INTRODUCTION:

In the last few decades, many natural plants have been reported as an alternative antibacterial agent because many antibiotics are no longer potent against bacterial^1,2. Morus or mulberry, the genus of plants belonging to the Moraceae family, is one of the plants which have widely been studied for its antibacterial activity. Species of this genus have also been used in traditional medicine as hepatoprotective, antipyretic, analgesic, diuretic, anemia, and arthritis^3,4. A variety of phenolic compounds contained in this genus could be used as alternatives in herbal medicines⁵.

Root bark extract from Morus nigra (MN) contains deoxynojirimycin, which is effective against HIV⁶. Besides that, its root can decrease blood sugar in diabetic patients, and has some effect on the pancreas, and glycogenolysis. In addition , its fruit which contains high amounts of total flavonoids, phenolics, and ascorbic acid, can control blood cancer and has a positive impact on blood glucose levels^7,8,9.

Many compounds contained in MN have been isolated and show pharmacological activity. In addition, Cyanidin 3-O-‚-D-glucopyranoside from its fruit can inhibit the damage that occurs during cerebral ischemic, which caused by deprivation of oxygen and glucose in PC12 cells¹⁰. Anthocyanins which have also been isolated from its fruits can inhibit the co-oxidation of linoleic acid and β-carotene, and copper-induced peroxidation of liposome^8,11. While Morin can reduce the level of cyclosporin in tissues, which acts as a potent immunosuppressive agent, and decrease nitric oxide production¹².

MN has been reported to have antimicrobial and antiinflammatory properties¹³. The MN fruits extract showed antibacterial activity against Staphylococcus epidermidis and Propionibacterium acnes, at MIC of 2.5%14. The MN leaves extract has an antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Streptococcus fecal, Escherichia coli, and Pseudomonas aeruginosa¹⁵.

In this study, the antibacterial activity and MIC of the MN stem bark extract against Streptococcus mutans (S. mutans), which is commonly found in the oral cavity of humans and formed biofilm on tooth surfaces^16,17,18, and a contributor to tooth decay was evaluated. This evaluation conducted using the microdilution method. In addition, its mode of action was observed using cell morphological observation, protein, nucleic acid leakage, and ion leakage analysis.

MATERIAL AND METHODS:

Materials:

The stem bark of MN was obtained from Cibodas, Maribaya-Lembang, and authenticated by the Department of Biology, Universitas Padjadjaran, Bandung, Indonesia. Furthermore, Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) were purchased from Sigma Aldrich, Germany, and all other chemicals used were of technical grade. S. mutans was obtained from the Laboratory of Microbiology, Universitas Padjadjaran.

Extraction:

The extraction was carried out on 400g of the stem back of this plant, with a maceration method, using 96% ethanol (4 L) as a solvent, for 72 h at room temperature. The liquid was then concentrated using a rotary evaporator (IKA RV 10, IKA Company, Germany), at 50°C to obtain the crude extract.

Phytochemical Screening:

Phytochemical Screening was conducted to investigate the presence of secondary metabolites, such as flavonoids, alkaloids, tannins, saponins, polyphenols, quinones, steroids/triterpenoids, monoterpenes/ sesquiterpenes^19,20,21.

Antibacterial Activity:

The antibacterial activity of this extract was ascertained using the agar diffusion method, which involved first, the mixing of Mueller Hinton Agar (MHA) media with the bacterial suspension, and pour into a Petri dish²⁵. Second, the dissolution of the extract in 1% v/v DMSO, and the addition of various concentrations of the mixture to the Petri dish. The mixture was then incubated for about 24 h at 37°C. Amoxicillin was used as positive control, while 1% v/v of dimethyl sulfoxide (DMSO) solution, as a negative control²⁶.

Minimum Inhibitory Concentration (MIC) Evaluation:

MIC evaluation was performed through a microdilution method. 100μL MHB media was mixed with 100μl of MN stem bark extract, of various concentrations. Furthermore, 10μl of the bacterial suspension adjusted to McFarland turbidity standards²², which is equivalent to 3.0 x 10⁸ CFU/mL, was added into each media, and then incubated at 37°C for 24 h. Amoxicillin was used as positive control, while 1% v/v of Dimethyl sulfoxide (DMSO) solution, as a negative control^23,24.

Cell Morphological Observation:

A clear zone of the medium was soaked in a solution of 2% glutaraldehyde overnight. This solution was then centrifuged, and its supernatant was discarded. The residue was dispersed into a solution of 2% tannic acid, soaked for a few hours, centrifuged, and disposed of, in a fixative solution. After this, cacodylate buffer was added to it, and the mixture was soaked for 20m. Furthermore, this mixture was centrifuged, and the supernatant was discarded. 1% osmium tetroxide was added to the residue and soaked for 1h. This solution was then centrifuged and the supernatant discarded. The remaining sample was soaked in 50% alcohol for 20 m, and then consecutively dried using 70%, 80%, 95%, and absolute alcohol for 20m, respectively. the resultant sample was dispersed into butanol and soaked for 20m. Finally, the sample was placed on a microscope slide, covered with a coverslip, dried, coated and observed using Scanning Electron Microscopy (SEM) (JEOL JSM-5310LV^®)²⁷^,²⁸.

Protein and Nucleic Acid Leakage Analysis:

The bacterial suspension which was grown in MHB medium for 24 h was taken and centrifuged for 20m, at 3500RPM. the sample was filtered, placed into a tube, washed, and suspended in phosphate buffer (pH 7) solution, and shaken for 24 h. MN stem bark extract, and the control were then added into suspension. This was immediately followed by centrifugation at 3500 RPM, for 15m. The resultant sample was filtered, and its supernatant was analyzed spectrophotometrically at 260nm and 280nm (Shimadzu UV-VIS 1800)²⁴^,²⁸.

Ion leakage analysis:

Samples of the bacterial pellet were prepared using a similar method to protein and nucleic acid leakage analysis. Furthermore, using atomic absorption spectroscopy, the samples were analyzed for the presence of Ca²⁺ and K⁺ (Shimadzu)²⁹.

RESULT AND DISCUSSION:

The phytochemical screening of MN stem bark extract is shown in table 1. this species is known to be rich in phenolics, such as kuwanon E and U, morusin, anthocyanin, vanillic acid hexoside, and chalcone. The presence of flavonoids and phenolics in this plant indicates that its stem back extract could have antibacterial activity, since these compounds are known to be capable of inhibiting metabolism in bacterial cells³⁰.

Table 1: Phytochemicals Screening of MN Fruit Extracts

Compound group	Crude drug	Extracts
Alkaloids	-	-
Flavonoids	+	+
Saponins	+	-
Polyphenols	+	+
Monoterpenoids/sesquiterpenoids	+	+
Tanins	+	+
Quinons	+	+
Steroids/triterpenoids	-	-

Over the last decade, the use of plants for therapeutic purposes has greatly increased. Furthermore, today, some antimicrobial drugs are derived from natural products which has undergone clinical trials and are available on the market. The antibacterial activity of MN stem bark against S. mutans was investigated. In addition, its antimicrobial activity was demonstrated by measuring the diameter of the inhibition zone. The result showed that MIC of the extract was 8 mg/mL as shown in figure 1. This indicates that the extract can inhibit the growth of S. mutans. Moreover, its antibacterial activity can be attributed to the presence of antibacterial compounds such as arylbenzofuran, and its derivatives³⁰.

A previous study reported that this extract, which contains moracin and artocarpecin, could inhibit the growth of Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, and Salmonella Typhimurium³⁰.

Figure 1: MIC result of MN stem bark extract against S. mutans

SEM analysis of S. mutans treated with this extract was carried out to observe morphological changes and damaged cell membrane. As shown in figure 2, the untreated group showed rod-shaped cells, with intact morphology, and uniform size and shape. In contrast, cells treated with MN at 3 × MIC showed severe morphological destruction. The signs of damage such as protruding bubbles and membrane stacks from the membrane cell of S. mutans may lead to DNA, RNA and protein leakage. This result indicates that MN stem bark extract could inhibit the growth of S. mutans and cause irreversible damage to the cell wall and membrane.

Figure 2: SEM micrograps of S. mutans scale in 10.000x (a) untreated, (b) bacteria treated with MN stem bark extract (3 x MIC).

The antibacterial mechanism of this extract against S. mutans was confirmed using the assay of DNA, RNA and protein leakage through the bacterial cell membrane as shown in figure 3. Furthermore, it was observed that the amount of nucleic acid from S. mutans treated with the extract of 1 × MIC and 2× MIC were 6.99 times, and 8 times higher than that of the control (untreated), respectively. These results indicated that this extract increased cell membrane permeability, and caused damage to cell membranes, which led to the loss of DNA and RNA. moreover, it can aggravate protein leakage through S. mutans cell membranes.

The amount of nucleic acid and protein leakage increased with increasing concentrations of this extract. This indicated that bacterial cell membrane integrity was compromised in response to increasing concentrations of the extract, and it could lead to cell death²⁷. Analysis of nucleic acid (RNA and DNA) and protein leakage can demonstrate the mechanism of antibacterial action of compounds against S. mutans. In addition, it was observed that this extract caused irreversible damage to the bacterial membrane, thus affecting its integrity. The damage of cell membrane is a key mechanism for antibacterial activity.

Figure 3: Nucleic acid and protein leakage from S. mutans treated with 1xMIC and 2xMIC of MN stem bark extract

When cells of S. mutans were incubated with MN stem bark extract, leakage of K⁺ from the cells was detected (Figure 4). The leakage of Ca²⁺ form S. mutans was significantly different, when compared to control. In addition, that of K⁺ increased with every increase in the concentration of the extract. Potassium is the cation required for charge balancing and protein synthesis, therefore, a small loss of K⁺ from a cell can lead to the development of large membrane potentials. An increase in the passive flux of ions across the membrane impairs its functions³¹. This indicates that this extract is a potential plant to inhibit the growth of S. mutans.

Figure 4. Potassium and calcium leakage from cells of S. mutans on treating with 1xMIC and 2xMIC of MN stem bark extract

CONCLUSION:

MN stem bark extract has antibacterial activity against S. mutans, at an MIC of 8 mg/mL. Furthermore, it was discovered to cause irreversible damage to the bacterial membrane, therefore, affecting their integrity. Severe morphological destruction of cells was observed after treatment with 4xMIC of the extract. Moreover, it can increase cell membrane permeability and cause damage to cell membranes, which lead to the loss of DNA, RNA and protein. The rate of K⁺ leakage simultaneously increased with every increase in the concentration of this extract. This indicates that the extract impaired the functioning of the membrane. Therefore, MN stem bark could developed as herbal medicines, especially for anticaries.

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Received on 22.08.2020 Modified on 09.10.2020

Research J. Pharm. and Tech. 2021; 14(8):4399-4402.

DOI: 10.52711/0974-360X.2021.00763