Antibacterial Activity of Combination Cold Plasma and Parijoto (Medinilla speciosa) Against Staphylococcus aureus and Pseudomonas aeruginosa on Diabetic Ulcer

This study aims to determine the potential of cold plasma (cold plasma) as an antibacterial if combined with Parijoto (Medinilla speciosa) extract against the dominant bacteria in diabetic ulcer infection, namely Grampositive Staphylococcus aureus and Gram-negative bacteria Pseudomonas aeruginosa with a concentration of 25%, 50 %, 75%, and 100%. This study was an experimental test in vitro using the well diffusion method. The well diffusion method uses an MHA given a 5mm diameter and inserted a 100uL sample then incubates 35 ± 2 ° C for 16-18 hours. The results showed antibacterial activity from Cold Plasma with a 10 mm shot distance for 3 minutes with inhibitory zones against S. Aureus 3 mm and against P. aeruginosa 3.35 mm, a combination of Cold Plasma antibacterial activity with parijoto extract with a zone of inhibition of S. aureus 11.7 mm (25%), 14.1 (50%), 16.5 (75%), 17.1 (100%) and against P. aeruginosa 7.7 mm (25%), 9.3 mm (50%), 10.1 mm (75%), 11.7 (100%). the results of the Cold Plasma inhibition zone and Cold Plasma combination with parijoto extract 100% concentration of S. aureus were greater than the inhibition zone for P. aeruginosa. Cold Plasma combination inhibition zones with starch extracts against S. aureus compared to Vancomicin (VA) antibiotics with a 19.9 mm inhibition zone included in the intermediate category, Cold Plasma combination inhibition zone with parijoto juice against P. aeruginosa bacteria compared with Meropenem antibiotics ( MRP) with a 9.7mm inhibition zone included in the sensitive category. Fajar Farra Anastasia, Itsna Risqii Aziz, Vanny Oktaviola, Arya Iswara 135


INTRODUCTION
The 2010 World Health Organization (WHO) reported that 60% of the causes of death of all ages in the world are due to Non-Communicable Diseases. Diabetes mellitus ranks 6th as a non-communicable disease that causes death. Indonesia is the 7th country with the highest prevalence of diabetes (Ministry of Health, 2013). Diabetes mellitus is a chronic metabolic disease characterized by an increase in blood glucose levels that leads to time and to serious damage to the heart, blood vessels, eyes, kidneys, and nerves. Uncontrolled diabetes mellitus can cause interference with the immune system with a cellular response that causes infection by bacteria and causes impaired macrophage function resulting in delayed healing and increased sensitivity to infection (Ammons, 2013).
Ulcers are a common problem in diabetics. Ulcers can cause inflammation or make the tissue damaged (Gardner dan Frantz RA, 2012). In this condition, Gram-positive substances such as Staphylococcus aureus and Gramnegative bacteria such as Pseudomonas aeruginosa can produce biofilms (Jenkins et al., 2014). Treatment of ulcers with bacterial infections is recommended by giving antibiotics. Antibiotics have the ability to inhibit bacterial growth. However, giving antibiotics for a long time and the wrong use can lead to resistance (Lipsky et al., 2012). Therefore, alternative solutions are needed to overcome problems in the treatment of diabetic ulcers infected by bacteria.
Since about ten years ago, in developed countries, efforts to implement plasma in the health sector were attracting attention. This effort has given birth to a new study called Plasma medicine. Plasma is the fourth phase of the substance after solid, liquid and gas. Plasma is commonly known as an ionized gas. Plasma produced in the air consists of reactive mixed atoms, excited molecules, charged particles, Reactive Oxygen, and Nitrogen Species (RONS). Cold plasma is used in sterilization applications, food decontamination, dermatology, and dysentery.
Cold plasma has become the spotlight as an alternative to non-systemic infection antibiotics, because plasma treatment shows renewable effectiveness of various microorganisms, including antibiotic and spore-forming biofilm strains (Wahyuningtyas et al., 2017). The mechanism of medical plasma work in influencing the biological function of cells is based on two main principles, namely: (1) That plasma biological effects are significantly affected by the cell fluid environment, (2) That RONS generated or transferred into the liquid phase plays a major role in biological response influenced by plasma (Weltmann, 2017).
Indonesia is rich in natural products which have an important role for health, for example, Ginger (Zingiber offcinale Rosc.), Noni (Morinda citrifolia), and Parijoto (Medinilla speciosa) which have the potential as natural antibiotics (Winarti et al., 2005). Parijoto (Medinilla speciosa) is a typical shrub plant, curved, single, and cross-faced leaves, the fruit is purplish pink and tastes sour and sate (Wibowo et al., 2012). Parijoto is a typical plant of the Colo region, Kudus Regency, Central Java (Wachidah, 2013).
This study combines cold plasma with Parijoto extract (Medinilla speciosa) which is used as an antibacterial alternative to diabetic ulcer infection.

Bacterial Culture
Pure cultures of P.aeruginosa and S. aureus bacteria were obtained from the Microbiology Laboratory of the Faculty of Nursing and Health Sciences at the University Muhammadiyah of Semarang. Rejuvenated in Brain Heart Infusion (BHI) media for 6 hours, then made a bacterial stock on the Heart Infusion Agar (HIA) media.

Making Bacterial Culture
Pure cultures of P. aeruginosa and S. aureus were standardized with standard Mc Farland 0.5 turbidity.

Antibacterial Activity Test
Suspension of bacteria P.aeruginosa and S.aureus, inoculated into MHA media. After 10 minutes, Argon Cold Plasma was fired indirectly with a voltage of 14.65 kV and a frequency of 12.72 kHz (Nasruddin, 2016). Spawning for 3 minutes at a distance of 10 mm. In other agar media 4 wells were made on MHA media with a depth of 0.5 cm, then each well was filled with 100 µl parijoto extract with concentrations of 100%, 75%, 50%, and 25%. The media was given 2 treatments, namely by firing Cold plasma and without shooting Cold plasma. MHA media were incubated 35 ± 2 °C for 16-18 hours. For positive controls, Vancomicin and Meropenem antibiotics were used, while negative controls used antibiotics Oxacilin and Metronidazole. The inhibitory zone activity is seen by measuring the diameter of the inhibitory zone of bacteria formed.

Data Analysis
processed using SPSS with the Kruskal Wallis Test..

Making Parijoto extract
Parijoto is cleaned then smoothed using a blender. Then filtered and squeezed use gauze to separate the extract from the pulp. After obtaining Parijoto juice with a concentration of 100%, Parijoto juice was made concentrations of 75%, 50% and 25% with the addition of aquabidest.

Antibacterial Activity Test
The results of the study of antibacterial activity of Cold plasma, Parijoto Juice and a combination of both showed the presence of inhibitory zones tested in vitro by measuring inhibitory zones in the agar juice so that the wells were filled with parijoto extract with concentrations of 25%, 50%, 75%, and 100% measurements using millimeters are presented in Table 1 and Figure 1.   Table 1 and Figure 1 above, it can be seen that the antibacterial activity against P.aeruginosa treated with Parijoto extract showed the best antibacterial activity with the largest inhibition zone produced by a concentration of 100% at 11.9 mm. While the S.aureus treatment of the combination of cold plasma with Parijoto extract has the largest bacterial inhibition compared to the antibacterial activity test with cold plasma and Parijoto juice with an inhibitory zone of 17.1 mm at a concentration of 100%. This shows that the higher the concentration and the duration of shooting affect the formation of inhibitory zones. The difference in inhibitory zones of P. aeruginosa and S.aureus bacteria can occur due to differences in cell wall structure between Gram negative and Gram positive bacteria which causes different responses to various treatments and content of a material (Astrini et al., 2014). The zone of Vancomicin (VA) positive control inhibition is 19.9 mm and Meropenem (MRP) is 9.7 mm. The inhibitory zone results of P.aeruginosa and S.aureus were compared with standard inhibitory zone diameters according to the Clinical and Laboratory Standards Institute (CLSI).

DISCUSSION
This study aimed to determine the effect of the combination of cold plasma with parijoto extract on P.aeruginosa and S.aureus wherein each bacterium was tested for antibacterial activity with Cold plasma, Parijoto extract and a combination of both. This research is based on the measurement of clear zones around the media so that the resulting antibacterial compounds diffuse into the agar layer and inhibit bacterial growth and are called inhibitory zones (Perdana, 2016). The ability of plasma to produce biological molecules, namely Reactive Oxygen and Nitrogen Species (RONS) plays a major role in the biological response influenced by plasma. The mechanism of medical plasma work in influencing the biological function of cells is based on two main principles, namely that plasma biological effects are significantly affected by the cell fluid environment and RONS that is generated or transferred into the liquid phase plays a major role in biological response (Wahyuningtyas et al., 2017). The presence of a water content in the ray juice acts as a liquid phase which results in an increases in the biological response of increased bacteria. Coupled with the activity of flavonoid compounds on bacterial growth carried out by damaging the bacterial cell wall that reacts with alcohol groups in flavonoid compounds so that flavonoids enter the cell nucleus, react with DNA so that the lysis bacteria and die (Rustama and lingga, 2005).

CONCLUSION
The results of testing the combination of cold plasma and Parijoto extract can be concluded that from concentrations of 25%, 50%, 75%, and 100% Parijoto extract has antibacterial activity against S.aureus and P. aeroginosa. The concentration of 100% has the best antibacterial activity with inhibitory zones of 11.7 mm and 17.1 mm.

ACKNOWLEDGEMENTS
Thank you to the Ministry of Research, Technology, and Higher Education for providing funding and the Muhammadiyah Semarang University which facilitated this research so that it could run well and smoothly.