This research utilized a randomized subject control experimental design with observational study. The researcher used a convenience sampling technique. The study was conducted in accordance with the Declaration of Helsinki and approved by the Research and Ethical Committee of College of Health Sciences, Niger Delta University. Animal handling was carried out according to Good Laboratory Practice (GLP) and in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.²²

For this investigation, 46 apparently healthy female rats (Rattus norvegicus) weighing between 120 and 210 g were used. The animals were separated into 5 groups of 8 animals per group based on body weight and housed in cages made of metal nesting under standard conditions with temperature range of 25 to 30 °C and relative humidity. They were fed with growers’ mash and distilled water, with 12-hour light and dark cycles. The beddings of the cages were changed, and the cages were cleaned daily with disinfectant. Groups A (negative control) received 2 ml/kg water and normal feed. Groups B (positive control), D, and E received 0.6 mg/kg body weight of combined oral contraceptive pills only once daily in 5-day cycles (4 days of treatment with a 1-day break) for 48 days. Group C received only 500 mg/kg body weight aqueous extract of Allium sativum once daily for 48 days. Groups D and E were post-treated (48 days after exposure to combined oral contraceptive pills) with 500 mg/kg and 700 mg/kg body weight of aqueous extracts of Allium sativum for 4 weeks, respectively. The rats were allowed to acclimatize for 2 weeks before the commencement of the experiment. Out of the 46 rats, 6 were used to determine the lethal dose (LD₅₀) of combined oral contraceptive pills.

A Levofem® tablet, which is a brand of combined oral contraceptive pill made up of 0.03 mg ethinyl estradiol and 0.3 mg levonorgestrel, manufactured by PT Harsen, Jakarta Timur-13750 (Indonesia) and imported and distributed by Deep K. Tyagi Foundation Nigeria was used for the study. The drugs were purchased from Danson Pharmacy Store Opolo, Yenagoa, Bayelsa State, Nigeria. The concentration of the combined oral contraceptive pills used was adopted in accordance with the method described by Toryila et al. ²³ The package contains 28 pills; each white tablet contains 0.03 mg ethinylestradiol and 0.3 mg levonorgestrel, and each brown tablet contains 75 mg ferrous fumarate. Twenty-one white tablets of the drug were dissolved in 100 ml of distilled water. The drug was administered daily by oral gavage syringe. A concentration of 0.6 mg/kg body weight was administered daily in 5-day cycles (4 days of treatment with a 1-day break) for 48 days. The dose and volume of the drug administered was calculated using the following formula:

Garlic bulbs were bought from a local market at Swali in Yenagoa, Bayelsa State, Nigeria. The plant bulbs were taken to the Department of Biological Sciences, Niger Delta University, Amassoma Nigeria, where they were identified by a taxonomist. The garlic aqueous extract was prepared according to the method described by Ghiasi.²⁴ Thirty grams of garlic were crushed and dispensed to 100 ml distilled water in a conical flask. The juice was obtained using a fruit juice extracting machine. The resultant homogenized mixture was then filtered 3 times with a cheese cloth and centrifuged for 10 minutes at 2000 rpm. The clear supernatant was collected in dark bottles and stored in a refrigerator at 2 to 8 °C until it was used. Based on weight of the starting material (30 g per 100 ml), the concentration of prepared garlic was considered to be 300 mg/ml. The animals were treated with garlic aqueous extract (1 ml per 100 g body weight) once daily via gavage for 48 days.

The drug was administered once daily via oral route in a 5-day cycles (4 days of treatment with a 1-day break) using a metal cannula attached to 2.0 ml syringe, for a period of 48 days. Groups B, D, and E were exposed to 0.6 mg/kg body weight except the control group, while group C was administered with 500 mg/kg body weight of aqueous extract of garlic. Groups D and E were post-treated (after 48 days) with 500 mg/kg and 700 mg/kg body weight of aqueous extract of garlic for 4 weeks, respectively.

The up-and-down method of acute toxicity studies was adopted to determine the LD₅₀ of the combined oral contraceptive pills. A total of 6 female albino rats were used (1 rat per dosage). The rats were administered with 0.4 mg/kg, 0.6 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.2 mg/kg, and 1.5 mg/kg, one at a time. The first dosage administered was 0.4 mg/kg, and it was steadily increased by 0.2 mg/kg after 2 days if each rat survived the initial dosage. Signs of toxicity were observed continuously for the first 2 hours after treatment in all rats, then frequently for 4 hours, and thereafter for a period of 48 hours. The surviving animals were monitored for delayed death for a period 7 days, and they were observed for physical signs of toxicity such as dullness, listlessness, tremor, deep breathing, decreased respiratory rate, and death.²⁵

After the required weeks of experimentation, the rats were anesthetized with chloroform and sacrificed. Blood samples were collected via cardiac puncture with syringe and dispensed into ethylene diamine tetra-acetic acid and plain sample containers for biochemical analysis. Thereafter, the rats were excised to collect heart tissues, which were fixed in 10% formal saline for histological investigations.

Serum creatine kinase myocardial band (MB) was estimated by kinetic ultra-violet (UV) method according to International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) as reported by Young²⁶with modification. Serum lactate dehydrogenase was estimated by kinetic UV method according to IFCC as reported by Cervellin et al.,²⁷ with modification. The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and NADH to form NAD . The rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is proportional to the lactate dehydrogenase activity in the sample. Serum cardiac troponin was estimated using enzyme immunosorbent assay (ELISA) as reported by Babuin and Jaffe²⁸ with modification.

The excised heart specimens from each rat were cut into slabs of about 6 mm³ in size and fixed immediately in 10% formal saline. A Histokinette automatic tissue processor- (LEICA TP 1020) was used for tissue processing, and all tissues were embedded in paraffin wax in a tissue embedder (LEICA EG 1160). The tissue blocks were sectioned in a rotary microtome (Heitz 150 Rotary Microtome) at 5 microns. The slides were stained with hematoxylin and eosin using the method described by Ochei and Kolhatkar²⁹ for general tissue architecture. The stained slides were examined under a high-resolution microscope (Olympus BX60MF, Japan), and photomicrographs were taken at a magnification of × 400.

For analysis of the data, Statistical Software for Social Sciences (SPSS), version 23.0 was used. One-sample Kolmogorov-Smirnov test indicated that the data were normally distributed. Two-way analysis of variance (ANOVA) was used to compare the groups. The Bonferroni multiple comparison. method was used for all post-hoc analyses. Data were presented using mean ± standard deviation for all quantitative values. The level of significance was set at 95% confidence interval, and p < 0.05 was considered significant.

Table 1 shows the results of the acute toxicity study carried out. The rats were observed for signs of toxicity (behavioral changes and death) continuously for first the 2 hours after the treatment with combined oral contraceptive pills, then frequently for 4 hours, and thereafter for a period of 48 hours. The surviving animals were monitored for delayed death for a period of 7 days. We observed no signs of toxicity and mortality in the rats administered with 0.4 mg/kg, 0.6 mg/kg, 0.8 mg/kg, and 1.0 mg/kg body weight of combined oral contraceptive pills. However, behavioral changes such as dullness, listlessness, tremor, deep breathing, redness of eye, and decreased respiratory rate were observed in the rats administered with 1.2 mg/kg and 1.5 mg/kg body weight of combined oral contraceptive pills. The LD₅₀ of combined oral contraceptive pills in female rats was estimated as a cut-off value, since no mortality occurred even at values above 1.2 mg/kg body weight.

Table 1

Weight (g)	Dosage (mg/kg)	Remark
150 g	0.4 mg/kg	No toxicity sign/death
180 g	0.6 mg/kg	No toxicity sign/death
150 g	0.8 mg/kg	No toxicity sign/death
180 g	1.0 mg/kg	No toxicity sign/death
180 g	1.2 mg/kg	Dullness, listlessness, tremor, deep breathing, and no death
150 g	1.5 mg/kg	Dullness, listlessness, tremor, deep breathing, and no death
		respiratory rate, reduced response to stimuli, lachrymation, redness of eye.

Table 2 shows the effect of aqueous garlic extract on some cardiac biomarkers and malondialdehyde in female Rattus norvegicus administered combined oral contraceptive pills. Serum troponin-1 (p = 0.000), lactate dehydrogenase (p = 0.0024), and creatine kinase MB (p = 0.001) levels were significantly elevated in the rats treated with only combined oral contraceptive (group B) when compared with the control (group A). Lipid peroxidation product malondialdehyde levels were significantly (p = 0.001) increased in the rats treated with only combined oral contraceptive (group B) when compared with the control (group A). However, there was a significant (p = 0.001) drastic reduction in the serum level of serum troponin-1, lactate dehydrogenase, and creatine kinase MB and malondialdehyde following treatment with 500 mg/kg and 700 mg/kg body weight of Allium sativum (groups D and E). This reduction was more pronounced in the rats treated with 700 mg/kg body weight of the garlic juice.

Table 2

Treatments	cTn-1	LDH	CK-MB	MDA
	(ng/L)	(IU/L)	(IU/L)	(nmol/L)
GRP A	0.11 ± 0.02	48.88 ± 11.53	10.00 ± 1.46	1.20 ± 0.24
GRP B	0.31 ± 0.03	69.36 ± 10.06	13.50± 1.06	2.46±0.66
GRP C	0.10 ± 0.03	47.32 ± 4.77	10.10 ± 0.96	1.06 ± 0.09
GRP D	1.15 ± 0.04	51.04 ± 7.44	9.20 ± 1.15	1.71±0.35
GRP E	0.09 ± 0.02	44.49 ± 5.55	10.30 ± 1.82	1.40 ±0.16
P value	0.000*	0.002*	0.001*	0.001*
F value	21.85	0.58	1.39	9.24

pP values < 0.05 are considered significant. CK-MB: creatine kinase MB; cTn1: cardiac troponin-1; GRP: group; LDH: lactate dehydrogenase; MDA: malondialdehyde. Group A (control) received feed and water. Group B received 0.6 mg/kg body weight of combined oral contraceptive pills. Group C received 500 mg/kg body weight of aqueous garlic extract. Group D received 0.6 mg/kg combined oral contraceptive pills + 500 mg/kg body weight of aqueous garlic extract. Group E received 0.6 mg/kg combined oral contraceptive pills + 700 mg/kg body weight of aqueous garlic extract. All post hoc testing was conducted using Bonferroni multiple comparison. *Significant difference observed, with p < 0.01. Significant differences were observed for the following: cTn1 concentrations between GRP A and GRP B (p = 0.000); cTn1 concentrations between GRP D and GRP B (p = 0.000); CcTn1 concentrations between GRP E and GRP B (p = 0.000); cTn1 concentrations between GRP D and GRP E (p = 0.046); LDH concentrations between GRP A and GRP B (p = 0.014); LDH concentrations between GRP D and GRP B (p = 0.036); LDH concentrations between GRP E and GRP B (p = 0.003); CK-MB concentrations between GRP A and GRP B (p = 0.007).

As shown in Figure 1, microscopic examination of hematoxylin and eosin-stained slides of heart tissues revealed that the transverse section of the control group (group A) showed normal histological features of the heart. The cardiocytes were normal, all suggestive of normal cardiac tissue architecture. Figure 2 shows the photomicrograph of a transverse section of the heart of an adult female rat administered with 0.6 mg/kg body weight of combined oral contraceptive pills orally for 48 days (group B). The section showed alteration in normal morphology of the heart with localized area of ischemic necrosis suggestive of obstruction in blood flow (ischemic heart disease). The substance administered is cardiotoxic at concentration and duration. Figure 3 shows the photomicrograph of a representative transverse section of an adult rat administered with 0.6 mg/kg body weight of combined oral contraceptive pills orally for 48 days and treated with 500 mg/kg body weight of aqueous garlic extract for 4 weeks after administration of oral contraceptive pills (group D). The section showed hypertrophy of the cardiocytes. The substance administered is non-toxic and shows potential ability to reverse the ischemic changes observed in group B. Figure 4 shows a photomicrograph of a representative transverse section of an adult rat administered with 0.6 mg/kg body weight of combined oral contraceptive pills orally for 48 days and treated with 700 mg/kg body weight of aqueous garlic extract for 48 days after administration of oral contraceptive pills (group E). The section showed normal histomorphology of the heart tissue. The substance administered is non-toxic and shows potential ability to reverse the ischemic changes observed in group B.

Figure 1

Figure 2

Figure 3

Figure 4