The THE POTENCY OF CINNAMON (Cinnamomum burmanni Blume) LEAF EXTRACT AS A BIOPRESERVATIVE AGENT FOR FOOD SAFETY OF SATE LILIT

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ARTICLE HIGLIGHTS
- Cinnamon leaf extract combats E. coli in traditional Balinese food.
- Natural preservative reduces harmful pathogens in sate lilit.
- Cinnamon extract offers a safer alternative to synthetic preservatives.
- Active compounds in cinnamon leaves inhibit bacterial growth.
- Effective biopreservative for enhancing food safety and quality.

ABSTRACT
Escherichia coli O157:H7 contamination of the meat used in preparing sate lilit, a favorite traditional food in Bali, Indonesia, has been a great concern for both local people and foreigners. Although C. burmanni has been included in its spice ingredients, active compounds that play a significant role in this pathogen have limitedly been elucidated. The main objectives of this research were to investigate the potency of this plant to control contaminants and elucidate possible compounds that prevent such contaminants by applying the disk diffusion method and LCMS analysis, respectively. The results showed that the leaf extract of this plant inhibited the in vitro growth of E. coli O157:7, with minimal inhibitory concentration (MIC) and LC50 values of 4% and 2.59%, respectively. The LCMS analysis chromatogram showed that the plant extract's most active fraction produced nine peaks, representing nine possible active compounds. Among those, three compounds (Azoxystrobin, Stigmatellin Y, and 2-arachidonoyl glycerol) were suspected of contributing to control contamination, especially by E. coli O157:H7.

INTRODUCTION

Being one of the tourist islands in Indonesia, Bali is rich in various types of ethnic foods. Such kinds of foods (traditionally served along the streets in small stalls or modern restaurants) can easily be found in areas of tourist destinations in Bali. A specific characteristic of Balinese ethnic foods is the spicy ingredients used in the preparation. Sate lilit is a traditional Balinese Kebab and a favorite food in Bali, enjoyed by both locals and foreigners. Sate lilit has also been part of offerings in the Balinese religious rituals, such as temple festivals, Balinese wedding ceremonies, etc. The meat (beef, pork, chicken, or fish as the primary raw material) of the sate lilit is frequently contaminated by pathogenic microbes (Palupi et al., 2019) that can damage the quality of the sate lilit. These contaminated meats often become a medium for the pathogen to spread and cause food poisoning (foodborne disease outbreak).

Some researchers found that beef (a primary raw material of sate lilit) traded in traditional markets in Bali is frequently contaminated by pathogenic bacteria. For example, (Purni et al., 2020) reported that 39 beef samples collected from some traditional markets located in Denpasar City, Badung, and Klungkung regencies were contaminated by E. coli. Among those samples, 25.6% were infected by E. coli O157:H7, the causative agent of hemorrhagic diarrhea, due to its ability to produce Shiga-like toxins (Patil et al., 2022). Similar results were also reported by (Pinatih et al., 2021), who stated that this serotype of E. coli O157:H7 has been well-known to produce potent toxins identical to those made by Shigella dysentery type 1, and therefore it is often referred to as Shiga like a toxin. As sate lilit is not fully cooked, it has the potential to spread pathogenic microbes that lead to an outbreak.

The application of synthetic chemical-based preservatives has not been encouraged to cope with such pathogenic microbial contamination due to their many harmful side effects on human health. According to the Indonesian Food and Drug Administration (BPOM), food poisoning was considered to be the highest case of poisoning in Indonesia in 2012. Approximately 66,7% of such poisoning cases were due to the consumption of foods containing poisonous preservatives, such as formalin, boric acid, or textile dyes (Utomo & Kholifah, 2018). Based on those food poisoning cases due to the application of inappropriate preservative agents, the possible use of natural preservative agents extracted from plants has been intensively researched so that some alternative and safe food preservations can be invented.

The bark and leaf extracts of C. burmanni Blume have been applied as flavor enhancers in various foods in Indonesia. C. burmanni Blume is a wooden plant. Its height may reach 50 meters (Sujarwo & Keim, 2020). According to (Djarot et al., 2023), this plant's leaves and bark contain high levels of flavonoids, saponin, tannin, and alkaloids. (Mohamed et al., 2020) reported that the bark extract of this plant has the potential as an antibacterial agent. This is due to the antimicrobial effect of its essential oil, containing several active compounds, such as cinnamaldehyde, eugenol, cinnamic acid, and cinnamate (Plumeriastuti et al., 2019). The potential of C. burmanni essential oil to inhibit Staphylococcus aureus, Enterobacter spp., Pseudomonas aeruginosa, and Candida albicans was also reported by (Novita & Sutandhio, 2021). This background has opened the door for its use as an alternative agent in developing food preservatives.

Based on the above rationale, it is urgently needed to elucidate active compounds in the C. burmanni leaf extract that may play an essential role in controlling E. coli O157:H7 contamination before its use as an organic food preservative. This study focused on the leaf extract of the plant, because this part of the plant has been widely used as an important ingredient of sate lilit preparation in Bali. The results of our current research expectedly can be used to develop alternative food preservation methods to control pathogenic microbes (E. coli O157:H7 in particular) in ethnic Balinese food products.

MATERIALS AND METHOD

Extraction of C. burmanni Blume leaves

Mature leaves (located on rows of 4-9 from tips of branches) of C. burmanni Blume were collected from Bedugul village, Tabanan-Bali. These leaf samples were chopped, air dried at ambient temperature (25 – 33^oC), powdered with a warring blender, macerated in 96% ethanol (Sigma Aldrich, Germany) in a ratio of 1:10 w/v, and filtered with 4 layers of Whatman no. 2 filter papers (pore size 8 µm). The filtrate was then evaporated in a vacuum rotary evaporator at 40ºC to obtain crude extract/pellet of the leaf extract and stored at 4^oC before being used in the subsequent experiments. The crude extract obtained from this evaporation was considered to be 100% concentration.

Preparation of C. burmanni Blume crude extract

The crude extract of the plant leaves was diluted with 96% ethanol (Sigma-Aldrich, Germany) to obtain final concentrations of 20% (w/v), 15% (w/v), 10%, and 5% (w/v) by applying the following dilution formula:

V₁. M₁ = V₂ . M₂

Where:

V₁ : Volume of the most concentrated extract (mL)

V₂ : Volume of the adjusted extract concentration (mL)

M₁ : Concentration of the most concentrated extract (% w/v)

M₂ : The adjusted extract concentration (% w/v).

Preparation of E. coli O157:H7 cell suspension

The strain of Escherichia coli O157:H7 was obtained from the stock culture collection of the Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Udayana University. Pure culture of E. coli O157:H7 (1 loopful) was inoculated into 100 mL of Nutrient Broth medium (Oxoid) and incubated for 24 hours at 37^oC. The density of this bacterial suspension was then adjusted to 0.5 McFarland scale or equal to 10⁸ cells/mL.

Bioassy (in vitro inhibition tests) of the C. burmanni Blume leaf extract on E. coli O157:H7

In vitro inhibition tests of the C. burmanni Blume leaf extract on E. coli O157:H7 were conducted by applying the method of Kirby Bauer (1966) with minor modifications. Suspension of E. coli O157:H7 amounted to 200 µL was homogenized in 15 mL nutrient agar medium (Oxoid) and poured into a sterile petri dish to produce bacterial lawn. Once the mix was settled, a preliminary bioassay was conducted on the crude extract of this plant at high concentrations (25%, 50%, and 75% w/v) to determine anti-bacterial activity of the crude extract. The lowest concentration of this crude extract that inhibited the bacterial lawn was then used as a reference to determine the crude extract concentrations applied in the main experiments. As the inhibition zone already happened at 25% (w/v), the C. burmanni Blume leaf extract concentrations used in the experiments were 5, 10, 15, and 20% w/v. In these bioassays, paper disks previously deposited with 20 µL C. burmanni Blume leaf extract at those various concentrations (5, 10, 15, and 20% w/v) were placed at equidistance on the surface of this bacterial lawn to allow active compounds of the plant extract to diffuse. The same volume of 96% ethanol (Sigma-Aldrich, Germany) and 1% w/v of chloramphenicol (a product of Kalbe Farma, Indonesia) in paper disks served as negative and positive controls, respectively. Four replicates were prepared in this bioassay to obtain representative data. All plates were incubated for 24 hours at 37^oC and measured for formation of inhibition zones around the paper disks, previously deposited with the plant extract. Measurements of inhibition zones around each paper disk were conducted from 4 different angles and the results were averaged.

Determination of minimal inhibitory concentration (MIC) value of the C. burmanni Blume leaf extract on E. coli O157:H7

MIC value of the leaf plant extract was determined by applying the same method (Kirby and Bauer, 1966) mentioned above. The concentrations of the plant extract were adjusted from 0% to 5% w/v with interval increment of 1% w/v. Four replicates were prepared in this bioassay to obtain representative data. All plates were incubated for 24 hours at 37^oC and measured for formation of inhibition zones around the paper disks (CT0998B) deposited with the plant extract. Measurements of inhibition zones around each paper disk were conducted from 4 different angles and the results were averaged.

Determination of Lethal Concentration 50% (LC50) of the C. burmanni Blume leaf extract on E. coli O157:H7

Bacterial suspension prepared above was diluted in serial dilution method so that cell with density of 10³ cells/mL was obtained. A volume of 100 µL of this diluted cell suspension (100 cells) was exposed in Eppendorf tubes for 15 minutes to the C. burmanni Blume leaf extract of the same concentrations with those applied in the MIC test. These exposed cell suspensions were then homogenized in nutrient agar medium (Oxoid), poured into petri dishes, incubated at 37^oC for 24 hours, and counted for colonies appeared on the surface of the medium. The results were then plotted in a graph that shows relationship between total colony counted and concentration of C. burmanni Blume leaf extract (% b/v). The value of the LC₅₀ was determined from the curve or calculated using the equation of the regression.

Phytochemical analysis of the C. burmanni Blume leaf extract

Alkaloid test

Leaf extract of the C. burmanni Blume was dissolved in 10 mL of chloroform-ammonia solution (Sigma-Aldrich, Germany), added with 0.5 mL 1N H₂SO₄ (1 N), homogenized, and let it to form 2 layers. The upper layer was collected and added with 1 drop of Meyer reagent (Sigma-Aldrich, Germany). The formation of precipitation indicates positive result (Dey et al., 2020).

Flavonoid and phenolic tests

These tests followed the method specified in (Aryal et al., 2019). The C. burmanni Blume leaf extract was dissolved in 70% ethanol, heated, and filtered. The filtrate was then placed on a plate with Mg and 1N HCL (Merck) for the flavonoid test and FeCL₃ (Merck) for the phenolic test. The formation of red color and green to purple ring indicated positive results for flavonoid and phenolic tests, respectively.

Steroid and terpenoid tests

The C. burmanni Blume leaf extract was added with chloroform, heated for 10 minutes, placed on a plate, and added with Liebermann-Burchard reagent. A positive result for the terpenoid test was indicated by formation of red, pink, or violet color, while the steroid test was indicated by the formation of green or pink color (Malik, 2023).

Saponin test

A weight of 0,1g leaf extract of the C. burmanni Blume was dissolved in 5 mL hot distilled water and shaken for 10 seconds. A positive test for saponin test was indicated by the formation of stable foam for 10 seconds (Depkes RI, 2009).

Tannin test

Leaf extract of the C. burmanni Blume amounted to 0,1 g and was added with FeCl_3, (Merck) and then it was left to react for 5 minutes. The positive test was indicated by the formation of a blue color (A et al., 2019).

Elucidation of active compounds contained in the Leaf extract of the C. burmanni Blume

Fractionation and purification processes of active compounds contained in the crude extract of C. burmanni Blume were conducted by applying column chromatography and thin layer chromatography (TLC), respectively. A 1g crude extract previously obtained was fractionated in column chromatography, where 300 g silica gel 60 (Merck, CT1.07733) was used as the stationary phase. The silica gel was first suspended in an eluent of n-hexane (Merck) as the mobile phase of the system. This silica gel suspension was poured into a column and acclimatized for 24 hours. The crude extract (1g) was dissolved in 1 mL, added with 1g silica gel, poured into the previously prepared column (length and diameter of 300 mm and 20 mm, respectively), and then eluted with a gradual change in the polarity of solvents. A volume of 25 mL eluent (with fractions of active compounds) flowing along the column was collected, evaporated, and analysed with a thin layer chromatography (TLC). Fractions with the same Rf values were composited and tested for their inhibitory activity against E. coli O157:H7 by disc diffusion (Kirby-Bauer, 1966). Fractions with the ability to produce inhibition zones were subsequently analysed using the LCMS (Liquid Chromatography Mass Spectroscopy, Shimadzu VP series SPD-10A VP Uv Vis Detector HPLC LC MS Chromatography) method, and the peaks that appeared were aligned with those produced by known compounds following the application of the MassLynx V4.1 software linked with the ChemSpider program.

Data analysis

Quantitative data was analyzed with analysis of variance (ANOVA) at p<0.05 using SPSS software for Windows version 17.0. When significant difference was indicated at p<0.05, the statistical test was further conducted by applying the Duncans Multiple Range Test (DMRT) at p<0.05.

RESULT AND DISCUSSIONS

In vitro inhibitory activity of C. burmanni Blume on E. coli O157:H7

All concentrations applied in the preliminary bioassay experiments were found to inhibit the growth of E. coli O157:H7 with various inhibitions. The diameter of inhibition zones was proportional to crude extract concentrations (Table 1 and Figure 1). These results provided us with information on the toxicity level of this plant crude extract on the E. coli O157:H7. As the negative control did not inhibit the growth of the E. coli O157:H7, the clear zones formed around the disks deposited with the plant crude extract were confirmed to be due to active compounds contained in the plant crude extract. As shown in Table 1, the diameter of inhibitions of the plant crude extract applied at concentrations of between 25% w/v – 100% w/v was slightly different from that produced by positive control, although they are statistically different at p<0.05. This indicated that the toxicity level of the crude extract was comparable with that of the 1% w/v of chloramphenicol.

The C. burmanni Blume leaf crude extract also inhibited the growth of E. coli O157:H7 when applied at concentrations lower than 25% w/v. The results are shown in Table 2 and Figure 1B. The inhibitory effect of the plant extract was still observed when the extract was applied at 5%w/v.

Table 1.Average diameter of inhibition of the C. burmanni Blume leaf extract in the preliminary determination of appropriate concentrations to control E. coli O157:H7

Extract concentrations (%)	Diameter of inhibition zones*
100	21.08±0.04e
75	19.53±0.05d
50	18.02±0.05c
25	17.21±0.06b
0	0.00 ±0.00a
Positive control	20.66±0.05f

Figure 1.Diameters of inhibition zones of the C. burmanni Blume leaf extract on E. coli O157:H7 obtained from preliminary bioassay (high concentrations of the plant extract) on nutrient agar medium (A); Diameters of inhibition zones of the C. burmanni Blume leaf extract on E. coli O157:H7 obtained from the main experiment of bioassay (concentrations of 5, 10, 15, and 20% w/v) on nutrient agar medium (B).

The result shown in Table 2 was consistent with those found in the preliminary study of the extract bioassay (Table 1). This led us to investigate the minimum inhibitory concentration (MIC) of the C. burmanni Blume leaf extract on E. coli O157:H7 by applying the same method with adjusted concentrations of between 0% and 5% w/v with an increment concentration of 1%, and the results are shown in Table 3. Based on this bioassay, the MIC value of the extract could be claimed at 4%, although it might fall between 3 and 4% w/v if the bioassay was continued by applying the plant extract concentrations between this range.

As the scientific evidence of this plant’s antimicrobial activity is still limited, we focused our study on elucidating active compounds (with the potential to inhibit the growth of E. coli O157:H7) contained in this plant extract. In vitro bioassays of this plant extract in our study showed that the crude extract of this plant inhibited the growth of this pathogen when applied at a low concentration of 5% or more (Table 1;Table 2;Table 3 and Figure 1). Scientists such as (Parisa et al., 2019) also reported a similar phenomenon, who found in vitro microbial activity of this plant extract inhibitory against E. coli and Staphylococcus aureus. (Didehdar et al., 2022) have also recently reviewed extensively the potential of this plant as a new therapeutic agent to inhibit biofilm formation by pathogenic fungi or bacteria in the early stages of their infection. This indicates that active compounds inhibitory to pathogenic bacteria or fungi exist in this plant extract.

Table 2.Inhibitory effect of the C. burmanni Blume leaf extract on E. coli O157:H7 when applied at low concentration (between 5% - 20% w/v) on nutrient agar medium

Extract concentrations (%)	Diameters of inhibition zones*
20	11.07± 0.05e
15	9.05± 0.05d
10	8.24± 0.05c
5	7.56± 0.06b
0	0.00 ± 0.00a
Positive control	20.66± 0.06f

Table 3.Averages of inhibition zone diameters of C. burmanni Blume leaf extract on E. coli O157:H7 when applied at a concentration of between 0% - 5% w/v on nutrient agar medium to determine MIC value.

Extract concentrations (%)	Diameters of inhibition zones*
5	7.56± 0.06b
4	6.11± 0.02 c
3	0.00±0.00 a
2	0.00± 0.00 a
1	0.00± 0.00 a
0	0.00 ± 0.00a

This plant extract appeared to be highly potent as its MIC value was very low (at 4% w/v concentration, which was deposited on filter paper at a volume of 20 µL). When applied at 100% concentration, the C. burmanni Blume leaf extract produced a diameter of inhibition higher than 20 mm, as shown by our study's positive control (1% chloramphenicol). Based on the diameter of the inhibition zone, the effectiveness of this plant crude extract in controlling E. coli O157:H7 can be categorized as very high, according to the criteria made by (Rastina & M, 2015). In all cases (Table 1; Table 2; Table 3), the negative control did not show an inhibition zone, indicating that the inhibition phenomenon shown by the crude extract of this plant (applied at various concentrations) must be due to the presence of compounds inhibitory against E. coli O157:H7.

Cinnamomum burmanni Blume (in Bali, traditionally named kayu manis) can abundantly be found in Indonesia as it is widely used as a component in many traditional cuisines in this country, including in the ethnic foods of Bali. In Bahasa, this plant is known as cinnamon or Padang cinnamon. This plant has been included in many popular Indonesian foods or beverages, such as rendang (the most popular slow-cooked meat from Padang, west Sumatra), Bali coffee, satay (skewered and grilled meat), Balinese spice paste, which is used as a base for a wide range of dishes, and sambal (spicy chili pastes or sauce which is a staple in Indonesian and Balinese cuisines). Recently, C. burmanni Blume has also been included in the production of herbal tonics. The main role of this plant part in all those traditional foods is to enhance their flavor and balance the aroma of the foods. Many people also believe that this plant has antimicrobial activity, so it is widely used as an alternative organic preservative agent to lengthen food shelf life.

Determination of LC50 value of the C. burmanni Blume extract on E. coli O157:H7

A lethal concentration of 50% (LC₅₀) is the concentration of the plant extract that kills 50% of bacterial population following exposure to such plant extract. The curve estimated this value (total viable cells vs extract concentration). The results are shown in Figure 2. The number of cell death was proportional to the increment of the plant extract concentration. At 5% w/v, this plant extract totally eliminated/killed the E. coli O157:H7 (Figure 2). The linear regression of the curve produces a formula of Y = -18.886X + 95.937, with r² value of 0.9908. From Figure 2 or the formula generated from the curve, the LC₅₀ value of the extract fell between 2 and 3% w/v (approximately 2.59% w/v, based on calculation using the displayed formula).

The values of the LC50 have been commonly used to assess the potential toxicity of substances exposed to living organisms (Gupta, 2020). According to this scientist, the values of LC50 could help scientists determine safe exposure levels of toxic substances for types of organisms, specifically humans. These values are often used to establish permissible exposure limits for chemicals and pollutants (Wolf & Segner, 2023). Therefore, LC50 values for toxic substances, including those produced by plants, are important to be determined prior to their application to control undesired microbes. This is aimed at reducing negative impacts following the application of those substances in any aspect of our lives.

Phytochemical analysis of the C. burmanni Blume leaf extract

From this analysis, the leaf extract of C. burmanni Blume contains several groups of compounds, such as alkaloid, steroid, phenolic, saponin, and flavonoid. In our study, terpenoids and tannin were not detected, and the results are shown in Table 4. These groups of compounds are very common in plant extract and may play an important role in inhibiting the growth of microbes. Inhibition zones formed on the lawn of E. coli O157:H7 might have been due to the presence of these groups of compounds.

Figure 2.Estimation of LC₅₀ value of the C. burmanni Blume leaf extract on E. coli O157:H7

Table 4.Groups of compounds detected in the leaf extract of C. burmanni Blume

Types of compounds tested	Results*
Alkaloids	+
Steroid	+
Terpenoid	-
Phenolic	+
Saponin	+
Flavonoid	+
Tannin	-

The phytochemical tests analyzed the active compounds that might have played important roles in inhibiting E. coli O157:H7 in vitro growth. These included alkaloids, steroids, phenolics, saponins, and flavonoids (Table 4). Our results are in line with those reported by (Raji et al., 2019), (Ullah et al., 2020), and (Huang et al., 2022). The mechanisms by which these compounds control microbial growth have also been extensively reviewed, such as by (Yuan et al., 2021), (Santiago et al., 2021), (Tungmunnithum et al., 2018), and (Kavya et al., 2021). The most common mechanism reviewed and reported is interference with the function of the cell membrane that leads to the release of important components (including proteins) from the cytoplasm of microbial cells. Other mechanisms have also been reported, such as enzyme inhibition, DNA binding, oxidative stress, iron chelation, or antioxidant activity of those compounds to interfere with microbial growth. According to (Raji et al., 2019), such compounds can also affect the swarming motility of bacterial cells.

The level of impact of alkaloid, steroid, phenolic, saponin, and flavonoid compounds to interfere with microbial growth is determined by many factors. These include types and concentrations of those compounds or microbial resistance developed over time (Palupi et al., 2019); (Biharee et al., 2020). Alkaloids, for example, may act in three different modes of action (disrupting cell membranes, interfering with enzyme activity, or binding on the DNA of the targeted cells (Jubair et al., 2021). Conversely, phenolic compounds may generate reactive oxygen species (ROS) that lead to oxidative damage to cellular components, such as proteins, lipids, and DNA. Oxidative damages caused by these ROS certainly result in cell death (Hajam et al., 2023).

Fractionation and elucidation of active compounds contained in the leaf extract of C. burmanni Blume)

On the completion of fractionation, 12 fractions were obtained, but only 4 of those fractions (fractions 3, 4, 5, and 6) showed inhibitory activity on E. coli O157:H7 (Table 5). As shown in Table 5, fraction 4 produced the biggest potential to inhibit the growth of this bacterial strain with a diameter inhibition of 25.23 mm (Table 5 and Figure 3).

It is clearly demonstrated in Figure 3 that some fractions inhibited the growth of E. coli O157:H7, which is indicated by clear zones around the paper disks deposited with each fraction. These results indicate that some active plant extract compounds exist in the fractions showing positive results in this in vitro bioassay (Table 5). In the subsequent stage of our research, only fraction 4 (F4) was elucidated in the Liquid Chromatography-Mass Spectroscopy (LCMS) analysis as it produced the highest inhibition zone in the in vitro bioassay, with a diameter inhibition zone of 25.23 mm (Table 5 and Figure 3).

In the LCMS analysis, fraction 4 of the plant extract was the main focus of the analysis as it gave the highest diameter of inhibition on this bacterium (Table 5). The chormatogram of this analysis (Figure 4) shows that nine peaks appeared in the chromatogram to represent compounds of N-(4-Methoxyphenyl)-4-methyl-1-piperazinecarbothioamide; azoxystrobin; 2-(2-Cyano-benzylsulfanyl)-4,6-diphenyl-nicotinonitrile; difenoconazole; Octoxinol-2; N-(4-Butylphenyl)-11-[4-(dimethylamino)phenyl]-10-methyl-8-phenyl-8,11 dihydropyrazolo [3',4':4,5] pyrimido [1,2-a] quinoxalin-6-amine; Stigmatellin Y; 2-arachidonoyl glycerol; and 1-(Butylamino)-3-methyl-2-octylpyrido[1,2-a]benzimidazole-4-carbonitrile. The chemical structures of these compounds are shown in Table 6.

Some compounds detected in our current study have been reported to have antimicrobial activity. Azoxystrobin was reported by (Sun et al., 2023) to inhibit the growth of Rhizoctonia solani, a fungal pathogen attacking tobacco plants. These authors also found this compound to affect the composition and diversity of microbes in tobacco plants. Stigmatellin Y, as detected in our study, has also been reported to have antimicrobial activity. (Boopathi et al., 2022) reported that this compound plays an important role in interfering with communication (quorum sensing) among cells of Pseudomonas aeruginosa cells, resulting in its use as an anti-virulence Pseudomonas. The compound of 2-arachidonoyl glycerol is another compound with the ability to inhibit the growth of microbes. According to (Chouinard et al., 2013), such compounds can stimulate neutrophils (a type of white blood cell) to release an antimicrobial effector that leads to growth inhibition on several bacterial species, such as Escherichia coli and Staphylococcus aureus. This result partially explains or elucidates the mechanism by which both leaf C. burmanni Blume crude extract and its fractions inhibited the in vitro growth of E. coli O157:H7 in our study. In other words, these three compounds (Azoxystrobin, Stigmatellin Y, and 2-arachidonoyl glycerol) contributed in the growth inhibition of the targeted bacterium (E. coli O157:H7)

The roles of the other six compounds to inhibit microbial growth have not been reported. They probably contribute to inhibiting the growth of microbes, although their contribution needs to be further elucidated.

Table 5.Inhibitory activity of each fraction in the fractionation of the crude leaf extract of C. burmanni Blume on E. coli O157:H7

Fractions	Diameters of inhibition zones on E. coli O157:H7 (mm)
F1	0.00
F2	0.00
F3	15.23
F4	25.23
F5	18.05
F6	9.40
F7	0.00
F8	0.00
F9	0.00
F10	0.00
F11	0.00
F12	0.00

Figure 3.Inhibition zones of each fraction of C. burmanni Blume on E. coli O157:H7 on nutrient agar medium

Figure 4.The chromatogram of LCMS analysis of the fraction 4 of C. burmanni Blume leaf extract. Arrowheads point out the suspected active compounds (Azoxystrobin, Stigmatellin Y, and 2-arachidonoyl glycerol) appeared at retention times of 10.06, 14.64, and 17.05, respectively, that might have contribution to inhibiting the in vitro growth of E. coli 0157:H7.

Table 6.Chemical structures of compounds detected in fraction 4 of the C. burmanni Blume leaf extract following application of LCMS MassLynx V4.1 software, which is linked to ChemSpider reference.

No	Retention time (Rt)	Molecular structure	Identified compound	Molecular formula	Molecular weight
1	10,53	Image	N-(4-Methoxyphenyl)-4-methyl-1-piperazinecarbothioamide	C13H19N3OS	266,1372
2	10,66	Image	azoxystrobin	C22H17N3O5	404,1246
3	10,81	Image	2-(2-Cyano-benzylsulfanyl)-4,6-diphenyl-nicotinonitrile	C26H17N3S	404,1237
4	12.20	Image	difenoconazole	C19H17N3O3Cl2	406,0725
5	13.41	Image	OCTOXYNOL-2	C18H30O3	295,2273
6	14.46	Image	N-(4-Butylphenyl)-11-[4-(dimethylamino)phenyl]-10-methyl-8-phenyl-8,11-dihydropyrazolo[3',4':4,5]pyrimido[1,2-a]quinoxalin-6-amine	C37H37N7	508,3189
7	14.64	Image	Stigmatellin Y	C29H40O6	485,2903
8	17.05	Image	2-arachidonoylglycerol	C23H38O4	391,2848
9	17.05	Image	1-(Butylamino)-3-methyl-2-octylpyrido[1,2-a]benzimidazole-4-carbonitrile	C25H34N4	391,2862

CONCLUSION

Leaf extract of C. burmanni Blume has the potential to control contamination of E. coli O157:H7 as it shows inhibitory activity in the serial in vitro bioassays, with minimal inhibitory concentration (MIC) and LC₅₀ values of 4% w/v and 2.59% w/v, respectively. The leaf crude extract of this plant contains groups of chemical compounds belong to alkaloids, steroids, phenolics, saponins, and flavonoid. Fraction of the extract with the highest inhibition zone (fraction 4) contains 9 compounds, namely N-(4-Methoxyphenyl)-4-methyl-1-piperazinecarbothioamide; azoxystrobin; 2-(2-Cyano-benzylsulfanyl)-4,6-diphenyl-nicotinonitrile; difenoconazole; Octoxinol-2; N-(4-Butylphenyl)-11-[4-(dimethylamino)phenyl]-10-methyl-8-phenyl-8,11 dihydropyrazolo [3',4':4,5] pyrimido [1,2-a] quinoxalin-6-amine; Stigmatellin Y; 2-arachidonoyl glycerol; and 1-(Butylamino)-3-methyl-2-octylpyrido[1,2-a]benzimidazole-4-carbonitrile. Among these compounds, three (Azoxystrobin, Stigmatellin Y, and 2-arachidonoyl glycerol) probably contributed to inhibiting the in vitro growth of E. coli O157:H7.

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