Anti-hypercholesterolemic potential of diet supplemented with Anchomanes difformis and Pleurotus tuberregium tubers in high cholesterol fed rats

Abstract

Background

One significant ethnomedicinal motivation behind Pleurotus tuberregium (PTT) and Anchomanes difformis (ADT) tubers is cardiovascular-related conditions treatment. This investigation is in this way planned for deciding the impact of PTT and ADT enhanced eating routine on key enzymes linked with hypercholesterolemia in elevated cholesterol fed rodents.

Methods

Rats were isolated into control group, hypercholesterolemic-prompted untreated group, hypercholesterolemic-treated groups with dietary routine containing PTT (5% and 10%), ADT (5% and 10%), 5% PTT and 5% ADT conbination and traditional medication, atorvastatin for 28 days. Ten rodents were utilized for every one of the groups.

Results

Feeding with PTT and ADT comprehensive eating regimen and their combination significantly (P < 0.05) diminished the AChE, HMG-CoA, ALT, AST, ALP, LDH and CK activities and levels of mevalonate, triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDLch), atherogenic indices, MDA and ROS yet significantly increased the SOD, CAT, GPx activities, and level of HDL, GSH when contrasted with HC-initiated untreated rodents. Likewise, histopathological of liver and heart demonstrated no obsessive changes in all the treated groups when contrasted with healthy control group. HPLC fingerprinting of the PTT and ADT aqueous concentrates uncovered the nearness of ferulic acid, vanillic acid, catechin, quercetin, chlorogenic acid, ellagic acid and gallic acid. Notwithstanding, aqueous concentrate of ADT contained plentiful convergences of the polyphenolics when contrasted with PTT concentrate.

Conclusions

The tubers HMG-CoA reductase inhibitory activity could additionally support their antihypercholesterolemic use in folk medication. Accordingly, the tubers may in this way be valuable as restorative nourishment for helpful treatment of clinical conditions related hypercholesterolemia with the ADT diet holding more guarantee.

Electronic supplementary material

The online version of this article (10.1007/s40200-020-00615-z) contains supplementary material, which is available to authorized users.

Introduction

With features, for example, low-density lipoprotein, elevated serum total cholesterol, and decreased high-density lipoprotein levels, hyperlipidemia is probably the highest factor that increase rate of coronary heart diseases [1]. Hyperlipidemia associated lipid issues are considered to cause atherosclerotic cardiovascular disease. As announced by Thiruchenduran et al. [2], pathogenesis of hypercholesterolemia and the resultant cardiovascular disease are because of reactive oxygen species (ROS). As progressively free radicals are shaped, there are trepidations in the status of antioxidant. The resultant impact is oxidative damage to cellular segments [2].

The main enzyme engaged with the metabolism of total cholesterol is 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. From the initial stage, cholesterol is synthesized by the enzyme through catalysis of mevalonate from HMG-CoA, consequently leading to total cholesterol through squalene [3]. The decreased HMG-CoA reductase and mevalonate kinase activities can successfully lessen total cholesterol generation [4–6]. Attempts have been made to create manufactured medications, for example, statins, bile acid sequestrants, and so on that are capable of lowering lipid and help to battle hyperlipidemia. In any case, these medications are not without their challenging reactions [7]. Attempts to lessen these impacts have prompted endeavors to new waves of research to utilize traditional and alternative medicines that are progressively compelling in diminishing cholesterol with next to zero reactions.

Tubers are parts of organs of plants that can be discovered underground, and contain some vital supplements. From the early age of human presence, tubers and natural products have been part of the mainstay of human nourishments, and are as yet being developed today. Tubers are easy to gather, wealthy in supplements, can be put away for future utilizations, and are important part of human eating routine [8].

The utilization of tubers as traditional medication in managing diseases, especially those that are not communicable in nature in Nigeria and many nations on the planet has been since days of yore [9]. World Health Organization affirmed the biodiversity nature of various fruits and tubers, even for those in the same condition [10]. This along these lines suggests that more varieties are added to human eating regimens, while also guaranteeing additional nutritional and health advantages, for example, antioxidative, hypoglycemic, hypocholesterolemic, antimicrobial, and immunomodulatory activities [9]. Various bioactive constituents, for example, phenolic mixes, saponins, bioactive proteins, glycoalkaloids, and phytic acids are answerable for the watched impacts [11].

Hardly any important examples of tubers that have great potentials in term of functional food and nutraceutical fixings, and are utilized by traditional medication practitioners for the reasons for decreasing the dangers of diseases are Anchomanes difformis (ADT) and Pleurotus tuberregium (PTT). As a sizable herbaceous plant, Anchomanes difformis (Blume) tuber is an individual from Araceae family, which is found for the most part in West Africa nations for example Benin, Ghana, Nigeria and Togo, and it develops horizontally, measuring 80 cm by 20 cm across. Bello et al. [12] also affirmed its utilization by various individuals across various culture in the treatment of various diseases.

In Nigeria, there have been reports of its uses in management of various diseases in the literature. Oyetayo [13] affirmed its utilization in type of invention to treat sickness, for example, cough, diabetes, dysentery and throat related problems. Similarly, Burkill [14] affirmed its topical utilization of the tuber and leaves as vesicants and rubefacient and a decoction for the management of oedemas, kidney pains, jaundice and as a diuretic in treating urethral discharge. There have also been various attempts to validate its utilization scientifically in biological investigations [15–20]. In any case, there have been no examinations on the antihypercholesterolemic properties of this vital, yet dismissed, wild vegetable tuber.

Similarly, with shapes that is spherical or ovoid, and of various sizes, Pleurotus tuberregium tuber, which is also alluded to as clerotia, is a storage tuber, with structures ranging from dark earthy colored to black on the surface, and a white underneath [21]. There have been reads on its utilization for medicinal purposes, and as food in Nigeria. Ude et al. [22] affirmed the nutritional prevalence of the tuber than its fruiting body. Similarly, however the tuber may be hard, yet when it is stripped and grounded, it is utilized for vegetable soup reason [21]. There have been concentrates in literature [21, 23, 24] about its lavishness in various medicinal properties, for example, antitumor, immunomodulatory, antigenotoxic, antioxidant, anti-inflammatory, antihypertensive, antihypercholesterolemic, antihyperglycaemic, antiplatelet-aggregating, antimicrobial, and antiviral activities.

Given the prior, investigating the therapeutic advantages of PTT and ADT is important given aforementioned health implication of hypercholesterolemia, and associated famous generation of reactive oxygen species (ROS). Evaluating and comparing the lowering abilities of the hypercholesterolemia properties of both PTT and ADT, as well as assessing their consequences for the activity of HMG-CoA reductase in hypercholesterolemic rats, is consequently convenient.

Materials and methods

Sample collection and preparation

The preparation began with the gathering of new samples of PTT and ADT (Plate 1) from Iropora Ekiti, Ekiti State, South Western Nigeria, its natural habitat. The samples were taken through certification forms with voucher numbers 2,020,099 and 2,020,100 separately in the Department of Plant Science and Biotechnology, Ekiti State University, Ado Ekiti, Nigeria. After an exhaustive washing of the tuber samples, a sanitized table blade was utilized to strip and cut them into pieces. They were along these lines air-dried for seven days at room temperature in a domain that was free from dust. They were thereafter powdered, and kept at -4⁰C in an airtight container before the analysis. So as to access the nutritional content of the tubers, the proximate analysis of the tubers was carried out, and this was utilized to formulate the diets. Thereafter hypercholesterolemic diet and hypercholesterolemic diet enhanced with various percentages of the tubers (drywt/wt) were formulated according to Agunloye and Oboh [25] as appeared in Table 1.

Plate 1.

(a) Pleurotus tuberregium tuber (b) Anchomanes difformis tuber.

Table 1.

Showing feed formulation (in g)

	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6	Group 7	Group 8
Skim milk	28	28	28	28	28	28	28	28
Corn starch	58	56	51	46	51	46	46	56
Premix	4	4	4	4	4	4	4	4
Oil	10	10	10	10	10	10	10	10
Cholesterol -		2	2	2	2	2	2	2
Sample	-	-	5%PTT	10%PTT	5%ADT	10%ADT	5%PTT + 5%ADT	Drug

Notes: Skim milk = 32% protein; The vitamin premix (mg or IU/g) h was the following composition; 3200 IU vitamin A, 600 IU vitamin D3, 2.8 mg vitamin E, 0.6 mg vitamin K3, 0.8 mg vitamin B1, 1 mg vitamin B2, 6 mg niacin, 2.2 mg pantothenic acid, 0.8 mg vitamin B6, 0.004 mg vitamin B12, 0.2 mg folic acid, 0.1 mg biotin H2, 70 mg choline chloride, 0.08 mg cobalt, 1.2 mg copper, 0.4 mg iodine, 8.4 mg iron, 16 mg manganese, 0.08 mg selenium, 12.4 mg zinc, 0.5 mg antioxidant. Group 1: Normal control rats placed on basal diet (Basal); Group 2: rats placed on High-cholesterol Diet (HC); Group 3: rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Pleurotus tuberregium tuber; Group 4: rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 10% Pleurotus tuberregium tuber; Group 5: rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Anchomanes difformis tuber; Group 6: rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 10% Anchomanes difformis tuber; Group 7: rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Anchomanes difformis and 5% Pleurotus tuberregium tuber; Group 8: rats placed on High‐cholesterol Diet (HC) and treated with standard drug (Atorvastatin 0.2 mg/kgBW)

Bioassays

Ethical clearance

The examination was carried out at the Biochemistry Department of University of Ado Ekiti, after the approval of the ethical council of the university has been looked for and gotten the approval. All the animals were appropriately handled all through the examination procedure in accordance with the standard strategies of the Guide for the Care and Use of Laboratory Animals prepared by the Science National Academy and the Health National Institute.

Quantification of compounds by HPLC-DAD

Based on the gradient conditions methods of Boligon et al. [26] and Reis et al. [27], the phenolic compounds by HPLC-DAD Reverse phase chromatographicanalysis was finished.

Experimental rats

There were eighty rats, altogether, utilized for the trial, all of them were adult male, Wistar rats, with weight ranging between 152 ± 2.83. They were all purchased from the Department of Anatomy, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria. They were taken care of and watered ad libitum, and reared and allowed to become accustomed to nature at 25 °C on a 12 h light/dark cycle for about fourteen days before the start of the examination. They were thereafter saved under the same condition for the entire time of the test.

Experimental protocol

Ten rats (n = 10) were utilized for the analysis in each group, making a total of 80 rats on the whole, The Basal group was the principal group, it was taken care of normal diet, the subsequent group was the HC group which was hypercholesterolemic-induced untreated group, while group 3 to group 8 were the hypercholesterolemic groups treated with 5% Pleurotus tuberregium tuber (HC + 5%PTT group), 10% Pleurotus tuberregium tuber (HC + 10%PTT group), 5% Anchomanes difformis tuber (HC + 5%ADT group), 10% Anchomanes difformis tuber (HC + 10%ADT group), a combination of Pleurotus tuberregium and Anchomanes difformis at 5% each (HC + 5%PTT + 5%ADT group) comprehensive diet, and conventional medication, atorvastatin 0.2 mg/kgBW (HC + AT group) individually (Table 2). The variation in the portions given to the treated groups was to recognize the variations in the biological impacts arising from variation in the intakes of the materials. While watching their feed intakes and weights, the formulated diets were given to the rats for 28 days, after which the feed were withdrawn the late evening going before the date of their sacrifice which was finished utilizing the decapitation method under gentle anesthesia. After gathering their blood, which was utilized to prepare serum, it was followed by determination of marker enzymes and lipid profile, while liver and heart tissues were also handled for biochemical and histological analyses.

Table 2.

Showing treatment table

Groups	Treatment
1	(Basal) healthy control rats fed basal diet
2	(HC-induced rats) hypercholesterolemic-induced untreated rats fed hypercholesterolemic diet that contained 2% cholesterol
3	(HC + 5%PTT group) hypercholesterolemic rats fed diet supplemented with 5% Pleurotus tuberregium tuber
4	(HC + 10%PTT group) hypercholesterolemic rats fed diet supplemented with 10% Pleurotus tuberregium tuber
5	(HC + 5%ADT group) hypercholesterolemic rats fed diet supplemented with 5% Anchomanes difformis tuber
6	(HC + 10%ADT group) hypercholesterolemic rats fed diet supplemented with 10% Anchomanes difformis tuber
7	(HC + 5%PTT + 5%ADT group) hypercholesterolemic rats fed diet supplemented with 5% Pleurotus tuberregium and 5% Anchomanes difformis tubers combination
8	(HC + AT group) hypercholesterolemic rats treated with atorvastatin 0.2 mg/kgBW by orally gavage.

Preparation of tissue homogenates

A cold saline solution was instantly used to rinse the liver and heart tissues separately, and was homogenized in phosphate buffer (pH 7.2; 1:5 w/v). Thereafter, the homogenate was centrifuged for 10 min at 5000 × g to yield a pellet that was disposed of, and a supernatant which was saved for resulting analysis. The liver and heart were also fixed in Bouin’s fixative for 6 h, separated and stained routinely with haematoxylin and eosin, at that point saw under a light microscope (Olympus/3H - Tokyo, Japan) for histopathological examination.

Biochemical determinations

Assessment of lipid profile and biochemical parameters

Total triglycride, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine transferase (ALT), aspartate transferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA) reductase activities and mevalonate were measured utilizing commercially available packs (Randox Laboratories Kits, St Louis, MO, USA) according to the manufacturer’s rule. The Atherogenic index (AI) and atherogenic index of plasma (AIP) were calculated following the strategy for Takasaki [28] and Onat et al. [29], individually. Cardiac risk ratio (CRR) was also calculated utilizing the technique for Ikewuchi and Ikewuchi [30]. acetylcholinesterase (AChE) activity was resolved utilizing the technique for Ellman et al. [31].

Assessment of antioxidant parameters

Utilizing the method proposed by Clairborne [32], catalase’s activity was resolved in accordance with the absorbance of hydrogen peroxide at 240 nm, pH 7.0, and 25 °C. To decide the activity of Superoxide dismutase (SOD), the strategy for Misra and Fridovich [33] was depended on, and this includes quantifying the hindrance of autoxidation of epinephrine at pH 10.2 at 30 °C. GPx was examined by estimating the vanishing of NADPH at 35 °C as indicated by Paglia and Valentine [34] and the unit is expressed as moles of NADPH oxidized per milligram of protein. Reduced glutathione (GSH) level was dictated by the technique for Ellman [35]. Reactive oxygen species (ROS) level (Hayashi et al., 2007), and lipid peroxidation was resolved as the formation of malondialdehyde (MDA) [36] level were resolved. Protein concentration was controlled by the technique for Lowry et al. [37].

Data analysis

The results were pooled and communicated as mean ± standard deviation, while one way analysis of variance was utilized for comparing the average. This was followed by a trial of Duncan’s different range and least significant contrasts were carried out, and accepted at P ≤ 0.05. The normality test for all the data were also analyzed through Prism (Graph pad, San Diego, CA, USA).

Results

Nutritional effects of PTT and ADT on consumption pattern, body and organ weights in the control and experimental animals

Table 3 shows that at the finish of the 28 days, the HC-induced untreated group had the highest extra body weight of 24%, indicating the impact of untreated hypercholesterolemic condition in the animals as against those in the treated groups. The gain in weight of the HC-induced untreated group is multiple times that of the HC + 5%ADT group, and more than that when compared to that of the HC + 10%ADT group. All the more importantly, when comparing with weight gain caused by hypercholesterolemic diet in HC-induced untreated group, treatment with ADT shows a lowest weight when compared with treatment with PTT and standard medication, with PTT having the lowest impacts on the animals’ weight loss, demonstrating that ADT is the best in decreasing the weight of the animals. A combination of both PTT and ADT at 5% each gives a similar outcome as individual intake of ADT at 10%. Similarly, Table 3 also affirms a higher liver and heart weights in the HC-induced untreated group when compared to those of the treated groups. The high fat diet gave 50% and 87% increament in liver and heart weights of HC-induced untreated group individually. Be that as it may, while treatment with PTT and ADT had lowering impacts on both the liver and heart weights, their individual impacts is lower than that of Atorvastatin treatment. Be that as it may, a combination of both PTT and ADT at 5% each gives a superior impact on liver weight than standard medication. Table 3 also reveals that the average daily feed intakes of all the groups was not significantly (P > 0.05%) not quite the same as each other. The outcome also shows that the tubers improved the food efficiency index by moderately, increasing weight gain through increasing food intake, as saw in the PTT, ADT and their combination enhanced diet-fed hypercholesterolemic animals.

Table 3.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on food intake, body and organ weights in the control and experimental animals

Variables	Basal	HC	HC + 5%PTT	HC + 10%PTT	HC + 5%ADT	HC + 10%ADT	HC + 5%PTT + 5%ADT	HC + AT
Initial weight (g)	150.35 ± 0.66	151.66 ± 0.39	153.73 ± 2.42	154.76 ± 1.23	153.32 ± 0.75	152.56 ± 0.34	151.60 ± 0.86	152.42 ± 0.66
Final weight (g)	166.24 ± 0.96	187.66 ± 0.28	174.97 ± 0.91	173.56 ± 1.47	162.79 ± 0.63	160.83 ± 0.28	159.44 ± 1.21	169.38 ± 0.82
Weight gain (g)	15.89a	36.00b	21.24d	18.80a	9.47c	8.27c	7.84c	16.96a
%Weight gain (%)	11.00a	24.00b	14.00d	12.00a	6.00c	5.00c	5.00c	11.00a
Liver weight (g)	4.72 ± 0.57a	7.06 ± 0.82b	6.61 ± 0.79b	5.55 ± 0.67a,b	5.54 ± 1.35a	4.35 ± 1.35a	4.14 ± 0.78a	4.26 ± 0.90a
Heart weight (g)	0.46 ± 0.13a	0.86 ± 0.15b	0.73 ± 0.09b	0.66 ± 0.14-b	0.54 ± 0.09a	0.40 ± 0.04a	0.43 ± 0.04a	0.46 ± 0.00a
Food intake (g/28 day)	1830.66 ± 155.89	1838.59 ± 144.62	1833.24 ± 193.27	1833.31 ± 113.40	1835.98 ± 165.97	1836.97 ± 153.02	1830.66 ± 252.14	1838.35 ± 119.21
Food efficiency	0.009 ± 0.001	0.019 ± 0.002	0.012 ± 0.001	0.010 ± 0.002	0.005 ± 0.002	0.005 ± 0.002	0.004 ± 0.001	0.009 ± 0.001

Values represent mean ± standard deviation (n = 10). Within a row, values with different superscript letters are statistically (P < 0.05) different. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Effect of dietary inclusion of PTT and ADT on marker enzymes in the control and experimental animals

Table 4 revealed increased activities of serum liver marker enzymes: ALT, AST, and ALP and serum cardiac marker enzymes: LDH and CK on account of the HC-induced untreated group as against those of the control group. Treatment with PTT and ADT significantly lowered the activities of ALT, AST, ALP, LDH and CK as against those of HC-induced untreated group. Notwithstanding, treatment with ADT was more compelling than that of PTT. Similarly, increasing the dosages of ADT had all the more lowering impacts on ALT, AST, and ALP, LDH and CK activities in the serum. On the contrary, Table 4 shows that a significant decrease happened in the activity of LDH and CK in the cardiac tissue homogenate of HC-induced untreated group as against that of the control group. A significant ascent was recorded in activities of LDH and CK because of taking care of with PTT and ADT as against that of the HC-induced untreated group. The addition of ADT to the hypercholesterolemic diet didn’t affect their cardiac capacity, because the activities of LDH and CK in the cardiac tissue homogenate were not significantly different when compared with normal control rats (Table 4).

Table 4.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on marker enzymes in the control and experimental animals

Variables	Basal	HC	HC + 5%PTT	HC + 10%PTT	HC + 5%ADT	HC + 10%ADT	HC + 5%PTT + 5%ADT	HC + AT
Serum
AST (IU/L)	10.87 ± 0.98a	30.12 ± 2.40b	23.75 ± 0.43c	22.80 ± 0.96c	21.57 ± 0.63c	16.15 ± 2.06d	20.11 ± 2.77c	12.53 ± 2.44a
ALT (IU/L)	21.68 ± 2.83a	47.42 ± 0.42b	44.40 ± 0.48c	40.98 ± 0.76d	38.92 ± 0.76d	32.06 ± 0.82e	34.00 ± 1.73e	31.42 ± 1.65e
ALP (IU/L)	6.10 ± 0.91a	16.93 ± 1.59b	11.27 ± 1.08c	9.53 ± 2.73c	8.85 ± 0.31c	6.08 ± 2.39a	6.18 ± 1.42a	8.43 ± 2.57c
CK (IU/L)	5.39 ± 0.29a	16.18 ± 0.28b	14.17 ± 0.45b	10.24 ± 0.43c	7.44 ± 1.66d	6.26 ± 0.48a	8.87 ± 0.45d	6.27 ± 0.24a
LDH (IU/L)	5.27 ± 1.38a	12.84 ± 1.65b	10.65 ± 1.42b	9.39 ± 1.86c	8.02 ± 2.41c	6.06 ± 0.53a	9.54 ± 1.94c	9.42 ± 2.59c
Cardiac homogenate
CK (IU/L)	6.50 ± 0.14a	3.09 ± 1.12b	3.62 ± 0.21b	5.84 ± 0.18a	6.35 ± 0.48a	6.45 ± 0.63a	5.32 ± 0.61a	5.67 ± 0.04a
LDH (IU/L)	14.97 ± 1.67a	7.10 ± 0.87b	9.50 ± 0.52c	11.01 ± 2.05c	13.53 ± 1.00a	14.73 ± 0.58a	13.50 ± 1.15a	13.63 ± 2.37a

Values represent mean ± standard deviation (n = 10). Within a row, values with different superscript letters are statistically (P < 0.05) different. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Effect of dietary inclusion of PTT and ADT on lipid profiles and artherogenic indices in the control and experimental animals

As revealed in Table 5, HC group had increased amounts of total triglycerides (TG), cholesterol (TC), LDL cholesterol (LDLc), decreased amounts of HDL cholesterol (HDLc), and had a high calculated atherogenic indicess (atherogenic index (AI), atherogenic index of plasma (AIP) and cardiac risk ratio (CRR)) compared with the control group. Also, the amounts of TG, TC and LDL cholesterol in the serum and the atherogenic indicess of treated groups were lowered while HDL cholesterol was increased by treatment with PTT and ADT supplementation and standard medication when compared to HC group. Shockingly, treatment with PTT and ADT enhanced diet caused a significant (P < 0.05) decrease in the aforementioned atherogenic indices. In like manner, a significant (P < 0.05) decrease in atherogenic records was obtained in drug-treated rats. By the by, 10% PTT and ADT enhanced diet displayed the higher decrease in lipid profiles and calculated atherogenic indices than 5% PTT and ADT enhanced diet. The impacts of high dosage of PTT and ADT on the lipid profiles were near those given atorvastatin, an intense medication of statin series (Table 5).

Table 5.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on lipid profiles and artherogenic indices in the control and experimental animals

Variables	Basal	HC	HC + 5%PTT	HC + 10%PTT	HC + 5%ADT	HC + 10%ADT	HC + 5%PTT + 5%ADT	HC + AT
Lipid profiles (mmol/L)
TG	6.17 ± 0.75a	16.87 ± 0.93b	14.58 ± 0.80c	12.12 ± 2.13d	12.17 ± 1.32d	9.07 ± 1.54e	12.02 ± 1.31d	10.00 ± 0.95e
TC	6.33 ± 1.15a	21.00 ± 2.10b	16.33 ± 2.30c	11.00 ± 2.64d	15.00 ± 1.60c	8.00 ± 1.20e	11.00 ± 1.15d	8.67 ± 1.15e
LDLc	6.13 ± 0.69a	14.89 ± 1.70b	11.73 ± 1.35c	9.70 ± 0.89c	7.97 ± 0.42c	6.96 ± 0.48a	7.94 ± 0.74c	7.21 ± 0.96a
HDLc	13.67 ± 1.66a	5.86 ± 1.34b	7.46 ± 0.11b	9.35 ± 0.12c	10.28 ± 0.18c	12.86 ± 1.08a	10.28 ± 0.19c	13.32 ± 0.18a
Artherogenic indices
AI	0.20 ± 0.03a	2.71 ± 0.81b	1.19 ± 0.11c	0.54 ± 0.05d	0.27 ± 0.12a	0.22 ± 0.10a	0.46 ± 0.22d	0.25 ± 0.08a
AIP	-0.31 ± 0.09a	0.47 ± 0.12b	0.28 ± 0.01c	0.19 ± 0.06c	0.08 ± 0.03a	0.02 ± 0.00a	0.07 ± 0.02a	-0.12 ± 0.04a
CRR	0.46 ± 0.05a	3.71 ± 0.81b	2.19 ± 0.31c	1.17 ± 0.27c	1.46 ± 0.37c	0.89 ± 0.07a	0.78 ± 0.10a	0.65 ± 0.08a

Values represent mean ± standard deviation (n = 10). Within a row, values with different superscript letters are statistically (P < 0.05) different. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW. AIP = Artherogenic index of plasma, AI = Artherogenic index, CRR = Cardiac risk ratio

Effect of dietary inclusion of PTT and ADT and their combination on liver and serum AChE activities in the control and experimental animals

As appeared in Fig. 1, a noticeable ascent in the serum and liver AChE activities was realized because of hypercholesterolemic diet as against those of control rats. Mmore specifically, a significant decrease was seen in the activity of AChE in serum and liver of rats fed hypercholesterolemic diet enhanced with 5 and 10% PTT, ADT and their combination. By the by, ADT enhanced diet showed the higher decrease (45% and 57%) in AChE activity than PTT enhanced diet (18% and 46%) at higher sample concentration both in the serum and liver separately.

Fig. 1.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on acetylcholinesterase activity in (1a) liver tissue homogenate and (1b) serum of control and experimental animals. Values represent mean ± standard deviation (n = 10). Values are significantly different at φp < 0.05 versus HC-induced untreated rats (HC), *p < 0.01 versus normal control group (Basal), #p < 0.05 versus 5% and 10% ADT supplemented diet. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Effect of dietary inclusion of PTT and ADT on 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA) reductase activity and mevalonate content in the control and experimental animals

As appeared in Fig. 2, HC-induced untreated group demonstrated increased HMG-CoA reductase activity both in the serum and liver homogenates when compared with the control group. The activity of HMG-CoA reductase of treated groups were significantly (P < 0.05) lowered by taking care of with PTT and ADT comprehensive diet when compared to HC-induced untreated group (Fig. 2). Moreover, a significant (P < 0.05) decrease in HMG-CoA reductase activity was obtained in drug-treated rats. Be that as it may, no statistical distinction was seen in the value obtained for HMG-CoA reductase activity among the treated groups. Expectedly, mevalonate content, which is a result of HMG-CoA reductase was significantly (P < 0.05) increased in the serum and liver homogenate of HC-induced untreated group when compared with the control group (Fig. 3). Treatment with PTT and ADT enhanced diet at higher sample concentration and drug caused a significant (P < 0.05) decrease in mevalonate content (serum: 4.91, 3.72; 4.57; liver: 9.31, 6.52, 6.73 individually) when compared to HC-induced untreated group (serum: 8.57; liver: 14.16) (Fig. 3). HMG-CoA reductase inhibitory activity of the PTT and ADT was comparable to atorvastatin, a standard HMG-CoA reductase inhibitor.

Fig. 2.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA) reductase activity in (2a) liver tissue homogenate and (2b) serum of control and experimental animals. Values represent mean ± standard deviation (n = 10). Values are significantly different at φp < 0.05 versus hypercholesterolemic untreated rats (HC), *p < 0.01 versus normal control group (Basal), #p < 0.05 versus 5% and 10% ADT supplemented diet. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Fig. 3.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on mevalonate content in (3A) liver tissue homogenate and (3B) serum of control and experimental animals. Values represent mean ± standard deviation (n = 10). Values are significantly different at φp < 0.05 versus hypercholesterolemic untreated rats (HC), *p < 0.01 versus normal control group (Basal), #p < 0.05 versus 5% and 10% ADT supplemented diet. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Effect of dietary inclusion of PTT and ADT on antioxidative status in the control and experimental animals

The antioxidative status of the treated rats was evaluated utilizing the activities of SOD, CAT and GPx as well as level of MDA, ROS and GSH (Figs. 4 and 5). As appeared in Fig. 4a-f, hypercholesterolemic diet caused a significant (p < 0.05) decrease in SOD, CAT and GPx activities in the liver and heart homogenates of HC-induced untreated rats compared to the normal control group. In hypercholesterolemic rats took care of hypercholesterolemic diet enhanced with 5 and 10% PTT, ADT and their combination and the group treated with atorvastatin, significantly (P < 0.05) higher activity of these enzymes were noted than those in HC-induced untreated rats, in any case, these enzyme activities remained significantly lower than those in normal control rats both in the liver and heart tissue samples (Fig. 4a-f). In addition, the degree of GSH in the liver and heart tissue samples from HC-induced untreated rats were significantly (P < 0.05) lower than those in normal control group (Fig. 5a and b). In any case, significantly (P < 0.05) higher GSH level was seen in hypercholesterolemic rats took care of hypercholesterolemic diet enhanced with 5 and 10% PTT, ADT and their combination and the group treated with atorvastatin. Curiously, there were no significant contrasts in the GSH level between hypercholesterolemic rats placed on 5 and 10% ADT enhanced diet and normal control group. In addition, high fat diet caused a significant (p < 0.05) increase in the liver and heart MDA and ROS levels compared to normal control group (Fig. 5b-f). Curiously, in hypercholesterolemic rats took care of hypercholesterolemic diet enhanced with 5 and 10% PTT, ADT and their combination and the group treated with atorvastatin, significantly (P < 0.05) lower level of MDA and ROS were noted than those in HC-induced rats, nonetheless, this decrease was not up to control level both in the liver and heart tissues (Fig. 5b-f).

Fig. 4.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on superoxide dismutase (SOD) (a and b), catalase (CAT) (c and d), and glutathione peroxidase (GPx) (e and f) activities in liver and heart tissue homogenates of control and experimental animals. Values represent mean ± standard deviation (n = 10). Values are significantly different at φp < 0.05 versus hypercholesterolemic untreated rats (HC), *p < 0.01 versus normal control group (Basal), #p < 0.05 versus 5% and 10% ADT supplemented diet. $p < 0.05 versus 10% PTT supplemented diet, &p < 0.05 versus 10% ADT supplemented diet. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Fig. 5.

Effect of dietary inclusion of Pleurotus tuberregium and Anchomanes difformis tubers on reduced glutathione (GSH) (a and b), malondialdehyde (MDA) (c and d), and reactive oxygen species (ROS) (e and f) levels in liver and heart tissue homogenates of control and experimental animals. Values represent mean ± standard deviation (n = 10). Values are significantly different at φp < 0.05 versus hypercholesterolemic untrexated rats (HC), *p < 0.01 versus normal control group (Basal), #p < 0.05 versus 5% and 10% ADT supplemented diet. $p < 0.05 versus 10% PTT supplemented diet, &p < 0.05 versus 10% ADT supplemented diet. PTT = Pleurotus tuberregium tuber. ADT = Anchomanes difformis tuber. AT = Atorvastatin 0.2 mg/kgBW

Effect of dietary inclusion of PTT and ADT on Histological changes

Representative photomicrograph of the liver and heart of healthy control and treated rats was appeared in Figs. 6 and 7. The photomicrograph of the liver of all groups demonstrated typically estimated halo spaced central vein encompassed by less thickly circulated hepatocytes. Sinusoidal space dilation is apparent (Fig. 6(1) to (8)). The normal histomorphological presentation of the heart tissue of all the groups demonstrated characteristic normal staininng properties and cellular delineation. The cellularity and morphological delineation apear normal (Fig. 7(1) to (8)). No pathological changes were seen in all the treated groups when compared with control group both in the liver and heart.

Fig. 6.

(1) Representative photomicrograph of the liver of healthy control rats fed basal diet, (2) rats placed on High-cholesterol Diet untreated (HC), (3) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Pleurotus tuberregium tuber, (4) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 10% Pleurotus tuberregium tuber, (5) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Anchomanes difformis tuber, (6) : rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 10% Anchomanes difformis tuber, (7) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Anchomanes difformis and 5% Pleurotus tuberregium tuber and (8) rats placed on High‐cholesterol Diet (HC) and treated with standard drug (Atorvastatin 0.2 mg/kgBW) showing a high-power magnification (x400 mag) of the tissue with normal histomorphological presentation of the liver showing typically sized halo spaced central vein surrounded by less densely distributed hepatocytes. Sinusoidal space dilation is apparent (dotted circle: Central vein, red arrow: Sinisoidal space, black arrow: Hepatocyte)

Fig. 7.

(1) Representative photomicrograph of the heart of healthy control rats fed basal diet, (2) rats placed on High-cholesterol Diet untreated (HC), (3) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Pleurotus tuberregium tuber, (4) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 10% Pleurotus tuberregium tuber, (5) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Anchomanes difformis tuber, (6) : rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 10% Anchomanes difformis tuber, (7) rats placed on High‐cholesterol diet (HC) and fed diet supplemented with 5% Anchomanes difformis and 5% Pleurotus tuberregium tuber and (8) rats placed on High‐cholesterol Diet (HC) and treated with standard drug (Atorvastatin 0.2 mg/kgBW) showing a high-power magnification (X400 mag) of the tissue with normal histomorphological presentation of the heart showing characteristic staininng properties and cellular delineation. The cellularity and morphological delineation apear normal (Black arrow- cardiomyocyte nuclei)

HPLC – DAD characterization of the phenolic constituents

The HPLC fingerprinting of the PTT and ADT aqueous extracts (Table 6) revealed the nearness of ferulic acid, vanillic acid, catechin, quercetin, chlorogenic acid, ellagic acid and gallic acid, while gallic acid was not detected in ADT. Generally, the ADT had ample degrees of the polyphenolics compared to PTT.

Table 6.

Phenolic constituents of aqueous extract of Pleurotus tuberregium and Anchomanes difformis tubers

	Composition (mg/g)
Constituents	Pleurotus tuberregium tuber	Anchomanes difformis tuber
Ferulic acid	3.87 ± 0.01a	8.99 ± 0.01b
Vanillic acid	12.16 ± 0.02a	12.47 ± 0.01a
Catechin	0.32 ± 0.00a	3.84 ± 0.02b
Quercetin	0.10 ± 0.00a	23.59 ± 0.02b
Chlorogenic acid	9.17 ± 0.02a	13.28 ± 0.01a
Ellagic acid	7.79 ± 0.01a	13.99 ± 0.02b
Gallic acid	11.77 ± 0.02	ND

Results are expressed as mean ± standard deviations (SD) of three determinations

Averages followed by different letters on the same row differ by Tukey test at P < 0.05. ND = Not determined

Discussion

Two important factors liable for rate of cardiovascular disease are hyperlipidemia and high cholesterol diets [38, 39]. Along these lines, finding another compounds and functional nourishments with abilities to manage HMG-CoA reductase activity, and thus help to forestall vascular diseases through decrease of cholesterol levels is imperative. This examination is an attempt to examine this by examining the comparative impacts of hypercholesterolemia lowering properties of PTT and ADT on the activity of HMG-CoA reductase in hypercholesterolemic rodents.

From the discoveries, there was astonishing impact in the body weight of rats took care of with hypercholesterolemic diets as the discoveries show a noticeable ascent in body weight of HC-induced untreated rats as against the results in the normal control rats. The ability of the tubers to lessen body weight was affirmed because of substantial fall in the body weight of rats that were given the tubers comprehensive diets. Although both ADT and PTT had decreasing impacts on the rats’ body weight, the impacts were more felt in the intake of ADT, proposing that ADT is more compelling than PTT. All the more along these lines, although a combination of both ADT and PTT at 5% each has a significant lowering impacts of body weight of the animals, the impacts is similar to that of 10% intake of ADT, recommending that increasing dosages of ADT or consolidating ADT and PTT at lower portions yields similar impacts.

Then again, the non-significant contrast saw in the feeding pattern across the various groups, indicate that increasing portions of each tuber diets or their combination has very little consequences for the eating habit of the hypercholesterolemic-induced treated rat. This isn’t anyway the case in the index of food efficiency which shows an improvement because of the tuber comprehensive diets utilization.

There have been attempts in literature to show the liver and cardiac damage results of hypercholesterolemia [38–42]. Elevation in the degrees of diagnostic liver and cardiac marker enzymes, for example, ALT, AST, ALP, LDH and CK activities in serum of HC-induced untreated rats is because of peroxide formation induced by hypercholesterolemia as reactive oxygen species (ROS) [42, 43]. This ROS formation increases cellular membrane permeability, intracellular liquid transfer on to intercellular space, bringing about liver and cardiac damage which leads to the leakage or release of liver and cardiac marker enzymes from the liver and cardiac tissues to serum and thus the degree of marker enzymes are raised in the serum while reduced in the cardiac homogenate in HC-induced untreated rats as saw in this examination [42, 43]. Treatment with PTT and ADT enhanced diet significantly reduced the activity of serum ALT, AST, ALP, LDH and CK to normal near levels. As appeared in the activities of the marker enzymes, there was improvement in the functioning of liver and cardiac of hypercholesterolemic rats because of utilization of PTT and ADT compared to HC-induced untreated group.

In the current investigation, the treatment of hypercholesterolemic-induced rats with PTT and ADT supplementation and medication brought about lower serum levels of total cholesterol, triglycerides, LDL-cholesterol, and higher serum levels of HDL-cholesterol than those in hypercholesterolemic-induced untreated rats. This impact was increasingly articulated in rats that had gotten 10% PTT and ADT enhanced diet than in rats that had gotten 5% PTT and ADT enhanced diet. The lipid-lowering activity of the PTT and ADT was perhaps because of restraint of cholesterol biosynthesis and additionally expanded fecal bile release release [44]. Also, serum total cholesterol didn’t just fall, there was also a diminishing in its LDL-cholesterol fraction, which is an essential supporter of cardiovascular infections. Raised serum levels of triglycerides are related with coronary artery disease [45].

A substantial drop in serum triglyceride levels was also realized in treated group because of the intake of PTT and ADT as against those of untreated hypercholesterolemic-induced rats. This outcome could have been an effect of increased catabolism of triglycerides, arising from an ascent in stimulation of plasma lipoprotein lipase activity, several investigations have attempted to utilize this occasion arrangement to explain the lipid-lowering properties of PTT in hyperlipidemic rats [42, 46–49]. As against the situation with HC-induced untreated group, an ascent in the degree of HDL-cholesterol was also found in the treated group. This finding is imperative given the ability of HDL-cholesterol to assemble triglycerides and cholesterol from the plasma to the liver, where these are catabolized and dispensed as bile acids [42]. The decreasing impact realized in lipid because of feeding on PTT and ADT probably won’t be detached to reactivation of lipolytic enzymes for early freedom of lipids from the flow in high fat diet instigated hyperlipidemia [50].

According to Fki et al. [51], an index that is usually used to access the danger of atherosclerosis is atherogenic records. Henceforth, attempt to diminish atherogenic indices is tantamount to lessening the atherosclerosis threat [51]. In the current examination, supplementation with PTT and ADT brought about lower atherogenic indices compared to hypercholesterolemic-induced untreated rats. Subsequently, lipid metabolism enhancements could be achieved through the assistance of the tuber intakes, and this could therefore assist with avoiding atherosclerosis and coronary heart diseases.

Cholinesterase is one of the family of enzymes that hydrolyze acetylcholine and other choline esters. There are two separate cholinesterases in human tissues; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Cholinesterase is combined mainly in hepatocytes and released into the blood [52]. High oxidative stress and free radicals can increase AChE activity [53, 54]. Dal-Forno et al. [55] proposed that an addition in reactive oxygen species (ROS) formation and a significant increment in lipid peroxidation could prompt the introduction of the more active locales of the AChE. The increase in AChE activity in hypercholesterolemic untreated rats may be because of increase in cholesterol, ROS and lipid peroxidation levels caused by the hypercholesterolemic diet in the untreated group. This increase in AChE activity may play a significant job in lipid disarrays. This outcome corroborates with the investigation of Duchnowicz et al. [56] who detailed that metabolic disorder which is a risk factor of hyperlipidemia caused a significant increase in the activity of AChE and BChE in the plasma of patients with metabolic condition when compared with normal subject. The decrease in AChE activity saw in the treated rats may be because of decrease in cholesterol, ROS and lipid peroxidation levels in the serum and liver of rats fed PTT, ADT and their combination enhanced diets.

All the more along these lines, the activity of HMG-CoA reductase was accessed so as to ascertain accurately the mechanism of the antihypercholesterolemic impact of the tubers. Cholesterol homeostasis has dual courses by which it is safeguarded, namely; cholesterol biosynthesis in which HMG-CoA reductase catalyzes rate restricting step and cholesterol absorption of both dietary cholesterol and cholesterol cleared from the liver through biliary emission [57]. Obtained results from the analysis shows that PTT and ADT utilization hinder the activity of HMG-CoA reductase significantly (P < 0.05), indicating the presence of interface with the enzyme and subsequently bringing about decreased cholesterol levels.

There are many factors, for example, thyroid hormone, hydrocortisone, magnesium, adenosine triphosphate, cholesterol and mevalonate levels, cyclic adenosine monophosphate and insulin, glucagon, which are answerable for the control of activity of HMG-CoA reductase [58]. In this examination, mevalonate the after effect of the response catalyzed by HMG-CoA reductase, was viewed as particularly influenced by hypercholesterolemia. A weighty rising was exhibited in the serum and liver amount of mevalonate of the HC-actuated untreated rodents, featuring the usage of HMG-CoA reductase for mevalonate creation which can be supported by the expanded activity of HMG-CoA reductase in the HC-prompted untreated group found in this examination [59]. Be that as it may, the dietary consideration of PTT and ADT and treatement with drug significantly decreased the HMG-CoA reductase activity and this thusly caused decrement in level of serum and liver mevalonate contents in the treated groups when compared with HC-induced untreated group [59].

Free radicals are delivered during hypercholesterolemic atherogenesis [60]. CAT, SOD, and GPx are innate antioxidant guard mechanisms that are contained in living tissues, through which superoxide anions and H₂O₂ are arranged [61]. A fall in the activity of these enzymes is associated with the development of free radicals that are amazingly reactive, bringing about harmful impacts, for example, loss of uprightness and capacity of cell membranes [60, 61]. From the discoveries of this investigation, the higher activities of CAT, SOD, and GPx noted in liver and heart tissue samples from hypercholesterolemic rats took care of hypercholesterolemic diet enhanced with 5 and 10% PTT, ADT and their combination and the group treated with atorvastatin than HC-induced untreated rats may perhaps because of the fact that the tubers and drug acted by improving the action of these antioxidant enzymes [62], as such neutralizing the free radicals produced during hypercholesterolemia [63]. A comparative improvement in the working of these antioxidant enzymes has been noted following the administration of the PTT extract [42].

Reduced GSH, an important non enzymic antioxidant in living frameworks, plays a key job in maintaining the structural uprightness of the cell membrane [64, 65]. In the current examination, the decrease in the degree of GSH saw in HC-induced untreated rats than those in normal control rats may be because of lipidemic oxidative pressure [66]. In any case, significantly (P < 0.05) higher GSH level was seen in hypercholesterolemic rats took care of hypercholesterolemic diet enhanced with 5 and 10% PTT, ADT and their combination and the group treated with atorvastatin. Strangely, there were no significant contrasts in the GSH level between hypercholesterolemic rats placed on 5 and 10% ADT enhanced diet and normal control group. The watched elevated degrees of MDA and ROS in HC-induced untreated rats may conceivably came about because of increased intensity of lipid peroxidation, which is accounted for to happen because of increased free radical creation [67]. The decrease intensity of lipid peroxidation, as encapsulated from the lower level of MDA caused by the dietary addition of PTT and ADT may potentially because of the fact that the tubers scavenged ROS, in this manner forestalling oxidative modification of lipoprotein, and in that leading to decrease in MDA level [68]. Also, this increase in ROS level caused by the hypercholesterolemic diet in the untreated group may probably explain AChE increased activity as earlier stated in the current examination. It has been accounted for that these tubers, often alluded to as rhizomes demonstrates to have significant antioxidant activity more than any different parts [12, 21, 22, 69].

The histological sections of the current investigation indicated that liver cellularity and morphological central vein, sinisoidal space and hepatocyte were very much safeguarded in HC-induced untreated rat and hypercholesterolemic rats fed diet enhanced with 5 and 10% PTT, ADT and their combination and the group treated with atorvastatin. The morphological characteristics of these liver tissues were comparable to those in control groups with no significant distinction watched. Also, no pathological changes were seen in the heart tissues of all the treated groups when compared with normal control group. No pathological changes saw in this investigation may be because of the duration of time (28 days) of introduction of the rats to high fat diet, we along these lines propose longer presentation.

Generally, the ADT had ample degrees of the polyphenolics compared to PTT. Nonetheless, quercetin is the most dominant polyphenol in ADT while vanillic acid is the most dominant polyphenol in PTT as appeared by HPLC finger printing. Quercetin, a dietary flavonoid had been accounted for by Khamis et al. [70] to diminish the serum cholesterol, triglycerides and LDL–cholesterol concentrations in tyloxapol-induced hypercholesterolemia rats. Be that as it may, the higher antihypercholesterolemic impact of ADT compared to PTT could be attributed to the higher degree of quercetin and the mechanism through which this compound potentially do this, could be by their antioxidant and HMG-CoA inhibitory abilities.

Conclusions

These results propose that PTT and ADT broadened insurance against various biochemical changes in hypercholesterolemic rats. The tubers HMG-CoA reductase inhibitory activity could additionally brace their antihypercholesterolemic benefits in local medication. Along these lines, the tubers may in this manner be valuable for therapeutic treatment of clinical conditions associated hypercholesterolemia. Be that as it may, ADT appeared to be the most encouraging. Although, further clinical investigations are required to affirm these results.

Author contributions

ASF and OOA carried out the analyses, analyzed and deciphered the data, and drafted the manuscript. OFL and ASF planned the investigation and participated in analysis and interpretation of data. OFL coordinated the investigation, overhauled the manuscript and approved the final form to be submitted for publication and aided in the analysis and interpretation of data. All authors read and approved the final manuscript.

Funding

This investigation was self-supported and no fund was gotten for the work.

Data availability

The data analyzed and materials utilized in this investigation are available from the comparing author on reasonable solicitation.

Compliance with ethical standards

Ethics approval and consent to participate

This examination was approved by the Ethics Committee of Ekiti State University, Ado Ekiti, Nigeria.

Consent for publication

Not applicable.

Conflict of Interest

The authors declare that they have no contending interest.