We demonstrated that the methanol extract of Mitrephora thorelii Pierre's branches and leaves in Lam Dong, Vietnam is anti-Staphylococcus aureus strain (Sta1) of Phenikaa University, Hanoi, Viet Nam and Staphylococcus aureus ATCC 29213 strain (Sta2) from Thermo Fisher Scientific, USA. Invitro anti-Sta1 and Sta2 activity of the methanol extract of Mitrephora thorelii Pierre's branches and leaves were not significantly different. With a rise in extract content, the inhibitory zone's created diameter grew as well. The extract content of 100 mg/mL demonstrated the greatest antibacterial activity with a 25 mm inhibition zone diameter against Sta1 and 24 mm inhibition zone diameter against Sta2. Bacteria were not resistant to the extract at concentrations of 20; 80 and 100 mg/mL after 48, 72, and 96 hours. The lowest inhibitory concentration (MIC) was between 312.5 and 625 µg/mL. The minimum bactericidal concentration was found to be 1.25 mg/mL.
Keywords: Antibacterial activity; Lam Dong; Mitrephora thorelii Pierre; Plants extract; Staphylococcus aureus
Staphylococcus aureus (S. aureus) is a member of the family Staphylococcaceae, the genus Staphylococcus, the order Bacillales, and the class Bacilli [1]. There are several different types of Staphylococcus. 30% of people on the planet carried S. aureus on a permanent basis [2, 3]. Along with osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections, S. aureus is a major contributor to bacteremia, infectious endocarditis, and these conditions. S. aureus is a common pathogen that lives everywhere and permanently resides in the upper respiratory, gastrointestinal, and urogenital tracts of 20% to 30% of individuals [4, 5]. S. aureus can colonize several animal skin surfaces, including the teat and canal skin. It can cause diseases ranging from mild skin infections to fatal conditions such as sepsis, endocarditis, necrotizing pneumonia, toxic syndrome, plantar fasciitis, necrotizing fasciitis, bone and joint duplication associated with thromboembolic disease, prohibition of fulminant hemorrhage with or without Waterhouse Friderichsen syndrome, orbital cellulitis and endophthalmitis, central nervous system infection predisposing factors different toxins [5-6]. Antibiotic strains of S. aureus have been resistant to methicillin, vancomycin, cephalosporins, penicillin, and linezolid. Due to the antimicrobials' selective pressure, S. aureus has the capacity to acquire drug resistance more quickly [5]. Finding medications that can effectively prevent and treat diseases from natural herbal sources is a major concern of medical professional biochemists and medics throughout the world due to the risk of drug resistance and adverse effects when using pharmaceutical drugs.
The research results at Tay Nguyen Institute for Scientific Research, VAST, Vietnam have developed the list of medicinal plants of 1003 species collected in Lam Dong [7]. Among those species is Mitrephora thorelii Pierre., according to the research results of Hoa and Yen, (2022) showed that they have anti-Helicobacter pylori activity [7] , Thuan et al. (2014) isolated four flavonoid compounds from Mitrephora thorelii leaves are astragalin, juglanin, quercetin, and quercitrin [8]. Mitrephora thorelii Pierre is on the list of endangered plant species in Pu Mat National Park [9]. According to the Vietnam Red Book, Mitrephora thorelii Pierre is on the list of endangered species. The realize their potential, in this study, we evaluated the in vitro anti- S. aureus activity from Mitrephora thorelii's branches and leaves in Lam Dong province, Viet Nam. The research results will contribute to providing scientific evidence on their anti- S. aureus activity as well as contribute to the conservation and development of medicinal plants in Vietnam's Lam Dong province.
Mitrephora thorelii Pierre leaves and twigs were collected in Duc Trong district of Vietnam's Lam Dong province in October 2013. Plant samples were classified by Dr. Nong Van Duy from Tay Nguyen Institute for Scientific Research, VAST. They are designated No. TN3/044. The sample is stored at the plant resources department of Tay Nguyen Institute for Scientific Research, VAST, Vietnam.
Two S. aureus strains: S. aureus strain (Sta1) was provided by Dr. Nguyen Hong Minh, Phenikaa University, Hanoi. Staphylococcus aureus ATCC 29213 (Sta2) imported from Thermo Fisher Scientific, USA. The strains were kept at the Microbiological Technology Department of Tay Nguyen Institute for Scientific Research, VAST, Vietnam.
Methanol, DMSO, Muller Hinton Agar and Muller Hinton Broth (Merck, Germany).
’ Mitrephora thorelii Pierre s branches and leaves (25 g) were air-dried, powdered, and then extracted three times at room temperature using methanol (200 mL/time). To get methanol residue, the methanol solutions were filtered, mixed, and concentrated while under reduced pressure. Until they were employed, the raw extracts were kept at -20°C.
On the day of the assay, an aliquot of the frozen extract was weighed and diluted in DMSO (10%) at 100 mg/mL. By serial dilution, the extract was then diluted to reach level of 80 mg/mL and 20 mg/mL.
The agar plate diffusion method was used to test the extract's antibacterial properties [10]. The sterile Petri-plates were filled with sterilized Muller Hinton Agar medium (15 mL) and allowed to harden. The bacteria was grown in Muller Hinton Broth medium and maintained culturing for 24 hours 8 at 37°C. The bacterial population was adjusted to 10 CFU/mL. Then, the bacterial broth (100 μL) was swabbed individually on an agar plate using a sterile bud. The wells (6 mm in diameter) were drilled into the agar with a sterile hole. Plant extract was placed into each well aseptically and maintained for 24 hours at 37°C. The positive control was ofloxacin (5 μg) and the negative control was DMSO (10%). The inhibitory zone's size was calculated. The tested bacterium's diameter of the inhibition zone (DIZ) was used to evaluate its antibacterial activity. Millimeters were used to measure the DIZ. Each experiment was performed in triplicate using the methods described in a previous study.
Minimum Inhibition Concentration (MIC) was determined according to the method of Sarker et. al. [11]. In this experiment, these extracts were diluted secondary to an initial concentration of 40 mg/mL. The bacterial extracts 6 was cultured overnight and diluted at a density of 10 CFU/mL. Add to each well 50 μL of bacterial extract and 50 μL of methanol extract at different dilutions. The control wells contain the bacterial extracts and the medium. Each o treatment was repeated 3 times. Incubate at 37 C, overnight. After 24 h, 30 μL of 0.15% resazurin (Across, USA) reagent o was added to each well. Incubate at 37 C and observe the color change of the mixture in the well. The MIC value is the lowest concentration in the test range of extracts that can inhibit bactericidal growth (wells do not discolor resazurin).
Spreading plate method was used to calculate the Minimum Bactericidal Concentration (MBC): 100 μL of test solution on wells without resazurin discoloration was spread onto agar plates containing Muller Hinton Agar and grown at 37ºC, checking for bacterial viability after 24 hour. The MBC value is the lowest concentration in the concentration range of extracts that can kill all bacteria (no bacterial colonies appear on the agar plate). The control medium plate
contains only bacterial fluid and medium, without the extract, bacterial colonies appear [11, 12].
The data were analyzed in SPSS 16 software using the ANOVA test. Statistical testing was performed at the level of significance p ≤ 0.05, using the LSD test.
The test findings indicated the methanol extract from ’ Mitrephora thorelii Pierre s branches and leaves is anti-S. aureus strain (A1) of Phenikaa University, Hanoi, Viet Nam and S. aureus strain ATCC 29213 (A2) imported from Thermo Fisher Scientific, USA.
The outcomes demonstrated that for the same concentration of extracts, the inhibitory impact of methanol extract on Sta1 and Sta2 was not substantially different. Using an extract 8 dosage of 100 mg/mL and a cell density of 10 CFU/mL for Sta1 and Sta2, the extract's inhibitory area measured 25 mm and 24 mm, respectively.
The diameter of the inhibitory zone varies depending on the concentration of extract used, while the strain, cell density, and amount of extract in each agar hole are all the same. The extraction concentration has a direct correlation with the inhibitory ring diameter. When the extract concentration increased from 20 to 100 (mg/mL), the inhibitory ring diameter increased from 16 to 25 mm for Sta1 and 15 to 24 mm for Sta2.
With the inhibition zone's size of 24 to 25 mm, anti-Sta1 and anti-Sta2 of the extract at 100 (mg/mL) were comparable to the positive control ofloxacin (5 g) (Figure 1, Table 1).
To evaluate the antibacterial ability of the extract over time, we continued to measure the width of the inhibition zone in 3.1 experiment after 48, 72, and 96 hours and compared it with the diameter of the inhibition zone after 24 hour. The findings demonstrated that when we continued to culture the S. aureus, the diameter of the inhibitory zone remained unchanged during 48, 72, and 96 hours S. aureus. This may indicated that the bacteria are not resistant to the extraction concentrations we used (20 to 100 mg/mL) (Figure 2).
The minimum inhibitory concentration (MIC) is the lowest concentration in the test concentration range of extracts that can inhibit bacterial growth (without discoloring resazurin). Therefore, the lower the minimum inhibitory concentration (MIC), the higher the antibacterial activity. Based on the color change of the resazurin reagent on the 96-well plate, it was possible to determine the minimum inhibitory concentration (MIC) of extract from leaves and twigs of Mitrephora thorelii Pierre against S. aureus. The results showed that in wells with the extract level of 312.5 μg/mL, the color of the resazurin reagent changed from blue to pink indicating the growth of bacteria in the well. Thus, the methanol extract from the leaves and twigs of Mitrephora thorelii Pierre has a value of 312.5 μg < MIC ≤ 625 μg/mL for S. aureus.
The minimum bactericidal concentration (MBC) is the lowest level in the extract's concentration range that can kill all bacterial in the well, with no bacterial colonies appearing on the agar plate. Wells without resazurin discoloration were spread onto Muller Hinton Agar plates, incubated at 37ºC, after 24h of observing the bacterial’s survival.
According to the results, S. aureus was totally eliminated at an extraction concentration of 1.25 mg/mL because no bacterial colonies could be seen growing on the agar plate. Therefore, the minimum killing concentration of the extract for Sta1and Sta2 was 1.25 mg/mL. Tables 2 and 3 present the findings.
Numerous scientists investigated various plant extracts for their ability to inhibit the growth of bacteria, yeast, and noticed their antibacterial properties. Numerous active chemical components found in medicinal plant extracts have antibacterial effects. The MICs value has been used for evaluating the extract's antibacterial activity [13]. If the MIC values are less than 500 g/mL, the activity is regarded as significant, and when they range between 500 and 1500 g/mL, it is regarded as moderate. Under these experimental conditions, Mitrephora thorelii was found to have significant against Sta1 and Sta2 activity. Furthermore, the smaller the MBC/MIC ratio, the higher the bactericidal effect. MBC/MIC ≤ 4, the extracts have good bactericidal ability [14]. In this instance, the extract from Mitrephora thorelii should be noted for its bactericidal properties.
For thousands of years, people have used medicinal herbs to treat various diseases. Isolation and biochemical characterization of pharmacologically active compounds from medicinal plants continue to this day. Numerous investigations have demonstrated that a variety of herbal species are anti-S. aureus. The methanol extract from the leaves of Ficus carica is resistant to S. aureus ATCC 29213 with MIC 2.5 mg/mL [15], anti-S. aureus activity of Pouzolzia zeylanica: 40 μg/mL < MIC < 80 μg/mL [16]. Hammer et al. [17] investigated the antimicrobial activity of 52 essential oils and extracts. The results have demonstrated that many essential oils have antifungal and antibacterial activities. In which, essential oil of Vetiveria zizanioides was able to inhibit the growth of S. aureus NCTC 6571 with MIC is 0.008 % (v/v). Vetiveria zizanioides against S. aureus was the strongest among the plants tested by the authors. Recently, various reports have confirmed the S. aureus activity of essential oils and medicinal extracts [18, 20]. Many experimental studies have shown a correlation between the antibacterial activity of plant extracts and secondary compounds found in plants [22]. Some researchers have suggested that the antibacterial components present in plant extracts such as terpenoids, alkaloids, and polyphenols interact with the enzymes and proteins of the bacterial membrane causing the dispersion of proton flux towards the outside of the cell. leads to cell death or can inhibit bacterial amino acid biosynthetic enzymes [23]. Antibiotic resistance is a worldwide public health threat, affecting the health and lives of people and the overall sustainable development of an entire country. The majority of S. aureus strains are resistant to a wide range of medicines. The World Health Organization (WHO) has recommended that research priorities need to be found, finding new methods against these bacteria. The current research results are the basis to encourage further studies on the chemical composition, toxicity, and pharmacology of the Mitrephora thorelii Pierre to apply it in the treatment of S. aureus effectively.
According to the study's findings, the methanol extract of Mitrephora thorelii Pierre, which was found in Lam Dong province, Viet Nam had anti-S. aureus species activity. These results are the basis to encourage further studies on the chemical composition, toxicity, and pharmacology of this species in order to apply it in effective treatment against S. aureus.
The authors declare that there is no conflict of interests regarding the publication of this research article.
All the authors have contributed equally in designing, drafting the manuscript as per the journal submission format. All authors read and approved the final manuscript.
This study does not involve experiments on animals or human subjects.
There is no funding to report.
All data generated or analyzed during this study are included in this published article.