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Comparative Studies on the Responses of Some Biochemical Markers in Immuno-Suppressed Rats Treated with Gongronema latifolium and Vernonia amygdalina Ethanol Leaf Extract



Joshua Parker Elijah, Eze Chuma, Ukegbu Chimere Young, Akabike Chigozie Adrain and Igwe Chinagolum Arinzechukwu
 
ABSTRACT
Background and Objective: Gongronema latifolium and Vernonia amygdalina are medicinal plants that have been used in the treatment of nausea, hypertension, diabetes, stomach ache, high cholesterol level etc. However this research was set out to access comparatively the immuno-stimulatory effect of ethanol leaf extract of Gongronema latifolium and Vernonia amygdalina in cyclophosphamide immuno-suppressed rats. Materials and Methods: Thirty Wistar albino rats of 5 rats per group were used for this study and were divided into 6 groups. Group 1 rats were normal control; group 2 (positive control) rats were administered cyclophosphamide only, at the dose of 50 mg kg–1 b.wt. i.p. Group 3 rats were administered low dose (400 mg kg–1 b.wt.) of the Vernonia amygdalina leaf extract after cyclophosphamide induction while group 4 rats were administered high dose (600 mg kg–1 b.wt.) of the extract after cyclophosphamide induction. Group 5 rats were administered low dose (400 mg kg–1 b.wt.) of Gongronema latifolium leaf extract after cyclophosphamide induction while group 6 rats were administered high dose (600 mg kg–1 b.wt.) of the extract after cyclophosphamide induction. The results were analyzed using one way analysis of variance (ANOVA) using SPSS version 20. Results: Treatment with Vernonia amygdalina and Gongronema latifolium leaf extract (group 3, 4 and group 5, 6 respectively) showed a significant increase (p<0.05) in SOD activity, glutathione and vitamin C concentrations when compared with group 2 rats treated with cyclophosphamide only. More so treatment with Vernonia amygdalina and Gongronema latifolium leaf extract (group 3, 4 and group 5, 6 respectively) showed a significant decrease (p<0.05) in the liver marker enzymes (ALT, AST and ALP) when compared to group 2 rats treated with cyclophosphamide only, however group 2 rats showed a significant decrease (p<0.05) in total protein concentration and a significant increase (p<0.05) in the total cholesterol concentration and triacylglycerol concentration when compared to the treatment groups. Conclusion: Comparatively, these findings indicate that Vernonia amygdalina may have better antioxidant properties and also exhibited more potency in inhibiting liver damage by lowering the liver marker enzymes compared to Gongronema latifolia.
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Joshua Parker Elijah, Eze Chuma, Ukegbu Chimere Young, Akabike Chigozie Adrain and Igwe Chinagolum Arinzechukwu, 2017. Comparative Studies on the Responses of Some Biochemical Markers in Immuno-Suppressed Rats Treated with Gongronema latifolium and Vernonia amygdalina Ethanol Leaf Extract. Research Journal of Immunology, 10: 8-15.

DOI: 10.3923/rji.2017.8.15

URL: http://scialert.net/abstract/?doi=rji.2017.8.15
 
Received: October 11, 2016; Accepted: November 30, 2016; Published: December 15, 2016

INTRODUCTION

Medicinal plants have formed the basis of health care throughout the world since the earliest days of humanity and have remains relevant in both developing and the developed nations of the world for various chemotherapeutic purposes1. It has been medically proven that the rate of consumption of vegetable is concurrent with the state of health of an individual. This has been clearly observed among people in Asia and some part of Africa. In South-Eastern Nigeria, the inhabitants are known for high consumption of vegetables. These vegetables are not only eaten as food but also during ill health and times of convalescence2. Thus they are referred to as herbal medicine.

The increasing use of herbs in the treatment of diseases in developing countries has been proven to be due to their historical and cultural significance3. However, this also suggests that aside from culture, other factors such as poor governance, drug resistance, poverty, emergence and re-emergence of infectious diseases, have exacerbated the interest in ethno-pharmacological research. Also these plants contain secondary metabolites that influence biological processes and reverse disease states and unlike some conventional drugs, they seem to have lesser side effects and are also inexpensive2. For this reason and because they are also available they are taken by the majority of the population4. Among the numerous herbal plants used in traditional medicine Gongronema latifolium and Vernonia amygdalina have been well investigated. These plants have been reported to contain alkaloids, carbohydrate, tannins, saponins, flavonoids and non-cyanogenic glycosides5. Their extracts have been identified with certain properties such as anti-bacterial, anti-fungal, anti-microbial and antioxidant.

Cyclophosphamide is a potent immuno-suppressive agent, capable of inhibiting both humoral and cell mediated immune responses6. The main use of cyclophosphamide is together with other chemotherapy agents in the treatment of lymphomas, some forms of brain cancer, leukemia and some solid tumors7. It is a chemotherapy drug that works by slowing or stopping cell growth. In animal models, this cytotoxic agent can suppress graft-vs-host disease, prevent development of certain auto-allergic conditions and promote tolerance to certain antigens. Due to this activity, cyclophosphamide is being evaluated in treatment of certain human disorders believed to result from aberrant immunity and as an agent useful in preparing recipients for marrow transplant6. A previous study showed that the biological actions of cyclophosphamide are dose-dependent. At higher doses, cyclophosphamide undergoes biotransformatio by hepatic microsomal cytochrome P450 mixed function oxidase system to metabolites that enter the circulatory system8. Phosphoramide mustard and acrolein are the two active metabolites. The cytotoxic effects such as necrosis, cell death and oncosis are associated with acrolein, while the antineoplastic activity of cyclophosphamide is linked with phosphoramide9.

Both immuno-suppressants and stimulants have serious side effects ranging from inflammatory disorders, hypersensitivities, immunodeficiency disorders etc; thus complementary or traditional remedies are other options to overcome this problem10. However the search for immune-stimulants from natural source with little or no side effect continues. Thus this project is a comparative study on the antioxidant properties and Immuno-stimulatory effect of Gongronema latifolium and Vernonia amydalina ethanol extract on cyclophosphamide immuno-suppressed rats. The results of this research will give a clue on immunological abilities of the different plants and give an insight on their mode of actions.

MATERIALS AND METHODS

Plant materials: Fresh leaves of Gongronema latifolium and Vernonia amydalina were purchased from Ogige market in Nsukka, Enugu State of Nigeria on the 10th day of June 2015 and were identified by Mr. Alfred Ozioko of the Herbarium Botany Department, University of Nigeria, Nsukka.

Extraction of the leaves of Gongronema latifolium and Vernoniaamydalina: The leaves of Gongronema latifolium and Vernonia amydalina were air-dried separately at room temperature, then grinded into powdery form using electrical grinding machine. The ground samples were extracted with 95% ethanol solution, using cold maceration techniques. The samples were filtered using Whatman filter paper No. 1. The filtrates concentrated to solid matter using rotary evaporator at the temperature of 45̊C. The extracts were stored in the refrigerator until further use.

Animals: Adult male Wistar albino rats of 10-16 weeks old and average weight of 160±15 g were obtained from the Animal House of the Faculty of Biological Sciences, University of Nigeria, Nsukka. The animals were acclimatised for duration of 7 days under standard environmental conditions with a 12 h light/dark cycle maintained on a regular feed (vital feed) and water ad libitum. Clearance and approval for the humane use and handling of laboratory animals were given by the ethical committee of Department of Biochemistry, University of Nigeria, Nsukka which is in accordance to CIOMS11.

Drug (cyclophosphamide): Cyclophosphamide (potent immuno-suppressant) was purchased from Elophez Pharmacy, Nsukka in Enugu State of Nigeria.

Administration of cyclophosphamide: The rats were administered intraperitoneally with a dose of 50 mg kg–1 b.wt., of cyclophosphamide (potent immuno-suppressant).

Chemicals/reagents/samples: All chemicals used in this study were of the analytical grade and products of May and Baker, England; BDH, England and Merck, Darmstadt, Germany. Reagents used for all the assays were commercial kits and products of Randox, USA; QCA, Spain; Teco (TC), USA; Biosystem Reagents and Instruments, Spain.

Experimental design: Twenty male albino Wistar rats were acclimatized at the same conditions of temperature and pressure and the same animal feeds were used for all the rats. The rats were divided into 6 groups of 5 rats each as shown below:

Group 1: Normal/negative control that were treated with normal saline only
Group 2: Positive control received cyclophosphamide intra-peritoneally at the dose of 50 mg kg–1 b.wt.
Group 3: Test group received cyclophosphamide intra-peritoneally first then a low dose (400 mg kg–1 b.wt.) of Vernonia amydalina ethanol extract
Group 4: Test group received cyclophosphamide intra-peritoneally first then a high dose (600 mg kg–1 b.wt.) of Vernonia amydalina ethanol extract
Group 5: Test group recieved cyclophosphamide intra-peritoneally first then a low dose (400 mg kg–1 b.wt.) of Gongronema latifolium ethanol extract
Group 6: Test group received cyclophosphamide intra-peritoneally first then a high dose (600 mg kg–1 b.wt.) of Gongronema latifolium ethanol extract

Biochemical investigation: The animals were treated for 7 days and on the 8 day the animals were sacrificed and blood was collected and used for biochemical analysis.

Assay of aspartate aminotransferase activity: A randox commercial enzyme kit according to the method of Reitman and Frankel12 was used in the study.

Assay of alanine aminotransferase activity: A randox commercial enzyme kit based on the method of Reitman and Frankel12 was used.

Assay of alkaline phosphatase activity: This was done using the QCA commercial enzyme kit which is based on the phenolphthalein monophosphate method of Klein et al.13.

Direct and total bilirubin: Direct and total bilirubin were measured using the method of Jendrasskik and Grof14.

Assay of superoxide dismutase activity: Superoxide dismutase (SOD) activity was assayed using the method as described by Fridovich 15 as contained in Randox commercial kit.

Assay of catalase activity: Catalase activity was assayed using the method of Aebi16.

Determination of glutathione concentration: The concentration of glutathione was determined according to the method of Habig et al.17.

Determination of vitamin C: The concentration vitamin C (ascorbic acid) was determined according to the method of Baker et al.18.

Determination of total cholesterol concentration: The concentration of total cholesterol was determined according to the method of Allain et al.19 as outlined in the QCA test kit.

Determination of triacylglycerol concentration (randox enzyme kit): The concentration of triacylglycerol was determined according to the method of Trinder,20 as outlined in the QCA test kit.

Determination of total protein concentration: The determination of protein concentration was carried out according to the method of Slater21.

Statistical analysis: Data obtain was expressed as Mean±SD, analysis for significance of disparity using a one-way analysis of variance (ANOVA). The Statistical Product and Service Solution (SPSS), version 20 was used for the analysis where p<0.05 was regarded as significant22.

RESULTS AND DISCUSSION

In the present study Table 1 and 2 show that administration of cyclophosphamide cause a significant increase (p<0.05) in the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) as shown in group 2 when compared to the normal control and this suggests possible case of necrosis or hepatocellular injury in the rats23.

Table 1:Effect of Vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on aspartate aminotransferarse and alanine aminotransferarse activity in immunosuppressed Wistar albino rats
Values are expressed as Mean±SEM; *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase

Table 2:
Effect of Vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on alkaline phosphatase activity in immunosuppressed Wistar albino rats
Values are expressed as Mean±SEM, *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance, ALP: Alkaline phosphatase

However treatment with treatment with Vernonia amygdalina and Gongronema latifolia leaf extracts (group 3, 4 and group 5, 6 respectively) showed a significant decrease (p<0.05) in the liver marker enzymes (ALT, AST and ALP) when compared to group 2 rats treated with cyclophosphamide only. The significant decrease (p<0.05) in the aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities of the rats treated with different doses (400 and 600 mg kg–1 b.wt.) of Vernonia amygdalina and Gongronema latifolium ethanol leaf extracts when compared to the group 2 animals indicates no form of myocardial infarction or viral hepatitis in the rats24. This is consistent with the works of Iweala et al.25 who studied the biochemical effects of leaf extracts of Gongronema latifolium in alloxan induced diabetic rats. However, Vernonia amygdalina leaf extract showed a higher potency when compared to Gongronema latifolium leaf extract.

From Table 3 it was observed that intra-peritoneal administration of cyclophosphamide caused a significant increase (p<0.05) in the concentration of direct and total bilirubin when compared to the normal control rats which is an indication of hyper-bilirubinemia might be as a result of an increase in breakdown of haemoglobin by the free radicals generated by acrolein or damage of the biliary duct. Treatment with different doses (400 and 600 mg kg–1 b.wt.) of Vernonia amygdalina and Gongronema latifolium leaf extracts showed a significant decrease (p<0.05) in both total and direct bilirubin concentrations. This suggests an effective control of bilirubin concentration which points towards an improved secretory mechanism of the hepatic cells by the extract. This study is in tandem with the works of Parker et al.,26 who demonstrated effective control of bilirubin by an alkaloid-rich fraction of an extract.

Administration of cyclophosphamide alone gave a significant decrease (p<0.05) in SOD, catalase, glutathione and vitamin C concentration of group 2 rats when compared with the normal control group 1 and other test groups as shown in Table 4 and 5. This might be because the activity of the metabolite of cyclophosphamide, acrolein, leads to the generation of ROS. These cause oxidative stress that may lead to the reduction or inactivation of these antioxidants. The oxidative stress also leads to the disruption of pro-oxidant and antioxidant balance as a result of an increase in Reactive Oxygen Species (ROS) generation, impairment of anti-oxidant defence systems or an insufficient capacity to repair oxidative damage27. This decrease observed in these antioxidants level is consistent with the results of Ismail et al.28. A study by Kedzierska et al.29 affirmed that immune-suppressants lead to generation of Reactive Oxygen Species (ROS) which cause oxidative stress. More so if due to decrease in SOD activity, hydrogen peroxide is produced in large amounts, catalase also takes part in its degradation. Also vitamin C an antioxidant nutrient plays an important role in removing ROS.

Furthermore, a study by Rajasekaram and Kalaivani30, confirmed that oxidant stress can be marked by LPO, SOD, GSH and catalase levels. While LPO increases in oxidative stress, GSH is reduced and SOD and catalase are inactivated.

The results also showed that treatments with the ethanol leaf extract of both Vernonia amygdalina and Gongronema latifolium gave significant increase (p<0.05) in SOD and catalase activities and glutathione and vitamin C concentrations.

Table 3:Effect of vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on direct and total bilirubin concentrations in immunosuppressed wistar albino rats
Values are expressed as Mean±SEM, *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance

Table 4: Effect of Vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on superoxide dismutase and catalase activity in immunosuppressed Wistar albino rats
Values are expressed as Mean±SEM, *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance, SOD: Superoxide dismutase

Table 5: Effect of Vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on glutathione and vitamin C concentrations in immunosuppressed Wistar albino rats
Values are expressed as Mean±SEM; *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance

This may be due to the antioxidants possessed by the plants extract. These antioxidants such as vitamin C, vitamin E, flavonoids, tannins, polyphenols, terpenes, phenolic acids and saponins mop up reactive oxygen species in the body. Studies by Nwanjo et al.31 and Ayoola et al.32 confirmed the presence of these antioxidant phytochemicals in these plants extract. A study by Buher and Miranda33 confirmed that flavonoids may help provide protection against diseases by contributing, along with antioxidant vitamins and enzymes, to the total antioxidant defence system of the human body. In addition, it was observed that Vernonia amygdalina treatments (group 3 and 4) show higher recovery in SOD activity, GSH and vitamin C concentrations compared to Gongronema latifolium treatments (group 5 and 6) while both plants show similar catalase activity recovery. This shows that Vernonia amygdalina might have better antioxidant property. This may be because Vernonia amygdalina has higher antioxidant content than Gongronema latifolium. A study by Farombi and Owoeye34 confirmed that in phytochemical screening, Vernonia amygdalina tested positive for most phytochemicals. They stressed that the presence of flavonoids and tannins in the plant is likely to be responsible for the free radical scavenging effects observed.

Also a study by Farombi and Owoeye34 showed that unlike Gongronema latifolium, Vernonia amygdalina contains phenolic acids. These phenolic acids, example ferrulic acid, have similar structures to flavonoids and tannins and thus contribute to their antioxidant property.

From Table 6, the results showed a significant increase (p<0.05) in the total cholesterol levels and triacylglycerol levels of group 2 rats that received only cyclophosphamide when compared with the normal group (group 1). The liver is central to the regulation of cholesterol levels in the body, not only does it synthesize cholesterol for export to other cells but it also removes cholesterol from the body by converting it to bile salts and excreting it into the bile. Levels of triglyceride in the serum or liver could increase due to several processes, including increased availability of free fatty acid, glycerol-phosphate, decreased VLDL in the serum and decreased removal of triglyceride and cholesterol from serum due to diminished lipoprotein activity35.

Table 6:
Effect of Vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on total cholesterol and of triacylglycerol concentrations in immunosuppressed Wistar albino rats
Values are expressed as Mean±SEM; *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance

Table 7:
Effect of Vernonia amygdalina and Gongronema latifolium ethanolic leaf extracts on total protein concentration in immunosuppressed Wistar albino rats
Values are expressed as Mean±SEM, *p<0.05 is considered significant when compared with positive control group using one-way analysis of variance

However, treatment with the extract showed a dose dependent reversal of such increased which could be as a result of the extract containing some phytochemical contents that could revive the functionality of the liver36.

Intra-peritoneal administration of cyclophosphamide cause a decrease in the total protein concentration of the group 2 animals when compared to normal control group as can be seen in Table 7. These low levels of total protein could be as a result of specific oxidative damage of some of the susceptible amino acids of the intracellular proteins of the body. However treatment with Vernonia amygdalina and Gongronema latifolium leaf extract was able to cause a significant increase (p<0.05) in the total protein concentration of the rats in the treatment group (group 3, 4, 5 and 6) when compared to the untreated control group 2. This points to the effectiveness of the extracts in inhibiting the breakdown of structural proteins of the body which could be by membrane stabilization effect or other unknown mechanism. More so stimulation of protein synthesis can be contributory to its effect which could accelerates the regeneration process and the production of liver cells. This improvement in the total protein level of the rats treated with the extracts is in tandem with the works of Kanchana and Sadiq37, where the hepato-protective effect of Plumbago zeylanica on positive induced liver toxicity in rats was determined.

CONCLUSION

The study showed that ethanol leaf extracts of Gongronema latifolium and Vernonia amygdalina possess an immune-stimulatory effect by its ability to revitalise hepatic functions and improve antioxidant properties of the immuno-suppressed rats. However, comparatively Vernonia amygdalina showed a better potency when compared to Gongronema latifolium.

SIGNIFICANCE STATEMENTS

The present research tends to discover the possible synergistic effect of Vernonia amygdalina and Gongronema latifolia ethanol extract in boasting cyclophosphamide immuno-suppressed rats. Although there are some inorganic immuo-stimulants but there is a continuous search for immuo-stimulants with little or no side effect. Thus the results of this research will give a clue on immunological abilities of the different plants and also give an insight on their mode of actions. Finally this research will take a lead in the discovery of new organic immunological agents with little or no side effects from both plants.

ACKNOWLEDGMENT

The authors would like to extend their sincere appreciation to Department of Biochemistry, University of Nigeria, Nsukka for for providing the animal house used in doing this work.

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