Antibacterial Activity of Leaf Extracts of Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. Against Streptococcus pneumoniae

Antibacterial resistance in Streptococcus pneumoniae has been increasing and is one of ongoing global concern. The need to find new antibacterial agents against Streptococcus pneumoniae is of paramount importance. Medicinal plants are prospective sources of antibacterial agents. The aims of the present study were to determine the activity of leaf extraxt of Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. against Streptococcus pneumoniae. Leaves of Anredera cordifolia (Ten.) Steenis were extracted using 96% ethanol, while the leaves of Muntingia calabura L were extracted using 100% methanol. The leaf extracts of the two plants obtained were bioassayed for antibacterial activity against Streptococcus pneumoniae ATCC 49619 and a clinical isolate Streptococcus pneumoniae PU 067. Results showed that leaf extracts of both Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. have antibacterial activity in vitro against Streptococcus pneumoniae ATCC 49619 at crude extract concentrations of 25%, 50%, 75% and 100% (w/v). Both plants extracts showed strongest activity against S. pneumoniae ATCC 49619 at extract concentration of 75%. In addition, the extracts of both plants have inhibitory activity against growth of the clinical isolate Streptococcus pneumoniae PU 067. Both plant extracts showed strongest activity against S. pneumoniae PU 067 at extract concentration of 100%. Therefore, leaf extracts of Anredera cordifolia (Ten.) Steenis and Muntingia calabura L. can potentially be used as a source of antibacterial agent for Streptococcus pneumoniae. .


INTRODUCTION
Streptococcus pneumoniae was discovered in 1881, by two microbiologists independently, George M. Sternberg in the United States and Louis Pasteur in France. The bacterium was described as roughly lancet-shaped pairs of coccoid bacteria (Watson et al. 1993). S. pneumoniae is a Gram positive bacterium, the most common cause of pneumonia in the elderly. The bacterium also causes of middle ear infections and meningitis in children. Currently, vaccine for the pneumococcus is available consisting of a mixture of 23 different capsular polysaccharides. It is very effective in young adults, but only about 60% effective in the elderly and infected children under 2 years old. This is because children at this age group are unable to mount an antibody response to the pneumococcal polysaccharides. Antibiotics such as penicillin have played important roles in diminishing the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets (Jedrzejas 2001). However, the emergence of drug-resistant S. pneumoniae, especially multidrugresistant (MDR) strains, decreases the effectiveness of antibiotics.
Multidrug-resistant S. pneumoniae has spread worldwide. In addition, the spread of extensively drug-resistant (XDR) S. pneumoniae has also been reported (Cho et al. 2014).
Increased antimicrobial resistance in S. pneumoniae has been a worldwide concern mainly due to increase used of antibacterial agents (Golden et al. 2015). The excessive use of antibacterial agents causes selective pressure that maintains strains harboring resistance genes once introduced into communities. It is well known that pneumococci have the ability to acquire resistance genes through natural transformation, even from other bacterial species (Vilhelmsson et al. 2000). Globally, S. pneumoniae causes ~582000 to 926000 deaths annually and antibacterial agents have been the first option for treating these infections. Therefore, it is critical to search for new effective antibacterial agents against S. penumoniae.
Plant extracts have been investigated for use as a source of novel antibacterial agents (Iauk et al., 2003;Njimoh et al., 2015;Rahmawati et al., 2017). A. cordifolia (Ten.) Steenis, commonly known as "binahong" in Indonesia, is one of the medicinal plants that has been shown to have pharmacological activities. In addition to its antibacterial activities, binahong has been reported to have antidiabetic, antiobesity,antihyperlipidemic,vasodilator and wound healing activities (Leliqia et al. 2017). Previous studies have shown that ethanolic extract of A. cordifolia leaves showed antibacterial activities against Bacillus subtilis, Escherichia coli, methicillin-resistant coagulase-negative staphylococci (MRCNS), and Pseudomonas aeruginosa (Garmana et al. 2014). Other studies have also shown that an ethanolic extract, n-hexane and ethyl acetate fractions of A. cordifolia leaves have antibacterial activities against S. aureus, methicillin-resistant S. aureus (MRSA), Bacillus subtilis and Bacillus cereus (Leliqia et al. 2017). Similarly, Muntingia calabura L., commonly known as "kersen" in Indonesia, has been shown to have antibacterial acitivities and other pharmacological potential such as cytotoxic, antiproliferative, insecticidal, antioxidant, anti-inflammatory, hepatoprotective, hypotensive, antinociceptive, cardioprotective, antipyretic, antiplatelet aggregation, antiulcer and antidiabetic potential (Mahmood et al. 2014;Zakaria et al. 2019). The ethyl acetate partition of Muntingia calabura L. leaves methanol extracts were found to effectively inhibit growth of Staphylococcus aureus 25923 and S. aureus 33591 (a multi-drug resistant S. aureus, MRSA) (Zakaria et al. 2010). The methanol extracts of Muntingia calabura L. leaves and of Muntingia calabura L. fruits were also found to inhibit growth of Escherichia coli (C600) and S. aureus (209 P) (Mahmood et al. 2014). The present study was aimed to test the actibacterial activities of leaf extracts of A. cordifolia (Ten.) Steenis and Muntingia calabura L. against S. pneumoniae. To the best of our knowledge, this is the first study exploring the antibacterial activity of A. cordifolia (Ten.) Steenis and Muntingia calabura L. leaf extracts against S. pneumonia.

2014)
Samples in the form of A. cordifolia (Ten.) Steenis leaves were collected from Bogor, West Java, Indonesia. Leaves were cleaned and washed under running water and then air-dried at room temperature protected from the sun. The samples were then dried in the oven at 50 0 C. The dried samples were then ground and extracted.
The sample preparation powder was soaked in the extracting solvent with sample:solvent ratio of 1:10 (w/v) for 24 h. The sample preparation powder of Muntingia calabura L. leaf was extracted using 100% methanol with sample:solvent ratio of 1:4 (w/v) for 24 h. Both extracts were then concentrated using rotary vacuum evaporator. Solvent free-extracts of A. cordifolia (Ten.) Steenis and Muntingia calabura L. were obtained, and then dissolved in ddH20. Extract with concentration of 25%, 50%, 75%, and 100% (w/v) was obtained by dissolving 0.025 g, 0.050 g, 0.075 g, and 0.100 g of each extract in 100 mL ddH2O, respectively.

Preparation of bacterial cultures (Safari et al. 2014)
Bacterial strains, S. pneumoniae ATCC 49619 and a clinical isolate of S. pneumoniae, PU 067, which were then used for the extract susceptibility test, were firstly activated by growing them in bloodagar medium under anaerobic conditions at 37 0 C, CO2 level less than 5%, for 24 h. The bacterial growth was then observed.

Extract Susceptibility Testing
Extract susceptibility test for the pneumococcus isolates was carried out using the disk diffusion method according to Bauer et al. (1966). Following incubation for 24 h, the testing bacteria were inoculated into an Eppendorf tube containing 2 mL of NaCl. The bacterial suspension was vortexed and its turbidity was then compared to the McFarland 0.5 turbidity standard. Using sterile cotton buds, the bacterial suspension was inoculated onto a blood-agar plate. An extract-containing 6 mm disk was then applied onto the bacterial culture and the plate was incubated at 37 o C for 24 h under anaerobic conditions. The antibacterial activity was assayed by measuring the inhibition zone around the disk.

Antibacterial activity of extract of Anredera cordifolia (Ten.) Steenis
Extract of A. cordifolia (Ten.) Steenis was obtained in the form of a paste. The yield of the extract was 9.53%. The extract was then dissolved in ddH2O and used for antibacterial activity test. Results of antibacterial activity test of the extract of A. cordifolia (Ten.) Steenis at various concentrations are shown in Table 1. The leaf extracts of A. cordifolia (Ten.) Steenis inhibited the growth of S. pneumoniae ATCC 49619 at crude extract

Muntingia calabura L.
Extract of Muntingia calabura L. was also obtained in the form of a paste with a yield of 9.54%. The extract was then dissolved in ddH2O and used for antibacterial activity test. Results of antibacterial activity test at various extract concentrations are shown in Table  2.
The optimum Muntingia calabura L extract concentration to inhibit S. pneumoniae ATCC 49619 and S. pneumonia PU 067 was 75% and 100%, respectively. The inhibitory activity of the Muntingia calabura L extract at all concentrations tested was also found to be lower than that of chloramphenicol (30 μg) against the two strains.

DISCUSSION
We report experimental data of antibacterial activity of leaf extracts of binahong (Anredera cordifolia (Ten.) Steenis) and kersen (Muntingia calabura L.) at various concentrations against S. pneumoniae in attempt to search for novel sources of antibacterial agents against S. penumoniae including the MDR and XDR strains. This is important as resistant strains of S. pneumoniae have been reported to be present in Indonesia (Safari et al., 2014). Two S. pneumoniae strains were used in this study, the S. pneumoniae ATCC 49619 and a multidrug resistant clinical isolate, S. pneumoniae PU 067. The strain S. pneumoniae PU 067 is resistant towards penicillin group antibiotics, oxacillin, sulfamethoxazole/trimethoprim, and tetracycline (Saputro 2013).
In the present study, the extracting solvents used were ethanol 96% to produce leaf extract of Anredera cordifolia (Ten.) Steenis and methanol 100% to produce leaf extract of Muntingia calabura L. For antibacterial assays, ddH2O was used as a negative control and chloramphenicol (30 μg) was employed as a positive control. Our results showed that the leaf extracts of binahong and kersen have inhibitory activity against both S. pneumoniae ATCC 49619 and S. pneumoniae PU 067. The activity, however, is lower than the activity of the antibiotic chloramphenicol. The inhibitory activity against S. pneumonia PU 067 tended to increase with the increase concentration of both Anredera cordifolia (Ten.) Steenis and Muntingia calabura L extracts.
Compared to the Anredera cordifolia (Ten.) Steenis extract, the Muntingia calabura L extract seemed to be slightly more potent in inhibiting the growth of the multidrug resistant S. pneumonia PU 067. Interestingly, both plants extracts showed a higher optimum concentration to inhibit the S. pneumoniae PU 067. The reasons for this have yet to be elucidated but may be associated with the resistant characteristics of the S. pneumoniae PU 067 strain. Further studies, therefore, are required to elucidate the compounds responsible for the antibacterial activity and the molecular mechanism underlying the S. pneumoniae growth inhibitory activity.
In Indonesia, the availability of data on the spread and disease level of S. pneumoniae are limited among the Indonesian population. It was reported that S. pneumoniae spread in Lombok Island in 2001 was 48% in healthy children.
Similarly, S. pneumoniae spread in Semarang in 2010 was reported to be 43% in children aged 6-60 months and and 11% in adults aged 45-75. In HIV-infected group of children in Jakarta the S. pneumoniae it was 46%, and 3% in elderly age 60-80 years attending routine visits at the Geriatric Clinic, Dr Cipto Mangunkusumo Hospital, Jakarta (Safari et al. 2015). The serotypes of S. pneumoniae identified from the HIV infected children in Jakarta include serotype 19F, 19A, 6A/B and 23F. Most of the isolates were susceptible to chloramphenicol, clindamycin, and erythromycin, but resistant to penicillin (Safari et al. 2014). Globally, there has been a dramatic increase in the incidence of penicillin-resistant and multiplyantibiotic-resistant pneumococci worldwide since 1967 (Charpentier and Toumanen 2000). The mechanism of penicillin resistance in S. pneumoniae has been suggested to involve the alteration of Penicillin-binding proteins (PBPs) which leads to reduced affinity of the PBPs to the antibiotic molecule. Mutations leading to penicillin resistance usually occur in the transpeptidasepenicillin-binding domain. Multiple mutations are required for high-level resistance to occur. In pneumococcus, five PBPs of high molecular weight (PBPs 1a, 1b, 2x, 2a and 2b) and one PBP of low molecular weight have been reported. Mutations in PBP2x and PBP2b bring about low-level resistance and are the prerequisite for high-level resistance mediated by mutations in other PBPs, like PBP1a. In many pneumococcal clinical isolates resistance is caused by changes in these three PBPs only (Charpentier and Toumanen 2000). Binahong is a medicinal plant belonging to the family of Basellaceae, and is considered to have high potency for phytopharmaceutical sources. Binahong has been reported to contain chemical constituents such as flavonoid, oleanolic acid, protein, saponins, steroids, terpenoids, phenols, polyphenols, alkaloids tannin, ascorbic acid, and mono polysaccharides including L-arabinose, D-galaktose, and L-rhamnose (Astuti 2011;Wijayanti and Esti 2017;Yuniarti and Lukiswanto 2017). The antibacterial activity of binahong leaf has been reported (Miladiyah and Prabowo 2012;Yuniarti and Lukiswanto 2017). The bioactive compounds of binahong have been indicated to play roles in its antibacterial activities against both Gram-positive and Gram-negative bacteria including those causing sexuality transmitted diseases (Astuti 2011 (Buhian et al. 2016).
Extraction is a critical step in the evaluation of antibacterial activity medicinal plants. It is important to ensure that the chemical components having antibacterial activities are extracted from the plant materials. The basic steps in preparing plant extract include prewashing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Care must be taken to prevent lost of potential active constituents. If the plants are selected on the basis of traditional uses it is necessary to prepare the extract as described by the traditional healer in order to mimic as closely as possible the traditional 'herbal' drug. Extraction methods using polar solvents such as methanol, ethanol or ethyl-acetate are generally used to extract hydrophilic compounds. For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol can be used (Sasidharan et al. 2011). In the present study, binahong leaf was extracted by maceration using 96% ethanol and the kersen leaf was extracted by the same technique using methanol 100%. The extracts showed antibacterial activity against S. pneumoniae ATCC 49619 and a clinical isolate of MDR S. pneumoniae. Future studies employing a more systematic and comprehensive approach is required to confirm the potency of A. cordifolia (Ten.) Steenis) and M. calabura L. leaves as a source of novel antipneumococcal agents.
Modern extraction techniques such as solid-phase micro-extraction, supercritical-fluid extraction, pressurized-liquid extraction, microwave-assisted extraction, solidphase extraction, and surfactant-mediated techniques, possess certain advantages (Sasidharan et al. 2011) and may be applicable for extracting compounds with antibacterial activities from A. cordifolia (Ten.) Steenis and M. calabura L.