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RESEARCH ARTICLE | MAY 08 2023
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Online Citation
Antibacterial activity of fermented katuk leaves (Sauropus androgynus (L.) Merr.) against gram positive
bacteria and its free radical inhibitory
AIP Conference Proceedings (January 2023)
Evaluation of antioxidant activity of formulated functional drinks derived from katuk (Sauropus
androgynous) leaf extracts: Optimization using response surface methodology (RSM)
AIP Conference Proceedings (November 2019)
Abstract. Katuk plant is a plant that is often found in the community, this plant contains compounds containing alcaloids,
triterpenoids, saponins, tannins, polyphenols, glycosides and flavonoids. This katuk plant has considerable benefits, namely
to increase breast milk, overcome constipation, lose weight, anti hypertension, anti hyperlipidemia, constipation, and can
treat infections. This plant has not been widely used by the community in the field of medicine as an antioxidant and
antibacterial, so this study aims to make the best formulation of katuk leaf extract more widely used by the community as
an antioxidant and antibacterial. This type of research is purely experimental. The method used in the manufacture of tablets
is wet granulation, tablet formulations are made with various concentrations of PVP 5% (F1), 7.5% (F2), and 10% (F3).
The evaluation of lozenges included organoleptic test, weight uniformity test, friability test which was analyzed
descriptively and flow time test, angle of repose and tablet hardness test which were analyzed using one-way ANOVA.
The result of this research is that the best formulation of leaf extract tablets is formula 3 which contains PVP with a
concentration of 10%. Katuk leaf tablets are proven to be effective as antioxidants that can counteract free radicals with an
IC50 value of 106,91μg/mL which is a moderate antioxidant. Katuk leaf extract tablet which has an inhibitory power against
Staphylococcus aureus is the third formula with an average inhibition zone of 31.5 mm.
INTRODUCTION
Weight, anti-hypertension, anti-hyperlipidemia, constipation, and can overcome infections caused by bacteria
which proves that the positive katuk plant contains alkaloid compounds, triterpenoids, saponins, tannins, polyphenols,
glycosides and flavonoids [2]. Antioxidants are compounds that in small amounts compared to the substrate are able
to delay or prevent the oxidation of easily oxidized substrates. Free radicals are atoms or molecules that are unstable,
free radicals have 1 or more unpaired electrons so that to become stable they tend to take electrons from other
molecules which then cause abnormal compounds and start chain actions that can damage tissues [2]. Antibacterial is
a substance that can interfere with the growth or even kill bacteria by interfering with the metabolism of harmful
bacteria [13].
Katuk leaves contain seven active compounds, namely five groups of polyunsaturated fatty acids which are a group
of eicosanoids, including octadecanoic acid; 9-eicosine; 5,8,11- heptadecatrienoic acid; 9,12,15-octadecatrienoic acid;
and 11,14,17- eicosatrienoic acid which acts as a precursor and is involved in the biosynthesis of eicosanoid
compounds (prostaglandins, prostacyclins, thromboxanes, lipoxins and leukotrienes). In addition, there are also
compounds from the biosynthesis of steroid hormones, namely Andostran-17-one and 3-ethyl-3-hydroxy-5alpha
which act as precursors in the synthesis of steroid hormone compounds (progesterone, estradiol, testosterone and
glucocorticoids. Katuk is a variety of phytochemical compounds that are useful as drugs, such as progesterone,
estradiol, testosterone and glucocorticoids. Katuk leaves also contain elcosanoids, prostaglandins, and prostacyclin,
thromboxane, lipoxin and leukotriene compounds [1].
020225-1
Granules are lumps of small particles. Generally, granules are made by moistening powder or a mixture of ground
powder and passing the moistened dough through a sieve slit with a sieve hole size corresponding to the desired
granule [6].
METHOD
Equipment
The tools used in this research are rotary evaporator, UV-Vis spectrophotometry, water bath, incubator
(memmertR), oven (memmertR), analytical balance, electric stove (maspionR), blander (miyakoR), filter paper, granule
sieve no. 16 and 18, single punch tablet machine, hardness tester, friability tester, disintegration tester, mortar and
stamper, pan, stand and clamp, ruler, funnel, stirring rod, aquadest, beaker, measuring cup (pyrexR), micro pipette
(socorexR) ), knife, glass jar, porcelain cup, watch cup, test tube and dropper, mask (sensi maskR) and gloves (sensi
gloveR).
Sampling
Katuk leaves were obtained from plantations in Kroya District, Cilacap Regency. Katuk leaves were washed and
dried without direct sunlight so that the compounds contained in the sample were not damaged by the sun's UV rays.
The dried samples were mashed with a blender.
Flavonoid test which aims to determine the chemical content in the form of secondary metabolites of a sample or
as an antioxidant test DPPH free radicals will oxidize flavonoids to form radicals with low reactivity. red or orange In
plant extracts it can be seen if there are flavonoid compounds when adding Mg and HCl will form flavilium salts,
these salts can give a red or brownish red color [12].
Tannin
Tannin test aims to determine the antibacterial compounds in the sample. The tannin test on the ethanolic extract
of katuk leaves with FeCl3 produced a green-black color, because the reaction between tannins and FeCl3 formed a
020225-2
complex compound. The results obtained produce a blackish green color which means that it contains tannin positive
[12].
Saponins
The saponin test aims to determine the effectiveness of antimicrobial compounds, a positive test for the presence
of saponins is evidenced by the formation of permanent foam and does not disappear with the addition of 2 mL 0.1 N
HCl [12].
Terpenoids
The terpenoid test is as an anti-cancer. In the terpenoid test, the fraction was reacted with chloroform and H2SO4,
showing positive results with a change in color to brownish red. This reaction is based on the ability of triterpenoid
and steroid compounds to form color by H2SO4 in acetic acid solvent. The reaction results obtained produce a blue
ring which means there is no terpenoid reaction.
020225-3
Evaluation
Granule’s evaluation
Tablets evaluation
a. Organoleptic Test
The organoleptic test was carried out by direct observation of the shape, color, smell, and taste of the tablets.
b. Weight Uniformity Test
The weight uniformity test was carried out by weighing 20 tablets one by one, calculating the average weight of
each tablet. There should be no more than 2 tablets, each of which deviates from the average weight [4].
c. Hardness Test
The tablet hardness test was carried out using a hardness tester on 20 tablets taken at random, then placed one by
one horizontally on the hardness tester tool, then the clamp will move towards the tablet and press the tablet until it is
crushed and the tool will display the hardness measurement on the display. The results are then calculated on average.
The tablet hardness test was determined using the Carr Index. The Carr index is a fast indirect method for measuring
the relative strength between particles and the frictional forces of bulk powders.
TABLE 2. The Carr Index
Index Carr Score
5 – 15 Excellent
16 – 18 Good
19 – 21 Fair
22 – 35 Poor
36 – 40 Very Poor
> 40 Worst
d. Friability Test
The tablet friability test was carried out by weighing 20 tablets that had been cleaned of dust particles attached to
the analytical balance, then inserted into the friabiliator tester at a speed of 25 rpm for 100 rotations for 4 minutes.
Then the tablet is removed and cleaned of loose dust. Calculate the percentage of weight loss before and after treatment
[4].
e. Disintegration Test
Disintegration time test was carried out using a disintegrator tester. Six tablets were prepared and then put in each
tube as many as one tablet in the basket, one disc was inserted in each tube and the device was run. Aquades is used
020225-4
as a medium with a temperature of 370C. After 15 minutes, the basket was removed and all tablets were observed
[10].
Antioxidant Activity
In this study, an antioxidant effectiveness test was conducted using the DPPH method (1,1-diphenyl-2
pikrylhydrazil) is a nitrogen radical compound DPPH will take up hydrogen atoms contained in a compound, for
example flavonoid compounds. Quantitative measurement of antioxidant activity is by measuring DPPH radical
capture by a compound that has antioxidant activity using UV-Vis spectrophotometry with a wavelength of 517 nm
[7].
According to Hartanto's research, the reason for being incubated for 30 minutes was because the reaction was slow
and samples containing antioxidants were optimum in reducing DPPH free radicals at that time and to obtain stable
results. The incubation process was also carried out at 37°C because it was the optimum temperature so that the
reaction between DPPH radicals and antioxidant compounds was faster and optimal. This can be seen from the
relationship between temperature and reaction rate, where temperature is one of the factors that can accelerate the
Antibacterial activity
Antibacterial power is the ability of a substance or compound to kill bacterial production, this antibacterial works
in four ways, namely the first inhibition of cell membrane function, the cytoplasmic membrane which acts as a
selective permeability barrier, functions as active transport. If the functional integrity of the cytoplasmic membrane is
disturbed, macromolecules and ions can leave the cell and cause cell damage [14].
020225-5
Evaluation of Granules
Flow Time Test
From the research data, it can be seen that F1 produces a flow time of 6.51 seconds, F2 produces a flow time of
6.46 seconds. Meanwhile, F3 has a faster flow time of 6.36 seconds. The difference in the results of the flow time test
can be caused by several factors, namely the difference in the granule surface of each different formula. This test
shows that all formulations meet the flow time requirements of not more than 10 seconds.
From the research data, it can be seen that formula I has an angle of repose of 27,36° and formula II has an angle
of repose of 27,16°, and formula III has an angle of repose of 23,43°. meet the requirements of <30°.
Compression Test
Based on the table shows the results of the Haunser ratio, namely formula I with a yield of 0.94, formula II 0.94,
and formula II 0.98, in formula III is different because the binder used more so affects the flow properties of the
granules, the three formulas meet the condition that is less than 1.25, it is said that the calculated value obtained is
adjusted to the Hausner ratio index versus flow properties.
Evaluation of Tablets
Organoleptic observation
The organoleptic test showed that, in F1 it was mottled green which was caused by damage to the manufacture of
tablets, the uneven color of the tablets had light or dark spots protruding on the surface of the tablet walls, while in F2
and F3 they had the shape, taste, smell, and taste. the same color is light green because when mixed evenly, the tablet
is green because the katuk leaf extract is green.
From the results obtained, it is known that the tablets in formulas I, II, and III have good weight uniformity and
meet the weight uniformity test according to the Indonesian Pharmacopoeia IV because they have CV values below
5% [4].
020225-6
Hardness Test
Based on the Carr Compressibility Index (Table 3), it was found that the tablets F2 and F3 which met the standard
of tablet hardness were very good. In general, the higher the Carr index value, the worse the powder flow. PVP is a
binder that is often used in the manufacture of tablets. Granules with polyvinylpyrrolidone have good flow properties,
minimum angle of repose, produce fewer fines and have better compactibility. PVP as a binder can be used in the
form of an aqueous solution or alcohol. PVP is also capable as a dry binder. The use of PVP at a concentration of 0.5-
2% in the manufacture of plant extract tablets can produce tablets that have sufficient hardness, low friability and long
disintegration time.
TABLE 3. Hardness test results
Formula Tablet 1 Tablet 2 Tablet 3 Tablet 4 Tablet 5 Average
F1 5 4.8 5 4.9 4.8 4.9
F2 6 5 6.5 6.8 7.9 6.4
F3 7.8 8 7.9 7.8 8 7.9
The tablet friability test showed that the three formulas met the requirements for the friability of lozenges, namely
friability less than 1%. Friability values greater than 1% are considered less good [4].
Disintegration Test
Disintegration time test on formulation 1 with the result that is 5 minutes 11 seconds, formulation 2 is 6 minute 72
seconds, formulation 3 that is 9 minutes 2 seconds, The results show that all three formulas meet the requirements of
disintegration time which is less than 15 minutes.
Antioxidant Activity
Test of Antioxidant Activity of Katuk Leaf Extract Tablets with DPPH Method. Based on the IC50 results table,
it shows that based on the IC50 value, katuk leaf extract has antioxidant activity of 106,91 μg/mL while katuk leaf
extract tablets have antioxidant activity of 107,66 μg/mL which means moderate anti-free radical compounds, Vitamin
C tablets as a comparison because Vitamin C is already known as an antioxidant, as an antidote to free radicals with
antioxidant results of vitamin C showing 4,88 μg/mL which is categorized as very strong so it is used as a comparison.
According to Nurdianti (2017), states that a substance has a very strong antioxidant if the IC50 value is <50 μg/mL,
strong if the IC50 value is between 50-100 μg/mL, while if the IC50 value is between 100-150 μg/mL, weak if the IC50
value is between 100-150 μg/mL. IC50 values range from 150-200 μg/mL and are inactive when IC50 is above 250
μg/mL.
TABLE 4. Antioxidant Test results
Sample Concentration (%) Inhibition (%) IC50 (µg/mL) Antioxidant Activity
Katuk Leaf Extract 40 19.2 106.91 medium
80 47.88
120 60.68
160 62.4
Katuk Leaf Extract Tablets 40 32 108.66 medium
80 43.2
120 56.34
160 60.8
Vitamin C tablets (Comparison) 40 28.99 4.88 high
80 38.93
120 52.89
160 83.31
020225-7
Antibacterial activity
In this study, using the extract of Sauropus androgynus katuk leaf as a test material and using Agar media which
is a medium commonly used in antibacterial tests where all bacteria can live in the media. The antibacterial test method
used is the paper disc method. The results of this study were an inhibition zone for the growth of Staphylococcus
aureus bacteria which was marked by a clear zone around the paper disc. The clear zone was measured using a ruler
in millimeters (mm) [13].
In the antibacterial test the katuk leaf extract showed the magnitude of the antibacterial activity as evidenced by
the diameter of the clear zone increasing with the increase in the concentration of the katuk leaf extract, namely a
concentration of 50% which showed a clear zone, thus proving that the katuk leaf extract contains flavonoids, tannins,
saponins, and alkaloids. This is because at high concentrations it means the ability of the inhibition zone is getting
bigger [3]. The antibacterial activity test aims to determine whether the tablet preparations of katuk leaf extract have
greater antibacterial activity than the antibacterial test results of pure katuk leaf extract with a concentration of 50%,
the results obtained in the study can be seen in table 5 and 6.
TABLE 5. Antibacterial result pure katuk leaf extract
The results of the antibacterial power test of pure extract of katuk leaf with 96% ethanol solvent with a
concentration of 50%, namely 20 mm, were carried out using the paper disc diffusion method, the results showed the
presence of inhibition with Staphylococus aureus bacteria and was characterized by the presence of a clear area or
inhibition zone around the paper disc. at a concentration of 50% it can be seen compared to the area of the inhibition
zone. The resulting katuk leaf extract tablets are F1 paper disc 1 which produces an inhibition zone of 25 mm, paper
disc 2 is 25 mm, F2 paper disc 1 is 26 mm, paper disc 2 is 28 mm, F3 paper disc 1 is 32 mm paper disc 2 is 31 mm.
According to Mukhriani, the higher the concentration of antimicrobial substances, the greater the ability to kill
microorganisms. In the positive control, the inhibition zone area was 35 mm, categorized as very strong. The
antibacterial activity of ciprofloxacin positive control was stronger than the three tablet formulas used in this study.
This is because ciprofloxacin is an antibacterial drug belonging to the quinolone class which is used for the treatment
of various types of bacterial infections [3]. Binders and disintegrants in the manufacture of tablets that can affect the
area of inhibition in the antibacterial test.
CONCLUSION
The results of the evaluation of the 3rd tablet formula, tablets from katuk leaf extract formulas 3 which had good
tablet evaluation test results. Katuk leaf tablets are proven to be effective as antioxidants that can counteract free
radicals with an IC50 value of 106.91μg/mL which is a moderate antioxidant. Katuk leaf extract tablet which has an
inhibitory power against Staphylococcus aureus is the third formula with an average inhibition zone of 31.5 mm
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020225-8
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