Immunomodulator effect of Moringa oleifera Leaves Fermented by Lactobacillus plantarum FNCC 0137 on Salmonella typhi infected Balb/C Mice

 

Rizky Dzariyani Laili¹, Erryana Martati¹, Muhaimin Rifa’i²*

¹Department of Food Sciences and Technology, Faculty of Agricultural Technology, University of Brawijaya, Malang, Indonesia.

²Laboratory of Animal Physiology, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Indonesia.

 

ABSTRACT:

This study aimed to investigate the immunomodulatory effect of Moringa oleifera leaves (MOL) extract fermented by Lactobacillus plantarum FNCC 0137 in mice infected with Salmonella typhi. This research employed ten groups of mice. Group (K-) received distilled water without bacterial infection, group (K+) received distilled water and infected by bacteria, and six groups of bacteria infected mice that pre-treated with three different doses (14, 42, and 84mg/kg BW/day) of fermented or non-fermented of MOL extract. Bacteria are intraperitoneally injected on the 29^th day. The administration of extract was continued for one week after injection. The lymphocyte cell isolated from lymph nodes was analyzed with flow cytometry. Statistical analysis was performed using SPSS 22.0 ANOVA (p<0.05) and followed by Duncan’s test. The result showed that fermented extract of MOL could act as an immunomodulatory effect in mice indicated by the increase of the expression of  CD4⁺IFN-γ⁺, CD4⁺TNF-α⁺ and, CD11b⁺IL-6⁺. However, at a dose of 84 mg/kg BW, non-fermented and fermented of MOL extract showed immunosuppression of cellular response in mice. In summary, non-fermented and fermented MOL extract act as an immunomodulatory agent in mice infected with Salmonella typhi. Fermented MOL extract is more effective as an immunomodulatory agent compared to non-fermented MOL extract.

 

 

 

INTRODUCTION:

The key to typhoid fever prevention is anticipating the infection of Salmonella typhi by increasing the humoral and cellular immune system. Lately, the use of antibiotics is known to cause resistance to bacterial infection is hard to terminate. Therefore, it is necessary to find alternative solutions, such as by using herbal medicines that have potential as an immunomodulator.

 

Moringa oleifera leaves (MOL) has been proved to have immunomodulatory effects that can increase the activity of non-specific immune systems such as lymphocytes, NK cells, and stimulation of macrophages¹, interferon released and interleukin², increasing the level of serum immunoglobulin³. MOL has immunostimulatory properties due to its complex nutrients and phytochemicals phenolic acids and flavonoids^4,5,6,46.

 

Lactobacillus plantarum produces lactic acid as the final product of carbohydrate metabolism, hydrogen peroxide and bacteriocins as antimicrobial agents⁷. The growth of pathogenic bacteria such as Salmonella and Escherichia coli can be inhibited by antimicrobial substances and acid compounds formed^8,9,47. The study of fermented MOL with Lactobacillus plantarum showed an increased bioavailability of iron, total phenol, and total protein content^10,11, and reducing antinutritional substances of tannin and phytate^12,13.

 

Thus, it is necessary to study the fermentation of MOL with Lactobacillus plantarum as an immunomodulator against Salmonella typhi. Immunomodulatory effects of fermented MOL extract can be determined by observing the relative amounts of CD4⁺IFN-γ⁺, CD4⁺ TNF-α⁺ and CD11b⁺IL-6⁺ on mice infected with Salmonella typhi.

 

MATERIALS AND METHODS:

Materials:

MOL is obtained from Pamekasan, Madura, East Java, Indonesia. MOL was taken from the Moringa tree aged 3-12 months. Salmonella typhi was obtained from the Laboratory of Microbiology, Faculty of Medicine, University of Brawijaya. Lactobacillus plantarum FNCC 0137 was obtained from the Food and Nutrition Study Center (PSPG), Gajah Mada University. A marker (Biolegend, USA) for immune analysis using flow cytometry was obtained from the Laboratory of Animal Physiology, University of Brawijaya.

 

Extraction and Fermentation of MOL:

Collected plant material was air-dried for 3 days and continued to dry at 40° C for 3 h in the oven. The dried leaves were stored at room temperature before further analysis. The dried MOL was milled in a blender and sieved 100 mesh.  The MOL powder was macerated with 70% of ethanol for 72 h. The slurry was then filtered through Whatman paper No.1. The solvent was evaporated to dryness in a rotary evaporator at 50°C¹⁴. The concentrated extract was inoculated with 10⁸ CFU/g of Lactobacillus plantarum and incubated at 37°C for 120 h¹⁰. The fermented MOL was added 10% sucrose and 5% NaCl and then freeze-dried¹⁵.

 

Physicochemical Analysis:

The moisture content was analyzed using (AOAC, 1999). Total acid and total sugar were analyzed with reference to the AOAC method (1995).

 

Total Flavonoid Content:

Total flavonoid content was determined using the aluminum chloride colorimetric assay^16,48,51. Briefly, 1 ml extract was added to 4 ml of distilled water and 0.3 ml of NaNO₂ 5%. The solution was vortexed and incubated at room temperature for 5 min, then added to 0.3 ml of 10% AlCl₃. The mixture was mixed well and incubated at room temperature for 6 min, then added to 2 ml of 1M NaOH. The absorbance of the sample was measured at 510 nm. Quercetin was used for the preparation of a standard curve. Total flavonoid content was expressed as mg quercetin equivalent (QE) per gram extract (DB). All experiments were performed in triplicate and results reported as mean values and standard deviation.

 

Animal Experiments:

Six-week-old female mice (20-30 g) were housed in a controlled room  (25°C, RH 60%) and with a 12 h light–12 h dark cycle. Animals accessed freely to feed and water at all times. After 1 week of acclimation, they were randomly assigned to 10 groups (7 animals/group).  Control negative (K-) and control positive (K+) were orally gavaged with distilled water every day. Test groups animals (6 groups) were pre-treated with the increasing doses of non-fermented or fermented MOL extract (14, 42 and 84 mg/kg BW/day, respectively) for four weeks prior to Salmonella typhi injection. The administration of MOL extract was continued for one week after injection. Salmonella typhi was injected intraperitoneally (0.5ml/10g BW) with a concentration of 10⁷ CFU/ml  (except a group of K-). The protocol of the animal experiment was approved by the ethical committee on animal experiments of University of Brawijaya, Malang, Indonesia (No: 829-KEP-UB).

 

Confirmation Test of Salmonella typhi:

Confirmation test was carried out on the 30^th day, one day after injection with Salmonella typhi.  Mice serum of 50mL was taken from the tail, then added with 450 µL sterile physiological NaCl. Serum samples were then planted in Luria Broth media and incubated at 37°C and 120 rpm for 24. The results of incubation on Luria Broth media were then inoculated on Salmonella selective media, Xylose-Lysine-Deoxycholate (XLD) media. Positif results showed Salmonella typhi formed colonies marked with a black core¹⁷.

 

Isolation of Lymphocyte from Mice Lymph node:

The lymph node was taken and washed using phosphate buffer saline (PBS) then crushed to obtain a homogeneous sample. The homogenate obtained was then transferred to a propylene tube and PBS was added to the volume reached 3 mL, then centrifuged at 2500 rpm for 5 min at 10°C. The supernatant is removed and the pellet obtained is added with 1 ml PBS, then resuspended using the vortex to homogenize. Homogenates were divided into two analysis of intracellular and extracellular. Homogenate of 50 µL was taken and put into a 1.5 mL tube containing 500 µL PBS. Those homogenates were stained using anti-human CD4 FITC monoclonal antibodies (Biolegend, USA) for extracellular. The homogenate was added fixation buffer 50 µL and then incubated at 4°C for 20 min in dark conditions. Then, added 200 µL permeabilization buffer. The mixture was mixed well and centrifuged at 10°C at 2500 rpm for 5 min. Pellet was added with 50 µL antibody of intracellular (Biolegend, USA) and incubated at 4°C for 20 min in dark conditions. Those samples were ready to be analyzed using flow cytometry (BD FACS Calibur, USA)¹⁸.

 

 

Data Analysis:

Data obtained from Flow Cytometry were analyzed using BD Cellquest Pro^TM software. The result was the relative cell numbers of CD4⁺IFN-γ⁺, CD4⁺ TNF-α⁺, and CD11b⁺IL-6⁺ cells. The number of the relative cells obtained (%) was analyzed statistically using Two-Way ANOVA at the 95% of confidence level (α = 0.05). If the ANOVA calculation showed the significant differences (p <0.05), then continued with  Duncan's test at 95% confidence level (α = 0.05).

 

RESULTS:

Characteristic of the MOL extract and its Fermented:

The characteristic of the MOL extract and its fermented is presented in Table 1. The moisture content, total sugar, total acid, and total flavonoid were different (p<0.05). The moisture content for MOL extract (12.09±0.56% DB) was significantly lower compared to fermented MOL (25.85±1.09% DB). There is decreased the total sugar of fermented MOL (5.97±1.06% DB) compared with the MOL extract (7.89±0.46% DB). The total acid of the fermented MOL (0.13±0.00% DB) was significantly higher than MOL extract (0.02±0.01% DB). Total flavonoid in fermented MOL (97.49±0.50 mg QE/g DB) than that of MOL extract (44.15±1.05% DB). Values are means of triplicate samples.

 

Cell Population of CD4⁺IFN-γ⁺:

The percentage of CD4⁺IFN-γ⁺ cells in the lymph node in a group of fermented MOL extract was significantly higher than the group of non-fermented MOL extract (p<0.05). The group administered with fermented or non-fermented MOL extract at a dose of 42 mg/kg BW was significantly different from those of 14mg/kg BW.  The group treated with  84 mg/kg BW had a mean of CD4⁺IFN-γ⁺ number that was not significantly different from the control group (K-).  Based on (Fig. 1), at a dose of 84 mg/kg BW the percentage of CD4⁺ IFN-γ⁺ is lower than that of 14 and 42 mg/kg BW. It also shows the decreasing the average percentage of CD4⁺ IFN-γ⁺ at a dose of 84 mg/kg BW compared to the treatment dose of 14 mg/kg BW and 42 mg/kg BW.

 

Table 1. Characteristic of the MOL extract and Fermented

Chemical Composition	Treatment
	Extract	Fermented Extract	Freeze-Dried Fermented Extract
Moisture Content (%) db	12.09 ± 0.56a	25.85 ± 1.09b	9.69 ± 1.11c
Total Sugar (%) db	7.89 ± 0.46a	5.97 ± 1.06b	5.66 ± 0.61b
Total Acid (%) db	0.02 ± 0.01a	0.13 ± 0.00b	0.07 ± 0.00c
Total Flavonoid  (mgQE/g) db	44.15 ± 1.05a	97.49 ± 0.50b	82.28 ± 1.97c

Note : Different letters demonstrate a significant difference (p<0.05) in the same rows according to Duncan’s multiple range test.

 

 

Fig. 1: The results of flow cytometry analysis of CD4⁺IFN-γ⁺ cells in lymph node. a. Representative flow cytometry expression of CD4⁺IFN-γ⁺ in lymph nodes. b. Percentages increasing of CD4⁺IFN-γ⁺ cells in lymph nodes cells was measured on the 36^th day after treatment of fermented and non-fermented MOL extract. There is a significant difference (p<0.05) between the group of fermented and non-fermented MOL extract.

Cell Population of CD4⁺TNF-α⁺:

The result of the statistical analysis demonstrated a significant difference (p<0.05) between the treatment group of MOL extract and fermented MOL extract. Based on Duncan's test, the highest percentage of CD4⁺ TNF-α⁺ percentage was found in the treatment dose of 42 mg/kg BW (1.45%) in the group of MOL extract and 1.82% in fermented MOL extract (Fig. 2.). The treatment dose of 14 mg/kg BW and a dose of 42 mg/kg BW has a significant difference with the treatment dose of 84 mg/kg BW. The increase in the mean number of CD4⁺ TNF-α⁺ in the group of 14 mg/kg BW and a dose of 42 mg/kg BW.

 

Fig. 2: Stimulation activity of flavonoid induced CD4⁺TNF-α⁺ expression in lymph node cells. a. Increasing cell expression of CD4⁺TNF-α⁺ that analyzed using flow cytometry. b. The result of percentage and statistical analysis demonstrated a significant difference (p<0.05) between the treatment group of MOL extract fermented and non-fermented on Duncan's test.

 

 

Cell Population of CD11b⁺IL-6⁺:

The result of the statistical analysis showed a significant difference (p<0.05) between the treatment group of MOL extract and fermented MOL extract. Based on Duncan's test, the results showed that the mean percentage of the number of CD11b IL-6 in the treatment group dose 42 mg/kg BW were not significantly different with the treatment dose of 14 mg/kg BW in the extract treatment and fermentation groups. In addition, there was a significant difference in fermentation treatment at a dose of 42 mg/kg BW (50.46%) and a dose of 84 mg/kg BW (35.36%). Based on Figure 3, it can be seen that the treatment of fermented MOL extract can increase the exposure of CD11b⁺ IL-6⁺ at a dose of 14 mg/kg BW and a dose of 42 mg/kg BW. There was also a decrease in CD11b⁺ IL-6⁺ expression in a dose of 84 mg/kg BW compared to a dose of 42 mg/kg BW.

 

Fig. 3: Immunomodulatory effect of fermented and non-fermented MOL extract to the expression of CD11b⁺IL-6⁺ after 36 days of treatment of infected mice with Salmonella typhi. a.  Lymphocyte cells were isolated from lymph nodes of mice and analyzed using flow cytometry, the cells labeled using anti-IL-6. b. Percentage of CD11b⁺IL-6⁺ in different doses and different treatment, data are means ± SD. Different notation for significant results (p<0.05).

 

DISCUSSION:

Fermentation of MOL extract with L plantarum decreased the total sugar which is used as energy sources for bacteria growth. The increasing of total acid was due to lactic acid produced during the metabolic activity of L. plantarum. This result agrees with the study of^[19] reported that L. plantarum increased the acidity of fermented sweet lemon juice. The increasing of total flavonoids in the fermented extract was due to the conversion of flavonoid glycosides to aglycone flavonoids by β-glucosidase of L. plantarum^20,21,22. Aglycone flavonoids are absorbable in the human intestine^23,24.

 

MOL contain active compounds of alkaloids, saponins, tannins, and flavonoids²⁵. Flavonoid compounds have been shown to increase IL-2 and lymphocyte proliferation²⁶, will affect CD4⁺ cells, which cause Th1 cells to be activated²⁷. IFN-γ activates macrophages, hence that macrophages will have increased phagocytic activity to inhibit bacteria growth. Flavonoids can activate NK cells to stimulate IFN-γ production, which is the main cytokine of MAC (Macrophage Activating Cytokine). This will activate macrophages and stimulate the increase of phagocytic activity^28,50. Previous studies showed that flavonoids in MOL extract have a role in improving the immune quality of the system by activating lymphocytes, increasing humoral immune response,  increasing phagocyte activity^29,3 and antioxidant^52,54. Flavonoids as immunostimulants can provide intracellular stimuli such as macrophages and T cells to work better and can eliminate incoming infections^30,53.

 

However, a flavonoid in MOL extracts fermented and non-fermented also act as immunosuppressants at a dose of 84mg/kg BW, which is by triggering the Mitogen-Activated Protein Kinase (MAPK) activity which can trigger the secretion of IL-10 and IL-4 cytokines^31,32. IL-10 can suppress Th1 cells so they can suppress IFN-γ, IFN-γ is a pro-inflammatory cytokine secreted by macrophages due to antigenic stimulation of Salmonella typhi; meanwhile, IL-4 plays a role in regulating Th1 and Th2 activation so that the immune system balance can be maintained³³, it is in line with studies conducted by¹⁷ which shows that the higher the dose is given, then the average amount of cytokines declined, it is associated with the activity of CD4⁺ T cells producing IFN-γ and TNF-α. Mice that lack of CD4⁺ cells fail to produce IFN-γ and TNF-α so that it is not able to provide a constant Salmonella from the body, besides the existence of a decrease in the number of CD4⁺ T cells that present the IFN-γ and TNF-α is inseparable in the apoptosis process the cell to maintain balance³⁴. This result is also in line with the study in mice dosed with 200 mg/kg BW of ethanolic seed extract of MOL³⁵. The immunosuppressant activities are characterized by a decrease in phagocytic activity by macrophages³⁶.

 

The infection with S. typhimurium significantly increased the expression of surface receptors of macrophage cells, thus spurring macrophages to excrete IL-6 and TNF-α. The increased cell capacity of macrophages or known as activated macrophages represent the morphological, metabolic, and functional abilities in eliminating infectious agents in the body. This increased ability is characterized by increased activity of macrophages, macrophage phagocytic capacity, and interleukin production³⁷. This increase in IL-6 will induce B cell differentiation into plasma cells that produce immunoglobulins³⁸. Conversely, increased IL-6 will stimulate B cell differentiation and T cell activation. Increasing the number of B cells circulating in the body's circulation will increase antibody production³⁹.

 

The high percentage of IFN-γ is presumably due to the role of Lactobacillus plantarum. Several studies reported that consuming Lactobacillus-lactic acid bacteria can increase the population and the proliferation of lymphocyte cells, the production of cytokines IFN-γ, IL-12, IL-10, Th immune cells, and immunoglobulin (Ig) A, IgE, IgG, and IgM⁴⁰. Lactic acid bacteria provide positive benefits for health, especially in maintaining the balance of the microflora and digestive tract. These health benefits associated with lactic acid bacteria are controlling pathogenic bacteria in the digestive tract^41,55.

 

Lactic acid bacteria in mice can increase the expression of TLR2, TLR4, and TLR9, and increase the secretion of TNF α, IFN-γ, and IL-10 in Peyer patches⁴². The study of mulberry leaves fermented by Lactobacillus plantarum can increase the total phenol²⁰. This is allegedly caused during the fermentation process in which the flavonoid glycoside of mulberry leaves is hydrolyzed and removed from the tissue or leaves cells by the enzymes of Lactobacillus plantarum such as glucosidase, amylase, and cellulase⁴³.

 

Organic acids, hydrogen peroxide, diacetyl, and other antimicrobial component produced by Lactobacillus plantarum can inhibit the growth of the pathogenic microorganism is by reducing the pH of the environment^44,49. The antimicrobial effect of organic acids resulted from the organic form of non-dissociated acid. Therefore, it can diffuse through the cell membrane which then pushes the internal pH down and results in a disruption which ultimately prevents the transport mechanism of the substrate⁴⁵.

In conclusion, MOL extract and its fermented by Lactobacillus plantarum at a dose of 14 mg/kg BW and 42 mg/kg BW can work as an immunomodulator indicated the increasing of  CD4⁺IFN-γ⁺, CD4⁺TNF-α⁺, and CD11b⁺IL-6⁺ in the lymph nodes of mice infected with Salmonella typhi. At a higher dose of 84 mg/kg BW, it suppressed the immune cells.

 

CONFLICT OF INTEREST:

All the authors have no conflict of interest

 

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Accepted on 18.05.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(8): 3595-3601.