M01743
Antibacterial Activity and Phytochemical Responses of
Tempuyung (Sonchus oleraceus) under Cr(VI), Cu(II),
Pb(II), dan Cd(II) Stress Condition
Foni Adilla Setyanti1, a), Sri Kasmiyati1, b), and Sucahyo1, c)
1
Faculty of Biology, Satya Wacana Christian University, Diponegoro Street 52-60, Salatiga, Indonesia
a)
Corresponding author: 412011014@student.uksw.edu
b)
c)
Kas@staff.uksw.edu
Sucahyo_2009@gmail.com
Abstract
Environmental pollution is able to discompose humans’ health, the quality of humans’ life, and also the
natural function of ecosystem. The presence of excessive amounts of heavy metals in the environment can cause
the environmental pollution and can interfere the plant growth. Plants that experienced stress, scientifically will
synthesize the secondary compound of metabolites. In this research, tempuyung (Sonchus oleraceus) is being
used. Nowadays, tempuyung (Sonchus oleraceus) is widely studied that mainly focused on its chemical content
and its utilization as a material of medical treatment. In this research, tempuyung (Sonchusoleraceus) will be
given the heavy metals’ stress of chromium (Cr (IV)), copper (Cu (III)), lead (Pb (II)), and cadmium (Cd (II)).
The research’s purpose is to comprehend and analyze the response of tempuyung (Sonchus oleraceus) toward
the exposure of heavy metals through the phytochemistry content’s test and antibacterial activity as well as its
correlation with the levels of total phenols, flavonoids, tannins, and anthraquinone. Phytochemical screening
results of tempuyung (Sonchus oleraceus) with various treatments showed similar results that are bioactive
compounds alkaloids, flavonoids, tannins, and anthraquinone. Antibacterial activity with a paper disk diffusion
method which is a tempuyung (Sonchus oleraceus) leaf extract showed that the exposure to heavy metals and
controls that were tested had greater inhibitory toward the growth of E. coli bacterium rather than S. aureus
bacterium. This proves that the antibacterial compounds in tempuyung (Sonchus oleraceus’s) leaf extracts are
giving more effect on gram-negative bacterium. The contents of total phenols, flavonoids, and tannins are
positively correlated with antibacterial activity. From all tests, the treatment of exposure to Cr(IV) shows the
influence of antibacterial activity with the contents of secondary metabolites that produced are phenols,
flavonoids, and tannins, which also can be interpreted that the plants experienced the highest condition of stress.
Key Words: Antibacterial activity, heavy metal stress, seconda ry metabolites, environmental pollution,
tempuyung (Sonchus oleraceus).
Introduction
Environmental pollution is not a new case, even some of us already understand the effect that caused
by environmental pollution toward the viability and ecosystem equilibrium. Pollution can be defined as
environmental contamination by components or compounds contained in the environment (soil, air, and water)
that exceeds the threshold and have the toxic characteristic which can disrupt humans health, quality of life, and
also the natural function of ecosystems (Wang et al 2006). One of these compounds which is heavy metal. The
heavy metal is an element - a chemical element with a density greater than 5 g / cm3, located in the lower right
corner of the periodic system, has a high affinity for elements of S and typically numbered 22 to 92 atoms of a
period 4 to 7 (Susana et al 2010). The use of heavy metals is very widespread among industries, for instances,
mixed metal industry, metal plating, leather tanning, wood preservation; paint, textiles, synthetic chemicals as
well as several brands of pesticides use this kind of heavy metal in its products (Husnan et al 2012).
The presence of heavy metals in excessive amounts inside the soil can destabilize the ecosystems inside
the soil, for examples soil organisms and disrupt plants growth, especially plants that are growing in
contaminated areas such as in the industrial area. Plants that experienced stress scientifically will synthesize
secondary metabolites. Secondary metabolites are metabolites that are not essential for the growth of organisms
and found in a unique form or different between one to another species. Each organism usually produces
different secondary metabolites, these compounds are not always produced, but only produced when these are
needed or in a particular phase. The function of secondary metabolites is to defend themselves from unfavorable
environmental conditions which one of the functions is to defend themselves in a polluted environment (Baron
and Stasolla 2008).
The secondary metabolites are classified into three main groups, namely: terpenoids, phenolic, and
compounds that contain nitrogen. Most of terpenoids compounds contain carbon and hydrogen that are
synthesized through the mevalonate acid metabolism, for examples monoterpene, sesquiterpene, diterpenes,
triterpene, and terpene polymers. Phenolic is a compound made from simple sugars and has a benzene ring,
hydrogen, and oxygen in a chemical structure, such as phenolic acids, coumarin, lignin, flavonoids, and tannins.
Compounds that are contain nitrogen, such as alkaloids and glucosinolates (Baron and Stasolla 2008).
Tempuyung (Sonchus oleraceus) is a wild plant that is widely spread. This plant is included in the
phylum of Magnoliophyta , class of Magnoliopsida , order of Asterales and family of Asteracea. Tempuyung is
included as annual plants; the height is reached up to 1 meter with a 2 cm diameter of trunk, single leaf lancetshaped, sharp edge, the edge of the irregular share pinnate, bright yellow flowers, and dark colored seeds
(Winarto 2004).
Based on a research that has been conducted on a number of plant extracts, tempuyung is commonly
used as a traditional medicine; some of them are potential as a source of antioxidants and antibacterial. In
Chinese pharmacology mentioned that this tempuyung leaf has the quality to reduce the heat, bitter taste, and is
widely used for the detoxification process that can be used as an indication of antioxidants’ contents, and
antibacterial in the plant (Xia, Z et al 2010). An antimicrobial agent is a biological or chemical compound that is
able to inhibit the growth and microbial activity (Nuraini 2007). The class of flavonoid and tannin has
antibacterial activity. Mohamad et al (2012), explained that flavonoids damage to the permeability of the
bacterial cell rilem bane, microsomes, and lysosomes that caused by an interaction between flavonoid with
bacteria DNA. The characteristic of lipophilic owned by the class of flavonoid causing this compound damaging
the bacteria’s cell membranes. Then, tannin allegedly associated with its ability in inactivated adhesion
microbes, enzymes, and protein transport in the cell membrane.
In this research, tempuyung will be given stress from heavy metal chromium (Cr VI), copper (Cu II),
lead (Pb II), and cadmium (Cd II). The stress that is given in tempuyung is expected to increase the contents of
secondary metabolites, one of which is flavonoid and tannin that has antibacterial activity. The research’s
purpose is to comprehend and analyze the response of tempuyung toward the stress from heavy metals through
testing the antibacterial and the phytochemical contents and its correlation with the levels of phenols,
flavonoids, and tannins from the total of the ethanol extract of the medical plant (Sonchusoleraceus) leaves. The
phytochemical contents test is using the phytochemical screening method, while the antibacterial activity is
using the disc diffusion method.
Materials and Method
Experimental Design
The research carried out experimentally using a Complete Randomized Design (CRD) with four
treatments and five replications. The treatment given is using stress of heavy metal types Cr, Cd, Cu, and Pb
with a concentration of 10 ppm.
Planting the Seeds of the Tempuyung (Sonchus oleraceus)
Seeds obtained from the agricultural field or land of Faculty of Agriculture, Satya Wacana Christian
University in Salaran, Kopeng are germinated in soil as a medium of the seeds that is put into the pot with 50
cm’s diameter. The land used as the medium is also taken from the agricultural field or land of the Faculty of
Agriculture. Furthermore, the soil is mixed with compost within the ratio 1: 1. The plants tested for 30 days long
is moved in a poly bag with the size of 15x20 cm which already filled with the soil that also from agricultural
field or land of Faculty of Agriculture as much as 500 grams, where each polybag filled with 3 plants and
acclimatized for 7 days. Previously, the land used as the medium in the polybag has been dried for 7 days and
does not mix with the compost.
Treatment of the Heavy Metal Stress
The treatment of heavy metal stress Cr, Cd, Cu, and Pb carried out for 30 days after a period of
acclimation. The heavy metal compounds used are K2Cr2O7, CdCl2, CuSO4, and Pb (CH3COO)2 obtained from
the Laboratory of Biochemistry and Molecular Cell Biology, Faculty of Biology, Satya Wacana Christian
University, Salatiga. The 10 ppm heavy metal’s concentration is given as much as 50 ml to 500 grams of soil.
The treatment carried out for 1 month, at the time of treatment plants are watered with 20 ml of water taps
(PDAM) in the morning (08.00 am) and afternoon (17:00 pm) where the watering system is done every day.
After one month, the leaves are harvested and analyzed. Each treatment is including controlling 5 replicates with
the place arrangement that is done randomly and moved again randomly every once a week during the treatment
times or days.
Preparation Sample of Tempuyung (Sonchus oleraceus) Leaf
According to Mohamad et al (2012), the Tempuyung leaf that has been harvested are cleaned with
water, then dried for 6 hours, next dried in an oven for 6 hours at a temperature of 400C, furthermore pulverized
with a blender to form powder, and lastly stored in a container.
Phytochemical Screening
According to Harbone (1987) and Department of Health (1995) modified by Tarin et al (2008),
phytochemical screening of simplicia powders with tube test method includes examining compounds alkaloids,
flavonoids, terpenoids / steroids, tannins, saponins, and anthraquinone. Each inspection of chemical compounds
repeatedly made as much as 3 times.
Alkaloid Examination
The simplicia powder is weighed as much as 0.5 grams then it is added with 1 ml of 2 N hydrochloric
acid and 9 ml of distilled water, heated on water bath for 2 minutes, cooled and filtered. Filtrate that is derived
from it used to test alkaloid with the reagent of Dragendorf and Wagner . A positive reaction with the
Dragendorf is characterized by the red color’s formation. Thus, the Wagner ’s reagent characterized by the
formation of brown precipitate.
Flavonoids Examination
The simplicia powder is weighed as much as 1 gram and then added 20 ml of ether for 2 hours, filtered
through a filter paper and then examined with flavonoids examination within concentrated H2SO4 reagent. A
positive reaction is characterized by yellowish green color.
Terpenoids/ Steroid Inspection
As much as 1 gram of the simplicia powder filtered with the ether for 2 hours, filtered and then
examined with terpenoids / steroid examination. Filtrate as much as 3 drops along with 2 drops of reagent
salkowsky (concentrated H2SO4). The positive reactions are marked with red color formed.
Tannins Examination
The simplicia powder is weighed as much as 1 gram, filtered with 10 ml of distilled water and then
filtered. The filtrate is diluted with water until colorless. The solution is taken as much as 2 ml and added 1-2
drops of reagent iron (III) chloride 3%. The positive reaction is characterized by blue or blackish green.
Saponin Examination
The simplicia powder is weighed as much as 0.5 grams and put into a test tube, then add 19 ml of hot
water, cooled it down, then shake it strongly for 10 seconds. If the foam is formed stable as high as 1-10 cm, no
less than 10 minutes, and does not disappear with the addition of 1 drop of hydrochloric acid 2 N; this indicate
the presence of saponin.
Anthraquinone Examination
As much as 1 gram of the simplicia powder is concentrated with 20 ml of ether for 2 hours, filtered,
and then examined with anthraquinone examination. 10 ml filtrate was added with 5 ml of benzene and then
added 5 ml of ammonia and shake. The positive reactions are marked with red color.
Extraction Sample of Tempuyung (Sonchus oleraceus) Leaf
According to Macari et al (2006), the simplicia powder obtained is being extracted by maceration using
96% ethanol for 3 x 24 hours and then evaporated. The ratio of the weight of the sample and the extracting
solvent is 1: 13.
Determination of Total Phenol Content
According to McDonald et al (2001), as much as 0.1 ml of extract is diluted (1: 1 mg / ml methanol),
then added 3.9 ml of distilled water, and 0.5 ml of Folin-Ciocalteu reagent (1: 10 in distilled water). The
solution is being settled down for 3 minutes, and then added 2 ml of 20% Na2Co3 and absorbance is measured at
765 nm with a UV-VIS spectrophotometer after being incubated for 30 minutes at a temperature’s room. The
test is carried out up to 5 repetitions. Gallic acid with a percentage concentration range between 12.5 to 200 mg /
L is used in making the calibration curve to determine the levels of total phenols. The total phenol contents are
stated in milligrams of tannic acid / gram of dry powder.
Determination of Total Flavonoid Content
According to Pourmorad et al (2006), as much as 0.5 ml of extract is diluted (1: 1 mg / ml methanol)
then added with 1.5 ml of ethanol, 0.1 m AlCl3 10%, 0.1 ml of 1 M sodium acetate, and 2.8 ml of distilled water.
The solution mixture is being left for 30 minutes, after it is measured with its absorbance at 417 nm with a UVVIS spectrophotometer. The tests are carried out as much as 3 replications. Quercetin within percentage
concentration ranging between 4.125 to 100 mg / L is used in making the calibration curve to determine the total
flavonoid contents. The total flavonoid contents in the ethanol extract stated as milligrams of quercetin
equivalents / gram of dry powder (EK mg / g).
Determination of Total Tannin Content
According to Polshettiwar et al (2007), as much as 0.1 ml of extract is diluted (1: 1 mg / ml methanol)
then added 3.9 ml of distilled water and 0.5 ml of Folin-Ciocalteu reagent and vortex, added with 2 ml of 20%
Na2CO3 and vortex again. The absorbance is read at 765 nm with a UV-VIS spectrophotometer after incubation
for 30 minutes at the temperature’s room. The tests are performed five repetitions. Tannic acid with a percentage
concentration range between 12.5 to 200 mg / L is used in making the calibration curve to determine the total
tannin levels. The total tannin content is stated in milligrams of tannic acid / gram of dry powder.
Antibacterial Activity Testing
According to Gariga et al (1993), the method used is disk diffusion method. Breeding the bacteria
(Escherichia coli and Staphylococcus aerus) are inserted into the medium of nutrient broth (NB) and then
incubated at 37° C for 24 hours for rejuvenation. As much as 0.1 ml of bacteria in NB is put in a nutrient agar
(NA) medium, which has already been solid. Furthermore, the paper discs which have filled 20 ul of ethanol
extract of the tempuyung leaves with a concentration of 400 mg / ml; 20 ultetrasiklin with a concentration of
0.5 mg / ml as a positive control, and 20 ul distilled water as negative control placed on the NA. Petri dish which
is contained NA incubated at 37° C for 24 hours and then measured in the diameter of inhibitory. The tests are
carried out as much as 3 replications.
Data analysis
Statistical analysis used was testing analysis of variance (ANOVA) with the SPSS Software for
Window Release 17.0. Homogeneity of variance (Levene's test) and normality) (Shapiro-Wilk test) will be
checked before the analysis of variance. Test followed by Tukey's test if it obtained the real effect of the
treatment. The test is performed to determine differences in the levels of phenol, total flavonoids and tannins in
each extract in each treatment, as well as to determine significant differences from the average diameter of the
inhibition formed each treatment. As for seeing the relationship variables for each treatment with phenol, total
flavonoids and tannins to the anti-bacterial activity were analyzed using Pearson correlation analysis.
Correlation value is 1 if there is a positive linear relationship, worth -1 if there is a negative linear relationship.
The closer to -1 or +1, the stronger the correlation between the two variables.
Results and Discussion
Phytochemical screening
Phytochemical screening is a qualitative test that can be known compounds in the leaves tempuyung
(Sonchus oleraceus). Results of phytochemical screening of the leaves tempuyung with various heavy metals
treatment (control, Cr, Cd, C, Pb) are listed in TABLE 1. As follows:
TABLE 1. Results of phytochemical screening tempuyung leaf (Sonchus oleraceus )
Chemical Content
Method
Result
Description
Alkaloids
Dragendorf
Red
+
Wagner
Sediment reddish brown
+
Flavonoids
H2SO4
Yelowwish green
+
Terpenoids
H2SO4
Yellow
Tannins
feCl3
Blackish green
+
Saponins
Uji Forth
Not formed foem
Anthraquinone
Brontrager
Brownish red
+
Description: (+) = exist, (-) = did not exist.
Based on the results in TABLE 1., it is known that the leaves tempuyung with various treatments of
heavy metals (Cr, Cd, Cu, Pb) and the controls have a similar phytochemical content. Tempuyung on the leaves
contained alkaloids, flavonoids, tannins and anthraquinone. Flavonoids and tannins are phenolic compounds
known to possess antibacterial activity. By because the levels of flavonoids, tannins and total phenols contained
in the leaves tempuyung with various treatments need to be measured in order to obtain quantitative results from
these compounds and analyzed the correlation between these compounds with antibacterial activity.
Levels of total phenols
Total phenol content measurement results on leaves tempuyung with various treatments of heavy
metals (Cr, Cd, Cu, Pb) and the control showed the following results:
FIGURE I. Average levels of total phenols from leaves tempuyung with various treatments of heavy metals (K, Cr, Cd, Cu,
Pb) (* Different letters indicate significantly different results)
Levels of total phenols in FIGURE 1. shows that in all treatments showed different results. Control
treatment was obtained levels of total phenols average of 87.755 mg of acid equivalent galatg dry powder,
treatment of heavy metal Cr showed the highest value that is equal to 102.273 mg equivalent gallic acid / g dry
powder, treatment of heavy metals Cd showed a value of 98.701 mg equivalent gallic acid / g dry powder, in the
treatment of heavy metals Cu showed a yield of 89.076 mg gallic acid equivalents / g of dry powder and the
treatment of heavy metals Pb obtained yield was 90.710 mg gallic acid equivalents / g of dry powder. The
results obtained show that in all treatments influence on the test plants. The treatment of heavy metals Cr, Cd,
Cu and Pb at 10 ppm has effects on phenolic secondary metabolites produced by the test plants can be seen from
all treatments significantly different from controls.
Levels of total flavonoids
The total flavonoid content measurement results on tempuyung leaves with various treatments of heavy
metals (Cr, Cd, Cu and Pb) and the control showed the following results:
FIGURE 2. Average levels of total flavonoids from the leaves tempuyung the treatment of heavy metal (K, Cr, Cd, Cu, Pb)
(* Different letters indicate significantly different values)
In FIGURE 2. the results of measurements of the levels of total flavonoids were significantly different
between the control and Cr and control with Cu, whereas the treatment of heavy metals Cd and Pb did not show
significantly different results with Cr, control and Cu. The total flavonoid in the control treatment was obtained
levels of total flavonoids average of 86.778 mg EK / gram of dry powder, in the treatment of heavy metal Cr
gained the results of the most high at 100.778 mg EK / gram of dry powder, treatment of Cd obtained yield was
97.306 mg EK / gram of dry powder, Cu treatment obtained yield was 88.972 mg EK / gram of dry powder and
in the treatment of Pb obtained yield was 89.693 mh EK / grams of dry powder.
Analysis of variance in total flavonoid contained extracts produced by measuring the levels of total
flavonoids were significantly different between the control and Cr and control with Cu, whereas the treatment of
heavy metals Cd and Pb did not show the same results, which means there is no effect on levels of total
flavonoids in the provision of heavy metals Cu, this was due to the heavy metals Cu is an essential compound
that plants need in small amounts, the granting of Cu with a concentration of 10 ppm has not effect on plants or
can be tolerated so as to control there is no difference (Husnan et al 2012). In the treatment of Cr showed
significantly different results with the control, it is because Cr is the most toxic among the four metals in the
treatment so that the concentration of 10 ppm is already generating effects on the plant, the plant was seized
then the plant will produce secondary metabolites higher also and flavonoids is one of the secondary metabolites
in accordance with the statement of Naron and Stasolla (2008).
Levels of Total Tannins
Results of measurements of total tannins in the leaves tempuyung with various treatments of heavy
metals (Cr, Cd, Cu, Pb) and the control showed the following results:
FIGURE 3. Average levels of total tannin from tempuyung the treatment of heavy metal (K, Cr, Cd, Cu, Pb) (* Different
letters indicate that the value of real berbda)
FIGURE 3. shows the results of measurements of the average total tannin levels are significantly
different from each treatment of heavy metal. Test plants without treatment (control) gained an average content
of tannin of 0.0270 mg equivalent tannic acid / g dry powder, in the treatment with exposure to heavy metals Cr
diproleh highest yield in the amount of 0.0445 mg equivalent tannic acid / g dry powder, treatment of heavy
metals Cd obtained yield was 0.0351 mg tannic acid equivalent / g dry powder, in the treatment of heavy metal
Cu was obtained a yield of 0.0290 mg tannic acid equivalent / g of dry powder and the treatment of heavy
metals Pb obtained yield was 0.0307 mg tannic acid equivalent / g of dry powder. The results obtained show that
in all metal exposure showed different effects. The provision of heavy metals Cr, Cd, Cu, Pb and 10 ppm has
given effect on the test plants against tannin content.
Antibacterial activity
Antibacterial compounds of extract of tempuyung leaves with various treatments of heavy metals (Cr,
Cd, Cu, Pb) and without heavy metal treatment (control) tested the activity using diffusion method against two
types of test bacteria. Both of the test bacteria Escherichia coli and Staphylococcus aureus. The effectiveness of
the extract in inhibiting the growth of test bacteria by the size of the clear zone that forms a circle around the
paper disc that can be calculated diameter of the inhibiting (Nuraini 2007). From observations on the diameter
of inhibition formed on all treatments including the control. On this test is used tetracycline as a positive control
and distilled water used as a negative control. In FIGURE 4. is the result of an average diameter of inhibition on
all treatments Cr, Cd, Cu, Pb and control treatment.
FIGURE 4. Average diameter of the inhibition of leaf extracts tempuyung with various heavy metals treatment against
Escherichia coli and Staphylococcus aureus (* Different letters indicate significantly different values).
According Nostro et al. (2000), the diameter of the minimum inhibitory showed antimicrobial activity
is ±6mm. Tempuyung leaf extract on all treatments were tested on Escherichia coli showed antibacterial activity
kaena on all treatments showed a value above 6mm namely, the average diameter of the control inhibition of
7,2mm, treatment of 10mm Cr, Cd for the treatment of heavy metal 8,4mm, Cu treatment of 7,8mm and the
treatment of heavy metals Pb by 8mm. Cr treatment showed the highest value and significantly different from
the control treatment and Cu. And Cu control treatment showed no difference, it can be interpreted that the
treatment of heavy metals Cu does not give effect to the antibacterial activity of the antibacterial activity of the
leaf extract tempuyung.
Tempuyung leaf extract were tested on Staphyloccocus aureus bacteria are not all showed antibacterial
activity. In the control treatment the average diameter of less than 6mm resistor is equal 5,8mm, it indicates that
no antibacterial activity in control when tested in Staphyloccocus aureus, the antibacterial activity was only seen
in the heavy metal treatment, while under normal conditions there is no activity or activity the bacteria are very
small. In the treatment of Cr look significantly different from the controls but did not differ by treatment of
heavy metals in the amount of 7,8mm, in the treatment of heavy metal Cd of 7,4mm, in the treatment of heavy
metal Cu 6,4mm and the treatment of heavy metals Pb obtained a value of 6,6mm.
Based on the values obtained indicate a difference in antibacterial activity against each bacteria tested.
In Escherichia coli obtained an average diameter of inhibition higher and the dick can be seen already showed
activity, whereas Staphycoccus aureus gained an average diameter of inhibition is lower and control treatment
had not occurred antibacterial activity or activity occurs but low visible of the average yield of less than 6mm.
The results obtained show that in all of heavy metal treatment raises clear zone. At control and Cr treatment
well against Escherichia coli and Staphyloccocus aureus seen no significant difference was shown with
different letters. This can occur due to exposure to heavy metals Cr can be seen the highest levels of phenolic
compounds and there are differences between the treatment and control Cr. Phenolic compounds are compounds
that when tested antibacterial activity of the compounds ii bacteria was instrumental in the clear zone is formed,
it is consistent with the statement Mohamad et al. (2012), explained that flavonoids have a lipophilic nature that
can destroy the cell walls of bacteria and Nuraini (2007) which states that the group of flavonoids and tannins
have antibacterial activity.
Antibacterial Activity Correlation with Phenolic Components
From the results of the antibacterial activity test paper disc diffusion method, it is known that in all
treatments to inhibit the growth of bacteria Escherichia coli and Staphycoccus aureus. Results of the analysis of
metals (Cr, Cd, Cu, Pb) including the control treatment were tested on Escherichia coli and Staphycoccus
aureus are shown in TABLE 2. and TABLE 3. Based on the correlation analysis obtained correlation values for
the content of total phenols, flavonoids and tannins to the antibacterial activity were tested on Escherichia coli
(TABLE 2.) amounted to 0.647; 0701 and 0.680, whereas in Staphycoccus aureus at 0,0,636; 0.681; 0.608
(TABLE 3.).
TABLE 2. Correlation of antibacterial activity with total flavonoids, tannins and total phenols in Escherichia coli by
Pearson correlation analysis.
Antibacterial Activity
Antibacterial Activity Total Phenols Total Flavonoids TotalTannins
1
0.647**
0.701**
0.680**
Total Flavonoids
0.647**
1
**
**
0.988**
0.951**
**
Total Tannins
0.701
0.988
1
0.944
**
**
**
Total Phenols
0.680
0.951
0.944
1
**. Correlation is significant at the 0.01 level (2-tailed).
Based on the correlation analysis, total phenol, total flavonoids and tannins with antibacterial activity
in bacteria Eschercia coli and Stephycoccus aureus both showed a significant positive correlation. From these
results it can be stated that the antibacterial activity of the leaf extract tempuyung with various treatments metal
is positively correlated with the content of total flavonoids, tannins and total phenols total in leaves tempuyung
seen also that phenol has a significant correlation with totla tannins and flavonoids membuktukan that total
phenol in tempuyung leaves derived from total flavonoids and tannins.
TABLE 3. Correlation with the antibacterial activity of total flavonoids, tannins and total phenols in Staphycoccus aureus
based on the analysis of Pearson
Antibacterial Activity Total Flavonoids Total Tannins Total Phenols
Antibacterial Activity
1
0.636**
0.681**
0.608**
Total Flavonoids
0.636**
1
0.988**
0.951**
**
**
Total Tannins
0.681
0.988
1
0.944**
Total Phenols
0.608**
0.951**
0.944**
1
**. Correlation is significant at the 0.01 level (2-tailed).
Conclusion
Based on phytochemical screening leaves tempuyung (Sonchus oleraceus) with a variety of metal
treatment contains alkaloids, flavonoids, tannins and anthraquinone. At antibacterial activity test, it can be
concluded that the extract of leaves tempuyung the treatment of heavy metal or the controls have inhibitory
greater in extracts were tested on Eschercia coli than were tested in Staphycoccus aureus , this proves that the
leaf extract tempuyung more effect on gram-negative bacteria, The content of flavonoids, tannins and total
phenols were positively correlated with antibacterial activity. Of all the trials showed that the treatment of heavy
metal Cr influence on secondary metabolites phenolic flavonoids and tannins that affect the antibacterial
activity, it is proved that the treatment of heavy metal Cr plants experience stress most high that produce high
levels of secondary metabolites is highest among treatments Another and significantly different from controls.
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Tempuyung (Sonchus oleraceus) under Cr(VI), Cu(II),
Pb(II), dan Cd(II) Stress Condition
Foni Adilla Setyanti1, a), Sri Kasmiyati1, b), and Sucahyo1, c)
1
Faculty of Biology, Satya Wacana Christian University, Diponegoro Street 52-60, Salatiga, Indonesia
a)
Corresponding author: 412011014@student.uksw.edu
b)
c)
Kas@staff.uksw.edu
Sucahyo_2009@gmail.com
Abstract
Environmental pollution is able to discompose humans’ health, the quality of humans’ life, and also the
natural function of ecosystem. The presence of excessive amounts of heavy metals in the environment can cause
the environmental pollution and can interfere the plant growth. Plants that experienced stress, scientifically will
synthesize the secondary compound of metabolites. In this research, tempuyung (Sonchus oleraceus) is being
used. Nowadays, tempuyung (Sonchus oleraceus) is widely studied that mainly focused on its chemical content
and its utilization as a material of medical treatment. In this research, tempuyung (Sonchusoleraceus) will be
given the heavy metals’ stress of chromium (Cr (IV)), copper (Cu (III)), lead (Pb (II)), and cadmium (Cd (II)).
The research’s purpose is to comprehend and analyze the response of tempuyung (Sonchus oleraceus) toward
the exposure of heavy metals through the phytochemistry content’s test and antibacterial activity as well as its
correlation with the levels of total phenols, flavonoids, tannins, and anthraquinone. Phytochemical screening
results of tempuyung (Sonchus oleraceus) with various treatments showed similar results that are bioactive
compounds alkaloids, flavonoids, tannins, and anthraquinone. Antibacterial activity with a paper disk diffusion
method which is a tempuyung (Sonchus oleraceus) leaf extract showed that the exposure to heavy metals and
controls that were tested had greater inhibitory toward the growth of E. coli bacterium rather than S. aureus
bacterium. This proves that the antibacterial compounds in tempuyung (Sonchus oleraceus’s) leaf extracts are
giving more effect on gram-negative bacterium. The contents of total phenols, flavonoids, and tannins are
positively correlated with antibacterial activity. From all tests, the treatment of exposure to Cr(IV) shows the
influence of antibacterial activity with the contents of secondary metabolites that produced are phenols,
flavonoids, and tannins, which also can be interpreted that the plants experienced the highest condition of stress.
Key Words: Antibacterial activity, heavy metal stress, seconda ry metabolites, environmental pollution,
tempuyung (Sonchus oleraceus).
Introduction
Environmental pollution is not a new case, even some of us already understand the effect that caused
by environmental pollution toward the viability and ecosystem equilibrium. Pollution can be defined as
environmental contamination by components or compounds contained in the environment (soil, air, and water)
that exceeds the threshold and have the toxic characteristic which can disrupt humans health, quality of life, and
also the natural function of ecosystems (Wang et al 2006). One of these compounds which is heavy metal. The
heavy metal is an element - a chemical element with a density greater than 5 g / cm3, located in the lower right
corner of the periodic system, has a high affinity for elements of S and typically numbered 22 to 92 atoms of a
period 4 to 7 (Susana et al 2010). The use of heavy metals is very widespread among industries, for instances,
mixed metal industry, metal plating, leather tanning, wood preservation; paint, textiles, synthetic chemicals as
well as several brands of pesticides use this kind of heavy metal in its products (Husnan et al 2012).
The presence of heavy metals in excessive amounts inside the soil can destabilize the ecosystems inside
the soil, for examples soil organisms and disrupt plants growth, especially plants that are growing in
contaminated areas such as in the industrial area. Plants that experienced stress scientifically will synthesize
secondary metabolites. Secondary metabolites are metabolites that are not essential for the growth of organisms
and found in a unique form or different between one to another species. Each organism usually produces
different secondary metabolites, these compounds are not always produced, but only produced when these are
needed or in a particular phase. The function of secondary metabolites is to defend themselves from unfavorable
environmental conditions which one of the functions is to defend themselves in a polluted environment (Baron
and Stasolla 2008).
The secondary metabolites are classified into three main groups, namely: terpenoids, phenolic, and
compounds that contain nitrogen. Most of terpenoids compounds contain carbon and hydrogen that are
synthesized through the mevalonate acid metabolism, for examples monoterpene, sesquiterpene, diterpenes,
triterpene, and terpene polymers. Phenolic is a compound made from simple sugars and has a benzene ring,
hydrogen, and oxygen in a chemical structure, such as phenolic acids, coumarin, lignin, flavonoids, and tannins.
Compounds that are contain nitrogen, such as alkaloids and glucosinolates (Baron and Stasolla 2008).
Tempuyung (Sonchus oleraceus) is a wild plant that is widely spread. This plant is included in the
phylum of Magnoliophyta , class of Magnoliopsida , order of Asterales and family of Asteracea. Tempuyung is
included as annual plants; the height is reached up to 1 meter with a 2 cm diameter of trunk, single leaf lancetshaped, sharp edge, the edge of the irregular share pinnate, bright yellow flowers, and dark colored seeds
(Winarto 2004).
Based on a research that has been conducted on a number of plant extracts, tempuyung is commonly
used as a traditional medicine; some of them are potential as a source of antioxidants and antibacterial. In
Chinese pharmacology mentioned that this tempuyung leaf has the quality to reduce the heat, bitter taste, and is
widely used for the detoxification process that can be used as an indication of antioxidants’ contents, and
antibacterial in the plant (Xia, Z et al 2010). An antimicrobial agent is a biological or chemical compound that is
able to inhibit the growth and microbial activity (Nuraini 2007). The class of flavonoid and tannin has
antibacterial activity. Mohamad et al (2012), explained that flavonoids damage to the permeability of the
bacterial cell rilem bane, microsomes, and lysosomes that caused by an interaction between flavonoid with
bacteria DNA. The characteristic of lipophilic owned by the class of flavonoid causing this compound damaging
the bacteria’s cell membranes. Then, tannin allegedly associated with its ability in inactivated adhesion
microbes, enzymes, and protein transport in the cell membrane.
In this research, tempuyung will be given stress from heavy metal chromium (Cr VI), copper (Cu II),
lead (Pb II), and cadmium (Cd II). The stress that is given in tempuyung is expected to increase the contents of
secondary metabolites, one of which is flavonoid and tannin that has antibacterial activity. The research’s
purpose is to comprehend and analyze the response of tempuyung toward the stress from heavy metals through
testing the antibacterial and the phytochemical contents and its correlation with the levels of phenols,
flavonoids, and tannins from the total of the ethanol extract of the medical plant (Sonchusoleraceus) leaves. The
phytochemical contents test is using the phytochemical screening method, while the antibacterial activity is
using the disc diffusion method.
Materials and Method
Experimental Design
The research carried out experimentally using a Complete Randomized Design (CRD) with four
treatments and five replications. The treatment given is using stress of heavy metal types Cr, Cd, Cu, and Pb
with a concentration of 10 ppm.
Planting the Seeds of the Tempuyung (Sonchus oleraceus)
Seeds obtained from the agricultural field or land of Faculty of Agriculture, Satya Wacana Christian
University in Salaran, Kopeng are germinated in soil as a medium of the seeds that is put into the pot with 50
cm’s diameter. The land used as the medium is also taken from the agricultural field or land of the Faculty of
Agriculture. Furthermore, the soil is mixed with compost within the ratio 1: 1. The plants tested for 30 days long
is moved in a poly bag with the size of 15x20 cm which already filled with the soil that also from agricultural
field or land of Faculty of Agriculture as much as 500 grams, where each polybag filled with 3 plants and
acclimatized for 7 days. Previously, the land used as the medium in the polybag has been dried for 7 days and
does not mix with the compost.
Treatment of the Heavy Metal Stress
The treatment of heavy metal stress Cr, Cd, Cu, and Pb carried out for 30 days after a period of
acclimation. The heavy metal compounds used are K2Cr2O7, CdCl2, CuSO4, and Pb (CH3COO)2 obtained from
the Laboratory of Biochemistry and Molecular Cell Biology, Faculty of Biology, Satya Wacana Christian
University, Salatiga. The 10 ppm heavy metal’s concentration is given as much as 50 ml to 500 grams of soil.
The treatment carried out for 1 month, at the time of treatment plants are watered with 20 ml of water taps
(PDAM) in the morning (08.00 am) and afternoon (17:00 pm) where the watering system is done every day.
After one month, the leaves are harvested and analyzed. Each treatment is including controlling 5 replicates with
the place arrangement that is done randomly and moved again randomly every once a week during the treatment
times or days.
Preparation Sample of Tempuyung (Sonchus oleraceus) Leaf
According to Mohamad et al (2012), the Tempuyung leaf that has been harvested are cleaned with
water, then dried for 6 hours, next dried in an oven for 6 hours at a temperature of 400C, furthermore pulverized
with a blender to form powder, and lastly stored in a container.
Phytochemical Screening
According to Harbone (1987) and Department of Health (1995) modified by Tarin et al (2008),
phytochemical screening of simplicia powders with tube test method includes examining compounds alkaloids,
flavonoids, terpenoids / steroids, tannins, saponins, and anthraquinone. Each inspection of chemical compounds
repeatedly made as much as 3 times.
Alkaloid Examination
The simplicia powder is weighed as much as 0.5 grams then it is added with 1 ml of 2 N hydrochloric
acid and 9 ml of distilled water, heated on water bath for 2 minutes, cooled and filtered. Filtrate that is derived
from it used to test alkaloid with the reagent of Dragendorf and Wagner . A positive reaction with the
Dragendorf is characterized by the red color’s formation. Thus, the Wagner ’s reagent characterized by the
formation of brown precipitate.
Flavonoids Examination
The simplicia powder is weighed as much as 1 gram and then added 20 ml of ether for 2 hours, filtered
through a filter paper and then examined with flavonoids examination within concentrated H2SO4 reagent. A
positive reaction is characterized by yellowish green color.
Terpenoids/ Steroid Inspection
As much as 1 gram of the simplicia powder filtered with the ether for 2 hours, filtered and then
examined with terpenoids / steroid examination. Filtrate as much as 3 drops along with 2 drops of reagent
salkowsky (concentrated H2SO4). The positive reactions are marked with red color formed.
Tannins Examination
The simplicia powder is weighed as much as 1 gram, filtered with 10 ml of distilled water and then
filtered. The filtrate is diluted with water until colorless. The solution is taken as much as 2 ml and added 1-2
drops of reagent iron (III) chloride 3%. The positive reaction is characterized by blue or blackish green.
Saponin Examination
The simplicia powder is weighed as much as 0.5 grams and put into a test tube, then add 19 ml of hot
water, cooled it down, then shake it strongly for 10 seconds. If the foam is formed stable as high as 1-10 cm, no
less than 10 minutes, and does not disappear with the addition of 1 drop of hydrochloric acid 2 N; this indicate
the presence of saponin.
Anthraquinone Examination
As much as 1 gram of the simplicia powder is concentrated with 20 ml of ether for 2 hours, filtered,
and then examined with anthraquinone examination. 10 ml filtrate was added with 5 ml of benzene and then
added 5 ml of ammonia and shake. The positive reactions are marked with red color.
Extraction Sample of Tempuyung (Sonchus oleraceus) Leaf
According to Macari et al (2006), the simplicia powder obtained is being extracted by maceration using
96% ethanol for 3 x 24 hours and then evaporated. The ratio of the weight of the sample and the extracting
solvent is 1: 13.
Determination of Total Phenol Content
According to McDonald et al (2001), as much as 0.1 ml of extract is diluted (1: 1 mg / ml methanol),
then added 3.9 ml of distilled water, and 0.5 ml of Folin-Ciocalteu reagent (1: 10 in distilled water). The
solution is being settled down for 3 minutes, and then added 2 ml of 20% Na2Co3 and absorbance is measured at
765 nm with a UV-VIS spectrophotometer after being incubated for 30 minutes at a temperature’s room. The
test is carried out up to 5 repetitions. Gallic acid with a percentage concentration range between 12.5 to 200 mg /
L is used in making the calibration curve to determine the levels of total phenols. The total phenol contents are
stated in milligrams of tannic acid / gram of dry powder.
Determination of Total Flavonoid Content
According to Pourmorad et al (2006), as much as 0.5 ml of extract is diluted (1: 1 mg / ml methanol)
then added with 1.5 ml of ethanol, 0.1 m AlCl3 10%, 0.1 ml of 1 M sodium acetate, and 2.8 ml of distilled water.
The solution mixture is being left for 30 minutes, after it is measured with its absorbance at 417 nm with a UVVIS spectrophotometer. The tests are carried out as much as 3 replications. Quercetin within percentage
concentration ranging between 4.125 to 100 mg / L is used in making the calibration curve to determine the total
flavonoid contents. The total flavonoid contents in the ethanol extract stated as milligrams of quercetin
equivalents / gram of dry powder (EK mg / g).
Determination of Total Tannin Content
According to Polshettiwar et al (2007), as much as 0.1 ml of extract is diluted (1: 1 mg / ml methanol)
then added 3.9 ml of distilled water and 0.5 ml of Folin-Ciocalteu reagent and vortex, added with 2 ml of 20%
Na2CO3 and vortex again. The absorbance is read at 765 nm with a UV-VIS spectrophotometer after incubation
for 30 minutes at the temperature’s room. The tests are performed five repetitions. Tannic acid with a percentage
concentration range between 12.5 to 200 mg / L is used in making the calibration curve to determine the total
tannin levels. The total tannin content is stated in milligrams of tannic acid / gram of dry powder.
Antibacterial Activity Testing
According to Gariga et al (1993), the method used is disk diffusion method. Breeding the bacteria
(Escherichia coli and Staphylococcus aerus) are inserted into the medium of nutrient broth (NB) and then
incubated at 37° C for 24 hours for rejuvenation. As much as 0.1 ml of bacteria in NB is put in a nutrient agar
(NA) medium, which has already been solid. Furthermore, the paper discs which have filled 20 ul of ethanol
extract of the tempuyung leaves with a concentration of 400 mg / ml; 20 ultetrasiklin with a concentration of
0.5 mg / ml as a positive control, and 20 ul distilled water as negative control placed on the NA. Petri dish which
is contained NA incubated at 37° C for 24 hours and then measured in the diameter of inhibitory. The tests are
carried out as much as 3 replications.
Data analysis
Statistical analysis used was testing analysis of variance (ANOVA) with the SPSS Software for
Window Release 17.0. Homogeneity of variance (Levene's test) and normality) (Shapiro-Wilk test) will be
checked before the analysis of variance. Test followed by Tukey's test if it obtained the real effect of the
treatment. The test is performed to determine differences in the levels of phenol, total flavonoids and tannins in
each extract in each treatment, as well as to determine significant differences from the average diameter of the
inhibition formed each treatment. As for seeing the relationship variables for each treatment with phenol, total
flavonoids and tannins to the anti-bacterial activity were analyzed using Pearson correlation analysis.
Correlation value is 1 if there is a positive linear relationship, worth -1 if there is a negative linear relationship.
The closer to -1 or +1, the stronger the correlation between the two variables.
Results and Discussion
Phytochemical screening
Phytochemical screening is a qualitative test that can be known compounds in the leaves tempuyung
(Sonchus oleraceus). Results of phytochemical screening of the leaves tempuyung with various heavy metals
treatment (control, Cr, Cd, C, Pb) are listed in TABLE 1. As follows:
TABLE 1. Results of phytochemical screening tempuyung leaf (Sonchus oleraceus )
Chemical Content
Method
Result
Description
Alkaloids
Dragendorf
Red
+
Wagner
Sediment reddish brown
+
Flavonoids
H2SO4
Yelowwish green
+
Terpenoids
H2SO4
Yellow
Tannins
feCl3
Blackish green
+
Saponins
Uji Forth
Not formed foem
Anthraquinone
Brontrager
Brownish red
+
Description: (+) = exist, (-) = did not exist.
Based on the results in TABLE 1., it is known that the leaves tempuyung with various treatments of
heavy metals (Cr, Cd, Cu, Pb) and the controls have a similar phytochemical content. Tempuyung on the leaves
contained alkaloids, flavonoids, tannins and anthraquinone. Flavonoids and tannins are phenolic compounds
known to possess antibacterial activity. By because the levels of flavonoids, tannins and total phenols contained
in the leaves tempuyung with various treatments need to be measured in order to obtain quantitative results from
these compounds and analyzed the correlation between these compounds with antibacterial activity.
Levels of total phenols
Total phenol content measurement results on leaves tempuyung with various treatments of heavy
metals (Cr, Cd, Cu, Pb) and the control showed the following results:
FIGURE I. Average levels of total phenols from leaves tempuyung with various treatments of heavy metals (K, Cr, Cd, Cu,
Pb) (* Different letters indicate significantly different results)
Levels of total phenols in FIGURE 1. shows that in all treatments showed different results. Control
treatment was obtained levels of total phenols average of 87.755 mg of acid equivalent galatg dry powder,
treatment of heavy metal Cr showed the highest value that is equal to 102.273 mg equivalent gallic acid / g dry
powder, treatment of heavy metals Cd showed a value of 98.701 mg equivalent gallic acid / g dry powder, in the
treatment of heavy metals Cu showed a yield of 89.076 mg gallic acid equivalents / g of dry powder and the
treatment of heavy metals Pb obtained yield was 90.710 mg gallic acid equivalents / g of dry powder. The
results obtained show that in all treatments influence on the test plants. The treatment of heavy metals Cr, Cd,
Cu and Pb at 10 ppm has effects on phenolic secondary metabolites produced by the test plants can be seen from
all treatments significantly different from controls.
Levels of total flavonoids
The total flavonoid content measurement results on tempuyung leaves with various treatments of heavy
metals (Cr, Cd, Cu and Pb) and the control showed the following results:
FIGURE 2. Average levels of total flavonoids from the leaves tempuyung the treatment of heavy metal (K, Cr, Cd, Cu, Pb)
(* Different letters indicate significantly different values)
In FIGURE 2. the results of measurements of the levels of total flavonoids were significantly different
between the control and Cr and control with Cu, whereas the treatment of heavy metals Cd and Pb did not show
significantly different results with Cr, control and Cu. The total flavonoid in the control treatment was obtained
levels of total flavonoids average of 86.778 mg EK / gram of dry powder, in the treatment of heavy metal Cr
gained the results of the most high at 100.778 mg EK / gram of dry powder, treatment of Cd obtained yield was
97.306 mg EK / gram of dry powder, Cu treatment obtained yield was 88.972 mg EK / gram of dry powder and
in the treatment of Pb obtained yield was 89.693 mh EK / grams of dry powder.
Analysis of variance in total flavonoid contained extracts produced by measuring the levels of total
flavonoids were significantly different between the control and Cr and control with Cu, whereas the treatment of
heavy metals Cd and Pb did not show the same results, which means there is no effect on levels of total
flavonoids in the provision of heavy metals Cu, this was due to the heavy metals Cu is an essential compound
that plants need in small amounts, the granting of Cu with a concentration of 10 ppm has not effect on plants or
can be tolerated so as to control there is no difference (Husnan et al 2012). In the treatment of Cr showed
significantly different results with the control, it is because Cr is the most toxic among the four metals in the
treatment so that the concentration of 10 ppm is already generating effects on the plant, the plant was seized
then the plant will produce secondary metabolites higher also and flavonoids is one of the secondary metabolites
in accordance with the statement of Naron and Stasolla (2008).
Levels of Total Tannins
Results of measurements of total tannins in the leaves tempuyung with various treatments of heavy
metals (Cr, Cd, Cu, Pb) and the control showed the following results:
FIGURE 3. Average levels of total tannin from tempuyung the treatment of heavy metal (K, Cr, Cd, Cu, Pb) (* Different
letters indicate that the value of real berbda)
FIGURE 3. shows the results of measurements of the average total tannin levels are significantly
different from each treatment of heavy metal. Test plants without treatment (control) gained an average content
of tannin of 0.0270 mg equivalent tannic acid / g dry powder, in the treatment with exposure to heavy metals Cr
diproleh highest yield in the amount of 0.0445 mg equivalent tannic acid / g dry powder, treatment of heavy
metals Cd obtained yield was 0.0351 mg tannic acid equivalent / g dry powder, in the treatment of heavy metal
Cu was obtained a yield of 0.0290 mg tannic acid equivalent / g of dry powder and the treatment of heavy
metals Pb obtained yield was 0.0307 mg tannic acid equivalent / g of dry powder. The results obtained show that
in all metal exposure showed different effects. The provision of heavy metals Cr, Cd, Cu, Pb and 10 ppm has
given effect on the test plants against tannin content.
Antibacterial activity
Antibacterial compounds of extract of tempuyung leaves with various treatments of heavy metals (Cr,
Cd, Cu, Pb) and without heavy metal treatment (control) tested the activity using diffusion method against two
types of test bacteria. Both of the test bacteria Escherichia coli and Staphylococcus aureus. The effectiveness of
the extract in inhibiting the growth of test bacteria by the size of the clear zone that forms a circle around the
paper disc that can be calculated diameter of the inhibiting (Nuraini 2007). From observations on the diameter
of inhibition formed on all treatments including the control. On this test is used tetracycline as a positive control
and distilled water used as a negative control. In FIGURE 4. is the result of an average diameter of inhibition on
all treatments Cr, Cd, Cu, Pb and control treatment.
FIGURE 4. Average diameter of the inhibition of leaf extracts tempuyung with various heavy metals treatment against
Escherichia coli and Staphylococcus aureus (* Different letters indicate significantly different values).
According Nostro et al. (2000), the diameter of the minimum inhibitory showed antimicrobial activity
is ±6mm. Tempuyung leaf extract on all treatments were tested on Escherichia coli showed antibacterial activity
kaena on all treatments showed a value above 6mm namely, the average diameter of the control inhibition of
7,2mm, treatment of 10mm Cr, Cd for the treatment of heavy metal 8,4mm, Cu treatment of 7,8mm and the
treatment of heavy metals Pb by 8mm. Cr treatment showed the highest value and significantly different from
the control treatment and Cu. And Cu control treatment showed no difference, it can be interpreted that the
treatment of heavy metals Cu does not give effect to the antibacterial activity of the antibacterial activity of the
leaf extract tempuyung.
Tempuyung leaf extract were tested on Staphyloccocus aureus bacteria are not all showed antibacterial
activity. In the control treatment the average diameter of less than 6mm resistor is equal 5,8mm, it indicates that
no antibacterial activity in control when tested in Staphyloccocus aureus, the antibacterial activity was only seen
in the heavy metal treatment, while under normal conditions there is no activity or activity the bacteria are very
small. In the treatment of Cr look significantly different from the controls but did not differ by treatment of
heavy metals in the amount of 7,8mm, in the treatment of heavy metal Cd of 7,4mm, in the treatment of heavy
metal Cu 6,4mm and the treatment of heavy metals Pb obtained a value of 6,6mm.
Based on the values obtained indicate a difference in antibacterial activity against each bacteria tested.
In Escherichia coli obtained an average diameter of inhibition higher and the dick can be seen already showed
activity, whereas Staphycoccus aureus gained an average diameter of inhibition is lower and control treatment
had not occurred antibacterial activity or activity occurs but low visible of the average yield of less than 6mm.
The results obtained show that in all of heavy metal treatment raises clear zone. At control and Cr treatment
well against Escherichia coli and Staphyloccocus aureus seen no significant difference was shown with
different letters. This can occur due to exposure to heavy metals Cr can be seen the highest levels of phenolic
compounds and there are differences between the treatment and control Cr. Phenolic compounds are compounds
that when tested antibacterial activity of the compounds ii bacteria was instrumental in the clear zone is formed,
it is consistent with the statement Mohamad et al. (2012), explained that flavonoids have a lipophilic nature that
can destroy the cell walls of bacteria and Nuraini (2007) which states that the group of flavonoids and tannins
have antibacterial activity.
Antibacterial Activity Correlation with Phenolic Components
From the results of the antibacterial activity test paper disc diffusion method, it is known that in all
treatments to inhibit the growth of bacteria Escherichia coli and Staphycoccus aureus. Results of the analysis of
metals (Cr, Cd, Cu, Pb) including the control treatment were tested on Escherichia coli and Staphycoccus
aureus are shown in TABLE 2. and TABLE 3. Based on the correlation analysis obtained correlation values for
the content of total phenols, flavonoids and tannins to the antibacterial activity were tested on Escherichia coli
(TABLE 2.) amounted to 0.647; 0701 and 0.680, whereas in Staphycoccus aureus at 0,0,636; 0.681; 0.608
(TABLE 3.).
TABLE 2. Correlation of antibacterial activity with total flavonoids, tannins and total phenols in Escherichia coli by
Pearson correlation analysis.
Antibacterial Activity
Antibacterial Activity Total Phenols Total Flavonoids TotalTannins
1
0.647**
0.701**
0.680**
Total Flavonoids
0.647**
1
**
**
0.988**
0.951**
**
Total Tannins
0.701
0.988
1
0.944
**
**
**
Total Phenols
0.680
0.951
0.944
1
**. Correlation is significant at the 0.01 level (2-tailed).
Based on the correlation analysis, total phenol, total flavonoids and tannins with antibacterial activity
in bacteria Eschercia coli and Stephycoccus aureus both showed a significant positive correlation. From these
results it can be stated that the antibacterial activity of the leaf extract tempuyung with various treatments metal
is positively correlated with the content of total flavonoids, tannins and total phenols total in leaves tempuyung
seen also that phenol has a significant correlation with totla tannins and flavonoids membuktukan that total
phenol in tempuyung leaves derived from total flavonoids and tannins.
TABLE 3. Correlation with the antibacterial activity of total flavonoids, tannins and total phenols in Staphycoccus aureus
based on the analysis of Pearson
Antibacterial Activity Total Flavonoids Total Tannins Total Phenols
Antibacterial Activity
1
0.636**
0.681**
0.608**
Total Flavonoids
0.636**
1
0.988**
0.951**
**
**
Total Tannins
0.681
0.988
1
0.944**
Total Phenols
0.608**
0.951**
0.944**
1
**. Correlation is significant at the 0.01 level (2-tailed).
Conclusion
Based on phytochemical screening leaves tempuyung (Sonchus oleraceus) with a variety of metal
treatment contains alkaloids, flavonoids, tannins and anthraquinone. At antibacterial activity test, it can be
concluded that the extract of leaves tempuyung the treatment of heavy metal or the controls have inhibitory
greater in extracts were tested on Eschercia coli than were tested in Staphycoccus aureus , this proves that the
leaf extract tempuyung more effect on gram-negative bacteria, The content of flavonoids, tannins and total
phenols were positively correlated with antibacterial activity. Of all the trials showed that the treatment of heavy
metal Cr influence on secondary metabolites phenolic flavonoids and tannins that affect the antibacterial
activity, it is proved that the treatment of heavy metal Cr plants experience stress most high that produce high
levels of secondary metabolites is highest among treatments Another and significantly different from controls.
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