic content of 313.31mg/g and 60 mesh sizes [10].
phenolic compounds [1, 11].
2.3.2 Extraction of phenolic compounds
The ethanol extract of Chomolaena odorata leaves can
be further processed, namely making a gel preparation
Extraction of phenolic compounds from 100 grams of
formula. Gel preparations are preparations that are kept
Chomolaena odorata leaf powder was carried out by
in the community. The goal is to make good quality
solvent extraction method using 1000 mL 70% ethanol
antioxidant gel preparations and have strong
solvent, then stirred for 6 hours using an electric stirrer
antioxidant activity. Antioxidant activity test using
DPPH method. DPPH method has the advantage that it and allowed to stand for 12 hours. The ratio of
is easy and can be directly refined with antioxidant Chomolaena odorata leaf powder to solvent is 1:10 (b /
v). After the extraction process, separation of solids
agents. Chomolaena odorata leaf extract gel can
and liquids is carried out using a vacuum oven. The
function as an aging therapy agent on the skin. In
results are then evaporated with a rotary evaporator at a
addition, the economic value of the plant is dehydrated.
temperature of 400C. Then the extract was dried using
Waterbath [7].
2. Methodology
2.3.3 Identification of phenolic compounds
2.1 Tools

UV-Vis (UV-1700) Spectrophotometry, Halogen Identification of phenolic compounds of ethanol extract

Moisturizer Analyzer (HB43 Metler Toledo), analytical 70% of Chomolaena odorata leaves was carried out
balance (Ohaus), vacuum pump, buchner funnel, rotary using thin layer chromatography. This is done to
evaporator, separating funnel, cuvette, aluminum foil, confirm the presence of phenolic compounds which are
micropipette, filter paper, a set of maceration tools efficacious as antioxidants in the extract. The test was
propipet, drop pipette, volume pipette, oven, glassware, carried out qualitatively using silica gel F254 nm as a
and gel physical properties. stationary phase and toluene mixture: ethyl acetate:
formic acid with a ratio (6:4:0.8) as the mobile phase.
Comparators used gallic acid standards.15 After the
2.2 Materials
elution process is complete, the silica plate is sprayed
using a DPPH 0.004% solution in ethanol. Positive
Chomolaena odorata leaves obtained from Kalasan, extracts contain phenolic activity compounds when
Sleman, DIY; DPPH 0.15 mM, 70% ethanol, aquadest, yellow spots with purple background on KLT plates
CMC-Na, glycerin, propylene glycol, methyl paraben, are obtained [2].
and ethanol.
2.3.4 Determination of water content
2.3 Methods

Water content of Chomolaena odorata leaf ethanol

2.3.1 Raw material for Chomolaena odorata extract was determined by a Halogen Moisture
leaves Analyzer. Extracts were tested as much as 1 grams
Fresh leaf extracts of 500 grams were taken, then with a temperature of 105oC for 15 minutes [12].
the leaves were dried using an oven at 600 C. Simplicia 2.3.5 Gel formulation

Table I. Chomolaena odorata leaf extract gel formula

Component F1 F2 F3
Chomolaena odorata leaf
IC80 1.5 x IC80 3 x IC80
ethanol extract
CMC-Na 0.3 grams 0.3 grams 0.3 grams
Glycerin 2 grams 2 grams 2 grams
Propylene glycol 1 grams 1 grams 1 grams
Methyl paraben 0.03 grams 0.03 grams 0.3 grams
Water 20 grams 20 grams 20 grams
*Note: IC80 value = 28 mg/mL
*Corresponding author: [email protected]
43
 44

Optimization of gel formula was carried out based on flask and then adding p.a ethanol to the boundary
the effect of different concentrations of ethanol extract markers.
70% of Chomolaena odorata leaves on the physical
properties of the gel and antioxidant activity. The b. Making Chomolaena odorata leaf extract gel
formula refers to the composition of the gelling sample
ingredients that have optimal physical properties Extraction of the gel extracts of Chomolaena
according to Maulina and Sugihartini (2015) with few odorata leaf ethanol extract was made by
changes (Table 2). Making the gel begins with weighing 500 mg of extract gel from each
developing CMC-Na in 10 mL of water at 70˚C, then formula dissolved in ethanol p.a to 50 mL to
the extract is added according to concentration (Table make a concentration of 10000 µg/mL. Then 2.5
2). This mixture is called Mixture 1. Methyl paraben as mL of pipette is extracted from a solution of
a preservative is dissolved in a little water then a 10000 µg/mL into a 25 mL volumetric flask, and
mixture of glycerin and propylene glycol is added as added to ethanol p.a to a boundary mark so that a
humectant. The mixture then it is called Mixture 2. The concentration of 1000 µg/mL is obtained. Then
two mixtures are put together, after which they are pipette 3 mL from a solution of 1000 μg/mL and
stirred and water is added to 20 grams then the mixture put in a 10 mL volumetric flask so that the
is stirred for 5 minutes at speed 500/rpm until concentration becomes 300 µg / mL. Each
homogeneous. concentration in ad with ethanol p.a to make from
formula 1, formula 2, and formula 3.

2.3.6Test the physical properties of the gel c. Making negative controls

a. Organoleptic test and homogeneity Negative control solution was made by mixing 1
mL DPPH (0.15 mM) with 1 mL ethanol p.a.
Organoleptic tests are carried out by direct
observation of the color, clarity, and odor of d. Making positive controls
the gel. Homogeneity testing is done by
applying gel on a piece of glass. A positive control solution was prepared by
dissolving 50 mg of vitamin C with ethanol p.a to
b. PH test 50.0 mL. Then from the mother's vitamin C
PH testing is done using a pH meter. solution a concentration of 8 µg / mL was made.
Each concentration of ad up to 10 mL with
c. Spread power test ethanol p.a [5].
The testing of the dispersing power begins
with as much as 0.5 grams of gel placed in a
round glass, the other glass is placed on it, and 2.3.8 Reading antioxidant activity
left for 1 minute. After that, 150 grams of load
were added, allowed to stand for 1 minute,
a. Determination of maximum DPPH
and measured a constant diameter [8].
As much as 1 mL DPPH (0.15 mM) was
d. Adhesion test reacted with 1 mL of ethanol p.a, then allowed to
Adhesion testing begins with a 0.25 grams gel sit in a dark place for 30 minutes. After that the
sample placed between 2 glass objects in the solution was measured at a wavelength of 450-
sticky power test, then pressed using a 1 kg 650 nm using a UV-Vis spectrophotometer [17].
load for 5 minutes. The load is lifted and
given a load of 80 grams on the tool then the b. Determination of operating time
gel release time is recorded [13]. Each extract gel sample and standard
vitamin C solution were added with 1 mL DPPH
solution (0.15 mM) then the absorbance was
2.3.7 Determination of antioxidant activity with observed for 0-75 minutes at a wavelength of 517
DPPH nm [9].
c. Testing of antioxidant activity
a. Preparation of 0.15 mM DPPH solution From each test solution and standard 1 mL
pipetted then put into flakon, the solution was
DPPH solution is made by DPPH stock
added 1 mL DPPH (0.15 mM) and shaken until
solution (1 mM) diluted with ethanol solvent p.a.
to obtain a concentration of 0.15 mM DPPH homogeneous. The solution is left in a dark place
during operating time. Then the absorbance test
stock solution (1 mM) was made by weighing 9.8
measured at the maximum wavelength. The blank
mg of DPPH powder into a 25 mL measuring
solution used is ethanol p.a [16].

*
Corresponding author: [email protected]
 INTERNATIONAL CONFERENCE ON PHARMACEUTICAL RESEARCH AND PRACTICE
ISBN: 978-979-98417-5-9 | Universitas Islam Indonesia

ethanol extract were obtained. Qualitative analysis of

phenolic compounds in the extract of thick leaves of
2.3.9 Data analysis
Chomolaena odorata It is important to do this process
The absorbance of Chomolaena odorata leaf extract to prove the presence of phenolic compounds in the
gel and vitamin C was changed in the form of percent extract. Identification of phenolic compounds was
capture, namely the amount of DPPH radical captured carried out using Thin Layer Chromatography (TLC)
by the sample. DPPH radical capture percentages can with comparable gallic acid. The results of the
be calculated using the following formula: identification test found yellow spots with purple
% DPPH Radical Capture = [(AbsControl-AbsSample) / background on the TLC plate (Figure 2). This shows
AbsControl] x 100 (1) that the extract contains phenolic compounds. In
addition to the qualitative analysis of the compound,
water content was also measured in the extract. Water
Information: content must be below 10% to maintain the quality of
AbsControl : Absorbance of controls the extract so that it is not easily contaminated with
AbsSample : Absorbance of test samples [3]. microbes when in storage [4].The average water
content of ethanol extract is 70% of Chomolaena
odorata which is an average of 7.85%±0.38, so it is in
To see differences in the results of antioxidant activity accordance with the requirements of Indonesian Herbal
and physical properties of gel between formulas an Pharmacopoeia.
analysis was performed using one way ANOVA. The
unpaired t test was used to see the difference in the
results of antioxidant activity between the optimal gel
formula and positive control of vitamin C.

3. Results and Discussion

Chomolaena odorata leaves were obtained in

Purwomartani Village, Kalasan District, Sleman
Regency, Yogyakarta Special Region. Leaves are taken
from the same location to minimize variations in the
active substance content which is strongly influenced Fig 2. Identification of phenolic compounds
by plant varieties, age, and plant location. The weight
of the Chomolaena odorata leaf obtained was 4.8 kg.
After 4.8 kg of wet leaves dried with an oven (60oC), The thick leaf extract was then formulated in gel form
the results of 880 grams of dried simplicia were with the formula listed in Table I. The extract dosage
obtained. Dry simplicia which has been mashed using a was based on the results of the study by Radava, et al.,
blender so that the resulting simplicia powder is 660 (2011) which obtained IC80 values of 28 mg. IC80 is
grams in the form of dry powder. The process of percent inhibition at 80%. Variation of dosage has been
refining the dried simplicia using a blender aims to made based on the research, which is as much as
reduce the particle size, so that the surface area of the 10xIC80, 15xIC80, and 30xIC80. The desired gel
powder which is in contact with the solvent when preparation of ethanolic extract of Chomolaena
extraction is greater. This will optimize the process of odorata leaves as antiaging was easily poured so that
withdrawing the desired chemical compound. before making gel, CMC-Na optimization was carried
out. The CMC-Na concentration needed in making gel
preparations is 0.3 grams.

Before testing the physical properties and further

processing, the gel antioxidant activity was tested by
DPPH method to really ensure that the gel had good
activity. Determination of OT is a step to find out the
reaction time between the active substance and the
radical agent, DPPH. Whereas the max lamda is done
to find out the lamda from DPPH solution which has
Fig 1. Chromolaena odorata leaves 517±2 nm value. DPPH is a stable free radical and is
used to evaluate the reduction of free radicals in natural
A simplicia is then extracted by maceration method ingredients. The principle of the reaction of this
and 70% ethanol solvent to attract phenolic compounds method is DPPH will be reduced by the hydrogen or
which are efficacious as antioxidants. After the electron donation process so that the color changes
macerate was evaporated, 121.61 grams of thick from violet to yellow with changes in color intensity
*Corresponding author: [email protected]
45
 INTERNATIONAL CONFERENCE ON PHARMACEUTICAL RESEARCH AND PRACTICE
ISBN: 978-979-98417-5-9 | Universitas Islam Indonesia

which is proportional to the number of electron the antioxidant activity. The results of the measurement
donations followed by DPPH absorbance [18]. The of antioxidant activity can be seen in table II.
greater the decrease in DPPH absorbance, the stronger

Table II. Results Measurement of antioxidant activity of gel of ethanol extract of Chomolaena odorata leaves using DPPH method

Absorbance Of Sample Average

Formula Replication % Inhibition
DPPH Control Absorbance % Inhibition
1 0.765 8.49
I
2 0.836 0.758 9.33 8.77±0.39
(10 x IC80)
3 0.765 8.49
1 0.758 9.33
II
2 0.836 0.759 9.21 9.33±0.09
(15 x IC80)
3 0.757 9.45
III 1 0.367 56.1
0.836 56.6±0.13
(30 x IC80) 2 0.364 56.5

The measurement results (Table II) show that with an inhibition of free radicals of 1.5x (p<0.05) significantly
increase in the dose of Chomolaena odorata leaf extract greater than a strong antioxidant of vitamin C. These
in gel preparations, the absorbance value produced results proved great potential for Chomolaena odorata
decreases so that % inhibition increases and is leaf extract gel as a product that has strong antioxidant
significantly different (p<0.05); p=0.00). The results of activity.
the formula III were the best results which had %

Table III. The results of measuring the antioxidant activity of vitamin C

Vitamin C Absorbance of Sample The Average of
Replication % Inhibition
Concentration DPPH Control Absorbance % Inhibition
8 µg/ml 1 0.581 30.50
2 0,836 0.517 38.16 35.29±3.41
3 0.525 37.20

To guarantee the good quality of the gel physical homogeneity, pH, dispersion, stickiness. Organoleptic
properties were tested including organoleptic, test results. Homgenity, and pH are stated in table IV.

Table IV. Organoleptic test and homogeneity

Organoleptic Test
Formula
Color Smell Homogeneity pH test

F1 Greenish brown Typical extract Homogeneous 6.09

F2 Greenish brown Typical extract Homogeneous 5.95

F3 Greenish brown Typical extract Homogeneous 5.74

Average :
5.93±0.17

The results of the third organoleptic test consistency not cause irritation to the skin, because the pH of the skin
formula, namely greenish brown color, characteristic of is normal at 5-6. The pH value produced so that the pH
extracts and have good homogeneity. The pH of the gel of the gel meets the requirements.
preparation meets the requirements of 5.93 so that it does

*Corresponding author: [email protected]

46
 INTERNATIONAL CONFERENCE ON PHARMACEUTICAL RESEARCH AND PRACTICE
ISBN: 978-979-98417-5-9 | Universitas Islam Indonesia

Table V. Results of the gel dispersion test

Replication I Replication II Replication III Average
Formula
D (cm) L (cm ) 2
D (cm) L (cm ) 2
D (cm) L (cm ) 2
D (cm) L (cm2)
F1 5.7 27.86 5.76 26.04 5.72 25.68 5.7±0.03 26.47±1.07
F2 5.74 25.86 5.46 23.40 5.8 26.41 5.6±0.18 25.22±1.60
F3 5.4 22.89 5.5 23.75 5.36 22.55 5.4±0.07 23.06±0.61
*Note: D = Diameter ; L = Large

Table VI. The test results of the gel adhesion strength

Time (second)
Formula
Replication I Replication II Replication III Average
F1 1.70 1.75 1.75 1.73±0.03
F2 1.56 1.55 1.59 1.56±0.02
F3 1.21 1.24 1.48 1.31±0.14
The good dispersion of gel preparations is gels that adhesion while a slightly higher dose increases. The
have a diameter of 5-7 cm [21]. Statistical analysis of results of statistical tests showed that the data were
scattering power shows the difference in results normally distributed 0.68 (>0.05) and not
between F1, F2, and F3. This is shown by statistics homogeneous 0.03 (<0.05) so that it used the kruskal
with one way ANOVA having a significance of 0.03 wallis statistical test which obtained a significance
(<0.05), so that H0 is rejected which means that there value of p <0.05. These results indicate that there are
are significant differences with the use of different significant differences in the effect of different doses
doses that can affect the physical properties of gel used on the sticky power of the gel ethanol extract gel
preparations. Increasing doses show a decrease in leaf extract.

4. Conclusion [2] A.D. Navitri, Maria, S.B.W. Monica, Uji

Aktivitas Antiradikal Bebas Ekstrak Buah
Jeruk Bali (Citrus maxima Burm.Fz) Dengan
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can be formulated in gel form. gel preparations have 1, 2, 1-6 (2012)
good physical properties. gel especially formula 3 has [3] A.M. Rawat, P.S. Mohsin, A.N. Negi, Sah, S.
strong antioxidant activity compared to vitamin C so Singh, Evaluation of poliphenolic contents
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