Abstract: Background and Objective: Premna pubescens blume popularly known as buasbuas belonging to the family Lamiaceae, wide-spread in the forests of Sumatra and Malaya peninsula. The preliminary screening of the ethanol extract of P. pubescens revealed the presence of alkaloids, steroids, flavonoids and phenolic. Previous studies revealed that there is no systematic study regarding the antimicrobial activity of leaf extracts of P. pubescens. The aim of this study was to assess the in vitro effect of leaf extracts of P. pubescens against Bacillus cereus and Escherichia coli causative agent of diarrheal diseases. Materials and Methods: Fresh leaves were procured from personal P. pubescens plant collections aged 5-7 years. Crude ethanol extract of leaves from P. pubescens were tested in vitro against B. cereus and E. coli at concentrations 10, 20, 30, 40 and 50 μg μL–1. Results: The B. cereus and E. coli showed concentration-dependent susceptibility towards the ethanol leaf extracts from P. pubescens. The degree of susceptibility varied depends on the concentrations, 50% concentration of P. pubescens leaf extracts showed the highest inhibition zone (12.5 and 13.6 mm diameter). Conclusion: Based on the current findings, it can be concluded that P. pubescens has antimicrobial activity which is as potent as standard antimicrobial drugs against B. cereus and E. coli. The antibacterial properties of P. pubescens leaves were not as effective as the commercial antibiotics chloramphenicol, enrofloxacin and penicillin. Nevertheless, future studies with higher extract concentrations, different method of extraction, properties from flowers, fruits, barks and roots extracts may be useful to evaluate the actual antibacterial performed to other pathogenic bacteria.
INTRODUCTION
Premna pubescens blume is a plant belonging to the family Lamiaceae. Premna pubescens is native to Southeast Asia and is one of the most wide-spread large shrubs in the forests of Sumatra and Malaya peninsula (Leeratiwong et al., 2009). It grows as ornamental plant. It is popularly known as buasbuas in Malay ethnic system of culinary and medicine. Most of the plant parts of P. pubescens have been used in the traditional system of medicine in Malay ethnic to treat various infectious diseases. Leaf forms an ingredient in well-known bubur pedas formulation which is used for variety of food for sultan (Goud, 1995). The other uses of P. pubescens are as an antioxidant, anticancer, antiviral, antifungal and antibacterial (Saeed et al., 2015; Selvam et al., 2015).
Like the other Premna, P. pubescens are rich in a wide variety of secondary metabolites such as alkaloids, flavonoids and terpenoids (Shukri et al., 2011; Hasanah et al., 2015). Of these metabolites, phenolic compounds such as flavonoids contained in plants is usually beneficial as an antioxidant, anticancer, antiviral, antifungal and antibacterial (Singh et al., 2011; Saeed et al., 2015; Dongamanti et al., 2015). The P. pubescens predicted has anti-coagulant, anti-inflammatory, anti-parasitic, antioxidant and antimicrobial properties. Flavonoids have received increased attention as useful pharmaceuticals in managing diseases like diarrhea (Mustafa et al., 2010; Fratiwi, 2015).
A previous investigation revealed that ethanol extract from P. pubescens leaves contained potential anti-inflammation (Marbun and Restuati, 2015). Previous studies revealed that there is no systematic study regarding the antibacterial activity of the leaf of P. pubescens. In the present study, an attempt has been made to evaluate the anti-bacterial activity of the ethanol extract of leaves of P. pubescens againts B. cereus and E. coli. Bacillus cereus and also E. coli was able to produce an exotoxin that causes gastrointestinal disorders and diarrhea (Fratiwi, 2015; Lakshminarayanan et al., 2015). Bacillus cereus is a gram-positive bacteria and Escherichia coli is a gram-negative (Kirk et al., 2015). Thus, the aim of this current investigation is to evaluate the ethanol extracts of leaves from P. pubescens against a gram-positive bacteria (B. cereus ) and a gram-negative (E. coli ) in vitro.
MATERIALS AND METHODS
Plant materials and extraction: The whole plants of P. pubescens were collected from personal P. pubescens plant collections aged 5-7 years in Medan, North Sumatra, Indonesia (Fig. 1a). The botanical identification of the collected materials was done in herbarium of bogor botanical garden. The leaves were separated and oven dried at 28°C room for 1 week (Fig. 1b). The leaves were grounded into powder form using the grinder (Fig. 1c). Extraction using Soxhlet apparatus with 95% (v/v) ethanol as solvent for 12 h was performed. The resultant extraction was frozen and freeze dried for 24°C/48 h. Yield of ethanol leaves extracts: 30% (Villalobos et al., 2016).
Microorganisms and medium: The microorganisms used in this present study were bacteria (Bacillus cereus and Escherichia coli ). The microorganisms were from clinical isolates. The B. cereus and E. coli were inoculated into in nutrient agar (NA, Merck, Germany), diluted 1:10 with sterile normal saline and then streak onto NA (Villalobos et al., 2016).
| Fig. 1(a-c): | Plant materials and extraction (a) The whole plants of P. pubescens, (b) P. pubescens leaves and (c) P. pubescens grounded leaves |
Antimicrobial sensitivity test: Sterile 6.0 mm diameter blank disc (Becton Dickinson Microbiology System, USA) were used to impregnate 6 different dilutions of the extracts as follows: 0, 10, 20, 30, 40 and 50 μg μL–1 extract ethanol. Discs were stored at 5°C prior to use. Tests were performed by the disc diffusion method. Extract impregnated discs were placed on agar and incubated either at 37°C for 24-48 h. Antibacterial activities were then measured indicated by the clear zones of inhibition (Dettman et al., 2015).
Comparison with antibiotic drug: Antibiotic discs of chloramphenicol (50 μg μL–1), enrofloxacin (50 μg μL–1) and penicillin (10 U) were purchased from Oxoid Ltd., UK. All discs were stored at -5°C prior to use. The antibiotic discs were then placed onto the B. cereus and E. coli cultures for 24-48 h. Antibacterial activities were then measured indicated by the clear zones of inhibition (Bai et al., 2015; Mahboubi et al., 2015).
Statistical analysis: Results were expressed as Mean±SD of four separate experiments. Statistical significance was determined using analysis of variance or Student’s t-test (Kuppusamy et al., 2016).
RESULTS
The results for the antibacterial activity test f various leaf extracts concentration of P. pubescens are shown in Table 1. The in vitro test on antibacterial activity revealed that ethanol of leaves of P. pubescens inhibited the growth of B. cereus and E. coli in a concentration dependent manner. The antibacterial activity was detected at 10 μg μL–1 leaves extract concentration for the ethanol extraction (Table 1). There was no antibacterial activity in control (Table 1). The growth of B. cereus and E. coli were inhibited by all concentrations of extracts. The E. coli was more sensitive to all various leaf extracts concentration of P. pubescens than B. cereus (Fig. 2).
The antibacterial activity of P. pubescens leaf extracts on B. cereus and E. coli were detected, the higher concentration exhibited higher antibacterial activity than the lower. The B. cereus was sensitive to all 3 commercial antibiotics tested, chlorampehenicol (50 μg μL–1), enrofloxacin (50 μg μL–1) and penicillin (10 U) (Table 2). Anyway, the growth of E. coli was inhibited only by chlorampehenicol and enrofloxacin but not penicillin. The E. coli was less sensitive to the commercial antibiotics chloramphenicol, enrofloxacin and penicillin than B. cereus (Table 2). The antibacterial properties of P. pubescens leaves (inhibition zones of 6.00-13.6 mm diameter) were not as effective as the commercial antibiotics chloramphenicol, enrofloxacin and penicillin (inhibition zones of 16.29-36.29 mm).
DISCUSSION
Almost all genus premna has been used to treat microbial infections (Sannomiya et al., 2015; Kchaou et al., 2016). Previous studies reported P. schimperi and P. oligotricha inhibited many types of bacteria including E. coli but not B. cereus (Mahboubi et al., 2015). There is no reported regarding the antibacterial activity of the leaf of P. pubescens against B. cereus and E. coli.
This study showed antimicrobial sensitivity testing was carried out in vitro using paper disc method against the following test organisms, E. coli and B. cereus. The zones of inhibition were measured after 24 h of incubation. The result of antimicrobial screening (sensitivity testing) using serial dilution method at varying concentrations (0, 10, 20, 30, 40 and 50 μg μL–1) on the E. coli and B. cereus with zones of inhibition recorded in millimetre is as shown in Table 1.
| Fig. 2: | Antibacterial activity of P. pubescens leaf extracts on B. cereus and E. coli |
| Table 1: | In vitro antimicrobial activity of P. pubescens leaf extracts on B. cereus and E. coli (Determined by diameter of inhibition zones) |
| Values are Mean±SD (mm) of four separate experiments. a-dMeans within a row where non-significant no common superscripts differ significantly (p≤0.05) and x,yMean within a column no common differ significantly (p≤0.05) | |
| Table 2: | Inhibition zones of antibiotics |
| Values are Mean±SD (mm) of four separate experiments | |
The choice of use of chloramphenicol, enrofloxacin and penicillin as clinical standards is based on the fact that at low concentrations, chloramphenicol only inhibits growth of the bacteria through induction of prokaryotic ribosomes to misread mRNA 56. Enrofloxacin prevents initiation of protein synthesis and leads to death of microbial cells. Penicillin inhibits bacterial growth by inhibiting protein biosynthesis (Lakshminarayanan et al., 2015).
In vitro of the leaf extracts of P. pubescens against against both gram-positive (B. cereus ) and gram-negative (E. coli ) bacteria resulted in growth inhibition lower susceptibility than the commercial antibiotics chloramphenicol, enrofloxacin and penicillin (Table 1, 2). It was noticed that the leaf extracts of P. pubescens was more potent than penicillin against E. coli. So, future studies with higher extract concentrations and the different method of extraction may be useful to evaluate the actual antibacterial properties of P. pubescens leaves extracts. Further investigations must be performed to examine the antibacterial properties from leaves, flowers, fruits, barks and roots extracts to other pathogenic bacteria at a higher concentration. In the present study, 50 μg μL–1 of extract has the antibacterial activity against B. cereus and E. coli. Toxicological studies on the extract must also be performed to ensure the safety of the extract. The finding of a potent herbal remedy that is safe will be an advancement opportunities in bacterial infection therapies.
CONCLUSION
In conclusion, P. pubescens leaves extracts display antimicrobial properties was able to inhibit moderately B. cereus and E. coli. Furthermore, this study showed that the antibacterial properties of P. pubescens leaves was not as effective as the commercial antibiotics chloramphenicol, enrofloxacin and penicillin, but it was more potent than penicillin against E. coli. Further investigations must be performed to examine the antibacterial properties from leaves, flowers, fruits, barks and roots extracts and the different method of extraction evaluate the actual antibacterial properties of P. pubescens.
ACKNOWLEDGMENT
The authors would like to thank the Laboratory of Microbiology Medan Health Clinic, Medan-North Sumatra-Indonesia, Department of Biology, Faculty of Math and Natural Science, Research Institute of Medan State University and Ministry of Higher Education and Research and Technology, Republic Indonesia.