International Research Journal of Biochemistry and Bioinformatics

Comparative Biochemical Effects of Natural and Synethetic Pesticides on Preserved Phaseolus Vulgaris in Male Albino Rats

Nweze CC, Muhammad BY, Wandoo Tseaa^*, Rahima Yunusa, Happy Abimiku Manasseh, Lateefat Bisola Adedipe and William Eneh Department of Natural and Applied Sciences, Nasarawa State University, Keffi, Nigeria
¹Department of Natural and Applied Sciences, Nasarawa State University, Keffi, Nigeria
^*Corresponding Author:
Wandoo Tseaa, Department of Natural and Applied Sciences, Nasarawa State University, Keffi, Nigeria, Email: wandootseaa@gmail.com

Received: 16-Feb-2023, Manuscript No. IRJBB-23-89453; Editor assigned: 20-Feb-2023, Pre QC No. IRJBB-23-89453 (PQ); Reviewed: 06-Mar-2023, QC No. IRJBB-23-89453; Revised: 18-Apr-2023, Manuscript No. IRJBB-23-89453 (R); Published: 25-Apr-2023, DOI: 10.14303/2250-9941.2023.67

Introduction

Legumes are the third largest family A significant (P<0.05) decrease was observed in most of the kidney parameters in the groups except for the rats fed beans preserved with dichlorvos in sodium ion of flowering plants belonging to the family Leguminosae also called Fabaceae that produce seeds within a pod and consisting of over 20,000 species. Common legumes include kidney beans, lima beans, peas, clovers, lentils, soybeans, lupins, lotus, sprouts, tamarind green beans and peanuts. Legumes are very essential especially in terms of nutrition. They contain the essential amino acids that are needed for building proteins, complex carbohydrates, dietary fiber, unsaturated fats, vitamins and essential minerals for the needed for human diet (Abdul Salam, et al., 2016).

Phaseolus vulgaris also known as kidney beans is a major legume cultivated worldwide for its edible seeds and pods. It is a highly polymorphic warm season, herbaceous annual crop. It is used in a variety of traditional dishes and regarded as a healthy component of a well-balanced diet. It is a member of the legume family Fabacee, most of whose members acquire the nitrogen they require through association with rhizobia, a species of nitrogen-fixing bacteria. Phaseolus vulgaris contain several vitamins and minerals such as, molybdenum, folate, iron, copper, manganese and potassium. They also contain a variety of bioactive plant compounds such as, isoflavones, anthocyanins, phytic acid as well as Phytohaemagglutinin (PHAs) which is a toxic lectin only found in raw or improperly cooked P. vulgaris. PHAs are carbohydrate binding proteins and interact with mammalian cells (Adekunle, et al., 2005).

Pest is a major causative factor resulting in the low yields and damage of Nigeria crops. They are harmful to the tissue component and developmental stage of the plant. Pest and insect infestation have rendered stored foods worthless, creating the need for the use of pesticides and other alternatives for storage. The dire need for satisfactory result and ignorance of farmers and traders about pesticide toxicity has led to its misuse, abusive application, and inadequate usage (Agarwal, et al., 2008). Different types of pesticides such as aluminum phosphide and dichlorvos are been used to save grains and seeds from rodents as well as pests. The most common insect pest usually blamable for beans damage is known as the bean weevil, Acanthoscelides obtectus. The use of these pesticides is to increase protection against pest or insect attack on post-harvest crops and retain the current production as well as yield level (Alam, et al., 2023).

Natural preservatives such as pepper have been used for years to extend the shelf life of various foods, making them last longer and enhance the colour, taste and aroma of food. It has also gained more acceptances from consumers because of the antioxidant power of spices. Antioxidants are used as food additives to prevent and delay the negative effects of oxygen in food matrix. Also, in most climates where seeds are susceptible to microorganisms or insect infestation, storing seeds in wood ash has proven effective in preventing both rot and insect predation. Wood ash is composed of the organic and inorganic residue remains after the wood combustion, this is used as a means of biological control which is generally favored as a method of storing seeds because it does not have any of those disadvantages of chemicals and tend to be more durable in its effect (Almasieh, et al., 2011).

Dichlorvos and Aluminum phosphide are insecticides used to control households and stored products insects. It acts against insects as a contact and stomach poison.

Dichlorvos is a class of organophosphate used against household insects as well as agricultural pests. Dichlorvos exerts its toxic effect like other organophosphate insecticides by irreversibly inhibiting neural acetylcholinesterase which provokes the accumulation of acetylcholine in synapse with destruction of nerve function (Aprioku, et al., 2013).

Aluminum Phosphide (AlP) is a solid pesticide that rapidly became one of the most commonly used grain fumigants because of its properties which are considered to be near ideal; it is toxic to all stages of insects, highly potent and does not affect seed viability. The tablets or pellets gradually lose their potency on exposure to atmosphere as they release phosphine gas, which is the active pesticidal component and leave behind a nontoxic residue in the form of aluminum hydroxide (Bartels, et al., 1972). It emerges as a poison of suicide as this pesticide has no effective antidote and is freely available in the market. The mechanism of action in the case of an oral intake causes the phosphine gas released to be absorbed by the gastrointestinal tract with simple diffusion and excreted by the kidneys and lungs. Phosphine, like cyanide, is a highly toxic gas that inhibits mitochondrial cytochrome oxidase and cellular oxygen utilization. It rapidly perturbs mitochondrial conformation and inhibits oxidative respiration by 70%. This situation can result in a severe decrease in mitochondrial membrane potential. In presence of this gas cellular superoxide and peroxide radicals are generated, with subsequent cellular damage by lipid peroxidation (Bonilla, et al., 2011).

The primary aim of this study is to evaluate and compare the biochemical effect of Phaseolus vulgaris preserved with natural preservatives and pesticides (Chinedu, et al., 2019).

Materials and Methods

Materials

The P. vulgaris was gotten from the area of cultivation in Keffi, Keffi local government area, Nasarawa state, Nigeria. The sample was collected and transported to laboratory, where they were cleaned and sorted to remove stones and dirt. Natural preservatives used for the research were: Wood ash gotten from neem tree and Capsicum frutescens commonly known as bird eye pepper. Pesticides used for the research were: Dichlorvos and Aluminium phosphide (Hall, et al., 2012).

The chemicals and kits used in biochemical analysis were of analytical grade and purchased from reputable companies.

Methods

Beans preservation and preparation: The stem from neem tree was burnt to get ash. The cooled ash was sieved to remove dirt. Then 300 g was weighed and packed into nylon bags. Fresh birds eye pepper (Capsicum frutescens) was purchased from the local market and dried in the sun. Then 300 g of sun dried pepper was weighed and packed in to nylon bags. The cleaned P. vulgaris sample was divided into five parts, each bucket contained seeds that weighed 1000 g and the buckets were tightly sealed. A bucket of P. vulgaris with no preservative/treatment, the second bucket contained P. vulgaris preserved with 300 g of birds eye pepper, the third bucket contained P. vulgaris preserved with 300 g of ash, the fourth bucket contained P. vulgaris preserved with 2 mls of DDVP and the fifth contained P. vulgaris preserved with 4 tablets of Aluminium phosphide. The buckets containing the beans were properly labeled and stored for a period of six (6) months (Kim, et al., 2010).

After the storage period the sample was milled into powder, packed into clean polythene bags and used as dietary supplement for the rats.

Experimental animals

A total of 36 male wistar albino rats weighing between 150-200 grams were used for this study. They were purchased from university of Jos, animal house. The rats were housed in clean cages, maintained in an air conditioned experimental temperature and left to acclimatize to laboratory condition for two weeks prior to experiment. Standard poultry mash (vital growers mash) were used as a basal diet during the experimental period. The control and experimental animals were provided with drinking water (Kyznietsova, et al., 2015).

Experimental design

The 36 adult male albino rats were randomly selected and divided into six (6) groups with each group having six rats. The following dietary intervention was carried out at a ratio of seventy (70) grams preserved P. vulgaris to thirty (30) grams vital growers mash for duration of 60 days (Lin, et al. 2008).

Group A (Control): Vital growers mash+water. Group B: Vital growers mash+P. vulgaris without preservatives+water. Group C: Vital growers mash+P. vulgaris preserved with of ash+water. Group D: Vital growers mash+P. vulgaris preserved with pepper+water. Group E: Vital growers mash+P. vulgaris preserved with of dichlorvos+water. Group F: Vital growers mash+P. vulgaris preserved with aluminum phosphide+water.

At the end of the experimental period, the animals were euthanized and blood serum samples collected for biochemical analysis.

Biochemical analysis

The biochemical analysis was performed as shown below:

Serum Alanine Transaminase (ALT) and serum Aspartate Transaminase (AST) activity was carried out using teco kit according to the colorimetric method described by Reitman and Frankel.

Blood urea and serum creatinine was assayed using the Bartels and Bohmer method as outlined in the randox kit. Sodium ion was determined according to the method of Trinder and Maruna as described in the teco kit. While potassium ion was determined using the method of Terri and Sesin (Lotti, et al., 2001).

Superoxide dismutase activity was assayed according to the method described by the international federation of clinical and applied chemistry as outlined in the randox kit. Catalase activity was assayed with the randox kit according to the method described by Aebi. Glutathione peroxidase was measured according to the method of Ursini, et al. (Makker, et al., 2009).

The concentration of malondialdehyde was evaluated in the serum according to the method described by Wallin, et al.

The C-reactive protein test was carried out according to the method described by Smits, et al., using the welcome diagnostic kit for its qualitative and quantitative analysis (Maruna, et al., 1958).

Data and statistical analysis

Statistical analysis of the data was carried out with SPSS version 25.0 using One Way Analysis of Variance (ANOVA). The statistically analyzed data were reported as Mean ± SD. Significant difference was accepted at 95% confidence level of probability i.e., if p<0.05.

Results

Effects of consumption on liver enzymes

The result in Table 1 presents the assessment of the changes in the serum levels of liver enzymes of the experimental animals. The results shows that the PV group (24.1967 ± 2.77085) had significant (p<0.05) increase in the levels of serum Aspartate amino Transferase (AST) when compared with the control group (15.3767 ± 1.38721). The group PVA (20.3833 ± 2.93004) and PVP (33.3433 ± 3.12085) were also revealed to be significant (p<0.05) when compared with the control group. The results showed elevated levels of serum AST in PVD (18.1367 ± 1.09187) and PVAL (17.4000 ± 1.88724) groups in comparison with the control group (15.3767 ± 1.38721) even though there was no significance (p>0.05) in the changes. The levels of Alanine amino Transferase (ALT) were also reported in Table 1. The control group showed a significant (p<0.05) increase in the level of serum ALT (15.1167 ± 2.76841) when compared with the serum levels of ALT in groups PVP (30.8200 ± 1.74106) and PVD (18.2330 ± 2.89686). The serum ALT levels of group PV did not show any significant (p>0.05) difference to any other group except group PVP (30.8200 ± 1.74106) where elevations were observed (Mehrpour, et al., 2011).

Table 1. Effect of consumption on serum liver enzymes.

Effects of consumption on kidney function test

The results for the analysis of kidney function showed values that are reported in Table 2. The result shows that the levels of Na+ in control group showed significant (p<0.05) decrease to groups PV and PVP. There was no significant (p>0.05) difference between the control group and the PVA, PVD and PVAL groups. The levels of K+ in the groups PVA, PVAL and PVD showed a significant (p<0.05) decrease when compared with the control group. The results did not reveal any significant (p>0.05) difference between the control group and PV. The results for urea analysis recorded a significant (p<0.05) increase when the PV was compared to the control group. The PVP also recorded a significant (p<0.05) decrease when compared with the control group. When compared to the control group, PVD and PVAL respectively showed no significant (p>0.05) difference. Creatinine levels revealed that the control group was significant (p<0.05) to only PV. The other groups, PVA, PVP, PVD and PVAL showed no significant (p>0.05) difference when compared with the control group (Miliken, et al., 2015).

Table 2. Effect of Consumption on Kidney Function Parameters.

Effects of consumption on antioxidant activity and malondialdehyde concentration

The results for the analysis of antioxidants are presented in Table 3. The results in the table revealed changes in the levels of CAT, even though only the values in group PV (7.73 ± 1.70) and PVP (9.03 ± 1.45) showed significant (p<0.05) increase to the control group (5.78 ± 0.81). The serum levels of CAT in PV (7.73 ± 1.70) showed no significant (p>0.05) difference to the serum levels of CAT in PVP (9.03 ± 1.45). The GPx activity was only significant (p<0.05) in group PVA when compared with the control group. There was a significant (p<0.05) increase in SOD activity in only groups PV (19.60 ± 2.19) and PVP (19.45 ± 1.84) when compared with the control group (15.86 ± 1.84). The levels of MDA in the control group (3.45 ± 1.25) showed significant (p<0.05) increase to only the PVD group (4.98 ± 0.57).

Table 3. Effect of consumption on serum levels of antioxidants and malondialdehdyde.

Effects of consumption on C-reactive protein

Table 4 shows the results for the mean values of the levels of CRP. The table records changes in the values of the CRP in the test groups when compared to the control group. There was a significant (p<0.05) decrease in the levels of CRP in groups PV (57.134 ± 3.16), PVA (56.05 ± 3.20) and PVP (57.08 ± 1.74) respectively when compared with the control group (62.98 ± 4.74). The PVAL group (66.65 ± 2.86) and PVD group (71.35 ± 1.99) respectively recorded significant (p<0.05) increase in CRP levels when compared with the control group (62.98 ± 4.74).

Table 4. Effect of consumption on serum C-reactive protein.

Discussion

The preservation of food and food crops has come a long way. Preservatives are used to keep microorganisms and/or insects that would otherwise pose as danger to stored food and food crops, thereby reducing the integrity of the material. The screening of blood constituents of experimental animals for research plays a vital role in studying the biochemical effect of preservatives. The liver plays a primary role in the detoxification process of whatever gains access to the body. This role contributes to expose the liver to danger. A set of tests exists to check suspected hepatic disorder, measure the outcome of disease, or simply to evaluate the status of liver function. These tests involve the screening of blood, which gives information about a wide range of disease conditions. It is well understood that any destruction or damage to liver causes the release of enzymes from hepatocytes into the serum and later on the condition advances to necrosis or leads to alteration in cell membrane permeability. The present study reveals the changes in the levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT), two common aminotransferases. The group that was fed with beans preserved in pepper, PVP showed an even higher level of AST (33.34 ± 3.12) and ALT (30.82 ± 1.74) when compared to the control group with values of 15.38 ± 1.39 and 15.12 ± 2.77 respectively at p>0.05. This suggests that pepper may be hepatotoxic on its own or in combination with the effect of lectins found in Phaseolus vulgaris. The increase in serum AST and ALT level suggests leakage of these enzymes in the blood which could be from damage to liver. This finding is in agreement with the work by Mohammed and Saleh, on the biochemical effect of pepper on rabbits which shows that capsaicin the active compound of pepper, possesses some chemical and pharmacological properties similar to the classes of drugs that may cause liver damage. Although, this is also contrary to the study by Alam, et al., which suggested that capsaicin possess therapeutic potential against cyclophoshamide induced liver damage. The results of the group PVA shows lower levels of serum AST (20. 38 ± 2.93) than PV (24.20 ± 2.77). This suggests that wood ash that was used to preserve the beans has some therapeutic effects. Wood ash has many constituents, among which is potassium.

In liver damages, there is decrease in the level of potassium. Hence wood ash contains, potassium, it is being hypothesized that the potassium supply ameliorates the shortage of potassium alongside other micro-nutrients. The results also revealed an insignificant increase in the serum activities of AST in the groups PVD and PVAL when compared to the control group. While a significant increase in ALT for PVD that indicates hepatoxicity (Moghadamnia, et al., 2012).

The kidney is a very important organ in the detoxification of the body, its major work being the filtration of wastes. This exposes it to dangerous compounds. Knowledge of the health of the kidney gives an insight on the overall wellbeing of the biochemical activities in the body as well the overall health of the organism. The results emanating from the present study revealed the analysis of some markers of kidney health. The results shows that the rats fed with the P. vulgaris alone had a higher amount of urea. This implies utilization of protein as suggested by Nwaigwe, et al. This could also imply that they kidneys may not able to excrete enough amount of urea. Animals in group PVA and PVP did not show any significant difference in the levels of creatinine groups, this suggests that wood ash and pepper may not have any negative effect on the kidneys ability to clear urea. The significant decrease of urea in group PVP suggests that the amount of protein in beans preserved with pepper made available for digestion is small. Overall this may not stress the kidneys for clearance of urea. In comparison with the control group, PVP group showed a significant decrease in sodium. The osmolarity of body fluids is determined by the serum sodium ion concentration which has regulatory effect on the blood volume. This means that low levels of sodium ion may result to reduced blood pressure. In the case of potassium, PVA, PVD and PVAL groups revealed a significant (p<0.05) decrease when compared to the control group. The decrease from the PVD group is contrary to the study by Nwankoe, et al., which observed an increase in potassium ion in rats due to prolonged exposure of toxic dichlorvos.

Stress is a collective occurrence that attracts cascade of events and is implicated as a cofactor in the severity and development of numerous diseases, and threatens cellular homeostasis by neutralizing oxidants in various tissues of the body. Stress also plays vital role in aggravating several ailments, particularly hepatic inflammation. An excess in the reactive oxygen species production according to AbdulSalam, et al., can affect different tissues following excess lipid peroxidation, oxidation, and different lesions generated in DNA. Aprioku maintains that deviations within the human system from the normal physiological ranges of antioxidants may result in the progression of several diseases in respective tissues. Obviously, antioxidant systems functions by minimizing the over production of oxygen derived free radicals released in excess during stress to protect itself from oxidative damage. Stress response could be perceived as a compensatory mechanism to the disturbances in the living system caused by a stressor by increasing corticosterone levels.

Antioxidant system differs from tissue to tissue and cell type to cell type. Most important in-vivo enzymatic antioxidant defense system include; SOD, CAT, GPx, and GST. They act as antioxidant by inactivating pro-oxidants to scavenge the activities of the free radicals. Antioxidant protection system can also include; dietary antioxidants, endogenous enzymatic and non-enzymatic constituents that regulates’ the overproduction of these ROS. Perhaps, one of the markers of lipid peroxidase (end products of unsaturated fats) widely used in determining oxidative stress measurement in experimental animals is Malondialdehyde (MDA). Most important in-vivo enzymatic antioxidants of focus in experimental research are; Catalase (CAT) and Superoxide Dismutase (SOD) activities of the different tissues considering that they are vital ROS neutralizing antioxidant and index of peroxidation. The enzyme, Catalase (CAT) is an important enzyme in the elimination of hydrogen peroxide (H₂O₂) from tissues. Several suggestions have been made according to Yamada, et al., that catalase is the major enzyme responsible for the elimination of H₂O₂. The results from the present study revealed elevated levels of CAT in group PVP when compared to the control group at p<0.05. This suggests the consumption of PVP increases the antioxidant activity in the blood. This agrees with the claims made by Kim, et al., that the consumption of pepper may increase antioxidant activity. The Levels of MDA shows a significant increase between the PVD and control. The elevated levels of MDA recorded in PVD suggest the possible degradation of H₂O₂, possibly by CAT. This is in line with the claims made by Chinedu, et al., which suggested that the levels of MDA are elevated to confirm the possibility of oxidative stress suggested by decrease levels of CAT. This agrees with the study by Sharma, et al., that the consumption of kidney beans preserved with dichlorvos may induce oxidative stress which may lead to lipid peroxidation. Lipid peroxidation plays a vital role in arthritis and inflammatory diseases. The level of GPx from the study shows that there is statistically significant difference between the control group and the PVA group. This suggests that the consumption of kidney beans preserved with ash has no effect on the level of lipid peroxides. The PVD group had a significant decrease when compared with the control. This suggests that consuming the kidney beans preserved with dichlorvos may lead to oxidative stress. Superoxide dismutase is the primary defense against oxidative stress. The levels of SOD significantly increased in the PVP group against the control group. This suggests the antioxidant potentials of pepper as earlier noted. The results in the table revealed a decrease in SOD levels in group PVD despite it not being significant. This is in line with the results of the other antioxidants in this study which suggest that the preservation of beans with dichlorvos may lead to oxidative stress in the body (Mohamed, et al., 2010).

C-reactive protein is an acute phase inflammatory protein, a highly conserved plasma protein that was initially discovered in 1930 by Tillet and Francis while investigating the sera of patients suffering from the acute stage of pneumococcus infection and was named for its reaction with the capsular polysaccharide of pneumococcus. CRP has been classed as an acute inflammation marker. The present study yielded results of the levels of CRP, the rats in groups PVA and PVP were fed with Phaseolus vulgaris preserved in wood ash and bird pepper recorded values of CRP to be 56.05 ± 3.20 and 57.08 ± 1.74 respectively. This decrease in CRP is significant with the control group and suggests no sign of persistent inflammation. The groups that were fed with beans preserved with pesticides, PVD and PVAL showed very high levels of CRP, 71.35 ± 1.99 and 66.65 ± 2.86 respectively. These values are significantly higher than the values recorded in the control and PV group. This suggests events that would increase the activity of CRP. Pesticides may induce oxidative stress, leading to generation of free radicals and alteration in antioxidants. The most prominent clinical effects of poisoning with pesticides result from their inhibition of Acetylcholinesterase (AchE). Several studies have demonstrated oxidative stress induced by pesticides like dichlorvos and aluminum phosphide in rats and humans.

Conclusion

Despite the effectiveness of these pesticides as preservatives in the storage of legumes to prevent pest attack and the benefits associated with the usage of wood ash and pepper. This research suggests that the selected preservatives used in the experiment possess some toxic effects that may be lethal over a period of time. According to the findings from the study P. vulgaris preserved with pepper for a prolonged duration of time when consumed in excess showed some level of toxicity in the liver when compared to the control group. Despite the increase in liver enzymes, this effect did not reflect on their other biochemical parameters when compared with the control, as there was an increase in antioxidant and decrease in C-reactive protein in the group fed beans preserved with pepper. This suggests that the residual content on the beans may only be harmful to the liver. There was no significant damage to the kidney by the preservatives when compared to the control group. The group fed dichlorvos preserved beans and the animals fed aluminum phoshide preserved beans showed a decrease in antioxidant activity and an increase in malondialdehyde concentration which may cause oxidative damage. Also, the C-reactive protein from the P. vulgaris preserved with dichlorvos and the P. vulgaris preserved with alumunium phosphide showed that there may be severe health implications as it increased significantly when compared to the control group. This is to say that, although these preservatives have being useful and often used in small quantity by farmers, they may lead to cellular damage.

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