INTRODUCTION:

The immune system is a connection between cells, tissues and organs that work together to protect the body from outside attacks. This system is a complex interaction of biochemical components and cellular dynamics. The dynamics in the immune system homeostasis are influenced by various exogenous and endogenous factors which can cause pathophysiological conditions.

This pathophysiological state can be corrected by a substance called immunomodulator. The restorative and rejuvenating nature of immunomodulators can overcome the body's defense system in fighting diseases. Immunomodulators can act as a stimulator by increasing the immune response (immunostimulant), suppressing an immune response (immunosuppressant), or increase the efficacy of a vaccine (immunoadjuvant)¹.

Polysaccharide is one example of compounds that can affect the immune system. Polysaccharide is a polymer of sugar units. It occurs as storage polysaccharides and structural polysaccharides⁵. Polysaccharide, as a primary metabolite, widely distributed around the world and can be collected from various plants and animals. Compared to the amount of secondary metabolite in the plant, the source of polysaccharide is abundant. For the past 20 years, polysaccharide rapidly garnered attention, especially on the variety of its use in biotechnology^1,2.

Various studies show that polysaccharides have the activity in modifying the immune system. Polysaccharides were able to modulate certain components of the immune system, by both direct and indirect effect. One theoretical concept that supports polysaccharide activity in the immune system is its ability to influence lymphocyte proliferation and the activity of macrophages. Activity on macrophages and lymphocytes is affected by cytokines. Previous research has proven that polysaccharides contained in noni can affect lymphocyte proliferation, activity of macrophages and also cytokines^3,6–8. Some examples of cytokines that have a direct relationship with lymphocytes and macrophages are IL-2 and IFN-γ. Several polysaccharides have been known to influence the activity of macrophages by several mechanisms and in various signaling pathways, resulting in the modulation of cytokines. However, few research has focused on the structure/class of polysaccharides and their principles of action⁹.

Many kinds of research have been conducted to evaluate polysaccharide activity in the immune system. From all of them, Ediati has been developing noni fruit polysaccharide and obtain polysaccharide-rich syrup that has been standardized and could enhance lymphocyte proliferation³. Noni fruit (Morinda citrifolia) has been acknowledged for its efficacy and usefulness in promoting health. Noni fruit contains up to 90% water, with solid components were formed by fibers, metal, and protein/amino acids such as glutamic acid or isoleucine^10,11. Noni fruit also contains terpenoids, fat, alkaloid, phenolic^12,13, anthraquinone, scopoletin, α- and β-glucopyranose, iridoid glycoside^14–16, and of course polysaccharide^6,17,18.

Efficacy is an important aspect of a quality drug. Also, an equally important aspect is safety or toxicity. Most of the polysaccharides derived from plants are reported to have much better safety compared to polysaccharides from other sources (fungi, synthesis, etc.). In addition, there are no known side effects/allergies that occurred as a result of polysaccharides consumption. This is an important factor to note in order to support the quality of drugs affecting the immune system.

Ediati’s finding is a significant progress in getting a standardized herbal syrup drug. Recently a Standardized Polysaccharide Fraction (SPF) syrup formula that could increase lymphocyte proliferation, has a stable physical properties, stable under influence of extreme temperature changes and fulfill microbial contamination parameter has been successfully made and published³. Therefore, evaluating its molecular mechanism is necessary. This study aims to elucidate Standardized-Polysaccharide Fraction (SPF) syrup immunomodulatory mechanism by evaluating cytokines affecting macrophages and lymphocytes (IL-2 and IFN-γ) and its safety by evaluating the histology of spleen and skin allergy potency mediated by mast cell degranulation.

MATERIALS AND METHODS:

Samples:

The SPF syrup was a grant from Prof. Ediati from the research in 2017. SPF syrup is a standardized noni fruit syrup with various non-specific and specific standardization parameter, with 5% glucose as a marker⁴.

Experiment:

Wistar rats aged 9-10 weeks having a weight range of 150-200 g were used in this study. They were obtained from local breeding in the Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada. Rats were divided into 5 groups consisting of 5 rats. Groups 1 (K1) as a negative control (SPF vehicle), Group 2 (K2) as normal control (SPF syrup vehicle + vaccination), group 3-5 (K3-5) as a test group (SPF syrup + vaccination). Rats were placed in the cage and adapted to the environment for 7 days. Food and water were given ad libitum. At days 8 – 33 SPF syrup was given to rats in 3 different dosages (group 3,4,5; 25, 50 and 100 mg/kg body weight). Rats were vaccinated every 7 days (day 8, 15 and 22). Days 31 blood was taken from the eye vein and collected for ELISA assay. Rat dissected in days 33, spleen and skin were harvested and persevered in 10% formalin for histological evaluation purpose.

ELISA Assay:

Blood was centrifugated at 3000 rpm for 30 minutes. Supernatant isolated and used as assay samples or stored at -20^°C for 3 months or -80^°C or 1 year until use. Cytokines level (IL-2 and IFN-γ) was evaluated using IL-2 and IFN-γ ELISA kit (Fine Test), using method as described in the ELISA kit protocol^19,20.

Histology of Spleen:

Spleen safety was evaluated by histology of spleen organ with hematoxylin-eosin staining. The spleen was dissected, deparaffinated with xylene, hydrated with water, and nuclear staining with hematoxylin. Differentiation with acid alcohol, bluing with ammonia water and counterstaining with eosin, and dehydrated, and also cleared with xylene. After fixation, the sample was analyzed under the microscope with 400x magnification.

Histology of Skin Mast Cell:

Skin allergy potency was evaluated by counting mas cell degranulation on the dermal layer zone with Toluidine Blue staining. The skin was deparaffinized/dewaxed, stained with toluidine blue for 30’ at 37^°C. The section was differentiated until the background was clear, dehydrated, and fixated for analysis. Samples were analyzed under the microscope with 400x magnitude and 5 fields of view. Intact mast cells in the dermal layer were calculated for analysis.

RESULTS AND DISCUSSION:

Noni fruit is a well-known herb as a good herb for promoting human health. It is also a good and potential herb with immunomodulatory activity, especially its polysaccharide. This study aims to elucidate immunomodulatory activity of Noni polysaccharide (as SPF syrup) by evaluating its molecular mechanism in cytokines affecting macrophages and lymphocytes (IL-2 and IFN-γ). A preliminary study about the safety of SPF syrup on several organs needs to be conducted. In this case, histological evaluation of spleen and evaluation of skin allergy potency mediated by skin mast cell degranulation were suitable.

Effect of SPF Syrup on Cytokines Level:

This study aims to evaluate the immunomodulatory mechanism of SPF syrup in vivo on cytokines level (IL-2 and IFN-γ). Rats were given a Hepatitis-B vaccine to induce immune responses. Immunomodulatory mechanism of SPF syrup evaluated based on the increase or decrease of cytokines level compared to the normal group (Vaccine + SPF Vehicle).

Hepatitis-B vaccine can induce immune responses by increasing IL-2 level²¹ and indirectly modulate IFN-γ. Changed on IFN-γ level caused by viruses mainly mediated by type-I Interferon and NK (Natural Killer) Cell. Normally, viral infection induces type-I Interferon immune response and further induce NK cell to produce IFN-γ²². For the record, IFN-γ level does not always increase throughout the immune response period. The T cell immune response caused by immunization is very broad and complex, encompassing a much broader spectrum instead of single cytokine measurements²³. There have been several reports in patients with acute HBV infection who have decreased activity from NK cells at the peak of viremia, as well as decreased IFN-γ and TNF-α production in chronic HBV patients²⁴.

Another factor is the dynamic homeostasis between IFN (Interferons) type I and type II which work together protecting the body and fight against pathogenic infections over time. IFN types I and II levels will rise and fall alternately according to their function in defence. IFN-γ level, in particular, will increase before 48 hours of the infection phase and can decrease again within a matter of hours²⁵.

p from Morinda citrifolia on IFN-γ levels. P-value was determined through ANOVA and t-test with α = 0.05.

The results can be seen in Figure 1 (IL-2) and Figure 2 (IFN-γ). Administration of SPF syrup showed no significant effect on IL-2 levels of the treatment group (SPF syrup 25, 50 and 100 mg / kg body weight) compared to the normal group. Although, there was a tendency of decreasing IL-2 levels due to SPF syrup (the levels of the treatment group were lower than the normal group). There is a significant difference in IFN-γ levels of the treatment group (SPF syrup dose 100 mg / kg body weight) compared to the normal group. The graph in Figure 2 shows a significant increase in IFN-γ levels due to administration of 100 mg/kg body weight of SPF syrup. Although, there were no significant differences in IFN-γ levels in the administration of 25 and 50 mg / kg body weight of SPF syrup. Further research can be conducted in finding the optimal dose of SPF syrup that can significantly modulate IL-2 levels.

Polysaccharides are complex combinations of various saccharide compounds. Recent studies has shown a better understanding of structural aspects of polysaccharides, which is closely related to the regulation of biological functions and regulation of cytokine systems/tissues therefore affecting the immune system of certain organisms. Literature related to polysaccharides are quite abundant and diverse, but it is not enough to generalize the effects of polysaccharides and the relationship between the composition/structure of polysaccharides and the immune response⁹. Limited information related to structural and mechanistic aspects became a boundary on establishing further research of polysaccharides, especially in their clinical use^26,27.

Research related to the specific identity/marker compounds from end products of polysaccharides that are consumed orally is not widely known. Some polysaccharide components such as arabinogalactan, galactomannan, glucans (laminarin), glucomannan and a mixture of polysaccharide products can be partially degraded by human intestinal bacteria and further interact with intestinal epithelium causing local effects, as well as entering the blood vessels and causing systemic effect. There were several recent studies on finding of relationship between the bioconversion of a polysaccharide/its bioavailability and the downstream/further effects on host metabolism and physiology using a metabolomic and metagenomic approach²⁶. Other research studying the function of polysaccharides in the immune response related to the microbes in human. The result indicates that other factors can affect polysaccharides before they can modulate the immune response. Mainly, they have effects to the ecology of intestinal microbes, which can further affect nutrition and the immune system²⁶. A temporary conclusion that can be drawn is that the mechanism essentially related to the complexity of the digestive tract environment itself. Some studies also show a relationship between the frequency of polysaccharide consumption and loss of effect (the effect of tolerance) or an increase in the effects of polysaccharides on the immune system, either direct effect to the immune system or indirect effect through their activity on the body's probiotics.

Other research related to the connectivity architecture of cytokines shows that immune cells do not work individually / alone, but work collectively, integrated and have a hierarchy. Connectivity between cytokines and immune system cells is very rich and complex, not only working in defence against pathogens but also integrates maintaining the balance of the immune system²⁸.

Consistent with the previous findings, a deeper study of the polysaccharides of noni fruit needs to be conducted. Research about polysaccharides from various plants and their effects on IL-2 and IFN-γ have been carried out. Most studies show that polysaccharides from various plants can increase of IL-2 and IFN-γ levels, while some studies show varied results or a decrease in both cytokines. Among them are summarized in Table 1.

Table 1. Various research related to polysaccharides from various plants and their effects on IL-2 and IFN-γ level.

Plant Source	Result (#$)	Reference

Zizhypus jujuba	$ IL-2	Hsu et al., 2014
Astragalus membranaceus	# IL-2	Yang et al., 2013
Artemisia argyi	# IL-2	Bao et al., 2013

lhagi pseudalhagi Desv. Alhagi pseudalhagi Desv.	# IFN-γ	Wusiman et al., 2019
Ulva pertusa	$ IFN-γ	Gao et al., 2019
Cordyceps ghunii	# IFN-γ	Zhu et al., 2012
Lycium barbarum	# IFN-γ	Bo et al., 2016

Codonopsis pilosula Nannf. Var. modesta	# IL-2, # IFN-γ	Fu et al., 2018
Caltha palustris	$ IL-2, # IFN-γ	Suszko and Obmińska-Mrukowicz, 2013
Rehmania glutinosa	# IL-2, # IFN-γ	Huang et al., 2013
Tremella	# IL-2, # IFN-γ	Zhou et al., 2018
Camellia sinensis	$ IL-2, $ IFN-γ	Liu et al., 2018
Panax ginseng	# IL-2, # IFN-γ	Wang et al., 2015
Angelica sinensis	$ IL-2, $ IFN-γ	Wang et al., 2016

CONCLUSION:

Our study showed that SPF syrup in dosage 100 mg/kg body weight could increase IFN-γ level. There is a decreasing tendency in IL-2 level, although it is not statistically significant on all dosages. Histological evaluation showed that SPF syrup was safe to be consumed because there are no toxic effect/lesion between SPF and the control group in both spleen histology and mast cell degranulation in the skin.

In future studies, further research can be conducted to find the optimal dosage of SPF syrup that can significantly modulate IL-2. Furthermore, more research could be established, as well as finding the active compound of polysaccharide, characterization of polysaccharide component and their functional groups/structures, studies of synergism/antagonism effect between polysaccharide components, their effects on intestinal microbial, metabolism in certain body organs and metabolite activities, studies of anti-inflammatory activities, studies of antihistamine activities, further research based on chemotaxonomy and metabolomics aspect, and histological evaluation of other organs commonly affected by most drugs such as liver and kidney.

ACKNOWLEDGEMENT:

The author acknowledges to Kementerian Riset dan Pendidikan Tinggi (Kemenristek Dikti) through the research scheme of Penelitian Unggulan Perguruan Tinggi year 2017-2018 with contract number of 1804/UN1/DITLIT/DIT-LIT/LT/2018.

CONFLICT OF INTEREST:

The authors have declared “no conflicts of interest with respect to the research, authorship, and/or publication of this article”.

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Received on 28.08.2019 Modified on 11.10.2019

Research J. Pharm. and Tech 2020; 13(2):882-888.

DOI: 10.5958/0974-360X.2020.00167.5