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Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts



Azzah Ibrahim Alghamdi and Ibtisam Mohammed Ababutain
 
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ABSTRACT

Background and Objective: Choosing the appropriate antibiotics (bacteriostatic or bactericidal) considered the most important aspect of treatment. This study aimed to determine the type of inhibitory activity of Eucalyptus camaldulensis’s leaves and bark extracts whether it is bacteriostatic or bactericidal. Materials and Methods: The antibacterial activity of E. camaldulensis leaves and bark extracts were assessed against four bacterial isolates (Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, Bacillus subtilis and Staphylococcus aureus ATCC24213). Minimum Inhibitory Concentration (MIC) was estimated using a 2-fold dilution method and Minimum Bactericidal Concentration (MBC) was estimated using a pour plate method. The bactericidal effect was estimated by calculating the ratio between MIC and MBC. Qualitative phytochemical assays were performed using a standard method and GC-MS analysis. Results: The results showed that the E. camaldulensis extracts have inhibitory activity against all tested bacteria. The results also indicated that the MBC/MIC ratios of E. camaldulensis extracts were one to two-fold dilutions which are considered as a bactericidal effect. The result of qualitative phytochemical assays showed the presence of tannin, saponins, flavonoid, carbohydrate and protein compounds in all leaves and bark extracts. The results of the GC-MS analysis showed that E. camaldulensis extracts possess a variation in chemical constituents. Some of these compounds have an anti-microbial effect like Cyclononasiloxane, octadecamethyl-, 17-Pentatriacontene, 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl- and Phytol. Conclusion: E. camaldulensis may be used as a natural antibiotic for its bactericidal effect to treat serious infections caused by pathogenic bacteria.

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  How to cite this article:

Azzah Ibrahim Alghamdi and Ibtisam Mohammed Ababutain, 2019. Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts. Asian Journal of Scientific Research, 12: 202-210.

DOI: 10.3923/ajsr.2019.202.210

URL: https://scialert.net/abstract/?doi=ajsr.2019.202.210
 
Received: July 28, 2018; Accepted: October 16, 2018; Published: March 15, 2019


Copyright: © 2019. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

The Eucalyptus tree belongs to the Myrtaceae family and it is considered a large aromatic genus that contains about 900 species1. For a longtime, attention has been given to the medical characteristics of Eucalyptus species; it has been used to treat arthritis, asthma, burns, fever, influenza, sore throat, malaria, wounds and pharyngitis2. Also it was used in the soap and cosmetic industries3. It has been found that leaves extracts of Eucalyptus species have shown many properties and several studies have brought to light the antihyperglycemic, antioxidants, antibacterial activity, ulcer-healing, cytotoxic effect, anti-inflammatory and analgesic effects4-10. Some studies have also demonstrated the antimicrobials activity of Eucalyptus plants barks11,12.

Eucalyptus camaldulensis is a fast-growing tree and evergreen that can tolerate drought for long periods and withstand high temperatures13,14 up to 60°C. Therefore E. camaldulensis plants are distributed in eastern region of Saudi Arabia15.

Drugs that are generated from plants are considered to be relatively safer than chemical drugs16. In this regard, traditional medicinal plants are widely used to sustain primary health care needs by more than 80% of the world’s population WHO17. Moreover, the search of antibiotics (ATBs) is never ending and the study of active substances in plants could enable the modulation of new types of ATBs.

However, ATBs work in two different ways, either inhibiting the growth of the microbe (bacteriostatic) or killing the microbe (bactericidal). This is one of the most important aspects in choosing the appropriate antibiotics for treatment. Where, the bactericidal ATBs are used to treat patients of severe microbial infections and immuno-suppression. While the bacteriostatic ATBs need a healthy immune system to complete the process of eliminating the infection18,19.

Although the inhibitory activity of the E. camaldulensis plant was investigated by few studied, the inhibitory activity was not determined to be bacteriostatic or bactericidal. Therefore this study was aimed to compare between the inhibitory activity for bark and leaves extracts of E. camaldulensis and determine the bactericidal or bacteriostatic effect for this plant. Also the phytochemical components of bark and leaves extracts for the E. camaldulensis were investigated using standard methods and GC-MS analysis.

MATERIALS AND METHODS

Preparation of plant extracts: The bark and leaves of E. camaldulensis plant were collected from a local park in the city of Dammam in Saudi Arabia in June 2015 and were morphologically identified15. Plant materials were washed under tap water to remove all dirt and then distributed over filter paper separately to dry at room temperature (20°C±2). The dry plant materials were grinded to powder using an electric mixer. Fifty grams of powdered bark and leaves were transferred individually into 500 mL flasks and 250 mL of different solvents included distilled water, methanol (80%) and ethanol (80%) were added separately to final concentrations of 20% (g mL1). Flasks were placed in a shaker at 350 rounds per minute (rpm/min) at 15°C for two days. Bacterial filters were used to filter the extracts and 100 mL of each filtered extract was dried using oven at 80°C. Dimethyl Sulfoxide (DMSO) was used to restore the extracts to the final concentration 20%. The crude extracts were kept at 4 °C until used.

Test micro-organisms: Four bacterial species two Gram-negative bacteria; Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922 and two Gram-positive bacteria; Bacillus subtilis and Staphylococcus aureus ATCC24213 were kindly provided by King Fahd Hospital, Al Khobar-Saudi Arabia.

Qualitative phytochemical analysis: The presence of biological compounds was tested using standard methods20,21.

Gas chromatography-mass spectrometry: Gas Chromatography-mass spectrometer QP2010 SE (Shimadzu Made in USA) with 5 Sil MS 5% diphenyl/95% dimethyl polysiloxane capillary column (30 m, 0.25 mmID, 0.25-μm df) was used to identify the bioactive compound of E. camaldulensis (leaves and bark). Hundred micro-milliliters of each sample were diluted with 1400 μL of DMSO (100/1400, V/V). For GC-MS detection, electron impact ionization (EI) with ionization energy of 70 eV was used. Carrier gas was helium (99.999%) at a column flow 0.7 mL min1 and injection mode was split. Flow control mode was linear velocity of 29.6 cm sec1 and split ratio 1:10. Injector temperature was 250°C and ion-source temperature 250°C. The oven temperature was programmed from 50-300°C (hold time 3 min) and total run time was 29 min. Compounds were identified by National Institute of Standards and Technology (NIST 08) library match.

Antibacterial susceptibility assay: Seven discs of antibiotics were used Ciprofloxacin (CIP 5 mcg), Piperacillin (PRL 100 mcg), Novobiocin (NV 30 mcg), Ampicillin (AMP 10 mcg), Clindamycin (Cd 10 mcg), Streptomycin (S 10 mcg), Amoxicillin-Clavulanate (AMC 10 mcg). All antibiotic discs were purchased from Bioanalyse Company.

Screening for antibacterial activities: Antibacterial activity of plant extracts was estimated using agar well diffusion technique as described by National Committee for Clinical Laboratory Standards (NCCLS)22. Petri plates were inoculated by 1 mL of overnight bacterial broth using inoculum 1-2×108 CFU mL1 that equivalent to 0.5 McFarland standards and 15 mL of melted nutrient agar medium was poured over the inoculum and mixed well. After cultures were solidified three holes were made and filled with 50 μL of the plant extracts. The plates were then placed in the refrigerator for 1 h for proper diffusion of the plant extracts then plates were placed in incubator at 37°C for 24 h. DMSO was used as a negative control and erythromycin (E5 mcg) disc was used as a positive control. Free zones of bacterial growth appear around the holes were measured in millimeters using ruler. All experiments were conducted in three replicates.

Determination of Minimum Inhibitory Concentration (MIC): The Minimum Inhibitory Concentration (MIC) for plant extracts that showed antibacterial effects were evaluated using 2-fold dilution method23. Ninety six well microtiter plates were used and MIC value was calculated after incubation over night at 37°C by using microtiter plate reader at a wavelength of 600 nm. All experiments were conducted in three replicates.

Determination of Minimum Bactericidal Concentration (MBC): From MIC observation, the MBC was carried out. All concentrations of plant extracts which showed no turbidity were transferred in to plates and 12 mL of milted nutrient ager were poured24. The MBC was calculated after incubation for 48 h at 37°C. Results were recorded by using binocular microscope and plates with no bacterial colonies were recorded as MBC. All experiments were conducted in three replicates. The ratio between MIC and MBC was calculated.

Statistical analysis: One way ANOVA, SPSS Version25 17.0 was used to compare the antibacterial activities of plant extracts and significance was tested at p<0.01.

RESULTS

Plant yields: The results showed that the plant yield of leaves extract was higher than the bark extract with values of 40.2, 34.8 and 22.2 g for ethanol, methanol and water extract, respectively. For the bark extract the highest plant yield was with methanol (21.0 g) followed by ethanol (11.6 g) and the lowest was water extract with a value of 8.4 g (Table 1).

Standard methods: The current study showed the presence of saponins, tannins, flavonoids, carbohydrate and protein compounds in all leaves and bark extracts of E. camaldulensis (Table 2).

Gas Chromatography-Mass Spectrometry (GC-MS) analysis: GC-MS analysis of the ethanolic, methanolic and water extracts of E. camaldulensis leaves and bark had shown a variation in chemical constituents (Table 3). The results showed a total of 12, 10 and 11 compounds identified in the ethanolic, methanolic and water leaves extracts, respectively. The numbers of compounds in the bark extracts were 9, 6 and 5 in the ethanolic, methanolic and water, respectively. All compounds (%) were listed in Table 3.

Table1:

Plant yields of methanol, ethanol and water extracts of bark and leaves for Eucalyptus camaldulensis

Image for - Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts
*Plant extracts weight/Plant weight X100

Table2: Qualitative phytochemical analysis of leaves and bark of Eucalyptus camaldulensis using methanol, ethanol and water as solvents
Image for - Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts
+: Exist, ++: Highly exist and +++: Very highly exist

Table 3: GC-MS analysis compounds name, peak area (%) and biological activities of E. camaldulensis leaves and bark extracts
Image for - Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts
EL: Ethanol leaves extract, ML: Methanol leaves extract, WL: Water leaves extract, EB: Ethanol bark extract, MB: Methanol bark extract and WB: Water bark extract

Table 4: Anti-bacterial susceptibility assay, anti-biotic class, bacteriostatic or bactericidal effects and mode of action
Image for - Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts
R: Resistance, Ciprofloxacin (CIP 5 mcg), Piperacillin (PRL 100 mcg), Novobiocin (NV 30 mcg), Ampicillin (AMP 10 mcg), Clindamycin (Cd 10 mcg), Streptomycin (S 10 mcg), Amoxicillin-clavulanate (AMC 10 mcg)

Antibacterial susceptibility assay: The results showed that six out of seven tested ATBs discs inhibited the growth of Gram-positive bacteria, whereas Amoxicillin-Clavulanate (AMC 10 mcg) failed to inhibit the growth of S. aureus and B. subtilis. Gram-negative bacterium P. aeruginosa was inhibited by four out of seven ATBs discs, whereas E. coli was inhibited by only two out of seven ATBs discs (Table 4).

Screening for antibacterial activities: The results showed inhibitory effects of all E. camaldulensis extracts on the bacterial strains under study E. coli, P. aeruginosa, S. aureus and B. subtilis at varying inhibition zones from 1.7±0.2 to 6.7±0.3 mm (Table 5).

Determination of minimum inhibitory concentration and minimum bactericidal concentration (MIC and MBC): Overall results showed that the extract of E. camaldulensis leaves and bark had MIC values ranged between 0.391-25 μg mL1 (Table 6). For bark extracts’ MIC ranged between 3.125-25 μg mL1, while the leaves extracts values were ranged from 0.391-25 μg mL1. Moreover, the lowest MIC values of ethanol and methanol extracts were 0.391 μg mL1 compared to the lowest MIC values of water was 12.5 μg mL1compared to the leaves extracts’ were 0.781-50 μg mL1.

The values of MBC ranged between 0.781-50 μg mL1. Bark extracts’ MBC ranged between 12.5-50 μg mL1 compared to the leaves extracts’ were 0.781-50 μg mL1.

Table 5: Antibacterial activity of Eucalyptus camaldulensis leaves and bark extracts at concentration of 20% by using agar well diffusion assay
Image for - Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts
*1, E. coli ATCC25922. 2, P. aeruginosa ATCC27853. 3, S. aureus ATCC24213. 4, B. Subtilis, **: Dimethyl sulfoxide negative control, ***: Erythromycin (5 mcg) positive control, that target protein synthesis 50S inhibition and R: Resistant

Table 6: Minimal inhibitory concentration, minimal bactericidal concentration, bactericidal effect and MBC/MIC ratios for Eucalyptus camaldulensis leaves and bark extract
Image for - Phytochemical Screening and Antibacterial Activity of Eucalyptus camaldulensis’s Leaves and Bark Extracts
No. 1: E. coli ATCC2592, 2: P. aeruginosa ATCC27853, 3: S. aureus ATCC24213 and 4: B. subtilis

In addition, MBC values of ethanol, methanol extract and water were ranging between 0.781-50, 1.56-50 μg mL1 and 25-50 μg mL1, respectively. The MBC/MIC ratios of E. camaldulensis leaves and bark extract was one to two fold dilutions (Table 6).

DISCUSSION

Plant yields result showed that the ethanol and methanol were the best solvents to obtain the highest plant yields this result is in agreement to another study that found ethanol was the best solvent to obtain the highest plant yields37. Standard methods result showed that E. camaldulensis leaves and bark extracts are rich in bioactive compounds, this result is in line with another study38 indicated the presence of saponins, tannins, flavonoid and glycosides in extract of E. camaldulensis. Moreover this finding agreed with previous phytochemical studies39,40 showed that E. camaldulensis leaves and bark are one of the richest plants in secondary metabolism compounds like essential oils, cineol, cuminal, phellandrene, aromadendral, valeraldehyde, geraniol, cymen, catechol, tannins, terpenes, isoprenoids, phenolics, cardiac glycosides, sterols, saponins and flavonoids.

Results of GC-MS analysis illustrated that the number of the compounds varied according to the solvent types and the difference in the plant part this result is in agreement with several researchers41,42. Most of these phytochemical compounds have medical, physiological and antimicrobial activities as previously reported by several investigators as listed in Table 3. However, some of these compounds have no data or report. Thus more investigation is required.

Antibacterial susceptibility assay result showed that Gram-positive bacteria are more sensitive to the antibiotics than Gram-negative bacteria because they lack an outer membrane43. Moreover the result indicated that E. coli bacterium was resistant to most of ATBs this result was consistent with a previous research44. Although most ATBs investigated in this study are similar in their mechanism of action, their inhibitory activity is uneven. The most effective ATB was Ciprofloxacin (CIP 5 mcg), that inhibited both Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa). Most tested ATBs used in this study are bactericidal except clindamycin (Cd 10 mcg)18,45. Moreover, Novobiocin (NV 30 mcg) works as bactericidal or bacteriostatic depending on its concentration19.

Screening for antibacterial activities results confirmed that E. camaldulensis extracts possesses a broad spectrum against both Gram-positive and Gram-negative bacteria. This may be attributed to the presence of several important secondary metabolism compounds like saponins, tannins and flavonoids. Which, were mentioned in several studies to possess antibacterial properties46-51. Overall, inhibitory activity of methanol leaves extract was higher than methanol bark extract this maybe due to the presence of Phytol and 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl- which both have been reported as antimicrobial agents29,36. Vice versa, inhibitory activity of water bark extract was higher than water leaves extract despite the presence of 17-Pentatriacontene and 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl- that both have recorded antimicrobial agents31,36. This may be attributed to the presence of Propane, 3-chloro-1,1,1-trifluoro- at high concentration (87.01%) and it is also present in high concentration in ethanol bark extract at 79.62%. However, there is no information on the biological activity of this compound. Inhibitory activity of ethanol bark and leaves extracts were high on both Gram-positive bacteria whereas ethanol bark extract was higher on P. aeruginosa than ethanol leaves extract. While, they were equal in their inhibitory activity on E. coli Table 5). The present study is consistent with other studies12,14,38,39,52 that proved E. camaldulensis extracts possess the ability to inhibit the growth of Gram-positive and Gram-negative bacteria. Moreover, the ethanol extract in both bark and leaves had the highest values of 6.7±0.3 and 6.0±0.3 against S. aureus, respectively this is consistent with a prior study42.

Many species belong to Eucalyptus genus exhibited antimicrobial properties. An early study reported that the leaves extracts of E. viminalis, E. maculata and E. globulus significantly inhibit six Gram-positive bacteria7. Moreover, E. tereticornis bark and leaves extract inhibit the growth of Gram-positive and Gram-negative bacteria11.

In general, the current study observed that E. camaldulensis extracts inhibit the growth of Gram-negative bacteria, while this bacterial group was not affected by most of the ATBs under study. All the extracts affected the Gram- positive bacteria significantly while they were not affected by Amoxicillin- Clavulanate (AMC 10mcg). This maybe attributed to the presence of phytochemical compounds with antimicrobial properties52. Few studies reported the MIC and MBC activity of E. camaldulensis. The current study is in agreement with Abubakar52 who found that water, ethanol and acetone leaves extracts of E. camaldulensis had MIC and MBC activity against E. coli, P. mirabilis, K. pneumoniae, S. typhi and S. aureus.

The current study indicated that the MBC/MIC ratio of E. camaldulensis leaves and bark extracts was one to two fold dilution which is considered as a bactericidal53 (Table 6). There are no reports about the type of effect for E. camaldulensis extracts whether bacteriostatic or bactericidal. However, this result is consistent with previous studies that also determined the type of effect using the MBC/MIC ratio for some medicinal plants and found that this ratio does not exceed three to four fold dilutions54-56.

Overall, the results of the present study showed that E. camaldulensis leaves and bark extracts were rich in phytochemical compounds with biological properties. These extracts were able to eliminate the growth of Gram-positive bacteria and Gram-negative bacteria tested in varying degrees, which exceeded the effect of some antibiotics tested. Also, the results showed that all E. camaldulensis extracts possessed bactericidal activity against the tested bacteria. Therefore, this plant extracts may be used as a source of antibiotics to treat specific bacterial infections. Moreover, several compounds in high concentration have no report or information mainly Propane, 3-chloro-1,1,1-trifluoro-. Thus further studies are required to investigate their biological properties, if any. This may provide novel compounds, leading to the development of new drugs.

CONCLUSION

This study showed that E. camaldulensis leaves and bark extracts have a broad inhibitory effect against both Gram-negative and Gram-positive. Inhibitory activity of this plant maybe attributed to the presence of active substances. Moreover, results demonstrated that the solvent type and plant part had a significant effect on the presence, concentration and inhibitory capacity of plant extract. Also, observed that the all E. camaldulensis extracts possessed bactericidal activity against the tested bacteria. Based on these results this plant may be used as natural antibiotic to treat some severe infectious diseases.

SIGNIFICANCE STATEMENT

This study demonstrated the bactericidal activity of leaves and bark extracts of E. camaldulensis against Gram-positive and Gram-negative bacteria, which may be beneficial for the manufacturing of effective new antibiotics. This is the first report of bactericidal activity of alcoholic and aqueous leaves and bark extracts of E. camaldulensis. Also, this study demonstrates several compounds in high concentration have no report or information mainly Propane, 3-chloro-1,1,1-trifluoro. Thus further studies are required to investigate their biological properties, if any. This may provide novel compounds, leading to the development of new drugs.

ACKNOWLEDGMENT

The researchers would like to thank the research units at Al Rayyan campus-College of Science-Imam Abdulrahman Bin Faisal University for providing place and devices which were required for this experiment. We also would like to thank Dr. Ahmed Alsayyah, Dr. Reem AlJindan and Msr. Nouf Alromaihi from King Fahd Hospital, Al Khobar-Saudi Arabia, for providing us with the bacterial samples.

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