Cardio-haematological and oxidative assaults in Wistar rats: the roles of Nicotiana tabacum leaf extracts

Received 09 Mar, 2021

Accepted 13 Aug, 2021

Published 01 Oct, 2022

Background and Objective: Cow urine extract of Nicotiana tabacum leaf, “adimenu” is being used by a significant percentage of Nigerian youths to manage various conditions without enough scientific information to refute or support the usage. This research investigated the effect of single and repeated doses of oral administration of aqueous and cow urine extracts of Nicotiana tabacum leaf on the lipid profile and oxidative and haematological status of Wistar rats. Materials and Methods: Seven groups of five male rats each were used. The control group received 0.5 mL of distilled water, groups A and D received 8 mg kg‾¹ body weight of cow urine and aqueous extracts, respectively, orally, once daily for 28 days, groups B and E received 16 mg kg‾¹ body weight of cow urine and aqueous extracts, respectively, orally, thrice-a-day for 28 days while groups C and F received 32 mg kg‾¹ body weight of cow urine and aqueous extract, respectively, orally, thrice-a-day for 28 days. Biochemical parameters were analyzed in sera and blood samples. Results: Administration of single and repeated doses of the extracts revealed that single and repeated doses of the extracts significantly increased the activity of SOD and catalase as well as the level of LDL and atherogenic index while HDL, RBC, Hb, HCT, MCH and MPV significantly decreased. Conclusion: The administration of the extracts was found to induce oxidative stress, cause anaemia and impair heart functionality

INTRODUCTION

Nicotiana tabacum is an annual plant that is grown across many countries for its leaves, which can be refined into tobacco for commercial purposes¹. The average height of the N. tabacum plant is between 1 and 2 m and it is widely distributed throughout the world’s tropical regions². Except for the seed, almost every part of the plant has a varied concentration of nicotine that increases with age depending on factors such as species, type of land and weather conditions³. Tobacco can be consumed in two forms: Smoking and smokeless tobacco. Snuff (dry and moist) and chewing tobacco are smokeless tobacco varieties, whereas cigarettes, cigars, bidis, kreteks, pipes, water pipes (also known as shisha) and paper-rolled cigarettes are smoking tobacco varieties^4,5.

Cigarette smoking, the far more popular way of using tobacco, exposes both active and passive smokers to a wide range of toxins. This can result in increased oxidative stress, decreased antioxidant availability, increased inflammation, altered lipid profile and nicotine addiction⁶.

Nicotine, the primary component of tobacco, causes addiction to all tobacco products and alters brain chemistry, making abstinence impossible for heavy smokers and posing health risks⁷. Tobacco carcinogens cause a wide range of tumours, including lung, prostate, stomach, dental and bladder cancer, among others. Administration of the extract has been reported to cause liver damage⁸.

Despite the numerous health risk posed by Nicotiana tabacum consumption, people continue to use cow urine as a solvent of extraction of N. tabacum leaf popularly known as “Adimenu or Grade 1” for acclaimed therapeutic uses. This prompted an investigation into the effect of the administration of the cow urine and aqueous extracts of Nicotiana tabacum leaf on the lipid profile and oxidative and haematological status of Wistar rats to engender the assessment of potentially harmful effects from repeated and long-term use of the Nicotiana tabacum leaf-based concoctions.

MATERIALS AND METHODS

Study area: The research was carried out between November, 2019 and December, 2020.

Plant material and authentication: A dried leaf of N. tabacum was purchased in November, 2019 from Ganmo market (8.4190°N, 4.6086°E) in Ifelodun LGA of Kwara State, Nigeria. The plant was identified and authenticated by Mr. Bolu Ajayi of the Herbarium of Plant Biology, University of Ilorin and the voucher number UIL/002/11011 was assigned. The plant name, Nicotiana tabacum L. was checked (data supplied on 2012-03-26).

Experimental animals: Thirty-five albino rats weighing 150.50±8.20 g were obtained from the Animal House of the Department of Chemical Sciences, Summit University Offa, Kwara State, Nigeria.

Assay kits and reagents: Total cholesterol and triglycerides High-Density Lipoprotein (HDL) kits were products of Randox Laboratories Ltd., Antrim, UK. Other chemicals and reagents used were of analytical grade.

Preparation of aqueous and cow urine extracts of N. tabacum leaf: The dried leaf of N. tabacum (400 and 200 g) was extracted in pre-boiled cow urine (4 L) and distilled water (1.5 L), respectively for 24 hrs at room temperature. The mixtures were separately filtered using Whatman no 1 filter paper and the filtrates were freeze-dried (Labconco Freeze Drier, Model 64132, Kansas City, Missouri, USA) to give the aqueous and cow urine extracts of 42.76% and 34% yields, respectively. The extracts were reconstituted in distilled water to give the desired doses of 8, 16 and 32 mg kg^–1 body weight.

Ethical clearance: This study was carried out after ethical approval from the Summit University Ethical Review Committee with approval number SUERC/CONAS/2020/001.

Table 1:

Animals grouping and extract administration

Groups	Dosage (mL)
Control	0.5 mL of distilled water
A	8 mg kg–1 body weight of cow urine extract of N. tabacum leaf once daily
B	16 mg kg–1 body weight of cow urine extract of N. tabacum leaf thrice daily
C	32 mg kg–1 body weight of cow urine extract of N. tabacum leaf thrice daily
D	8 mg kg–1 body weight of aqueous extract of N. tabacum leaf once daily
E	16 mg kg–1 body weight of aqueous extract of N. tabacum leaf thrice daily
F	32 mg kg–1 body weight of aqueous extract of N. tabacum leaf thrice daily

Animal grouping and extract administration: Healthy male Wistar rats weighing 150.50±8.20 g were kept in well-ventilated cages with free access to rat feed and clean water ad libitum. Thirty-five animals were divided into 7 groups (A, B, C, D, E, F and Control) of 5 rats each as outlined in Table 1.

Preparation of serum and determination of biochemical parameters: The procedure described by previously⁹ was adopted for the preparation of serum. Briefly, the blood was collected by cutting the jugular vein and the blood was drained into sterile sample bottles (plain and anticoagulant). Blood samples in the plain sample bottles were left undisturbed at 25°C for 30 min to form clots after which the samples were centrifuged at 1282 g for 5 min. After centrifugation, the sera were collected by a means of Pasteur pipette into a clean sample bottle, appropriately labelled and stored in a freezer at 4°C not later than 72 hrs of preparation for the biochemical assays.

The methods employed¹⁰ were used to assay for the activity of catalase and superoxide dismutase while glutathione transferase and reduced glutathione were assayed¹¹. Cholesterol, triglyceride, high-density lipoprotein and low-density lipoproteins were determined using commercial assay kits (Randox Laboratory Limited, UK) ensuing the manufacturer’s method while, very low-density lipoprotein (VLDL-c) was estimated¹⁰. Haematology was done using an automated haematological analyzer SYSMEX KX-21 (a product of SYSMEX Corporation, Harrier, Japan).

Data analysis: Data were expressed as the Mean±SEM of five determinations. Data were analyzed using a One-way Analysis of Variance followed by Tukey’s post-hoc Test for multiple comparisons. Statistical significance was set at a 95% confidence interval (p<0.05) and graph Pad Statistical Package Version 6.0 was used for the statistical analyses.

RESULTS

Antioxidant enzyme activity: The activity of Superoxide Dismutase (SOD) significantly (p<0.05) increased from 2.28 to 3.11 (U mg^–1 protein) while, catalase activity significantly (p<0.05) increased from 0.36 to 0.43 (U mg^–1 protein) following oral administration of single and repeated daily doses of aqueous and cow urine extracts of N. tabacum leaf. Glutathione Peroxidase (GPX), Glutathione-S-Transferase (GST) and Glutathione Reductase (GSH) activities were not significantly (p>0.05) different from the control in Table 2.

Lipid profile: Oral administration of aqueous and cow urine extracts of N. tabacum leaf produced no significant (p>0.05) difference in cholesterol, triglyceride and VLDL of the animals administered all doses of aqueous and cow urine extract of N. tabacum when compared with the control. However, there was a significant (p<0.05) decrease from 16.32±2.22 to as low as 8.08±0.90 in HDL levels of wistar rats administered doses of aqueous and cow urine extracts of N. tabacum leaf except for the group administered 16 mg kg^–1 body weight of the aqueous extract of N. tabacum leaf three times daily that was not significantly different from the control group administered distilled water (16.32±2.22). Also, there was a significant (p<0.05) increase in LDL level from 11.25±2.94 to 22.12±1.04 and the atherogenic index of all groups administered doses of the extracts up to 2.71±0.53 when compared with the control in Table 3.

Table 2:

Antioxidant enzyme activity of rats administered aqueous and cow urine extracts of N. tabacum leaf

	Superoxide dismutase	Catalase	Glutathione peroxidase	Glutathione transferase	Glutathione reductase
Groups	(U mg–1 protein)	(U mg–1 protein)	(U mg–1 protein)	(U mg–1 protein)	(U mg–1 protein)
Control	2.28±0.17a	0.36±0.01a	4.71±0.73a	0.09±0.02a	21.00±3.24a
A	3.00±0.13b	0.42±0.02b	3.36±0.18a	0.09±0.00a	24.06±4.44a
B	3.05±0.85b	0.40±0.01b	2.99±0.71a	0.11±0.02a	24.70±3.84a
C	2.94±0.08b	0.42±0.02b	3.21±0.90a	0.08±0.01a	23.07±5.07a
D	2.79±0.16b	0.39±0.01b	3.41±0.65a	0.11±0.02a	27.03±7.11a
E	3.11±0.26b	0. 41±0.01b	3.86±0.82a	0.09±0.02a	27.24±5.41a
F	2.90±0.46b	0.43±0.02b	4.36±0.81a	0.08±0.01a	24.63±3.34a
Values are Means±SEM of five replicates and values with different superscripts are significantly (p<0.05) different from one another

Table 3:

Lipid profile of rats administered aqueous and cow urine extracts of N. tabacum leaf

Treatment	Cholesterol	Triglyceride	HDL	LDL-C	VLDL-C	Atherogenic index
groups	(mmol L–1)	(mmol L–1)	(mmol L–1)	(mmol L–1)	(mmol L–1)	(LDL-C/HDL-C)
Control	29.98±1.68a	12.00±2.26a	16.32±2.22a	11.25±2.94a	2.40±0.45a	0.71±0.25 a
A	29.99±1.58a	12.28±1.31a	9.94±1.50b	17.59±1.60b	2.46±0.26a	1.82±0.43b
B	31.34±2.15a	12.61±1.14a	11.78±0.92b	17.04±1.74b	2.52±0.23a	1.45±0.16b
C	33.90±1.01a	14.29±1.12a	08.92±0.64b	22.12±1.04b	2.86±0.22a	2.49±0.27c
D	31.07±2.80a	11.12±0.71a	10.97±1.70b	17.88±2.27b	2.22±0.14a	1.66±0.32b
E	35.82±1.46a	11.65±1.66a	14.52±1.13a	18.97±2.49b	2.33±0.33a	1.32±0.27b
F	31.86±2.23a	11.16±1.08a	08.08±0.90b	21.55±2.66b	2.23±0.22a	2.71±0.53c
Values are Means±SEM of five replicates, values with different superscripts in a column are significantly (p<0.05) different from one another, HDL: High-density lipoprotein, LDL-C: Low-density lipoprotein-cholesterol and VLDL-C: Very low-density lipoprotein-cholesterol

Table 4:

Hematological parameters of rats administered aqueous and cow urine extracts of N. tabacum leaf

Groups/ parameters	Control	A	B	C	D	E	F
RBC (×106)	6.27±0.15a	3.72±0.13c	5.53±0.28b	5.10±0.51b	5.50±0.14b	6.40±0.22a	6.25±0.38a
Hb (g dL–1)	12.6±0.35a	9.98±0.38b	8.67±0.55b	10.07±0.67b	10.58±0.85b	9.37±0.42b	9.37±0.21b
WBC (103 μL–1)	19.70±5.32a	15.73±2.34a	13.03±2.31a	22.13±7.25a	19.04±5.14a	15.37±4.79a	13.47±3.44a
HCT (%)	46.92±2.19a	39.1±4.01b	33.43±2.42b	38.93±0.15b	40.16±3.08b	34.33±0.70b	35.30±2.07b
MCV (fL)	64.04±3.21a	57.68±4.58a	55.97±1.17a	58.2±3.50a	57.2±3.67a	52.53±0.06a	53.47±1.03a
MCH (pg)	17.2±0.47a	14.73±0.38b	14.53±0.83b	15.00±0.92b	15.02±0.66b	14.3±0.46b	14.2±0.36b
MCHC (g dL–1)	26.92±1.88a	25. 68±2.52a	25.97±1.88a	25.83±1.63a	26.40±2.02a	27.27±0.86a	26.57±0.97a
PLT (103 μL–1)	761.40±132.8a	1065.00±154.22a	1216.00±327.38a	885.33±33.65a	995.6±227.77a	1032.76±108.36a	1105. 33±351.76a
RDW-SD (fL)	44.34±5.77a	41.35±9.76a	37.17±1.98a	39.80±3.42a	35.70±3.81a	34.50±2.05a	34.33±0.75a
RDW-CV (%)	761.40±132.8a	1065.00±154.22a	1216.00±327.38a	885.33±33.65a	995.60±227.77a	1032.67±108.36a	1105.35±351.76a
PWD (fL)	12.86±1.82a	10.80±1.09a	9.47±0.71a,b	11.50±0.30a	11.22±1.39a	9.73±0.38a,b	9.83±0.68a,b
MPV (fL)	9.00±0.70a	7.93±0.57b	7.57±0.31b	8.21±0.17b	8.06±0.32b	7.37±0.25b	7.40±0.10b
P-LCR (%)	20.54±4.96a	13.28±3.77a	10.47±2.65a,b	15.77±0.70a	14.08±3.04a	9.90±0.89a,b	10.20±0.62a,b
PCT (%)	0.69±0.13a	0.85±0.17a	0.92±0.23a	0.73±0.03a	0.80±0.22a	0.76±0.17a	0.82±0.25a
Values are Means±SEM of five replicates, values with different superscripts in a row are significantly (p<0.05) different from one another, RBC: Red blood cell count, HB: Haemoglobin, WBC: White blood cell, HCT: Hematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular haemoglobin, MCHC: Mean corpuscular haemoglobin concentration, PLT: Platelet, RDW-SD: Red blood cell distribution width standard deviation, RDW-CV: Red blood cell distribution width coefficient of variation, PDW: Platelet distribution width, MPV: Platelet volume, P-LCR: Platelet-large cell rate and PCT: Platelet crit

Hematological parameters: Administration of single and repeated daily doses of N. tabacum leaf extracts produced a significant (p<0.05) decrease in Mean Platelet Volume (MPV (fL)) from 9.00±0.70-7.37±0.25, Mean Corpuscular Hemoglobin (MCH (pg)) from 17.2±0.47-14.2±0.36, Hematocrit (HCT (%)) from 46.92±2.19-33.43±2.42, Hemoglobin (HB (g dL^–1)) from 12.6±0.35-8.67±0.55 and Red Blood Cell count (RBC (×10⁶)) from 6.27±0.15 to as low as 3.72±0.13 except the RBC of the repeated doses of 16 and 32 mg kg^–1 body weight of aqueous extract of N. tabacum leaf that were not significantly different from the control in Table 4. However, the White Blood Cell (WBC (10³ μL^–1)), Mean Corpuscular Volume (MCV (fL)), Platelet (PLT (10³ μL^–1)), Platelet Distribution Width (PDW (fL)), Platelet-Large Cell Rate (P-LCR (%)), Platelet Crit (PCT (%)), Red Blood Cell Distribution Width Standard Deviation (RDW-SD (fL)), Red Blood Cell Distribution Width Coefficient of Variation (RDW-CV %)) were not significantly different from the control.

DISCUSSION

The surge in the use of herbal medicines for healthcare¹² is attributed partly to its safety, acceptability and affordability by the poor masses. Patients in some cases became addicted to some herbal medications/concoctions, the most notable in Southwest Nigeria is the extracts of Nicotiana tabacum leaf popularly known as “adimenu”.

Endogenous antioxidants such as Superoxide Dismutase (SOD), catalase and glutathione peroxidase are known to attenuate the generation of Reactive Oxygen Species (ROS) by removing potential oxidants or by transforming ROS/RNS into relatively stable compounds. The cell protects itself from oxidative damage by recruiting GSH and scavenging enzymes such as SOD and GST as a first-line cellular defence in response to oxidative challenges to protect cellular integrity¹³. The significant increase in the antioxidant activities of SOD and catalase as evident in this study might be due to the presence of saponins, polyphenols and flavonoids that have been earlier reported to have prolific antioxidant capacity¹⁴. The increased activity of SOD and catalase suggests that there were a lot of superoxide radicals produced during the metabolism of nicotine.

The lipid profile assay presented a unique trend of results in the concentration of total cholesterol, triglyceride, very low-density lipoprotein and atherogenic index. The alterations in lipid profiles have also been linked to tobacco use. The significant increase and decrease in the level of LDL and HDL concentration can lead to heart disease, particularly atherosclerosis which is supported by a significant increase in the level of the calculated Atherogenic Index (AI)¹⁵.

Assessment of haematological parameters can be used to determine the extent of the harmful effect of the foreign compound on the blood and also explain blood relating functions of biochemical compounds¹⁶. Erythrocytes are particularly susceptible to oxidative damage as a result of high polyunsaturated fatty acid content in cell membranes and high concentrations of oxygen and haemoglobin, which promotes oxidative processes¹⁷. This was evidenced by the decrease in the number of Red Blood Cells (RBCs), haemoglobin, hematocrit, MCH and MPV level in rats administered extracts of N. tabacum leaf in this study. The observed decrease in these 16 parameters could be because nicotine interferes with vitamin C levels, which is crucial for functional iron absorption and inhibits iron uptake¹⁸. Also, the nicotine reduction of red blood cells may be due to peroxidative membrane damage via ROS production¹⁷ and/or by ROS reacting with haemoglobin, altering its structure and releasing free iron ions that, in turn, increase the generation of ROS¹⁹. The decreased RBC, MPV and Hb levels agree with the previous study on rats administered aqueous extract of N. tabacum leaf²⁰.

CONCLUSION

The cow urine and aqueous extracts of Nicotiana tabacum leaf alter the lipid profile, antioxidant and haematological status. This can result in oxidative stress, anaemia and heart disease. Based on this result, users of this concoction should do so with care and not take the extract daily.

SIGNIFICANCE STATEMENT

The study discovered that aqueous and cow urine extracts of N. tabacum leaf are capable of inducing toxicological assaults on the oxidative status and cardiovascular system of Wistar rats. The results of this research will serve to inform researchers of the toxicological effects of N. tabacum extracts and thus prompt research into the molecular mechanism of its toxicity.

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