Tropical Journal of Natural Product Research

plants contain abundant natural antioxidants, some of which are ellagic acid, lycopene, ascorbic acid, α-tocopherol, and β-carotene. These compounds are reported to be responsible for the anti-aging effect of trèvo. 13,14 Some of the reported biological activities of trèvo include hepatoprotective, neuroprotective, and anticancer activities. 13,15,16 However, there have been few scientific reports to support the health claims reported by the manufacturer. We hypothesized that trèvo can mitigate the cardiotoxicity of cyanide through antioxidant action and of cytochrome C oxidase modulation. The aim of this study is to determine the antidotal effect of trèvo on cyanide-induced cardiotoxicity.

electron donor, and binding to cyanide to reduce the cyanohemoglobin level in the blood. 10 The toxic effect of cyanide is also time-dependent, that is the physiological alteration of cyanide poison normally occurs within 1 h of exposure, however, under some severe cases it might extend beyond 1 h. Tremor peaks at 15 min and 2 h post-exposure, seizure peaks at 2 h post-exposure, difficulty and labour in breathing peak at 5 min and 60 min, respectively. 11 Thus, there is a strong link between the effectiveness of treatment and duration of exposure. In the search for an antidote against cyanide poison, chemicals such as antioxidants, detoxicants, and biochemical regulators are often tested against cyanide poisoning. 10 While common antidotes, such as methylene blue and sodium thiosulfate are recommended, further search for readily available and accessible antidotes are ongoing. 12 Trèvo® is a product packaged in Oklahoma City, USA. Some of the ingredients reported by the manufacturer to be present in trèvo were sourced from plants such as Mangosteen fruit, bacopa, green tea, grape seed, aloe vera, and turmeric. These plants contain abundant natural antioxidants, some of which are ellagic acid, lycopene, ascorbic acid, α-tocopherol, and β-carotene. These compounds are reported to be responsible for the anti-aging effect of trèvo. 13,14 Some of the reported biological activities of trèvo include hepatoprotective, neuroprotective, and anticancer activities. 13,15,16 However, there have been few scientific reports to support the health claims reported by the manufacturer. We hypothesized that trèvo can mitigate the cardiotoxicity of cyanide through antioxidant action and of cytochrome C oxidase modulation. The aim of this study is to determine the antidotal effect of trèvo on cyanide-induced cardiotoxicity.

Experimental Animals
A total of twenty-four rats weighing between 180-200 g were used for the experiment. They were housed at room temperature in plastic cages and were kept under constant healthy environmental and nutritional conditions. They were fed on rat pellets and water ad libitum. All protocols and design were conducted according to the guidelines provided by Basic and Clinical Pharmacology and Toxicology policy for experimental and clinical studies 17 18 induced moderate to severe toxicity with little lethality. The effective dose of trèvo (2 mL/kg bwt) was based on an unpublished pilot study. Administration of trèvo was based on method described by Ilesanmi and Ikpesu 18 .

Tissue preparation
The heart was excised, blotted, dried, weighed, and rinsed with icecold 1.5% KCl, before homogenizing in 0.1 M phosphate buffer, pH 7.4. The homogenate was centrifuged at 12,000 rpm for 10 min using a cold microcentrifuge (IEC: CENTRA-GP8R, DJB Labcare, UK model to obtain the mitochondria fraction that was used for biochemical assays.

Biochemical Assays Oxidative Stress
Assay kits for superoxide dismutase (SOD), catalase (CAT), and acetylcholinesterase (AChE) activities as well as malondialdehyde (MDA), and total protein (TP) were all procured from Abcam®.

Cytochrome C Oxidase
Complex IV (cytochrome c oxidase) was determined by following the oxidation of cytochrome c at 550 nm and was expressed as a firstorder decay rate constant. 19 p53 p53 was assayed, using Enzyme-linked Immunosorbent Assay (ELISA) kits, in heart tissue homogenates according to the methods of Yang et al. 20 Histology Slices of heart tissue were stained with eosin and hematoxylin and observed under an Olympus binocular research microscope (Olympus, New Jersey, USA) and captured 5.0MP Amscope Camera (Amscope Inc., USA) at x400 magnification.

Statistical Analysis
Data were organized, tabulated, and statistically analyzed using SPSS for Windows, Version 16.0. (SPSS Inc., Chicago, USA). For quantitative data, the mean and SD were calculated and were expressed as mean ± standard deviation and analyzed using Analysis of Variance (ANOVA). For comparison of means of more than two groups, the F-test was used. Statistical significance was taken at a P value of less than 0.05.

Effects of trèvo on AChE activity in cyanide-induced cardiotoxicity
The effect of trèvo on AChE activity in rats exposed to cyanide is presented in Figure 1. It was revealed that KCN caused a significant increase in AChE activity, but was reversed after treatment with trèvo at 5 and 60 min post exposure. Comparing the difference between time of trèvo administration showed no significant difference (P > 0.05).

Effect of trèvo on SOD and CAT activities in cyanide-induced cardiotoxicity
The effects of trèvo on SOD and CAT activities in cyanide-induced cardiotoxicity are presented in Figures 2 and 3, respectively. KCN caused a significant reduction in the activity of SOD (P < 0.05). Upon treatment with trèvo, there was a significant increase in SOD activity as compared to KCN group (P < 0.05). However, when the administration time for trèvo was compared, administration of trèvo after 5 minutes post-exposure to KCN had a significant increase in SOD activity as compared to administration of trèvo after 60 minutes post exposure to KCN. Similar to SOD activity, KCN caused a significant decrease in CAT activity (P < 0.05) as compared to the normal control. Treatment with trèvo caused a significant increase in CAT activity when compared to the negative control (P < 0.05). Trèvo administered at 5 min post exposure to KCN caused a higher increase in CAT activity as compared to trèvo administered after 60 min post exposure to KCN.

Effect of trèvo on MDA levels in cyanide-induced cardiotoxicity
The Effect of trèvo on MDA levels in cyanide-induced cardiotoxicity is presented in Figure 4 below. KCN caused a significant increase in MDA concentration as compared to the normal control (P < 0.05). Treatment with trèvo at different time of post exposure to cyanide showed a significant decrease in MDA concentration as compared to negative control (P < 0.05). Comparing the concentration of MDA at 5 min and 60 min post exposure shows that treatment with trèvo after 5 min caused a higher decrease in MDA concentration as compared to trèvo administered 60 min post exposure to KCN.

Effect of trèvo on p53 levels in cyanide-induced cardiotoxicity in rats
The effect of trèvo following cyanide exposure on the concentration of p53 in the heart is presented in Figure 5 below. The concentration of p53 was significantly increased by KCN when compared to the normal control (P < 0.05). Treatment with trèvo caused a significant reduction in concentration of p53 as compared to the negative control (P < 0.05). However, the late administration of trèvo to the rats was not as effective as early administration (P < 0.05).

Effects of trèvo on cytochrome C oxidase activity in cyanide-induced cardiotoxicity in rats
The effect of trèvo on CCO activity in cyanide-induced cardiotoxicity in rats is presented in Figure 6 below. It was observed that KCN caused a significant decrease in CCO activity as compared to normal control (P < 0.05). Treatment with trèvo significantly increased the activity of CCO as compared to the negative control (P < 0.05). However, administration of trèvo 60 min post-exposure to KCN could not reverse KCN-induced inhibition of CCO activity. Figure 7 shows the typical histomorphological manifestation of the cardiac myocytes. I. The myocytes appear striated and branching to join adjacent myocytes. The staining characteristic and nucleic histomanifestation are normal with no deducible pathological alterations. II: vertical sections of cardiomyocytes with striated muscular fibers, intact and typically stained nuclei. The cellular density and distribution are characteristically normal. III: appears characteristically normal with no deducible histopathological alteration. IV: The staining characteristic and nucleic histomorphology are normal with no deducible pathological alterations.  Results are expressed as mean ± SD (n = 6). *Significantly different at P ˂ 0.05 (control vs KCN), # significantly different at P ˂ 0.05 (KCN vs treatment group), ^significantly different at P ˂ 0.05 (trèvo 5 min vs trèvo 60 min).

Histology
Mechanism of cyanide poisoning is due to the inhibition of cytochrome C oxidase (CCO), an enzyme of the mitochondria respiratory chain. 20 This deprives the cell of its full compliments of ATP, resulting in cell dependence on the anaerobic source of ATP. 22,23 This affects various cardiac functions such as centrality and arrhythmia. 1, 23 In this study, the ability of trèvo to ameliorate the cardiotoxicity of cyanide and the effect of the time of administration was evaluated. Our results showed that trèvo reversed the cardiac poison of cyanide through its antioxidant, antiapoptotic, anticholinesterase and increase cytochrome C oxidase activity. Also, the early administration of trèvo was more effective than the late administration.  Results are expressed as mean ± S.D (n = 6). *Significantly different at P ˂ 0.05 (control vs KCN), # significantly different at P ˂ 0.05 (KCN vs treatment group), ^ significantly different at P ˂ 0.05 (trèvo 5 min vs trèvo 60 min).
Cyanide-induced oxidative stress occurs through the inhibition of CCO, an enzyme that catalyzes the reduction of oxygen to water with the concomitant production of ATP. 24,25 This singular act leads to partial oxidation of oxygen leading to the formation of superoxide anion and hydrogen peroxide. [26][27][28][29] This highly reactive species oxidize functional lipids and proteins, leading to cell injury and death. One of the key regulators of cell death is the tumor suppressor protein, p53, 30 this was also evaluated to check if cyanide poison can exert its cell death through p53. Our results showed that cyanide inhibits CCO, which could have been responsible for the observed increase in MDA concentration and expression of p53. 31   Cyanide has also been reported to inhibit antioxidant enzymes and molecules such as catalase and superoxide dismutase, working in a two-way mechanism to aggravate the oxidative stress condition. [32][33][34] In line with this report, our results also showed the inhibition of CAT and SOD activity by cyanide in the heart. Trèvo has been reported to improve the health status of the heart, prevent cardiovascular-related diseases through its antioxidant, immune boosting, and invigorating phytonutrients. While the presence of some of these phytonutrients have not been scientifically proven, the hepatoprotective, neuroprotective, and other therapeutic effects have been reported. 15,16 Our results showed that trèvo, through its antioxidant, hormone boosting, suppression of p53, and stability of mitochondria function was able to reverse the biochemical poison of cyanide in the heart. Antioxidants such as catalase and superoxide dismutase were I IV I III ISSN 2616-0692 (Electronic) 308 © 2020 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License increased, p53 was decreased, and AChE and MDA were decreased. All these biochemical activities showed that trèvo has the potential to be used as an antidote against cyanide poisoning. This antidote effect of trèvo might be linked to the abundance of various bioactive phytochemicals present in the product (as reported on its label). Some of these chemicals have been reported to be cardioprotective. Histological parameters showed that the heart of rats in the control group showed normal heart tissue with normal epicardial and myocardial layer, the cardiac muscles generally are normal. There was no pathological lesion seen in the heart tissue. Exposure to KCN had no observable pathological alteration to the heart tissue, this might be as a result of acute dose, which has been reported not to have much effect on organ morphology. 35 This antidote effect of trèvo might be linked to the abundance of various bioactive phytochemicals present in the product (as reported on its label). Some of these compounds have been reported to be cardioprotective. [36][37][38][39] Though the mechanism by which trèvo reverses the toxic effect of cyanide on biochemical parameters is not well understood, we can deduce based on other reports on some of the antidotes against cyanide, that trèvo might neutralize the toxic effect of cyanide by binding to it or enhance its clearance from the animals. 1,7,9 Also, some of the natural products present in trèvo such as vitamins, flavonoids, and polyphenols can improve the antioxidant state of the animals in countering the oxidative stress induced by cyanide.

Conclusion
From the results, it can be summarized that early administration of trèvo, which is rich in bioactive compounds can be a new therapy against toxic chemicals such as cyanide, through its antioxidant, anticholinesterase and improvement in the activity of CCO in the heart.