GREEN TEA ETHANOL EXTRACT EFFICACY AGAINST PSEUDOMONAS AERUGINOSA

Research about green tea ethanol extract efficacy against Pseudomonas aeruginosa has been done. The green tea leaves were sourced from tea distributor in Sidamanik District, North Sumatra, Indonesia. Green tea leaves extract was obtained by maceration technique in which green tea leaves were soaked with ethanol 96% for 24 hours. After that, the filtrate was concentrated to be thick extract with no liquid content again. After that, the concentrated extract was made by 5%, 10% and 15% as extract variations. The efficacy of green tea ethanol extract against Pseudomonas aeruginosa was signed by increasing the inhibitory diameter that using disc method, in which the distilled water was used as negative blank. The results obtained for blanks, 5%, 10% and 15% extracts were 0; 1.85; 2.9 and 4.45 mm. The conclusion of this study is that the concentration of the extract is increasing proportionally to its inhibitory power, the higher of extract concentration is higher of its inhibitory activity against Pseudomonas aeruginosa. The conclusion described the green tea ethanol extract was effective to be antibacterial agent against Pseudomonas aeruginosa.


INTRODUCTION
Pseudomonas aeruginosa is a gram negative bacteria which known as obligate aerobic bacteria. It has encapsulated and polar flagella. The flagella has function as motility organelle which measuring about 0.5-1.0 m. These bacteria do not produce spores and cannot ferment carbohydrates. (Wu et al., 2015) In biochemical tests, these bacteria produced a positive impact on the indole, Methyl Red, and Voges-Proskauer assays.
These bacteria are widely found in nature, for example in soil, water, plants, and animals. P. aeruginosa is an opportunistic pathogen. This bacteria is nosocomial pneumonia infection causing (Bassetti et al., 2018).
When P. aeruginosa colonies grow on suitable media, they produce a bluish nonfluorescent pigment, pyocyanin. Some strains of Pseudomonas are also capable of producing a green fluorescent pigment, namely pioverdin (Sulviana et al., 2017).
These bacteria are also often used to degrade pesticide substances. P.
aeruginosa is a bacteria that is able to adapt to conditions of low oxygen and nutrients.
It also grows well in a temperature range of  (Flayyih et al., 2013) Green tea has great benefits with few side effects. Therefore, more and more studies are focused on the effects of green tea on human health until now (Zhou et al., 2016).
Here, We study about the efficacy of green tea ethanol extract (Camelia sinensis) from a distributor labeled Juma in Sidamanik District, North Sumatra against P. aeruginosa because this sample is not analyzed the antimicrobial activity of P.
aeruginosa yet and to get information involve the activity of green tea.

MATERIALS AND METHODS
The type of research used was experimental laboratory. Green tea was from commercial product labeled JUMA from Sidamanik district in North Sumatra.

Sample Preparation and Extraction
The sample of green tea were dried leaves ready to be macerated. Amount 100g of simplicia powder was extracted with 96% ethanol by maceration method for 2 days in a macerator container with occasional stirring. Then, it was filtered using filter paper and then re-immersed on the filtered residue for 1 days with the same treatment as the previous stage. The macerate was evaporated in average temperature at 78 0 C to obtain a thick green tea ethanol extract (Fahmi, 2020).

Preparation of Bacterial Culture Stock
P. aeruginosa colonies were taken using a round needle that had been sterilized and implanted on the surface of the nutrient media so that it was slanted by scratching and then incubated at 37 0 C for 24 hours (Fahmi, 2020).

Bacterial Inoculum Making
P. aeruginosa isolates were grown as colonies and then taken from the culture stock using a sterilize ose needle first, after that suspended in a test tube containing 10 mL of nutrient broth media and incubated at 37 0 C for 24 hours which means fulfill to the standard (Mc. Farland) for the turbidity (Fahmi, 2020).

Inhibitory Efficacy
0.1 mL of P. aeruginosa as inoculum was put in a petri dish, after that the nutrient agar (NA) that boiled with distilled water was poured into 15 mL petri dish with size 15 mL, then the petri dish was shaken on the table surface so that the media and bacterial suspension were homogeneous and allowed to be solid agar.
Disc paper that has been given with the concentration of each test solution that placed on the surface of the media has solidified then, incubated at 37 0 C during 18 hours until 24 hours. Observed and measured the inhibition diameter which formed using a caliper (Fahmi, 2020).  Average ± SD 8,90 ± 0,00
The formation of the inhibition zone was due to the antibacterial content in green tea ethanol extract and increasing as the extract concentration too. Green tea mechanism can prevent the attachment of pathogenic bacteria on the cell membrane of host. Also, green tea can inhibit the bacteria adhesion on it. The green tea also able to act as a potential anti-adhesive agent (Lee et al., 2009).
Epigallocatechin gallate (EGCG), which is catechin derivative contained in green tea has also been reported to interact with the outer membrane bacterial and may prevent the adhesion to mammalian epithelial cells (Sharma et al., 2012).
Green tea may affect the activity of dihydrofolate reductase, an enzyme that is needed by pathogenic bacteria to synthesize purine and pyrimidine as well as increase the thickness of the epidermis.
Pseudomonas aeruginosa is binded by EGCG contained in green tea and the binding located in the bacterial cell membrane (Jeon et al., 2014;Xiong et al., 2017) CONCLUSION Green tea ethanol extract against pathogen bacteria of P. aeruginosa has been carried out. The results obtained for blanks, 5%, 10% and 15% green tea ethanol extracts variation using distilled water as solvent were 6 ± 0,00; 7,85 ± 0,21; 8,90 ± 0,00 and 10,45 ± 0,21 mm with the inhibition diameter 0; 1.85; 2.9 and 4.45 mm with concentration variations that used. The conclusion of this study is that higher extract concentration make the inhibitory activity is higher too and they are increasing proportionally against P. aeruginosa.