Indian Journal of Research in Homeopathy

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 6  |  Issue : 1  |  Page : 26--32

Immunomodulatory effects of hydromethanolic extract of Moringa oleifera leaf on male wistar rats


Augustus Obi1, Jude N Egwurugwu2, SO Ojefa1, MC Ohamaeme3, CN Ekweogu4, FU Ogunnaya5,  
1 Department of Human Physiology, College of Health Sciences, University of Port Harcourt, Port Harcourt, Nigeria
2 Department of Human Physiology, College of Medicine, Imo State University, Owerri, Imo, Nigeria
3 Department of Community Medicine, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria
4 Department of Medical Biochemistry, College of Medicine, Imo State University, Owerri, Imo, Nigeria
5 Department of Internal Medicine, Imo State University Teaching Hospital, Orlu, Imo, Nigeria

Correspondence Address:
Mr. Augustus Obi
Department of Human Physiology, College of Health Sciences, University of Port Harcourt, Port Harcourt
Nigeria

Abstract

Background: The current global economic recession has made modern drugs expensive, not easily accessible and affordable, thereby making alternatives such as medicinal plants including Moringa oleifera imperative. Aim: This study assessed the immunomodulatory effects of the leaf extracts of M. oleifera on male Wistar rats. Materials and Methods: Forty-eight male Wistar rats weighing between 180 and 250 g were randomly divided into eight groups of six rats each. Group I served as the positive control. Groups II, III, and IV were administered oral doses of 125, 250, and 500 mg/kg of M. oleifera leaf extracts, respectively, for 15 days. Cyclophosphamide (CTX) at a dose of 30 mg/kg body weight was administered orally to Groups V (negative control), VI, VII, and VIII for the first 3 days. Thereafter, 125, 250, and 500 mg/kg doses of the extract were administered, respectively, for another 15 days. On days 16 and 19, blood samples were collected through cardiac puncture and analyzed for red blood cell count, hemoglobin concentration, total white blood cell (WBC) count, percentage differentials of WBCs, and liver enzymes (alanine transaminase [ALT], aspartate transaminase [AST], and alkaline phosphatase [ALP]). Results: Results showed statistically significant dose-dependent increase in total WBC (TWBC) count and percentages of neutrophil, eosinophil, monocytes, and lymphocytes in Groups II, III, and IV compared to Group I. It also showed that the extract statistically reduced serum hepatic enzymes (ALT, AST, and ALP) in Groups II, III, and IV compared to Group 1 (P < 0.05). Administration of CTX significantly reduced TWBC count and percentages of neutrophil, eosinophil, monocytes, lymphocytes, and increased serum hepatic enzymes (ALT, AST, ALP) in Groups VI, VII, and VIII when compared to Group V (P < 0.05). The effects of CTX on blood and hepatic enzymes were reversed with administration of the extracts in a dose-dependent manner (P < 0.05). Conclusion: Our findings showed that M. oleifera leaf extracts have stimulatory effects on the production of WBCs and also have hepatoprotective potentials. Thus, it may have immune-modulatory properties.



How to cite this article:
Obi A, Egwurugwu JN, Ojefa S O, Ohamaeme M C, Ekweogu C N, Ogunnaya F U. Immunomodulatory effects of hydromethanolic extract of Moringa oleifera leaf on male wistar rats.Niger J Exp Clin Biosci 2018;6:26-32


How to cite this URL:
Obi A, Egwurugwu JN, Ojefa S O, Ohamaeme M C, Ekweogu C N, Ogunnaya F U. Immunomodulatory effects of hydromethanolic extract of Moringa oleifera leaf on male wistar rats. Niger J Exp Clin Biosci [serial online] 2018 [cited 2019 Jul 23 ];6:26-32
Available from: http://www.njecbonline.org/text.asp?2018/6/1/26/248005


Full Text



 Introduction



Immune system is a system of biological structures and processes within an organism that protects against diseases.[1] Thus, it can also be viewed as a network of cells, tissues, and organs that work together to protect the body from infection[2] The human body provides an ideal environment for many microbes, such as viruses, bacteria, fungi, and parasites, and the immune system prevents and limits their entry and growth to maintain optimal health.[3]

An immune modulator is any substance that affects directly or indirectly the immune response to external agents or therapeutics and prevents or reduces the development of degenerative diseases.[4],[5] They have broad effects on the entire immune system; both cell-mediated immunity and humoral immunity. Immune modulators achieve their effects by boosting specific areas of the immune system, most especially, the innate immunity and the activities of both T- and B-lymphocytes through the action of plasma cells.[6],[7]

Various allopathic drugs or medicines are used to modulate the immune system. However, these drugs are very expensive for poor people, they are not easily accessible, and in most cases, they are associated with adverse drug reactions. As a result, the majority of people, especially in the rural areas of the developing world turn to the use of alternative herbal medicines from medicinal plants such as Moringa oleifera that are widely accepted, accessible, cheaper, and assumed to have fewer side effects.[8]

M. Oleifera is a highly valued plant, distributed or found wild, and cultivated in many countries of the tropics, subtropics, and semi-arid climate.[9] It is a fast-growing, drought-resistant tree, native to the Southern foothills of the Himalayas in Northwestern India. Thus, it has an impressive range of medicinal usage and high nutritional value. Several researchers have indicated that M. oleifera is a highly valued plant with multipurpose effects; the tree ranges about 10–12 m (32–40 ft) in height, the trunk can reach a diameter of 45 cm (1.5 ft).[10] It is considered as one of the world's most useful trees, as almost every part of the tree has an impressive effect of food, medication, and industrial purposes.[11],[12]

Different parts of the plant contain a profile of important; minerals (such as potassium, calcium, iron, selenium, and magnesium), Vitamins (such as A, B-complexes, C, D, E, and K), β-carotene, proteins, and essential amino acids that are not commonly found in plants, good cholesterol, and various phenolics. It is a good source of natural antioxidants which can neutralize free radicals, this may account for its central importance in the prevention of carcinogenicity, cardiovascular diseases, and neurodegenerative changes associated with aging.[13],[14] Epidemiological studies have shown that the consumption of vegetables and fruits can protect humans against oxidative damage by inhibiting and/or quenching free radicals and reactive oxygen species.[15],[16] M. Oleifera also provides a rich and rare combination of zeatin with several flavonoid pigments. Its oil and micronutrients contain antitumor, antiepileptic, antidiuretic, anti-inflammatory, and venomous bite characteristics.[17],[18],[19] These activities of M. oleifera may be due to one or more phytochemical constituents such as gallic tannins, catechol tannins, flavonoids, steroids and triterpenoids, saponins, anthraquinones, alkaloids, and reducing sugars.[20]

Traditional medicine practitioners claim that some herbal preparations detoxify toxins in the body, cleanse the body of such toxins, and ultimately modulate the immune system[21] Early studies carried out in Africa revealed that Moringa powder supplementation might act as an immune stimulant for patients suffering from HIV infection.[22] As the plant is widely used in folklore for the treatment of suppressive conditions of immune system and so far no study has been carried out to prove the stimulatory actions of M. oleifera on immune system, therefore, the present work was undertaken using modern scientific techniques in experimental models of cellular and humoral immunity in animals.

 Materials and Methods



All experiments were conducted under a carefully designed good laboratory practice protocol, supervised by external auditors. The recommendations for quality standards for biomedical research were noted and implemented.

Experimental plant collection

M. oleifera leaves were collected from Rumuji, in Emuoha Local Government Area of Rivers State Nigeria. The plant was botanically identified and authenticated by a botanist in the Department of Plant Science and Biotechnology, University of Port Harcourt Nigeria. Herbarium specimen was prepared and deposited with a herbarium number UU/BY/H134. After which, it was rinsed in distilled water to remove the dirt and air-dried for 1 week to ensure proper removal of the moisture content while reserving the important constituents of the leaves.

Extraction process (80% hydro-methanolic)

The leaves of M. oleifera were separately rinsed in distilled water once again for proper removal of dirts, and were dried in an air oven at a temperature of about 35°C for 2 days and then pulverized or grinded into a fine powdered form, that passed through a 30-mesh sieve using a manual grinder and was weighed. The 300 g extraction of the plant material was macerated in a mixture of 80% methanol and 20% distilled water using a Soxhlet extractor and was concentrated at 40°C in vacuo using rotary evaporator.

The M. oleifera mesh was placed in the thimble of the extractor while the liquid (80% methanol and 20% distilled water) was placed in the reservoir of the extractor.

Heat was applied from the heating mantle of the extractor which resulted to vaporization of the liquid in the reservoir. The vaporized liquids were condensed by the condenser component of the extractor, the condensed liquid were later dropped into the thimble were the meshed leaves were placed in order to extract the Moringa component. Increase in condensation of the liquid (hydro-methanol) resulted to an overflow of the liquid extract from the thimble via the siphon tube back into the reservoir which was placed below the thimble. The liquid extract in the reservoir was subjected to heat for several minutes in order to vaporize the methanol content of the extraction. The sample was collected in a gel-like form and the yield was 80 g which was used to administer to the rats.

Acute toxicity studies

The acute toxicity study was carried out according to the limit test described in the OPPTS guidelines[23] and other studies were cited.[24] Briefly, a test dose of 2 g/kg and 5 g/kg were given orally to the mice. No mortality was found even at 5 g/kg; po. However, methanolic extract of M. Oleifera caused significant changes in the total red blood cells (RBC), packed cell volume, hemoglobin concentration (Hb), mean corpuscular volume, mean corpuscular Hb concentration, and total and differential White Blood Cell (WBC) when evaluated at 1.5 g/kg and 2 g/kg. Hence, we opted for half of 1.5 g/kg as high dose and 1/5th of 1.5 g/kg as low dose corresponding to 750 mg/kg and 100 mg/kg, respectively.

Drug used

Cyclophosphamide (CTX) was the drug used and it was of analytical grade. It was purchased from the German Remedies, Mumbai, India, and shipped to Nigeria by Institute of Animal Health and Veterinary Biologicals with control number IAHVB4-1382. The drug was dissolved in a normal saline (0.9% NaCl) and administered orally at a dose of 30 mg/kg body weight.

Experimental design

Forty-eight adult male Wistar rats weighing between 180 g and 250 g were procured and housed in Animal house of the Department of Human Physiology, University of Port Harcourt, Nigeria. The rats were randomly grouped into eight, comprising of six rats per group and were housed in cages made of wooden frames and metal netting. They were fed ad libitum with tap water and rat feed (Bendel Feeds and Flour Mills Ltd., Ewu, Edo State, Nigeria) with 12-h light/dark cycle. The cages were cleaned every morning and disinfected at intervals of 3 days. The rats were allowed to acclimatize for 14 days before extract administration was commenced. All doses of the leaf extract (125 mg/kg, 250 mg/kg, and 500 mg/kg) were administered orally once daily between 8 am and 9 am to Groups II, III, and IV, respectively, for 15 days. Group I was the Positive control group. CTX at a dose of 30 mg/kg body weight was administered orally to Groups V (Negative control group), VI, VII, and VIII for the first 3 days. Then, doses of the extract (125 mg/kg, 250 mg/kg, and 500 mg/kg) were administered orally to Groups VI, VII, and VIII, respectively, for the next 15 days. All administrations were done using cannula attached to a 2 ml syringe. On days 16 and 19, the rats were then fasted for 12 h and body weights determined before sacrifice using 25% urethane (ethyl carbamate) at the dose of 0.6 ml/100 g body weight. Blood samples were obtained through cardiac puncture for analyses of hematological parameters. The hematological parameters were analyzed using automated hematology analyzer E. Z Lyser (Sysmex K2x1: Sysmex Corporation, Japan), while the serum hepatic enzymes were analyzed using biochemical analyzing machine (Cel Tech CL3000m, Version: 4.21, Cel Tech Corporation, Japan).

Ethical consideration

The Ethical Committee on Animal Studies of the University of Port Harcourt approved the protocol for this research and their guidelines were strictly adhered to throughout the course of this work. Furthermore, “Principles of laboratory animals' care” (NIH publication No. 85, revised 1985), were followed as well as specific national laws where applicable.[25]

Statistical analysis

The results were expressed as mean of 5 replicates ± standard error of mean and were analyzed using Statistical Package for Social Sciences (SPSS) version 20.0 (IDM-SPSS, New York). Least significant difference post hoc (multiple comparisons) test was applied to determine the level of significance between the control and experimental groups. One-way analysis of variance was also performed to test the effect of each dose on the parameter under investigation at 95% level of confidence. Values were considered statistically significant at (P < 0.05).

 Results



Effects of administration of Moringa oleifera extracts on normal Wistar rats

The study reports a significant (P < 0.05) dose-dependant increase in the values of WBC count and lymphocyte (i.e., the higher the dose, the higher their serum levels). There is a statistically significant (P < 0.05) increase in monocytes, eosinophils, and neutrophils but not dose dependent as seen in [Table 1] below. However, there are variations in the values of serum levels of hepatic enzymes (alanine transaminase [ALT], aspartate transaminase [AST], and alkaline phosphatase [ALP]), particularly on ALT which was a dose-dependent decrease when compared with the control group and these variations were considered statistically significant at (P < 0.05). Also in [Table 2], the administration of the extract showed increase in RBC count at all doses (125, 250 and 500 mg/kg bw), though this increase was not considered statistically significant at (P < 0.05) when compared with the control group. The Hb concentration was slightly increased at all doses of the extract (125, 250, and 500 mg/kg bw), but these variations were not considered statistically significant at P < 0.005 when compared with the control group.{Table 1}{Table 2}

Effect of Moringa oleifera on immunosuppressed Wistar rats induced by cyclophosphamide

In the rats treated with CTX alone (i.e., Group 5), there were significant reductions in total WBC (TWBC) count, percentages of neutrophil and eosinophil with margins of 7.83 ± 1.40*, 7.63 ± 1.56*, and 1.00 ± 0.82, respectively, and insignificant reduction in monocyte percent at 1.00 ± 0.33 when compared with the positive control group (Group 1). These effects of CTX were reversed with the administration of the extract.

There are appreciable increase in the serum levels of hepatic enzymes (ALT, AST, and ALP), especially ALP at 700.67 ± 239.07* but was ameliorated with the administration of the graded doses of M. oleifera which was dose dependent as seen in [Table 3].{Table 3}

There is no visible significant change on both RBC count and Hb level when compared with both the positive and negative control groups as seen in [Table 2].

Note: In the tables below, Groups I and V are positive and negative control groups, respectively, Groups II, III, and IV were compared with Group I while Groups VI, VII, and VIII were compared with group.

 Discussion



This study investigated the immunomodulatory effects of M. Oleifera leaf extracts in male Wistar rats. Previous reports and phytochemical screening of the M. oleifera leaf extracts have shown the presence of flavonoids, alkaloids, tannins, thiamin, riboflavin, niacin, calcium, iron, proteins, carbohydrates, rhamnose, glucosinolates, glycosides, antioxidants such as vitamins C, E, A, caffeoylquinic acids, carotenoids-lutein, α and β-carotenes, kaempferol, quercetin, and rutin as potent immunomodulators.[26],[27],[28],[29],[30],[31]

The present work showed statistically significant increase in TWBC count, lymphocytes, eosinophils, monocytes, and neutrophils in the test groups administered M. oleifera extracts compared to positive control (P < 0.05). The observed increase was dose dependent. This same trend was also observed in the groups treated with a combination of the Moringa leaf extracts and CTX. This reported increase in the hematological indices shows that the plant extract may have hematopoietic as well as immunomodulatory potentials, probably due to the presence of some of the phytochemicals such as iron, calcium, protein, niacin, riboflavin, and carbohydrates.[32] In addition, compounds such as phenols, alkaloids, glycosides, polysaccharides, and saponins have been tested for their efficacy as both chemical and biological markers for immunomodulation.[33],[34],[35] M. oleifera extracts boost immune system at high doses and prolonged duration.[36]

This study also showed nonstatistically significant variations in RBC count and Hb concentrations in the test groups compared with the positive and negative controls. This little or no effect on the erythrocytes could be due to the presence of some phytochemicals such as saponins, anthraquinones, pterygospermin, and 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate that can act as stressors, reducing oxidative potentials of CTX and thus, normalizing the effects.[37],[38],[39]

This research efforts also noted statistically significant reduction in the serum concentrations of liver enzymes (ALT, AST, and ALP) assayed in the test groups compared with the positive and negative controls (P < 0.05). The decrease in the enzymes was dose dependent. That is, M. oleifera leaf extracts decreased serum liver enzymes of the rats treated with the extracts only and later ameliorated the elevated values caused by CTX. This hepatoprotective potentials of M. oleifera leaf extracts might be due its high antioxidant activity. The drumstick leaves of M. oleifera are known to be natural sources of antioxidants.[40],[41],[42] Furthermore, M. oleifera leaf extracts are rich in flavonoids such as quercetin and kaempferol. These major bioactive constituents exert anti-inflammatory actions by regulating inflammatory cells, blocking the T-lymphocyte proliferation, and pro-inflammatory cytokines.[43]

M. oleifera leaf extracts are also rich in alkaloids. Alkaloids exert immunomodulatory activities.[44] Some bitter alkaloids (tropane alkaloids) are metabolized in the liver into dimethylxanthine and finally methyl uric acid by CYP450 Oxygenase systems.[45] Methyl uric acid in the liver stimulates the expression of tumor necrosis factor (in the endothelia cells of the liver by macrophages), which modulates the immune system.[46] Saponins, which concentration was high, is also implicated in the modulation of the immune system by serving as adjuvant (saponins-cholesterol-phospholipid complexes) at low concentrations that stimulated cell-mediated immune system by inducing the production of interleukins, especially by the antigen-presenting cells in mast cells.[47],[48]

 Conclusion



This study shows that M. oleifera leaf extracts may have stimulatory effects on WBCs; exhibits hepatoprotective potentials and can have immunomodulatory properties. The leaf extract can be used to boost the immune system.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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