Changes of Blood Biochemical Parameters During Immune Deficiency Prevention with an Extract of Moringa oleifera in Wistar Rats

The aim of this work was to determine the evolution of some biochemical parameters during a preventive immunostimulation assay of the total aqueous extract of Moringa oleifera leaves in rats. To do this, 48 albino rats including 24 males and 24 females divided into 12 groups of weight between 107 g and 140 g were used. The different doses of the aqueous extract of Moringa oleifera leaves 200 mg/kg bw, 400 mg/kg bw, 800 mg/kg bw and 1600 mg/kg bw respectively were compared with controls subjected to distilled water and 50 mg/kg bw of levamisole for 14 days. At the end of this 14-day treatment, immunosuppression was performed by administration of 5 mg/kg bw intraperitoneally dexamethasone in all rats grouped for three days twice daily. Then a two-week observation was made to assess the behavior of some biochemical parameters after administration of dexamethasone. During the experiment five samples were taken on day 1, day 14, day 17, day 21 and day 28. These different samples allowed the determination of biochemical and nutritional parameters. These samples were used to determine urea, creatinine, transaminases (AST/ALT), PAL, LDH, total cholesterol, HDL, LDL, RRC, CA, RC, IAP, triglycerides and finally nutritional parameters (albumin and total proteins)


Introduction
Currently, worldwide, there is an increase in diseases especially infectious diseases that require effective defense mechanisms of the body to control them through the process of [16].The resurgence of immunosuppression has been a real public health problem in the world for several decades.This immunosuppression is caused by several factors, the most important of which are immunosuppressive drugs, excessive use of drugs such as corticosteroids [21], immunosuppressive diseases, undernutrition, deficiencies, cancer, including metastatic or infections of immune cells by viruses such as human immunodeficiency (HIV) or human T-cell lymphotropic virus [21].Also, the immune response is decreased at both extreme ages due to prematurity and decline in the functions of the organs of the body respectively.the use of immunomodulatory drugs (immunostimulants or immunosuppressants depending on the nature of the disease).These immunomodulators have their drawbacks; some are toxic, others are expensive and therefore beyond the reach of the average citizen.Cheaper and safer alternatives must therefore be found [20].In recent decades, there has been growing interest in the study of medicinal plants and their traditional use in different parts of Africa, particularly in Côte d'Ivoire.Today, about 3.3 billion people in emerging countries depend on traditional medicine, which consists mainly of medicinal plants.These plants have many pharmacological and nutritional applications that can help prevent life-threatening diseases.Among medicinal plants, Moringa oleifera commonly known as "Moringa" or drumstick tree in many parts of the world has been considered an alternative remedy [14].Moringa oleifera, also known as the "miracle tree" or "tree of life" [11] is a tree native to India.Drought tolerant [6], Moringa oleifera is a widely available plant in tropical and subtropical countries with great economic importance [10].It is a plant with very high nutritional values.Moringa oleifera is described as a natural anthelmintic, a mild antibiotic, a detoxifier, an exceptional immune builder.It is used in many countries to treat malnutrition and malaria.For [8] Moringa oleifera is the cheapest and most credible alternative to provide good nutrition, cure and prevent several disorders.However, there is very little information on the preventive use of Moringa oleifera leaves as an immunomodulator

Plant material
The plant material consisted of the leaves of Moringa oleifera which was harvested on the outskirts of the city of Bouaké in the middle of Côte d'Ivoire, 342.3 km from the city of Abidjan.The present study was carried out in the Physiology, Pharmacology and Pharmacopoeia Laboratory of the Natural Sciences Training and Research Unit.

Animal material
Rattus norvegicus rats of wistar strain weighing between 107 and 140 g, of both sexes were used for the experiment.These rats were bred in the animal facility of the Physiology, Pharmacology and Pharmacopoeia Laboratory of NANGUI ABROGOUA University.

Aqueous extract preparation
Dried leaves of powdered Moringa oleifera were used.For the preparation of the aqueous extract, 150 g of Moringa oleifera leaf powder were macerated in 500 ml of distilled water for a period of 24 hours on a magnetic stirrer.The macerated obtained was filtered and heated to the oven at 45 °C for 72 hours.

Experimental procedure
Female and male rats were separated and weighed to form homogeneous groups.Twelve groups of four rats including 24 females and 24 males were constituted as follows: Groups III (200), IV (400), V (800) and VI (1600) consisting of eight rats, four males and four females respectively, received 200 mg/kg bw, 400 mg/kg bw, 800 mg/kg bw and 1600 mg/kg bw.Then, another group II (LEV) of rats consisting of eight rats including four males and four females given 50 mg/kg bw of Levamisole hydrochloride and finally a group I (ED) of eight rats received distilled water.The rats were all fed exclusively on IVOGRAIN brand pellets.The adjustment period lasted five days.After these five days, the rats were treated for a period of 14 days with the aqueous extract and the reference molecule.The immunosuppressive period during which we induced immunosuppression by administration of intraperitoneal dexamethasone at a dose of 5mg/kg body weight twice daily for three days according to the protocol of [23].The observation period lasted two weeks.

Sampling and blood parameters determination
Five rounds of sampling were performed on rats fasted the day before during this study.On days 1, 14, 17, 21 and 28.Venous blood samples were taken from the retroorbital sinus of the eye using a sterile pasteur pipette.According to the method described by [30], each animal was previously anesthetized under a bell containing cotton wool soaked in ether for two to three minutes.The animal, removed from this bell, was held with one hand in a lateral position and held by the skin of the neck, between the thumb and forefinger.After disinfecting the eyelid with 70 °C alcohol, the tip of the pipette was inserted into the lateral corner of the eye, thanks to a slight rotation until reaching the venous plexus.Thus, 0.5 to 2 ml of blood collected was immediately collected in tubes containing the anticoagulant ethylene diamine tetraacetic acid (EDTA) for haematological analyses and in dry tubes for biochemical analyses.The samples were sent to the Institut Pasteur in Cocody (Côte d'Ivoire) for haematological analysis.The samples contained in the dry tubes were used to determine biochemical parameters in the Physiology, Pharmacology and Pharmacopoeia laboratory.

Statistical analyses
The statistical analysis of the results was performed with GraphPad prism Version 8.4.3 software (San Diego, California, USA).Results were averaged followed by standard mean error (M±MSS).The comparison of the means between control and treated rats was performed with ANOVA and the likelihood test was done with the BONFERRONI test.The different proportions observed of leukocyte and biochemical 47 parameters were compared by the significance level was set at p < 0.05.

Effect of treatment on initial blood parameters before immunosuppression
The effect of Moringa oleifera leaf extract on cellular immunity was evaluated after a 14-day treatment.Values for each group are given as the mean ± standard error of the mean (SEM).The p values are a withingroup and group-to-group comparison between periods 1 and 14.Lot I received only distilled water, lot II received 50 mg/kg bw of Levamisole.Groups III, IV, V and VI received 200 mg/kg bw respectively; 400 mg/kg bw; 800 mg/kg bw and 1600 mg/kg bw.

Functional exploration of the kidney
The exploration of the renal function takes into account the different possible damage to this organ in the animals subjected to the different substances studied.This exploration shows us, through Tables 1 (appendix) and 2 (appendix), that there is no significant difference (P>0.05) on day 14 after treatment and on day 17 of dexamethasone administration.This non-significant variation indicates good tolerance of the extract and dexamethasone in rats.During the observation phase, Figue I shows us there was a decrease in the concentration of urea and creatinine in the different groups treated on the 28th day compared to group I control ED.Functional exploration of the liver Exploration of liver function reflects possible liver damage.Tables 3 (appendix) and 4 (appendix) show the evolution of hepatic biomarkers during the different phases of the trial.After the treatment phase, there was a non-significant increase (P > 0.05) in the concentration of AST in all groups.At the level of ALT, we observed a non-significant increase (P > 0.05) in group II LEV and group III (200) compared to group I ED control.Also, there was a very significant increase (P = 0.007) in group VI (1600) compared to control group I ED.On the other hand, there was a less significant decrease compared to group I ED.With regard to the PAL concentration, we noted a non-significant increase in this parameter in all the treated and control groups, but this increase was more marked in group I ED.As for the concentration of LDH, there was a slight increase in the treated and control groups.On the other hand, in group IV (400), there was a decrease.Also at the level of the level of LDH secreted by the liver, there was a slight non-significant increase in all the treated and control groups with the exception of group IV (400) in which this rate decreased in a non-significant way.(P = 0.410).The administration of dexamethasone did not cause any significant variation in AST in group III (200), group V (800) and group VI (1600) compared to the control group I ED.At the same time, the ALT concentration decreased in groups III (200) and VI (1600) compared to the control group I ED.On the other hand, this rate significantly increased (P < 0.001) at the level of group IV (400).The concentration of PAL and LDH decreased in all the treated groups and in the control group I ED.The serum concentration of hepatic biomarkers decreased in all the groups treated with the aqueous extract during the observation period compared to group I ED from the 17th to the 28th day as shown in Figure 1.

Lipid profile
Tables 5 (appendix) and 6 (appendix) show the variations of lipid parameters as a function of different phases during the experiment.At the end of the treatment phase, no significant difference (P > 0.05) was observed outside group VI (1600) which records a significant increase (P = 0.019) on day 14 in cholesterol level total.Equally there was a non-significant increase in HDL and LDL concentrations in all treated groups and group I ED control.Concerning the triglycerides level there was a non-significant decrease in the level of group IV (400) and group V (800) compared to group I ED control.The induction of immunosuppression provoked a mild increase in total and HDL cholesterol levels at the level of group I ED, group II LEV, group III (200) and group VI (1600).On the other hand there was a non-significant decrease of these parameters at the level of group IV (400) and group V compared to group I ED control.The LDL rate noted a decrease in group III (200), group IV (400), group V (800) and group VI (1600) subjects compared to group I ED control.There was also an increase in triglycerides at the level of all groups subjected to testing.This increase was highly significant (P < 0.001) at the group II LEV level and highly significant (P = 0.008) at the group III level (200).During the observation phase, the concentrations of total cholesterol, HDL, LDL and triglycerides showed a nonsignificant decrease at the level of the groups treated with the aqueous extract of Moringa oleifera leaves compared to the control groups from the 17th to the 28th day of our experimentation.At the 14th day of treatment, no significant variation (P > 0.05) was observed in the level of RRC and CA among all the treated groups compared to the control group I ED.However, we have a non-significant increase of these indices among group III (200) and group VI (1600) contrary to IAP and RC which had highly significant difference (P < 0.001) compared to treated groups and the lot I witness ED.The administration of Moringa oleifera extract induced a significant decrease (P = 0.001) at the dose of 800 mg/kg bw in terms of RRC and CA.Equally, the extract induced a non-significant decrease in IAP in group IV (400) and group V (800) compared to the control group I ED.This decrease was significant (P = 0.023) and highly significant (P = 0.003) respectively in group II lev and group III (200) on the 14th day of treatment.On the 17th day after administration of dexamethasone, there was a reduction in the RRC, the CA and the RC in all the treated groups and the control group I.This decrease was significant in group I and group III (200) at the level of RRC and CA.

Nutritional status exploration
At the end of the treatment phase, there was an increase in the albumin level in all the groups treated with the aqueous extract and group II LEV compared to the control who received only distilled water which recorded an increase significant (P = 0.004).This increase was highly significant (P < 0.001) in lot II LEV, very significant (P = 0.009) in lot V (800) and significant (P=0.024) in lot III (200).After the administration of dexamethasone, apart from group IV (400) which decreased, the serum albumin level increased insignificantly in all the treated groups and the control groups.Regarding the variation in the total protein level, on day 14 there was an increase in all the groups apart from group IV (400) which decreased at the end of the treatment phase.After the immunosuppression phase on day 17, there was a decrease in the concentration of total proteins in all the treated groups compared to the control group I.The variation of these nutritional status parameters are recorded in tables 5 (appendix) and 6 (appendix).At the end of the test, there was an increase in the albumin level in all the groups treated on day 14 compared to the control group I ED, the concentration of which decreased significantly (P = 0.004).This increase was not significant at the level of groups IV (400) and VI (1600).On the other hand, this increase was significant at the level of groups II LEV, III (200) and V (800) compared to the control group I ED.However, there was an increase in the concentration of total proteins in all treated groups and group I ED.The administration of dexamethasone caused a decrease in the albumin level in all the treated groups, unlike the control group I ED whose albumin level increased insignificantly (P = 0.458).Also, the concentration of total proteins experienced a decrease at the level of groups II LEV, IV (400), V (800) and group VI 1600) compared to the control group I ED.

Renal and hepatic biomarkers evolution in reversibility phase.
During observation phase, the Figure 1 shows us that there was a non-significant decrease in the concentration of urea and creatinine in different groups treated on the 28th day compared to the ED control group I and within the different groups according to the observation periods.The serum concentration of hepatic biomarkers decreased in all groups treated with aqueous extract during the observation period compared to group I ED from the 17th to the 28th day as shown in the Figure 1.

Lipid biomarkers evolution in reversibility phase.
During observation phase, the concentrations of total cholesterol, HDL, LDL and triglycerides experienced a non-significant decrease in treated groups with aqueous extract of Moringa oleifera leaves compared to control groups from the 17th to the 28th day. of our experiment as shown in Figure 2. On the 17th day after administration of dexamethasone, there was a reduction in RRC, CA and CR in all the treated groups and the control group I.This reduction was significant in group I and group III (200) at the level of RRC and CA.On the 28th day of immunosuppression, there was a decrease in RRC, CA and IAP in all treated groups compared to controls as shown in Figure 3. Unlike the other indices, there was had a non-significant increase in group VI (1600) in CR and IAP on day 28.

Discussion
The aim of this work was to determine the evolution of some biochemical parameters during a preventive immunostimulation test of the aqueous extract of Moringa oleifera leaves in rats.Ingestion of the aqueous extract of Moringa oleifera leaves did not induce any significant variation in urea and creatinine during treatment, which reflects good tolerance of renal function.These results are in agreement with those of [1] who carried out an acute toxicity study by oral administration of a dose ranging from 100 to 5000 mg/kg of body weight of aqueous extract of leaves of Moringa oleifera, their results did not reveal any significant variation compared to the control, which makes it possible to affirm that the use of our extract has no negative impact on the renal function of the subjects.In the same vein, [27] showed that the use of Moringa oleifera leaf powder did not cause any disturbance of renal function in HIV-positive and HIV-negative subjects.The administration of dexamethasone did not induce any significant variation.During the reversibility period, there was no significant variation in the values of urea and creatinine in all the treated compared to the control from the 17th to the 28th day, which comforts us.e in our position.Transaminases (ASAT and ALT), alkaline phosphatase and lactate dehydrogenase are enzymes that are synthesized by the liver under normal conditions and the most specific marker being ALT makes it possible to assess the proper functioning of this organ.There was no significant variation in AST, PAL and LDH after treatment with Moringa oleifera extract.Our results are in agreement with those of [9] who showed that the administration of 200 and 800 mg/kg of hydroethanolic extracts of Moringa oleifera prevented liver damage induced by acetaminophen, leading to decrease in AST, ALT and ALP.IV 400: group IV 400 mg/kg bw.Group V 800: group 800 mg/kg bw.Group VI 1600: group VI 1600 mg/kg bw.IAP: Plasma Atherogenic Index; RC: Classic Report; CA: Atherogenic Coefficient; CRR: Cardiac Risk Report.Lipids (total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides) are synthesized by the liver and their synthesis above normal values is an indicator of the risk of cardiovascular disease.Our experimentation does not indicate any significant variation on the 14th day in all the groupes treated compared to the control.Nevertheless, the slight increase in these lower lipids compared to the control would be due to phytosterols such as β-sitosterol [15].These compounds are responsible for reducing the absorption of dietary cholesterol in the intestine in rodents, which would lead to a decrease in plasma cholesterol and an increase in faecal cholesterol [15].Our results are in agreement with these authors.This decrease remained progressive after the induction of immunosuppression until the 28th day due to the presence of chemical compounds.In the same vein, the administration of the aqueous extract of Moringa oleifera leaf at a dose of 400 mg/kg bw and 800 mg/kg bw induced a decrease in lipid indices such as RRC, CA and l IAP unlike group II submitted to Levamisole.Our results are in agreement with those of [24] who studied the antihyperlipidemic activity of the crude extract of Moringa oleifera leaves.It appears from their study that the administration of a crude aqueous extract had induced a decrease in lipids.Also [25] showed the antilipidemic effect of the aqueous extract of Moringa oleifera leaves.Oxidation of LDL to oxidized low-density lipoprotein (ox-LDL) indicates the first stage of atherosclerosis in cardiovascular disease, stimulating immune and inflammatory responses that initiate the process of atherosclerotic plaque accumulation.Oxidized LDL damages vascular endothelial walls, induces local hormone production from blood vessel walls, promotes inflammation, and attracts macrophages which take up ox-LDL via scavenger receptors and transform macrophages into foam cells.Elevated HDL reverses cholesterol transport by scavenging excess cholesterol from peripheral tissues to the liver for metabolism and excretion [7] and by inhibiting LDL oxidation.In addition, HDL can reduce or neutralize the atherogenic effects of oxidized LDL in arterial walls.Atherogenic indices are powerful indicators of risk of heart disease: the increase in indices is proportional to the increase in the risk of developing cardiovascular diseases and vice versa [18].The low level of certain atherogenic indices such as CR compared to the ED and lev control would be due to the capacity of our extract at dose of 1600 mg/kg of body weight to inhibit the oxidation of LDL and would promote the action of HDL at collect and transport lipid deposits to the liver for metabolism.The results obtained indicated an increase in all groups compared to control group I ED in the level of albumin and total protein after the 14-day treatment.The increase in these parameters is an indicator of good nutritional status.These results are in agreement with those of [27] who showed that the increase in albumin indicates the absence of malnutrition.However, these proteins synthesized by the liver decrease due to the onset of malnutrition or in the presence of an inflammatory reaction [27][28][29][30][31].

Conclusion
At the end of the experiment, all the parameters determined made it possible to evaluate the effect of the total aqueous extract of Moringa oleifera leaves on cellular and humoral immunity in rats.This study highlighted the preventive immunostimulant properties of Moringa oleifera leaves at doses of 800 mg/kg body weight and 1600 mg/kg body weight on haematological parameters.At the end of this work we can encourage the use of Moringa oleifera leaves in all people subject to an immune deficiency, in particular the elderly, people with HIV, kidney failure, people with cancer, etc. however it would be very important to determine the prevention mechanism of Moring oleifera leaf extract for wider use.