Antibacterial Activity and Phytochemical Responses of
Tempuyung (
Sonchus oleraceus
) under Cr(VI), Cu(II),
Pb(II), dan Cd(II) Stress Condition
Foni Adilla Setyanti
1, a), Sri Kasmiyati
1, b), and Sucahyo
1, c)1
Faculty of Biology, Satya Wacana Christian University, Diponegoro Street 52-60, Salatiga, Indonesia
a)
Corresponding author:412011014@student.uksw.edu
b)
Kas@staff.uksw.edu
c)
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).
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 lancet-shaped, 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.
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
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 UV-VIS 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
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)
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:
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 -
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)
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
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
TABLE 2. Correlation of antibacterial activity with total flavonoids, tannins and total phenols in Escherichia coli by Pearson correlation analysis.
Antibacterial Activity Total Phenols Total Flavonoids TotalTannins
Antibacterial Activity 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
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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