Free Radical Scavenging Capacities and Alleviating Actions of Polysaccharides Extract of Termitomyces le-testui on Methylprednisolone-Induced Immunodepression in Rats

Background Natural products have been said to show immunomodulatory and antioxidant activities. The research study was aimed to assess the immunomodulatory and free radical scavenging activities of crude polysaccharide from dry mushroom fruiting bodies of Termitomyces le-testui. Materials and Methods Hot water extract of polysaccharide extract of T. le-testui was prepared and tested in white albino Wister rats for its immunomodulatory activities effect on methylprednisolone-immunosuppressed animals. In addition, the radical scavenging activity of the polysaccharide was evaluated using nitrite and hydrogen peroxide. Results The result of the study showed that the polysaccharide T. le-testui increases the phagocytic index, energy metabolism of macrophages, spleen index, and nitric oxide in a concentration-dependent manner in immunosuppressed animals. Also, it was observed that the extract increased dose-dependent total oxidative stress and thymus index. Finally, the crude polysaccharide-rich extract showed nitrite and hydrogen peroxide scavenging activity in a concentration-dependent manner. Conclusion Polysaccharide-rich extract possesses immunomodulatory and antioxidant properties.


Introduction
Natural products, especially plants, are one of the immunomodulators that can provide an alternative to conventional chemotherapy for a large variety of diseases. As immunomodulators, they are especially important when the host defense mechanism has to be stimulated in cases of the impaired immune response. Some of them are important when a selective immunosuppression is desired. Mushrooms have been utilized in folk medicine since ancient times [1], but this concept gained little more credibility in the last decades as they exhibit significant therapeutic effect including antioxidant and immunomodulatory activities. is is partly due to a large number of substances with potential immunomodulatory activities that mushrooms possess [2,3].
Mushrooms, as immunomodulators, they can suppress or enhance the immune system activities [4], and this offers them a large use to treat infections [5]. Among derived mushroom substances, there is ample evidence that polysaccharides exhibit various bioactivities such as antitumor, anticancer, antiviral, antibacterial, antifungal, anticoagulant, and immunological activities [6,7]. Lentinan from L. edodes and G. lucidum polysaccharide, for example, through their immunostimulatory activities, showed important roles in maintaining tissue homeostasis and fighting diseases [8,9]. In addition to the immunomodulation, polysaccharides also play an important role in the control of oxidative stress [10][11][12], thus contributing to the improvement of the immune response.
T. le-testui is an edible mushroom commonly consumed in Cameroon. e dry matter of T. le-testui revealed that it is made up of 59.02% of total carbohydrates, 23.6% of total sugars, and 0.84% of reducing sugars [13]. Data from various studies reported the immunomodulatory activities of T. le-testui [3,14]. However, there is no data available on the active constituents of T. le-testui. Polysaccharides may be the bioactive constituents, but this remains to be demonstrated. e present study was therefore undertaken to assess the immunemodulatory activities against methylprednisolone-induced immunosuppression in rats and in vitro antiradical activities of polysaccharides extract of T. le-testui.

Preparation Polysaccharides.
Polysaccharides were extracted using hot water [15]. Fresh mushrooms were fully immersed in distilled water and incubated at 95°C in a water bath for six hours. e preparation was filtered and the filtrate was used to obtain the polysaccharides extract. e filtrate was mixed with ethanol (70%) at equal volumes and kept overnight at 4°C to precipitate the polysaccharides. After precipitation, the mixtures were then centrifuged at 1800 rpm during 10 min at 4°C temperature, and the pellets were then collected and dried at 30°C. e dried products representing the polysaccharide extracts were kept at about 4°C.

Determination of Total Carbohydrate Content.
e carbohydrate contents were determined by the slightly modified phenol-sulfuric acid method according to Masuko et al. [16]. Fifty microliters of crude polysaccharide solution was mixed with 150 μL of concentrated sulfuric acid and immediately with 30 μL of 5% phenol and then the mixture was kept at 90°C for 5 min. e absorbance of the mixture after cooling to room temperature was measured at 490 nm. e total carbohydrate content was calculated using a standard curve of D-glucose and it was found to be 75% of the dried extract.

Scavenging Activity Determination.
e antioxidant activities of polysaccharides extract of T. le-testui were analyzed by assessing the free radical scavenging activities.
e absorbance values measured in a UV-Vis spectrophotometer were used to determine the percentage inhibition of free radical scavenging activity by using the standard formula (F 1 ) as reported by Bahatti et al. [17]: where A 518 (control) is the absorbance of control and A 518 (sample) is the absorbance of free radical + sample extract/standard.

Nitrite-Scavenging Activity.
Nitrite-scavenging activity was evaluated based on the absorbance at 520 nm using a UV-spectrophotometer according to the method reported by Kato et al. [18]. One milliliter of 1 mmol/L NaNO 2 (Sigma Co.) solution was added to 1 mL of each sample, and the resulting mixtures were adjusted to a pH of 2.5 using 0.1 N HCl and 0.2 N citric acid solutions. Each sample was allowed to react at 37°C for 1 hour, after which 1 mL of each sample was taken from the solution and mixed thoroughly with 3 mL of 2% acetic acid and 0.4 mL of the Griess reagent. e solutions were stored at room temperature for 15 min. e Griess reagent was prepared by mixing an equal amount of 1% sulfanilic acid (Sigma Co.) and 1% naphthylamine (Sigma Co.), which were made with 3% acetic acid.

Hydrogen Peroxide Scavenging Activity.
e ability of the mushroom extracts to scavenge hydrogen peroxide was assessed according to Ruch et al. [19]. Mushroom extracts were added to 0.6 mL H 2 O 2 solution (40 mM H 2 O 2 was prepared with phosphate buffer, 0.1 M, pH 7.4) and the total volume was made up to 3 mL and mixed properly. en the absorbance was measured at 230 nm using a spectrophotometer. Phosphate buffer, without H 2 O 2 , was considered as blank solution.

Assay of Inhibition of Free Radicals Using
iobarbituric Acid (TBA) Method.
e TBA method was used to represent the inhibition of the production of carbonyl compounds degraded from the peroxides at a later stage. TBA test is used to measure the second product of peroxide oxidation such as aldehyde and ketone. e assay was done according to Kikuzaki and Nakatani [20]. To 2.0 mL of the sample solution, 1.0 mL of 20% aqueous trichloroacetic acid (TCA) and 2.0 mL of aqueous thiobarbituric acid (TBA) solution were added. e mixture was placed in a boiling water bath for 10 min. After cooling, it was centrifuged at 3000 rpm for 20 min and the absorbance was measured at 532 nm and recorded.

In Vivo Immunological
. Albino rats (Wistar strain) aging 7 to 8 months and weighing 100-250 g were obtained from the laboratory of physiology and pharmacology of the University of Dschang. e animals were housed in cages under controlled conditions of temperature and alternating 12 hours cycle of light and darkness. ey were given free access to standard rat pellet food and tap water ad libitum. All animals were acclimatized to the working environment 1 week before the beginning of the experiment. e experimental protocol was approved by the Ethics Committee of the Department of Biological Sciences of the University of Bamenda.

Grouping and Treatment of Animals.
After 1 week of acclimatization, rats were randomly distributed into 6 groups of 5 rats each. A group of 5 rats did not receive the immunosuppressive drug (methylprednisolone) (Group 1 or normal control). e remaining 5 groups of rats were all immunosuppressed by the injection of methylprednisolone via the tail vein (1 mL/kg of animal weight) before receiving the other treatments. One group of the immunosuppressed rats (Group 2 or negative control) received distilled water. Another group (Group 3 or positive control) received intraperitoneal injection of BCG (2.10 3 CFU/mL) for the entire treatment period. Finally, the remaining immunosuppressed groups of rats (Groups 4, 5, and 6) were orally treated by gavage with the different doses of the mushroom extract 25, 50, and 100 mg/kg, respectively. Treatment was done every day for 10 days.

Sample Collection.
For this part of the study, two animal groupings were done. e first grouping was to evaluate the phagocytic index and lymphoid organ index, and the second grouping was to evaluate the energy metabolism in intraperitoneal macrophages, production of nitric oxide, and total oxygen radical. e treatment was done for 10 days. ereafter, animals were administered a mixture of ketamine (dose) and Diazepam (dose) (ratio 1 : 3) and blood was collected by cardiac puncture in dry essay tubes to assess the serum nitric oxide and total oxygen radical or through the orbital venous to evaluate the phagocytic index. Peritoneal macrophages were obtained by washing the peritoneal cavity in the phosphate buffer. Spleen and thymus were collected and kept in formalin solution 10% for histological analysis.

Assessment of Phagocytic Index. Phagocytic index was assessed through the carbon clearance test as described by
Honghui et al. [21] with some modification. irty rats treated as described previously were intravenously injected with diluted India ink at 100 μL/10 g body weight. Blood specimens were collected at 2 min (t 1 ) and 10 min (t 2 ) from the retinal venous plexuses and 20 μL of the collected blood was then mixed with 2 mL 0.1% Na2CO3. e absorbance at 600 nm was measured on a UV-visible spectrophotometer with 0.1% Na2CO3 as the blank. e liver and the spleen were weighed, and the phagocytic index was calculated as follows: , where OD 1 was optical density for t 1 and OD 2 was optical density for t 2 ( where A is the body weight, B the liver weight, and C the spleen weight

Evaluation of Energy Metabolism in Peritoneal
Macrophages. Peritoneal macrophages were harvested by washing the peritoneum in 3 mL of phosphate buffer saline (PBS). Peritoneal cells were immediately counted using a hemocytometer (Malassez cell). A cell suspension (density; 1 × 10 3 cells/mL) was prepared and 2 mL of this suspension was mixed with 1 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) solution (1% in PBS). After 2 hours of incubation, 1 mL of 1N HCl was added and the mixture was allowed to stand for 15 min after which the optical density was read at 540 nm. e mean of the optical density in treated animal was compared to that of control to evaluate the energy metabolism of the cells (reference).

Evaluation of Radical Compounds Production.
e crude polysaccharides of T. le-testui were analyzed for their effect on radical compounds production. is test is based on iron catalyzed breakdown of hydroperoxides into alkoxyl (RO۰) and peroxyl (ROO۰) radicals which react with the chromogen (N, N-dimethyl -p-phenylenediamine sulphate) towards formation of a colored compound, the absorbance of which is photometrically detectable. e intensity of the color correlates directly with the quantity of radical compounds, according to Lambert-Beer's law, and it can be related to the oxidative status of the sample. is was done by the method described by Pinaki et al. [22]. e method makes use of chromogen reagent solution (23.5 mg of N,Ndimethyl-p-phenylenediamine sulphate (Aldrich, Sigma) in 10 mL of 20 millimolar PBS (pH 7.4)) and kept in 4-8°C. Briefly, 100 microliters of plasma diluted to 20 times in PBS was dissolved in 1 mL of acetate buffer. 25 microliters of working chromogen solution was added, and absorbance was taken at 505 nm by 6 min time-scan in UV-Vis spectrophotometer. e absorbance value was obtained for each Advances in Pharmacological and Pharmaceutical Sciences sample against blank and compared to the curve obtained using H 2 O 2 .

Evaluation of Nitric Oxide Production.
e crude polysaccharides of T. le-testui were analyzed for their effect on nitric oxide production as described by Pinaki et al. [22]. Griess reagent (made by mixing an equal amount of 1% sulfanilic acid and 1% naphthylamine which was made with 3% acetic acid) is the principal reagent used. e collected serum sample was made up to 1 mL using 20% phosphate buffer. To each sample of serum, 1 mL of the working reagent (Griess reagent) was added and allowed to stand for 5 min and absorbance was measured at 540 nm. is was done in duplicate for each sample, and the absorbance was recorded. A standard curve was produced using NaNO 2 as standard solution.

Histological Analysis of Spleen and ymus.
Spleen and thymus of sacrificed animals were collected, washed in cold phosphate buffer saline, and blotted dried. ey were weighted to obtain the absolute organ weight. e organ index was calculated as follows: Organs were immediately fixed in 10% formalin until the day of histological analysis. Later, the fixed tissues were dehydrated and then the samples were cleared in 2 changes of xylene. Samples were then impregnated and embedded in paraffin, then embedded, and blocked out. Sections of 4-6 μm thickness were cut using a rotary microtome and mounted on glass slides. After staining with Hematoxylin and Eosin (H&E), the sections were imaged using the Pannoramic Viewer (3DHISTECH, Budapest, Hungary) connected to a microscope.

Statistical Analysis.
Experimental data obtained were expressed as the mean ± standard deviation of the experiment done in triplicate. Data was analyzed using one-way analysis of variance, followed by post-Students tests. Values of p < 0.05 were considered as indicative of significance. All calculations were carried out using the GraphPad Prism ® V5.03 software (GraphPad Software Inc. ® , CA, USA).

Nitrite and Hydrogen Peroxide Scavenging Activity of Crude Polysaccharides of T. le-testui.
e polysacchariderich extract of T le-testui was tested for its antiradical activity by evaluating its effect on nitrite and hydrogen peroxide scavenging activity (Table 1). e result showed that a concentration-dependent increase of the inhibition percentage of free radical activity was observed with extract. is activity of polysaccharides was similar to the effect of vitamin C (100 mg) at 11.06 mg/mL polysaccharides extract while 8.3 mg/mL was significantly different from the effect of vitamin C.
e TBA method was used to evaluate the inhibition of the production of carbonyl compounds degraded from the peroxides at a later stage. e results showed that the polysaccharides active extracts of T. le-testui caused a decrease in the production of carbonyl compounds in a concentration-dependent manner (Figure 2). e production of carbonyl compounds was significantly less with the polysaccharides extract than the synthetic antioxidant vitamin C.

Effect of T. le-testui Crude Polysaccharide on Phagocytic Index and Peritoneal Cells Metabolism in Methylprednisolone-
Immunosuppressed Rats. In vivo administration of methylprednisolone resulted in significant decreases (p < 0.05) in the phagocytic index and energy metabolism of peritoneal macrophages (Table 2). However, BCG as well as polysaccharides extract of T. le-testui treatments prevented the increase and maintained these parameters near the normal level and even above normal for the dose of extract of 100 mg/kg. Polysaccharides extract treatments were also able to reverse the decreases in phagocytic index caused by methylprednisolone to near normal. No significant differences were observed with BCG treatment.

Effect of Crude Polysaccharide Extract of T. le-testui on Organ of Immune System.
e treatment with methylprednisolone significantly decreased the spleen and thymus weight ( Table 3). Administration of polysaccharides extract of T. le-testui significantly caused the increase of spleen index and thymus index at the doses of 100 mg/kg and 50 mg/kg, respectively. e reversal effect of the extract brought back the organs to normal. ere was no difference compared to the effect of BCG. methylprednisolone to rats significantly decreased the serum NO and radical compounds level compared to normal animal. In concentration-dependent manner, treatment with polysaccharides extracts of T. le-testui resulted in significant increase in the levels of NO (Table 4). e effect of the treatment with polysaccharides extract was similar to that of BCG. Moreover, polysaccharides extract of T. le-testui reversed the effect of methylprednisolone with the highest effect observed with the lowest dosage.

Effects of Polysaccharides-Enriched Extracts of T. le-testui on Lymphoid Cells Accumulation in Spleen and ymus
Tissues. Methylprednisolone resulted in significant reduction in richness of lymphocytes in spleen and thymus of rats. In thymus, polysaccharides extract of T. le-testui treatment also increased the richness of lymphocytes (thymocytes) as compared to that of animals not treated (negative control).
is was demonstrated by the dense thymus cortex observed in treated animals (Figure 3). e administration of polysaccharides extract of T. le-testui also affected the lymphoproliferative activity of spleen. e treatment has caused the white pulp to have a dense germinal center compared to the negative control (Figure 4).

Discussion
Since a long time ago, mushrooms have been known for numerous pharmacological activities.
is also includes immunomodulatory and antioxidant activities [23]. In the present study, we assessed the reversal effect of crude polysaccharide extract on methylprednisolone-immunosuppressed effect in white albino Wistar rats and its antioxidant activities. Nowadays, there is considerable effort to identify natural substances that can protect against oxidative stress or stabilize or deactivate free radicals in an immunosuppressed state. Nitric oxide radical is one of the potent radicals that acts by generating full name of NO 2 (NO 2 ) [24]. e polysaccharide extract of T. le-testui was found to decrease the amount of radical. Moreover, the polysaccharides extract of T. le-testui has demonstrated a potential to have a H 2 O 2 -scavenging activity demonstrating that T. le-testui has potent antioxidant properties. Hydrogen peroxide (H 2 O 2 ), a biologically relevant, nonradical oxidizing species, can be formed in tissues through oxidative processes. H 2 O 2 produced may result in the production of hydroxyl radicals (•OH) causing the lipid peroxidation [25] or alteration of the structure and function of many cellular components [26].
us, the polysaccharide-rich extract of T. le-testui may counteract the oxidative effect of H 2 O 2 . Further demonstration of the radical scavenging activity of T. le-testui was observed in TBA test assay. ough the scavenging activity of polysaccharide-rich extract of T. le-testui noticed in TBA assay test was lower compared to that of vitamin C, this result supports its radical scavenging potential. Together, the results of this study demonstrated the free radicals scavenging power of the polysaccharides extract of T. le-testui, as it has been reported for numerous natural products [27].
Several studies reported the immunomodulatory effects of the extract of Termitomyces species through a remarkable improvement of the antibodies production, and also the phagocytic activities [3,14]. In this study, the crude polysaccharides of T. le-testui were found to reverse the immunosuppressed effect of methylprednisolone. Firstly, this was demonstrated by increasing the phagocytic index towards the normal level in a dose-dependent manner. Secondly, the polysaccharides extract of T. le-testui may enhance the metabolism rate of peritoneal macrophages from the methylprednisolone-treated rats. Macrophages have a strong engulf particulate matter function in vivo or in   Advances in Pharmacological and Pharmaceutical Sciences  vitro [28], and macrophage metabolism has been also reported as a crucial indicator for the immune function [29]. is activity of macrophages is affected by the methylprednisolone [30,31]. us, the results of this study indicate the potential immunostimulatory effect of T. le-testui, through which it may stimulate the immune cells and improve the body's immune function [32,33]. Methylprednisolone is also known to cause lymphocytic depletion in spleen in rats [34]. e polysaccharide-rich extract of T. le-testui, furthermore, demonstrated that it can improve the thymus and spleen indices in rats treated with this immunosuppressive drug. is is evidence that T. le-testui improves the strength of the body's innate immune function [35][36][37]. is effect of T. le-testui is also supported by the lymphocytes colonization of thymus and spleen as demonstrated by the histological analysis of these organs reported for other mushrooms species such as Lentinula edodes [38]. As a result of phagocytes activation, reactive oxygen species and nitrogen reactive species are generally produced to participate in the intracellular killing of pathogens and mediate inflammatory reaction [39]. e results of this study also showed that polysaccharide-rich extract of T. le-testui has a potential to increase the NO and total radical compounds in methylprednisolone-treated rats. Once more, this demonstrated that polysaccharides of T. le-testui can boost the body's immune system and can reverse the symptoms of low immunity.
In conclusion, the polysaccharide-rich extract showed potent antioxidants and immunomodulatory effects on different components of the innate and adaptive immune response.
ese pharmacological activities can justify the traditional use of this mushroom for the treatments of several diseases in relation to the immune response.

Data Availability
Data will be available under request to the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.