Antimicrobial Activity of Terminalia Catappa L Leaves in Various Colors

Ketapang leaves, widely known for their antifungal properties, are traditionally extracted using organic solvents. However, this study aimed to demonstrate that it can be more accessible and practical to extract them with water. The study focused on the effect of leaf age (marked by its color) with water extraction on its antimicrobial ability. The results showed that ketapang leaf extract ( Terminalia catappa L.) can inhibit the growth of microbes, both gram-positive, negative, and fungal growth. The effectiveness of the inhibition was higher in the inhibition of fungi. Young leaves (color light green) and old leaves (color red) are very effective in inhibiting fungal growth, higher than the dichloro control. The content of phenols and antioxidants is high compared to the other two leaf colors. At a concentration of 20% extract, it inhibited 100% of the growth of the fungus A. oligosporus .

to make the application easier.In addition, the influence of leaf age was also studied based on leaf color (light green, dark green, yellow, and red).Meanwhile, Nadirah (et al., 2013) divide the age of leaves into young leaves (3rd leaf from the top) and mature leaves (9th leaf from the top).This study aimed to find the effect of leaf color on the inhibition of microbial growth and the minimum concentration of inhibition.

MATERIALS AND METHODS Material
Fresh ketapang leaves (Terminalia catappa L.) are taken from plants around campus.Bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Rhizopus oligosporus) are collections from the Food Biotechnology laboratory, UGM.Media used were Mueller Hinton Agar form Merch and Potato Dextrose Agar from Oxoid.The chemicals used are analytical grade.

Preparation of leaf extract
The leaves used in the study were of various colors, including light green, dark green, yellow, and red (Figure 1).To extract the active compounds, 100g of fresh leaves were thoroughly washed and blended with 100 mL of water.The resulting extract was separated from the pulp using Whatman paper no.42 And then centrifuged at 4000 rpm for 15 minutes to remove any precipitate.The clear extract obtained was stored at 4 o C until further use.

Growth inhibition analysis
Bacteria growth inhibition was analyzed using the method of well diffusion using Mueller Hinton Agar media, with a well diameter of 5 mm (20 ml leaf extract).Incubation was carried out at 37 o C for 24 h.Each petri dish contained 10 6 CFU bacterial cells inoculated by the pour plate method.The resulting clear zones indicating growth inhibition were measured Fungi growth inhibition was measured using a medium of Potato Dextrose Agar containing leaf extract (10 mL/Petri dish).Twenty mL of fungal spore suspension (105/mL) was inoculated in a well 5 mm in diameter (volume 20 mL) and then incubated at 30 o C for 4 d.Growth diameter was measured daily.As a control, 100 ppm Chloramphenicol (for bacteria) or 100 ppm Dichloran (for fungi) were used.In the same way the minimal concentration of leaf extract (0-50%), which inhibits the growth of the fungus R. oligosporus, was studied with various incubation times of 0-4 days.Every day the diameter of the colony was measured.The percentage of inhibition was calculated with the following formula: % Inhibition=(Ac -At)/Ac×100 Where Ac = control absorbance and At = test absorbance.The MIC was defined as the lowest concentration at which no fungal growth was observed.

Phenolic compound
The phenolic compound was measured using the method of Khoddami et al. (2013), and the results were compared with the standard curve of phenol.All tests were performed in triplicate.

RESULT AND DISCUSSION Inhibition of microbial growth
Changes in leaf color indicate the age of the leaves, starting from light green to dark green, getting older yellow and red before falling off.Ketapang leaf extract showed a clear solution, but the young leaf extract looked slightly cloudy.All extracts obtained were free from bacterial or fungal contamination; this indicates that the solution contains compounds that can inhibit or kill microbes.Tests on inhibiting the growth of Staphylococcus aureus bacteria showed that all types of leaves could inhibit growth, although light green and red leaf types inhibited more strongly (Table 1).On the other hand, the inhibition test on Escherichia coli bacteria, light green leaves, and red leaves did not show any inhibition.All these results were still below the inhibition of chloramphenicol (100 ppm).In other words, the ketapang extract was ineffective in inhibiting the growth of gram-positive and gram-negative bacteria.This result differed from the report of Nadirah et al. (2013), who found that the aqueous extract of ketapan leaves effectively inhibited the growth of Vibrio parahemolyticus.However, this study is also in line with the results of research by Nair and Chanda (2008), which states that the extraction of ketapang leaves with water is less effective in inhibiting bacterial growth.As stated by Cock (2015) that non-polar components are responsible for inhibiting bacteria.Extraction using organic solvents such as acetone and methanol is very effective in inhibiting (bacteriostatic)/killing (bactericidal) the growth of bacteria (Staphylococcus aureus, Proteus vulgaris, Salmonella typhi, Pseudomonas aeroginosa, and Bacillus subtilis) (Raveesha, 2021).Note: 1) control: chlorampenicol 100 ppm for bacteria and dichloran 100 ppm for fungi; 2) clear zone, indicating inhibition zone (the higher the number, the more inhibition); 3) diameter growth, indicating growth inhibition (the lower the number, the more inhibition).Fungal growth on media without the addition of dichlorine or leaf extract had a diameter of 91.5 mm.
Table 1 also shows the growth inhibition of the fungus Rhizopus oligosporus.All types of leaves (50% concentration, 5 g/mL ) inhibited 100% of fungal growth (zero diameter growth).The control in the form of 100 ppm dichlorane showed a diameter of 50.7 mm or only inhibited 44.6%.From these results, ketapang leaves extracted with water were very effective in inhibiting the growth of fungi.The same results were shown in ketapang extract in ethanol, which could inhibit the growth of Candidaspp (Terças et al., 2017).Previous studies showed that the most polar fraction obtained from the leaves of T. catappa was effective against fungi (Jagessar & Alleyne, 2011).The extraction of ketapang leaves was generally done using dry leaves that have been ground into a powder and then extracted.This study directly extracted fresh leaves with water, making it easier to prepare.

Phenol and antioxidant levels
The levels of phenol and antioxidants in young (light green) and red leaves were higher than in other types of leaves (Table 2).This may cause the leaf extract to inhibit the growth of S. aureus more strongly.Further chemical analysis showed that ketapang leaf extract contains tannins and favonoids (Purwani et al., 2015), as well as other antioxidants such as punicalagin, punicalin, terfluvina A and B, khebulis acid, benzoic acid, coumaric, and their derivatives (Chyau et al., 2006;Kinoshita et al., 2007).

Minimum inhibitory level
The effect of the concentration of ketapang leaf extract on the growth of the fungus R. oligosporus was analyzed at an extract concentration of 0-50%.The results can be seen in Figure 2. The higher the leaf extract concentration, the greater the inhibition on the growth of A. oligosporus.Young leaves that are light green in color and old leaves that are red are the most effective in inhibiting growth.At a concentration of 10% incubation for 4 days, it inhibited 35.81 and 39.07, respectively, compared to growth without leaf extract (Table 3).Concentration above it causes the fungus not to grow at all.This result differed from dark green and yellow leaf extracts, each of which were slightly less inhibitory, and inhibited 100% growth at 30% leaf concentration.This may be related to the levels of phenols and antioxidants (Table 3), which showed high levels in light green and red leaves.

CONCLUSION
Ketapang leaf extract (Terminalia catappa L.) can inhibit the growth of microbes, both gram positive, negative, and fungal growth.The effectiveness of the inhibition was higher in the inhibition of fungi.Young leaves that are light green and old leaves that are red are very effective in inhibiting fungal growth, higher than the dichloro control.This is because the content of phenols and antioxidants is high compared to the other two leaf colors.At a concentration of 20% extract, it inhibited 100% of the growth of the fungus A. oligosporus.

Figure 1 .
Figure 1.Changes in leaf color from young to late fall.A, young leaves (light green); B, dark green; C, old leaves (yellow); D, old leaves (red).Growth inhibition analysisBacteria growth inhibition was analyzed using the method of well diffusion using Mueller Hinton Agar media, with a well diameter of 5 mm (20 ml leaf extract).Incubation was carried out at 37 o C for 24 h.Each petri dish contained 10 6 CFU bacterial cells inoculated by the pour plate method.The resulting clear zones indicating growth inhibition were measured Fungi growth inhibition was measured using a medium of Potato Dextrose Agar containing leaf extract (10 mL/Petri dish).Twenty mL of fungal spore suspension (105/mL) was inoculated in a well 5 mm in diameter (volume 20 mL) and then incubated at 30 o C for 4 d.Growth diameter was measured daily.As a control, 100 ppm Chloramphenicol (for bacteria) or 100 ppm Dichloran (for fungi) were used.In the same way the minimal concentration of leaf extract (0-50%), which inhibits the growth of the fungus R. oligosporus, was studied with various incubation times of 0-4 days.Every day the diameter of the colony was measured.The percentage of inhibition was calculated with the following formula: % Inhibition=(Ac -At)/Ac×100 Where Ac = control absorbance and At = test absorbance.The MIC was defined as the lowest concentration at which no fungal growth was observed.

Figure 2 .
Figure 2. Fungal growth (in colony diameter) at various concentrations of leaf extract.Values are mean ± standard deviation in triplicates.