Maria Genesis G. Magleo
Danica Pearl M. Untiveros
Dawn Nichelle S. Medrano
Stephanie L. Solano
B.S. Biology Program
College of Arts and Sciences (CAS)
Maribel Beltran-Cruz
Department of Biology, College of Arts and Sciences (CAS)
How to Cite:
Magleo, M. G. G., Untiveros, D. P. M., Medrano, D. N. S., Solano, S. L., & Beltran-Cruz, M. (2017). Preliminary assessment of the phytochemical components and antibacterial activity of Cinnamomum mindanaense bark extract against Staphylococcus aureus. NEU Knowledge Journal: A Compilation of Researches of New Era University Faculty, Staff, Students, and Administrators, 1(1), 17–31. https://doi.org/10.64303/neu-urc-kj2017-PrAsPhCo
ABSTRACT
A pilot study was conducted to identify the bioactive phytochemical constituents and evaluate the antibacterial activity of Cinnamomum mindanaense. This research was done in line with the thrust of the Department of Environment and Natural Resources Administrative Order (DAO) to characterize and assess the potentials of the 19 Cinnamomum species endemic in the Philippines. Crude extract was tested for the presence of sterols, triterpenes, flavonoids, alkaloids, saponins, glycosides, and tannins. The antibacterial activity, as indicated by diameter of zone of inhibition, was tested against Staphylococcus aureus ATCC 25923 (a gram-positive bacterium) and Chloramphenicol (a commercial antibiotic) at 25 mg/ml, 50 mg/ml, 75 mg/ml, and 100 mg/ml extract concentrations in eight replications.
Phytochemical analysis showed that C. mindanaense bark is rich in flavonoids, alkaloids, and triterpenes. Bark extracts showed moderate antibacterial activity. ANOVA test showed significant group differences. Using post hoc Tukey’s Honest Significant Difference (HSD) test, antibacterial activity of Chloramphenicol treatment was significantly higher than those effected by 25 mg/ ml, 50 mg/ml, 75 mg/ml, or 100 mg/ml bark extract concentration (p<0.001). Extract concentrations of 75 mg/ml and 100 mg/ml were found not significantly different (p=0.564); also between 25 mg/ml and 50 mg/ml concentrations (p=0.059). Highly significant difference was found between concentrations of 100 mg/ml and 50 mg/ml, 100 mg/ml and 25 mg/ml, and 75 mg/ml and 25 mg/ ml (p<0.001). Plot of zone of inhibition (y) against treatment concentration (x) suggests linear relationship. Further study is recommended to address this observation and to validate findings by increasing the number of replications.
Keywords: Cinnamomum mindanaense, antibacterial activity, Staphylococcus aureus, lyophilization, disk diffusion
INTRODUCTION
There are 19 endemic Cinnamomum species in the Philippines. The majority of these species including the Cinnamomum mindanaense have not been fully evaluated as to their therapeutic value as envisioned to be accomplished by the Department of Environment and Natural Resources Administrative Order (DAO).
Plants have long been known for their medicinal properties long before the advent of synthetic commercial medicine. Because increasing number of pathogens has already developed resistance to existing antibiotics such as penicillin, researchers continue developing more potent drugs primarily from novel florae to combat new strain of pathogens. The discovery of new plant species possessing antibacterial properties such as cinnamon, paves way in crediting plants as potential source of antibiotics. Moreover, herbs and spices from various parts of the world have embellished human diet in terms of their distinctive taste, appealing color, and food aroma (Venugopal, Dasani, & Rai, 2009; de Souza et al., 2005).
Walker affirmed that compounds known as phytochemicals or secondary compounds are developed in plants to safeguard them against herbivorous insect vertebrates, fungi, pathogens, and parasites. Srinivasan reported that the growth of interest regarding herbs and spices has flourished not only because of their flavor-enhancing properties but also of their natural antioxidant properties (as cited in Shiney, Ramya & Ganesh, 2012).
Tepe et al. stated that plant extracts have been assessed for their potential contents as alternative medicine to treat infection diseases. Burt and Kordali et al. reported that plants are primary sources of chemicals known for their antibacterial, antifungal, antiviral, insecticidal, and antioxidant properties. According to Milhau et al. plants rich in secondary metabolites are used as food preservative (as cited in Al-dhaer, 2008). In the Philippines, plant extract of cinnamon bark is used traditionally in the same way as cinnamon is used in Ceylon (Guerrero, 1921). Filipinos use cinnamon in decoction with ginger, star anise, and sugar as stomachic beverage, particularly during breakfast. Cinnamon leaves have stimulant and carminative properties (Quisumbing, 1978).
Studies mentioned that the antibacterial activity of Cinnamomum was attributed mainly from its inherent active constituents, namely: cinnamaldehyde, eugenol, cinnamic acid, and cinnamate, all of which are major components of its bark (Sharma et al., 2016; Baria, 2015; Stefanovic et al., 2015; Sova, 2012).
This study was conducted to identify bioactive phytochemicals and determine antibacterial activity of Cinnamomum mindanaense bark extract against Staphylococcus aureus ATCC 2592 and Chloramphenicol.
MATERIALS AND METHODS
Plant material
The C. mindanaense bark was bought from a market in Barangay of San Antonio Boljoon, Cebu, Philippines. The cinnamon bark species was identified by a cinnamon specialist from the Center for Conservation Innovations-Philippines.
Decoction of Cinnamon Extract and Lyophilization
One hundred grams of cinnamon bark was pulverized using mortar and pestle. A grater can also be used to powder the bark. One thousand ml of distilled water was added and boiled for 3 hours. The mixture was cooled down for 30 minutes at room temperature. It was then filtered using 2 layers of sterilized 10 cm x 20 cm gauze. And 240 ml of crude extract was obtained. Through the use of ChemLab 7B4 Double Stage Freeze Dryer, the crude extract was lyophilized for one week until constant weight was achieved. (The duration of lyophilization varies depending upon the quantity of other extracts present in the freeze drier.) With the pressure maintained at 70 millibar and the temperature set between -40 to -50 °C, 18.01 grams of lyophilized extract was produced. The final dried extract was stored in an amber bottle and desiccators were used to keep it dry (Attieh et al., 2015; Parvazi, 2016).
Phytochemical Screening and Analysis
The procedure used was based on Trease & Evans (2002). The C. mindanaense was powdered using a Wiley mill grinder and soaked in 2.0L of methyl alcohol for 48 hours. The mixture was filtered and the filtrate obtained was concentrated using rotary evaporator at 60 °C for 2 hours. To obtain a semi-solid extract, the concentrated extract was further evaporated using water bath at 60 °C. The process yielded 32.0g (6.74%) of semi-solid extract.
Furthermore, the secondary metabolites of C. mindanaense bark extract were tested thru qualitative phytochemical analysis (Trease & Evans, 2002). The extract was also tested for its antibacterial activity against S. aureus and Chloramphenicol.
Bacterial Strains, Culture Media, and Growth Conditions
The bacterial culture was obtained from the culture collection of Medical Microbiology at University of the Philippines, Manila -College of Public Health. The bacterial strain used in the experiment was Staphylococcus aureus ATCC 25923, a gram-positive bacterium. The culture was streaked into sterile Mueller Hinton Agar (MHA) and was incubated at 37 °C for 24 hours prior to susceptibility testing. After 24 hours, the culture was transferred aseptically to 10 ml brain heart infusion broth. The tip of the inoculating loop was heated until red hot. The mouth of the test tubes was heated before and after opening it to inhibit contamination of other microorganisms on the test tubes. The subculture was adjusted to 0.5 McFarland turbidity standards using a nephelometer or paper with black lines. The turbidity of inoculum was regulated to conform with standard.
Kirby-Bauer Disk Diffusion Susceptibility Test
The culture of S. aureus was transferred aseptically to 10 ml brain heart infusion broth. The culture was adjusted to 0.5 McFarland turbidity standards using a nephelometer or paper with black lines. The resulting suspension for the isolate was swabbed on the surface of MHA plate using sterile cotton swab. Six paper disks (Whatman No. 1) of 6 mm diameter in each petri dish per trial (Gold Biotechnology, n.d) were formed. Each sterile paper disc was impregnated with 25 mg/ml, 50 mg/ml, 75 mg/ml, and 100 mg/ml concentrations of lyophilized bark extract of C. mindanaense using transferpette. The procedures were carried out in laminar flow hood. Disk impregnated with distilled water was used and designated as the negative control treatment. The paper disks were transferred onto the MHA plate and incubate at 37°C for 24 hours. (Maidment, Dyson, & Haysom, 2006).
Antibiotic Sensitivity Testing
The antibiotic sensitivity of S. aureus ATCC 25923 was tested against 30 μg of Chloramphenicol (a broad-spectrum antibiotic which kills gram-positive bacterium), designated as the positive control treatment. The turbidity of the culture was also adjusted to 0.5 Mcfarland’s turbidity standards equal to 106 CFU/mL. The test culture was swabbed aseptically in MHA plates. The antibiotic disc was carefully seeded in MHA plates with treatment group. The plates were incubated at 37 °C for 24 hours (CLSI, 2007).
Measuring the Zone of Inhibition
The antimicrobial activity was assessed through measuring the diameter of the zone of inhibition (in mm) and comparing it to that of S. aureus ATCC 25923. (Wong, Ahmad-Mudzaqqir, and Wan-Nurdiyana, 2014; Coyle, 2005; Lalitha, 2004). Zone Diameter Interpretative Standards for Staphylococcus spp. are coded as follows: S [sensitive], R [resistant], and I [intermediate] (CLSI, 2007).
Statistical Test
The experiment was replicated eight times and one-way ANOVA was used to find significant treatment difference at α=0.5. Post hoc Tukey’s Honest Significant Difference (HSD) test was performed to identify significant pairwise comparisons. All statistical tests were done using SPSS version 23.
RESULTS AND DISCUSSION
Phytochemical Analysis
Cinnamon bark extract revealed presence of phytochemicals specifically, sterols, triterpenes, flavonoids, alkaloids, saponins, glycosides, and tannins (Table 1). Triterpenes, saponins, and tannins are found in abundant quantities while sterols, alkaloids and glycosides in moderate amounts. Discovery of the presence of flavonoids, glycosides, alkaloids, tannins, and saponins in C. mindanaense bark extract supports previously reported results of Shiney Ramya and Ganesh (2012) and Mohamed Sham Shihabudeen, Hansi Priscilla, & Thirumurugan (2011).
Because of the antibacterial property of flavonoids, alkaloids, and tannins, cinnamon bark extract is recommended for eradicating bacteria. The plant extract’s hydrophobicity allows it to separate the lipids of bacterial cell membrane and mitochondria, in turn, increases cell permeability. Consequently, huge loss of essential molecules in bacterial cell leads to death (Sharma et al., 2016). Additionally, studies found that saponins, also found in C. mindanaense extract, possesses antidiarrheal property which prevents histamine release, possesses anticancer property as it has membrane permeabilizing contents, and disintegrates teguments of helminthic parasites. Glycosides have also antidiarrheal activity which restrains the discharge of autocoids and prostaglandins (Tiwari et al., 2011).
Antibacterial Activity
The antibiotic susceptibility test results were stated in term of diameter of zone of inhibition using the following standard:
≤12 mm -resistant [R]; 13 mm to 17mm -intermediate [I]; and ≥18 -susceptible [S] (CLSI, 2007) (Tables 2 & 3). Concentration of 100 mg/ml exhibited the greatest zone of inhibition compared to all lyophilized C. mindanaense bark extract concentrations. Chloramphenicol (positive control) provided the greatest zone of inhibition.
The antibacterial property of lyophilized C. mindanaense bark extract is influenced by the rate of diffusion of its chemical constituents into the culture medium. For instance, less soluble compounds have been found to diffuse gradually into the culture medium (Moreno et al., 2006). Also, according to Klancnik et al. (2009), the rate of diffusion of an antibacterial agent is affected by the zone of inhibition itself. Additionally, the hydrophobicity of plant extracts was found to inhibit even distribution of substances in agar media. Furthermore, the solvent used in preparing the extract affects the inhibitory effect of the bark (Ekwenye & Elegalam, 2005). In this research, water was used as solvent to prepare the different extract concentrations (Sharma et al., 2016; Mukhtar & Ghori, 2012).
The single membrane of gram-positive bacteria is considered more susceptible to permeation by antibacterial agent (Klancnik et al., 2009). Studies mentioned that the antibacterial activity of Cinnamomum was due to the presence of some active constituents such as cinnamaldehyde, eugenol, cinnamic acid, and cinnamate, all of which are major components of its bark (Sharma et al., 2016; research, water was used as solvent to prepare the different extract concentrations (Sharma et al., 2016; Mukhtar & Ghori, 2012).
The single membrane of gram-positive Baria, 2015; Stefanovic et al., 2015; Sova, 2012). Specifically, cinnamaldehyde is the active compound found in cinnamon which is responsible for its antibacterial activity (Sharma et al., 2016; Mukhtar and Ghori, 2012).
Statistical Analysis
Treatments produced zones of inhibition that were highly significantly different, F(4,35)=277.08, p<0.0001 (Table 4). Post hoc multiple comparison using Tukey’s Honest Significant Difference (HSD) test showed eight statistically significant pairwise treatment comparisons (p<0.05) (Table 5). For instance, zone of inhibition between 50 and 25 mg/ml was found not statistically significant (p=0.059); the same was true between 100 and 75 mg/ml (p=0.564). On the other hand, zone of inhibition produced by Chloramphenicol was highly significantly greater than that produced by 25, 50, 75, or 100 mg/ml bark extract (p<0.001). Positive control (Chloramphenicol) shows the 100 mg/ ml (12.63 mm) or by 75 mg/ml (11.75 mm) (Table 3). Summary of pairwise comparison is shown in Table 6. Plot of zone of inhibition (y) against treatment or dose of C. mindanaense (x) suggests relationship (Figure 1). However, because of the limited number of data points used in regression analysis, a statistically grounded conclusion or claim is withheld at this point. A more in-depth study is recommended to validate this observation.
CONCLUSIONS
Bark extract of C. mindanaense shows presence of antimicrobial secondary metabolites such as sterols, triterpenes, flavonoids, alkaloids, saponins, glycosides, and tannins. Bark extract has moderate antibacterial potency. Antibacterial property between 25 mg/ml and 50 mg/ml bark extract concentrations as well as between 75 mg/ml and 100 mg/ml is not significantly different. But there is significant difference between 75mg/ml and 25 mg/ml and also between 100mg/ml and 50 mg/ml concentrations. Antibacterial property of Chloramphenicol is highly significantly greater than that of any of the bark extract concentrations tested.
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