Abstract: Background and Objective: Despite numerous reports of medicinal uses of Thaumatococcus daniellii (T. daniellii) plant, there remains a dearth of information on the in vivo effect of the seed. In this study, the antioxidant and biochemical effects of T. daniellii seeds in the liver and kidney of male wister rats were assessed. Materials and Methods: Seeds were macerated with ethanol and filtrate was concentrated to yield an ethanolic crude extract. Rats were orally dosed with vitamin C, 500, 1000 and 1500 mg kg–1 of the extracts for 14 days. Antioxidant and biochemical parameters were evaluated. Liver histology was examined. Data were subjected to one-way analysis of variance and expressed as Mean±SEM. Results: It was observed that T. daniellii seed reduced the amount of total body weight gain but did not have any effect on organ weight (p<0.05). Treatment also resulted in significant (p<0.05) increase in hepatic SOD and GSH. It was only at the highest dose that renal GSH increased significantly (p<0.05). These antioxidant effects were associated with maintaining bilirubin concentration and reducing AST activity in the liver (p<0.05). Histopathological observations were in correlation with the biochemical results showing that there was no pathologic abnormality. Conclusion: The results of this study indicate that T. daniellii seed is a source of natural antioxidant and may be exploited for the treatment of kidney and liver diseases.
INTRODUCTION
Thaumatococcus daniellii (Benn.) Benth is a monocotyledonous herb found in rain forests and coastal areas of West and Central Africa1-3. This plant is a large rhizomatous flowering herb which grows to about 3-4 m in height with large papery leaves4. It bears pale purple flowers and a soft crimson coloured fruits containing a few shiny black seeds4. It is commonly referred to as miraculous fruit, miraculous berry, serendipity berry and katamfe/katempfe. In Nigeria, it is popular referred to as soft cane5. It was one of the underutilized and neglected plant species in Nigeria, which grows wildly mainly in cocoa-growing areas of Southwest Nigeria4,6. It is categorized as a non-woody fibre (NWF) plant used to supplement to wood fibre in paper manufacturing7. The plant, commonly known as "ewe eran" in Southwest Nigeria8, is popular for being the natural source of thaumatin, a globally traded protein sweetener9-11. This low-calorie sweetner is about 2000 times sweeter than sucrose and suitable for diabetic patients12. Thaumatin is found in the arils which are located at the top of the seeds9,13. The seeds are black, hard and impervious, wrapped around by a transparent sticky gel-like sac14. The seed was used by locals in ethnomedicine as an emetic and in treating pulmonary problems15. The jelly on the seed can be used as a substitute for agar16. Nonetheless, it has little to no antimicrobial effect17. More so, while on a field trip to the South Western part of Nigeria in a group of four researchers, it was discovered that the seed of the plant when grinded and added to food could have a positive effect on sight and total wellness of an individual though there is presently no scientific basis to support these claims. Several in vitro studies have been done on T. daniellii seed, but little was known on the in vivo effect of the seed14,16. This present study evaluated the biochemical, antioxidant and histopathological effects of T. daniellii seed in experimental rats.
MATERIALS AND METHODS
Chemicals and reagents: Tris-HCl, ethylenediaminetetraacetic acid (EDTA), pyrogallol, Ellman’s Reagent (DTNB) were obtained from Sigma–Aldrich, Germany. Sodium nitrate, thiobarbituric acid (TBA), trichloroacetic acid (TCA) were obtained from Fischer Scientific, UK. All other chemicals and reagents were of analytical grades.
Collection of plant samples and extracts preparations: Fresh fruits of T. daniellii were obtained from local farmers in Ekiti, Ekiti State, Nigeria and authenticated by Dr. J.O. Popoola in the Department of Biological Sciences, Covenant University, Ota, Nigeria. The seeds were removed, air-dried at room temperature (25°C) and pulverized. The powder was macerated in 80% ethanol (1:10 w/v) for 72 h. The resulting mixture was filtered using Whatman (No.1) filter paper and the filtrate was concentrated to dryness at 60°C on a rotary evaporator to give the crude ethanol extract.
Experimental animals and procedure: Thirty male wister rats, 4-6 weeks old, weighing between 100 and 150 g were purchased from the Nigerian Institute of Medical Research (NIMR), Lagos, Nigeria. The animals were maintained under standard laboratory conditions in the animal house of Covenant University in compliance with Covenant University Ethics Committee guide for care and use of laboratory animals. Animal feed and water were given ad libitum. The rats were acclimatized for 2 weeks after which they were randomly distributed into five (5) groups. Group 1 served as the control, Group 2 was dosed with vitamin C (10 mg kg–1 bwt) daily as drug standard, Group 3, Group 4 and Group 5 was daily administered 500, 1000 and 1500 mg kg–1 b.wt., dose of T. daniellii seed respectively for 14 days. At the end of 14 days, the animals were fasted overnight and sacrificed under light ether anaesthesia. Brain, heart, kidney, liver, testes and spleen was excised. Kidney and liver were biochemically and histologically examined.
Preparation of tissue homogenates: Tissue homogenates were prepared using the procedure of Amin et al.18 with slight modification. 0.2 g of hepatic and renal tissue were weighed in 1.8 mL phosphate buffer saline then minced by a homogenizer (Raider Professional, Hamburg, Germany) for 15 min followed by centrifugation for 10 min at 3000 rpm. The formed supernatants were collected and stored at -20°C for antioxidant and biochemical analysis.
Biochemical assay
Estimation of antioxidant parameters: Superoxide dismutase (SOD): 1 mL each of 75 mM of Tris-HCl buffer (pH 8.2), 30 mM EDTA and 2 mM of pyrogallol were added to 50 μL of tissue homogenate. Change in absorbance at 420 nm for 3 min in a spectrophotometer was noted. One unit of enzyme activity was equivalent to 50% inhibition of the rate of autooxidation of pyrogallol as determined by a change in absorbance min–1 at 420 nm19.
Reduced glutathione (GSH): Three hundred microliter of 10% TCA was added to 300 μL of tissue homogenate. It was centrifuged at 5000 rpm for 10 min before 500 μL of the supernatant was transferred to a separate tube. 250 μL of Ellman’ s reagent (19.8 mg of DTNB in 100 mL of 0.1% sodium nitrate) and 1500 μL of phosphate buffer (0.2M, pH 8.0) were added and absorbance was read at 412 nm20.
Malondialdehyde (MDA): One mL of tissue homogenate was added to 2 mL of (1:1:1 ratio) TCA-TBA-HCl reagent (thiobarbituric acid 0.37%, 0.24N HCl and 15% TCA). The mixture was boiled at 100°C for 15 min and allowed to cool. Flocculent materials were removed by centrifuging at 3000 rpm for 10 min. The supernatant was removed and the absorbance read at 532 nm against a blank21.
Estimation of liver function parameters: Commercial test kits obtained from Randox laboratories, England, UK were used for aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL) and direct bilirubin (DBIL) analysis according to manufacturer’s instruction.
Histopathology: Liver tissues were separated from two animals from each group and stored in 10% formalin. They were later sectioned using a microtome, dehydrated in graded alcohol, embedded in paraffin section and stained with hematoxylin and eosin (H&E). Specimens were evaluated with a light microscope. Histopathological changes were evaluated by a pathologist22.
Statistical analysis: Statistical analysis was performed using IBM Statistical Package for the Social Sciences statistical package V 23.0. Data were expressed as means of six replicates±SEM and were subjected to one-way analysis of variance (ANOVA) followed by Duncan multiple range test (DMRT). They were considered statistically significant at p<0.05.
RESULTS
Table 1 shows the amount of total body weight gain in rats treated with T. daniellii seed was significantly lower than the control (p<0.05). There was no difference in relative organ weight between all groups. Only the group dosed with 1000 mg kg–1 had their heart weight significantly (p<0.05) lower than the control.
Table 2 indicate groups treated with T. daniellii seed exhibited an increase in hepatic SOD activity and GSH concentration (p<0.05). There was no significant effect on renal SOD activity and GSH concentration. However, a significant (p<0.05) increase in renal GSH concentration was observed at the highest concentration. T. daniellii seed had no effect on hepatic TBARS level. Nevertheless, a significant increase was observed in renal TBARS level.
The effect of T. daniellii seed on liver function parameters were shown in Table 3. Though there was no effect of T. daniellii seed on TBIL and DBIL, ALT and AST were significantly increased and decreased respectively when compared with the control group (p<0.05).
Figure 1 shows the representative histological sections of liver tissue treated with 500-1500 mg kg–1 of T. daniellii seed where mild periportal infiltration by inflammatory cells was observed as a non-toxic sign of by the extracts concentration.
| Table 1: | Effect of T. daniellii seed on rat and relative organ weights |
Values are expressed as Mean±SEM of 6 replicates. Values with different superscripts in a row are significantly different at p<0.05 compared to the control, TBWG = Total body weight gain | |
| Table 2: | Effect of T. daniellii seed on antioxidant defence system in rat liver and kidney |
Values are expressed as Mean±SEM of 6 replicates. Values with different superscripts in a row are significantly different at p<0.05 compared to the control | |
| Fig. 1(a-e): | Hematoxylin and eosin-stained cross-sectional liver views. (a) Normal histology of control group (b) group administered with Vit. C, (c) Histology of group orally administered 500 mg kg–1 show very mild periportal infiltration by inflammatory cells (black arrow), (d) Histology of group orally administered 1000 mg kg–1 show very mild periportal infiltration by inflammatory cells (black arrow) and disseminated steatosis (green arrow) and (e) Histology of group orally administered 1500 mg kg–1 show mild periportal infiltration by inflammatory cells (black arrow) and congestion of vessels (blue arrow) (Magnification 400X) |
| Table 3: | Effect of T. daniellii seed on liver function parameters in rat liver |
Values are expressed as Mean±SEM of 6 replicates. Values with different superscripts in a row are significantly different at p<0.05 compared to the control | |
DISCUSSION
From the result, there was no decrease beyond 10% of the animal organs weight indicating that T. daniellii seed may be nontoxic to the organs.The observed difference in weight gain may be as a result of the high crude fibre, low fat content and fat yield potential of the seed which has been reported in previous studies15,16. It may also be as a result of the crude fibre present in the seed binding to cholesterol in the gut and delaying gastric emptying and cholesterol synthesis thus reducing body weight. This is in line with the study conducted by Iheagwam et al.23 and Elemo et al.24. Due to the metabolic processes that are simultaneously carried out in cells, reactive oxygen species (ROS) are produced25. Endogenous and exogenous antioxidants (enzymatic and non-enzymatic) protect cells from ROS attack26. Plants are made up of secondary metabolites and phytoconstituents with excellent antioxidant properties, which help in prevention and management of diseases27. SOD, an endogenous enzymatic antioxidant exerts its function by scavenging radicals while GSH (an endogenous non enzymatic antioxidant) conjugates these radicals preventing cellular injury through lipid peroxidation of cell membranes28. MDA is the consequent product of lipid peroxidation and an index of measuring the metabolic level of oxidative stress29.The observable increase in SOD and GSH after T. daniellii seed administration may be as a result of synergistic stimulatory effects of various active components present in the seed on the synthesis of these enzumes. Flavonoids possess antioxidant properties28 while these elements are cofactors responsible for the normal functioning of SOD’s four isozymes30. Chinedu et al.14 and Abiodun et al.16 have previously reported the presence of flavonoids and micronutrients (copper, nickel, manganese and iron) respectively in the seeds. Conversely, high oxalate content in the seed might be responsible for the increase in renal MDA as they are known to aid kidney stone formation31. Liver function bio markers are used to assess hepatotoxicity and hepatocyte integrity32. The significant decrease of AST and insignificant effect on DBIL and TBIL despite the high concentration of T. daniellii seed dosage suggests a hepatoprotective ability of the seed since liver membrane integrity was not affected. The antioxidant activity of this seed may consequently be responsible for the hepatoprotective effect of T. daniellii seed. This relationship has been previously reported by Kamisan et al33. Slight to mild periportal infiltration by inflammatory cells was observed in the liver histology. This observation has been reported as an indicator of positive immune response34. The result obtained in this study was in conformity with in vivo studies carried out on other parts of T. danielli particularly the leaves28,35.
CONCLUSION
This research has shown that Thaumatococcus daniellii seed is a natural source of antioxidant principles. It also showed that the in vivo antioxidant properties were favourable when compared to vitamin C. This seed can be exploited for antioxidant bioactive components for the amelioration of liver and kidney diseases.
SIGNIFICANCE STATEMENTS
This study indicates the possible antioxidant properties of Thaumatococcus daniellii seed on rats’ kidney and liver, which is highly comparable with vitamin C (a known antioxidant). The study could also lead to the discovery of a novel antioxidant lead compound, in the seed, for amelioration of liver and kidney diseases.
ACKNOWLEDGMENT
This study was supported by Covenant University Institutional grant (CUCRID RG 007.11.14/FS).