INTRODUCTION:

Salmonellosis is one of the most common bacterial infections in the world. It is responsible for a variety of clinical syndromes, including enteric fever, which is usually caused by typhoid or paratyphoid species, enterocolitis, bacteremia, and severe local infections¹_.Salmonella typhi (S.typhi) is a cause of systemic typhoid fever in humans, which is characterized by fever, enlarged spleen and liver (hepatosplenomegaly)^2,3_.Salmonella typhi is a facultative intracellular microorganism that can live and even it multiplies in the macrophage.

It is resistant to lysosomes, can to prevent and inhibit phagolysosome fusion. Therefore, it is difficult to dispatch⁴_.One way to remove these germs is by stimulating the function of macrophages with immunostimulants. Immunostimulants will stimulate the function of macrophages for killing through respiratory bursts. Activated macrophages will release various metabolites such as Reactive Oxygen Species (ROS). They are characterized by increased ROS. This substance is a crucial mediator of inflammation, microbicides, and tumoricidal. ROS plays important role in the killing and as one chemical lethal it can kill and eliminate bacteria. However, bursts of ROS can suppress the action of antioxidants which are responsible for blocking the excess ROS production, thereby, it causes oxidative stress in the liver⁵.Excessive ROS will react with biological macromolecules, proteins, and DNA and it can cause damage to hepatocytes⁶.Oxidative stress results from an imbalance between increased free radical production and decreased antioxidant capacity⁷.ROS is a major cause of oxidative stress. It can be in the form of superoxide (*O2), hydroxyl (*OH), peroxyl (ROO*), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), nitric oxide (NO*), nitric peroxide (ONOO*), hydrochloric acid (HOCl), and fat oxidation results⁸. In physiological conditions, oxidative stress will trigger the up-regulation of endogenous antioxidants and cytoprotective proteins to prevent or limit tissue damage. This process is mediated by Nrf-2 activation, which will activate the gene transcription rate of various antioxidant and detoxification enzymes^7,9and anti-inflammation¹⁰.Various external stimuli, including plants containing polyphenol through the expression of nuclear factor-erythroid-2 related factor 2 (Nrf-2), will inject Heme Oxygenase-1 (HO-1) and Superoxide Dismutase-2 (SOD-2)^{11,12,13,14,15}.Among the plants which can induce the activation of Nrf-2, there is M. Oleifera plant. M. oleifera leaves have the activities of antioxidant, hypolipidemic, anti-inflammatory^16,17 and anti-microbial ¹⁸capable of stopping free-radical chain reactions^19,20.L. plantarum can ferment the red M. Oleifera leaves and increase total phenol by producing β-glucosidase enzymes. The enzymes have an important role in biotransformation processes such as modifying primary and secondary metabolites which can add bioactive components such as polyphenols²¹.Total phenol in moringa leaves after 48 hours fermentation increased from 18.2±0.1 to 24.0±0.1 (mg/100g of extract)²².

MATERIAL AND METHOD:

Materials:

Dark green red M. oleifera leaves were obtained from Pamekasan, Madura, East Java, Indonesia. S. typhi bacteria were obtained from the Microbiology Laboratory, Faculty of Medicine, Brawijaya University, Malang, Indonesia. Lactobacillus plantarum FNCC 0137 was obtained from the Center for Food and Nutrition Studies (OSPG) of Gajah Mada University Yogyakarta, Indonesia. Balb/C mice were obtained from the Biosciences Laboratory of Brawijaya University, Malang. Anti-superoxide dismutase 1 antibody [EP1727Y] (Abcam), anti-NRF- 2 [EP1809Y] (Abcam), anti-oxygenase 1 [EP1391Y] (Abcam), Ms mAb to Rb IgG [2A9] -FITC (Abcam), cytometry flow analysis were conducted at the Laboratory of Animal Physiology, Structure and Development, Brawijaya University.

Preparation of red Moringa oleifera leaf fermentation extract:

Red M. oleifera leaves were dried in the air for 3 days, then put them in an oven at 40°C for 3h. They were stored at room temperature before further analysis. The dried M. oleifera leaves were ground with a blender and sieved 100 mesh. M. oleifera powder was macerated with 70% ethanol for 72 h. The maceration results were then filtered with Whatman paper size No. 1. M. oleifera solution was evaporated to dryness in a rotary with a temperature of 50°C.²³ The concentrated extract was inoculated with 10⁸CFU/g L. plantarum and the evaporator was subsequently incubated at 37°C for 120h²⁴.The fermented M. oleifera leaf extract was added by 10% sucrose and 5% NaCl and then it was put into a freeze dryer²⁵.

Preparation of experimental design:

This type of research is an exploratory study. It used 35 female white mice Balb/C strain with a bodyweight of about 20grams, ± 6 weeks of age obtained from Gadjah Mada University, Jogjakarta. Mice were grouped into five and each group consisted of 7 mice. There was positive control group (mice injected with S.typhi fed and drinking), negative control group (mice fed and drinking), and P1 treatment group (given fermented extract of M.oleifera leaves in a dose of 14mg/kg BW), P2 treatment group (given the fermentation of M. oleifera leaves at a dose of 42mg/kg BW), the P3 treatment group (given the fermentation of M.oleifera leaves at a dose of 84mg/kg BW). The treatment group was given M. oleifera fermentation extract for 28 days. On day 29 they were infected with S. typhi using a dose of 10⁷CFU/mL intraperitoneal (0.5mL/10g BW). After day 36 of post-treatment mice were neck dislocated, then surgery and cell isolation were performed.

Treatment of Animal Testing:

Six-week-old female mice (25-30g) were placed in the control room (25°C, RH 60%). They were given food and drink every day. After 1 week of acclimatization, they were randomly divided into 5 groups (7 individuals/group). Normal controls (K-) and positive controls (K+) were given orally distilled water every day. The test animal groups (3 groups) were each treated with a multilevel dose of M. oleifera fermentation extract (14, 42 and 84mg/kg BW/day) for 28 days before being injected by S. typhi. The administration of M. oleifera leaf fermentation extract was continued for 36 days after injection. S. typhi was intraperitoneally (0.5 mL/10g BW) with a concentration of 10⁷CFU/mL (except group K-). The animal testing protocol was approved by the ethics committee on animal experiments from Brawijaya University, Malang, Indonesia (No: 829-KEP-UB).

Test Confirmation of Salmonella typhi in the Blood:

In the group of mice infected by S. typhi, on day 30, a confirmation test was conducted to determine the success of S. tyhpi in infecting mice. The test was done by cutting off the tail to take the mice’s blood. The blood was then tested for pour plates and catalase tests. The pour plate test was carried out in xylose lysine deoxycholate agar (XLD agar) media while the catalase test used hydrogen peroxide (H₂O₂)²⁶.

Cell Isolation in the liver:

After day 36 of post-treatment, mice were neck dislocated, then surgery was performed to remove liver organs. The obtained liver organ was crushed, filtered, and suspended with Phosphate Buffered Saline (PBS). The obtained homogenate was transferred to a propylene tube and given PBS until the volume reached 3mL, then it was centrifuged at 2500rpm for 5 min at 10°C. The supernatant was removed and the obtained pellet was added with 1mL of PBS, then it was resuspended by using a vortex. Homogenates were mixed with 50μL of buffer and then they were incubated at 4°C for 20 min in the dark. Then, we added 200 mL of buffer. Homogenate was mixed well and centrifuged at 10°C at 2500rpm for 5 min. The pellets were added with 50μL of intracellular antibodies and incubated at 4°C for 20 min in the dark. Then the sample could be analyzed with flow cytometry (BD FACS Calibur, USA)²⁷.

Hematoxylin and Eosin (HE) Staining:

The liver tissues of mice were fixed in 10% neutral phosphate-buffered formalin solution, then dehydrated and embedded in paraffin to make conventional paraffin sections. The sections were cut into 4μm thickness and stained with Hematoxylin and Eosin (HE). The histopathological changes of liver tissues were observed under the optical microscope

Flow Cytometry analysis:

Flow cytometry analysis was performed to detect cell populations expressing HO-1, SOD-2, and Nrf-2. Flow cytometry was set to the acquiring state and parameter settings were analyzed. After incubating with the appropriate antibody, the sample was added with 500 mL of PBS and transferred to the flow cytometry cuvette for analysis. Then the acquire menu was selected and the flow cytometry counted the total cell count and detected fluorescents which were conjugated with antibodies. The obtained results were analyzed with BD cell quest Pro^TM.

DATA ANALYSIS:

The number of cells (%) expressing HO-1, SOD-2, and Nrf-2 were analyzed by flow cytometry and the obtained results were tested for normality. Furthermore, the obtained data were tested by ANOVA with SPSS 16.0 for windows, followed by Duncan Multiple Range Test (DMRT) using p-value significance values <0,05. Hepatic preparations stained with HE were analyzed by a microscope to determine the profile of hepatocyte cells.

RESULT:

Analysis of HO-1 expressions:

From each treatment group, the average percentage of HO-1 showed the result that a dose of 14 mg/kg BW did not provide a significant difference when compared to control (+). Meanwhile, a dose of 84 mg/kg BW gave increasing results but they were not significant. However, a dose of 42 mg/kg BW gave significant results compared to a dose of 14 mg/kg BW and a dose of 42 mg/kg BW.

Figure 1. Flow cytometric analysis of liver cells. a. HO-1 representative expression in liver cells. b. Increase of HO-1 percentage in liver cells for 36 days after treatment. Results were considered significant at p < 0.05.

Analysis of SOD-2 Expression:

The average percentage amount of SOD-2 from each treatment group showed that fermentation extract of red M. oleifera leaves at a dose of 42 mg/kg BW had significant results than those of 14 mg/kg BW and 84 mg/kg BW. However, the treatment dose of 14 mg/kg BW was not significantly different from the dose of 42 mg/kg

Figure 2. Flow cytometric analysis of liver cells. a. SOD-2 representative expression in liver cells. b. Increased percentage of SOD-2 in liver cells for 36 days after treatment. Results were considered significant at p < 0.05.

Analysis of Nrf-2 expression:

Statistical test results showed a significant difference (p-value < 0.05) between treatments. The mean percentage of Nrf-2 from each treatment group showed that the dose of 42mg/kg BW was not significantly different from the dose of 84 mg/kg BW. However, the administration of fermented extract of red M.oleifera leaves at a dose of 14 mg/kg BW was significantly different with doses of 42 mg/kg BW and 84 mg/kg BW by 27.66 %.

Figure 3. Flow cytometric analysis of liver cells. a. Nrf-2 representative expression in the liver. b. Increased percentage of Nrf-2 in the liver for 36 days after treatment. Results were considered significant at p < 0.05.

Liver Histological Analysis:

Histological features in the treatment group showed a significant effect in preventing necrosis. In healthy controls (-) there were a few cells that experienced necrosis and dense hepatocyte cells were visible. It indicated that hepatocyte cells were in a normal state. Whereas in pain control (+), there were very many necrotic cells accompanied by sinusoids undergoing dilation. Treatment with a dose of 14mg/kg BW showed changes in liver cells and some normal cells were seen in some parts although necrosis cells were still visible. In some fields of view, fat degeneration appeared and sinusoidal widening was again seen. It was degenerating and necrotic by an average of 27.77%. Treatment with a dose of 42mg/kg BW showed improvement in hepatocyte cells and the sinusoid was not widened. Degeneration and necrosis appeared by an average of 26.89%. Treatment with a dose of 84mg/kg BW improved liver, but the cell damage was higher than the doses of 14mg/kg BW and 84mg/kg BW, in which the degeneration and necrosis were by an average of 30.88 %.

Figure 4. HE staining in liver preparations. a. (K-) indicated normal hepatocyte cells and (K+) indicated abnormal hepatocyte cells. b. Group dosage of 14 mg/kg BW, group dosage of 42 mg/kg BW and a group of 84 mg/kg BW. c. The percentage results and statistical analysis showed a significant difference (P < 0.05) between the treatment groups of fermented M. oleifera leaves extract. Results were considered significant at p < 0.05.

DISCUSSION:

The liver is an important organ that plays a central role in the metabolism of proteins, carbohydrates, fats, vitamins and detoxification functions. The liver can suffer damage caused by bacterial and viral infections, alcohol consumption, pollutants, and toxic chemicals²⁸. S. typhi bacterial infections have been reported to cause liver enzymes to increase and their concentration is a marker of liver damage²⁹. In S. typhi infection, the liver’s histopathological evaluation usually shows mononuclear cell infiltration with a minimum focus of portal channel infiltration and focal necrosis areas²⁸.Oxidative stress on cells is associated with increased expression and antioxidant enzyme activity³⁰.This defense system is very important to detox ROS³¹. The appearing ROS is the chemical lethal which can kill and eliminate bacteria in phagocytic cells. ROS are the main causes of oxidative stress, including superoxide anions, hydroxyl radicals, and hydrogen peroxide³². Excessive ROS can cause oxidative stress in the liver, suppress the formation of antioxidant transcription pathways³³. It will also react with biological macromolecules, proteins, and DNA leading to hepatocyte damage⁵. Nrf-2 is a transcription factor responsible for the transcription of antioxidants and cytoprotective genes³⁴.The Nrf-2 pathway has an important role in acute liver damage³⁵.HO-1 is effective to prevent various oxidative injuries. Many studies have confirmed that HO-1 has a protective effect on the liver with several pathological conditions, such as ischemia, reperfusion injury, chronic liver disease, etc³⁶. Meanwhile, Superoxide dismutase 2 (SOD-2) is known as manganese-dependent superoxide dismutase (Mn-SOD). This enzyme plays an important role in the antioxidant defense system when dealing with oxidative stress³⁷. SODs act first in defense of antioxidant enzymes against ROS and especially superoxide anion radical³⁸.

The results showed that anti-oxidative compounds derived from the extract fermentation produced a powerful antioxidant and contributed to the hepatotoxicity protection associated with the activation of Nrf-2/HO-1 and SOD-2 signaling pathways. SOD-2 works in such a way to capture and inhibit ROS production by delaying or preventing the occurrence of free radical reactions or by arresting the antioxidative radical compounds (radical scavenging) as initial protection against oxidative damage³⁹.As an anti-oxidative stress regulator, Nrf-2 regulates the expression of antioxidant genes and phase II detoxification enzymes such as heme oxygenase-1 (HO-1), NAD (P) H quinone oxidoreductase 1 (NQO-1), and Glutamate Cysteine Ligase Catalytic subunit (GCLC) which counteracts oxidative stress by increasing the inhibition of ROS^40,41. Uncontrolled chronic inflammation can cause disease. Therefore, anti-inflammatory agents from natural or synthetic sources are needed as therapeutic agents to prevent the disease. The increase in flavonoid total during fermentation is thought to be due to the activity of lactic acid bacteria during the fermentation process. Lactic acid bacteria produce enzymes that can free phenolic compounds in Moringa leaves so that they can add to the phenol group of flavonoid compounds⁴².

In conclusion, it showed that anti-oxidative compounds derived from the fermented extract have a powerful antioxidant that acts as immunomodulatory agents that contribute to hepatotoxicity protection. The mechanism of action seems to be associated with the activation of Nrf-2/HO-1 and SOD-2 signaling pathways.

ACKNOWLEDGMENT:

The author would like to thank the members of Animal Anatomy and Physiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, and LPDP Ministry of Finance of Indonesia through the BUDI-DN scholarship for their supports in our research.

CONFLICT OF INTEREST:

All the authors have no conflict of interest.

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Received on 29.01.2020 Modified on 18.03.2020

Research J. Pharm. and Tech. 2020; 13(12):5947-5952.

DOI: 10.5958/0974-360X.2020.01038.0