Antibacterial Activity and Mode of Action of Black Mulberry ( Morus nigra ) Fruits Extract Against Streptococcus mutans

Several studies reported the antibacterial activity of the BM extract. However, the mechanism involved in this process has not been clearly understood. In this study, antibacterial activity of the extract against S. mutans was evaluated using the micro-dilution method. Then, analysis of protein, nucleic acid, and ion leakage, as well as cell morphological observation were also investigated. inhibitory concentration (MIC) of the extract. Protein and ion leakage from the bacterial cells induced by the extract was analyzed spectrophotometrically. The morphological change was determined using Scanning electron microscopy (SEM). Result: Black mulberry (BM) fruits extract showed antibacterial activity against S. mutans with MIC value of 8 mg/mL, as well as contributed to the aforementioned leakage. Meanwhile, cellular membrane damage was observed, indicating that the extract might inhibit growth of the bacteria. Conclusion: BM extract could be a potential raw material for herbal medicine, especially anticaries which has antibacterial activity.


INTRODUCTION
Morus or mulberry is the genus of plants belonging to the Moraceae family. They are widely grown in subtropic regions of Asia, Africa, and North America. 1 The fruits were commonly used as food ingredients such as juices, wines, and jams. 2 Various parts of the plants could also be used as cosmetic products and herbal medicines. 3 In addition, flavonoids, polyhydroxylated alkaloids, and benzofurans are the most representative bioactive compounds that have been identified from the root of mulberry. 4 The extract have shown several pharmacological activities, which include antimicrobial, anti-inflammatory, antifungal, antioxidant, skin-whitening, anticancer, antidiabetic, anti-hyperlipidemic, anti-platelet, and immunomodulation. 5,6 Black mulberry belongs to the genus Morus, which contains the highest total phenolic compounds compared to other species. 7,8 Furthermore, the extract exhibited an antibacterial effect on Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes. 10,11 The fruits are a rich source of flavonoids and anthocyanins which can be used for antioxidation. 8,11 Souza et al reported that black mulberry extract has strong antioxidant and antibacterial activities as well as weak cytotoxic effects. 12 Streptococcus mutans are gram-positive bacteria commonly found in the human oral cavity and are the main contributor to tooth decay, 13 while pharynx, mouth, and intestine are the primary habitats. 14 In addition, they can act as strong acid producers together with lactobacilli, causing the risk for cavities. 16 S. mutans and Streptococcus sobrinus can cause dental caries because of the ability to adhere to enamel salivary pellicle. 15 Several studies have reported the antibacterial activity of the BM extract. However, the mechanism involved in this process has not been clearly understood. In this study, antibacterial activity of the extract against S. mutans was evaluated using the micro-dilution method. Then, analysis of protein, nucleic acid, and ion leakage, as well as cell morphological observation were also investigated.

Materials
Black mulberry (BM) fruits were obtained from Cibodas, Maribaya-Lembang, and authenticated by the Department of Biology, Faculty of Science, Universitas Padjadjaran, Bandung, Indonesia. In addition, Muller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) were purchased from Sigma Aldrich, Germany, while all other chemicals used were of technical grade. S. mutans was obtained from the Laboratory of Microbiology, Faculty of Pharmacy, University of Padjadjaran. method thrice for 24 hours at room temperature. After which, the solvent used was removed by a rotary evaporator (IKA RV 10, IKA Company, Germany) at 50°C to obtain the crude extract.

Phytochemical screening
This was conducted to observe the presence of secondary metabolites such as alkaloids, tannins, saponins, quinones, steroids/triterpenoids, polyphenols, monoterpenes, and sesquiterpenes.

Antibacterial activity
This process was performed by using the disc diffusion method (Kirby Bauer). Furthermore, MHA was mixed with 5% sterile sheep blood as a bacteria growth medium. S. mutans were dissolved in 0.5 McFarland of Tryptic Soya Broth (TSB), and then poured into the media. The disc paper that soaked in the extract suspension with various concentrations was dried, then placed into the media, and incubated for about 24 h at 37°C.

Minimum inhibitory concentration (MIC) evaluation
This was evaluated through the micro-dilution method using a microplate. 100 μl of BM fruit extract with various concentrations was added into the media containing 100 μl MHB. Ten μl of the bacterial suspension was adjusted to McFarland turbidity standards which have equivalence of 3.0 x 108 CFU/ml, followed by addition into each well of the media. Furthermore, the microplate was incubated at 37°C for 24 h. Amoxicillin and 1% v/v of Dimethyl sulfoxide (DMSO) solution were used as positive and negative controls, respectively. 6

Analysis of protein and nucleic acid leakage
Ten mL of bacterial suspension grown in MHB medium for 24 hours was taken and centrifuged at 3500 RPM for 20 m. The filtrate was discarded and the pellets in the tube were washed with a solution of phosphate buffer at pH 7, then suspended in another 10ml of the buffer and shaken for 24 h. Furthermore, BM fruit extract (with a concentration of 1xMIC and 2xMIC) and control were added into the suspension. After which centrifugation was performed at 3500 RPM for 15 m, followed by filtration to separate the supernatant and cells. The supernatant was analyzed spectrophotometrically at 260 nm and 280 nm (Shimadzu UV-VIS 1800). 6

Analysis of ion leakage
The sample of the bacterial pellets was prepared using similar method with the proteins and nucleic acid that were analyzed. The leakage of cells was expressed with the presence of ions (Ca 2+ and K + ) in antibacterial activity evaluation, after BM fruit extract with 4xMIC concentrations was added. The leakage of the ions was determined by using Atomic absorption spectroscopy (Shimadzu). 17

Cell Morphological Observation
A clear zone of the medium from the antibacterial test was soaked into a solution of 2% glutaraldehyde overnight as a sample. This was centrifuged and the supernatant was discarded. The residue was dispersed into solution of 2% tannic acid, then soaked for few hours, followed by centrifugation and disposition of the fixative solution. After that chocodylate buffer was added and soaked for 20 m. The mixture was centrifuged and the supernatant was removed, then added 1% osmium tetraoxide, and soaked for 1 h. The resulting mixture was again centrifuged, followed by discarding the supernatant, and the remaining sample was soaked in 50% alcohol for 20 m. The residue was dried consecutively using 70%, 80%, 95%, and absolute alcohol for 20 m, respectively. Furthermore, the samples were suspended into butanol and soaked for 20 m. The suspension were placed into the coverslip, subjected to drying, coated and observed using Scanning Electron Microscopy (SEM) (JEOL JSM-5310LV®). 18

RESULT AND DISCUSSION
The phytochemical screening of BM fruit extract was carried out as shown in Table 1. Flavoniods and phenolics which attributed to the antibacterial activity were observed. There were many compounds in the extract which belong to phenolic groups such as chalcone, kuwanon E and kuwanon U, moracin, morusin, and anthocyanin. 19,20 In addition, they were predicted to be capable of denaturing bacterial cell proteins as well as inhibiting the cellular metabolism, while flavonoids and tannins can damage the walls.
The antibacterial activity of BM fruit extract was performed using the diffusion method as a result of being simple, rapid, inexpensive, and reproducible. 21 This method is not suitable for natural waterinsoluble antimicrobial compounds, such as essential oil steroids and terpenoids due to impossiblity for them to homogeneously diffuse into the medium. 22 DMSO was used to increase the diffusion rate with the suspension system. The MIC of the extract against S. mutans was 8 mg/ mL, while the MBC was 16 mg/mL (Figure 1).
The leakage of cell will occur if the extract has antibacterial activity. Meanwhile, this could be detected spectrophotometrically at 260 nm and 280 nm for nucleic acid and protein, respectively. 17 The result of the analysis can be seen in Figure 3. The amount of these molecules increases along with the concentration of BM fruit extract. When there is detection outside the bacterial cell, that indicates posibility of the extract to damage cell walls or change permeability of the membrane. The phenolics and flavonoids can diffuse through the membrane. This data is in agreement with the theory that flavonoids may demonstrate the antibacterial activity through the reduction of membrane fluidity causing fluid imbalance in the cells. 23 The detection of metal ions such as potassium (K + ) and calcium (Ca 2+ ) from bacterial cells could be attributed to the potential antibacterial activity of the compound. The K + plays an important role in the ribosome, while Ca 2+ is needed as a component of the cell wall of gram-positive bacteria. 24 The amount of Ca increases along with the concentration of BM fruit extract, while there was no significant change in the total K ( Figure 3). Therefore, this result indicated that BM fruit extract can damage the cell wall and / change the membrane permeability. The Ca 2+ can maintain the stability of the bacterial wall, while the release of these ions from the cell can disrupt the stability of bacterial wall and then resulting to death.
Morphological alteration was observed using scanning electron microscope. After treated the extract with 4xMIC, the cell of S. mutans was shrinking due to degradation of the cellular membrane as shown in Figure 2. This result indicated that the fruit extract is a potential antibacterial activity agent.