Spices find wide applications in homes and many industries. Clove and ginger are one of the most popular spices with numerous applications. Clove, Syzygium aromaticum, is a widely known spice and is the flower bud of a tree that belongs to the Myrtaceae family [1,2]. It originated from Indonesia and is widely cultivated in Asia and other parts of the world [3]. Clove is rich in bioactive compounds such as eugenol, eugenyl acetate, trans-caryophyllene, β-caryophyllene, polyphenols, tannins, and triterpenoids [2]. These rich bioactives make it a sought-after home remedy for illness from dental complications to inflammation of various kinds [2]. The active compounds found in clove and ginger can be effectively extracted using solvents such as water, ethanol, and methanol [2]. These compounds are responsible for the fragrance, antioxidant, antibacterial, antifungal, anticancer, and anti-inflammatory properties of clove [4].

Ginger (Zingiber officinale Roscoe, Zingiberaceae) is one of the most widely utilized spices and contains some bioactive compounds such as gingerol and shogaol [5]. These bioactive compounds are responsible for the antioxidant, antibacterial, antifungal, anticancer, and anti-inflammatory properties of ginger [5]. Clove and ginger are used in combination with other spices and herbs for the production of herbal tea. Apart from herbal tea, extracts from these two spices are used for different purposes. In some cases, the spices are used conventionally in diverse concentrations in homes, which might pose health risks. According to Nirmala et al. [2], the Food and Drug Administration (FDA) reported that phenolic extract of 1000 mg/kg did not have an adverse effect on Wistar rats. However, there is a need to study the safety of the extended doses of these extracts on humans. Therefore, the aim of this study was to assess the safety doses and evaluate hypolipidemic activities of clove and ginger ethanolic extract on albino rats.

Preparation of Ginger and Clove Extract

Preparation of the Aqueous Extract from Clove and Ginger

The clove was cleaned, sorted, washed, and oven-dried at 60°C and was ground into powder using a blender. The pulverized clove was then soaked in 70% ethanol and was kept for 48 hours at room temperature (26-30°C) and filtered. The filtrate was evaporated to dryness T using a rotary evaporator, and the extract was stored in a cool, dry place for further studies. The same procedure was adopted for the preparation of the ethanol extract of ginger.

The percentage yield of the extract was calculated using the formula:

%Yield = weight of the extract / weight of plant material × 100.

Acute Toxicity Study of the Ethanol Extract of Clove and Ginger on the Albino Rat

Toxicity Study of the Plant Extract

Sub-Chronic Toxicity Studies

Twenty-four rats were randomly divided into 7 groups, with Group I as the control group without any treatment. Group 2 to 7 was treated with low dose (1 mg/kg body weight of the albino rats) and high dose (5 mg/kg body weight of the albino rats) as below:

Experimental Design/Treatment for the Toxicology Studies of the Ginger and Clove Ethanolic Extract were Carried Out as Shown Below

Group 1: Normal control animal without the plant extract.

Group 2: low dose of the single clove ethanolic extract.

Group 3: High dose of the single clove ethanolic extract.

Group 4: low dose of the single ginger ethanolic extract.

Group 5: High dose of the single ginger ethanolic extract.

Group: 6 low doses of both (50% each) clove and ginger ethanolic extract.

Group 7: High dose of both (50% each) clove and ginger ethanolic extract

Acute Toxicity Test

A total of fifty-seven overnight-fasted mice (n = 57) were separately used for evaluating the acute toxicity of the plant extract. Groups I was used as the control group without any treatment. Group 2 to 19 was treated with varying doses of both the single and combined doses of the clove and ginger extract. The doses for the different groups are as shown below: 

Group 1: Normal control animal without the plant extract.

Group 2: 10 mg/kg of clove ethanolic extract.

Group 3: 100 mg/kg of clove ethanolic extract.

Group 4: 1,000 mg/kg of clove ethanolic extract.

Group 5: 1600 mg/kg of clove ethanolic extract.

Group 6: 2900 mg/kg clove ethanolic extract.

Group 7: 5000 mg/kg clove ethanolic extract

Group 8: 10 mg/kg of the ginger ethanolic extract

Group 9: 100 mg/kg of the ginger ethanolic extract.

Group 10: 1,000 mg/kg of the ginger ethanolic extract.

Group 11: 1600 mg/kg of the ginger ethanolic extract.

Group 12: 2900 mg/kg ginger ethanolic extract.

Group 13: 5000 mg/kg ginger ethanolic extract

Group 14: 10 mg/kg of clove and ginger (50% each) ethanolic extract.

Group 15: 100 mg/kg of clove and ginger (50% each) ethanolic extract.

Group 16: 1,000 mg/kg of clove and ginger (50% each) ethanolic extract.

Group 17: 1600 mg/kg of clove and ginger (50% each) ethanolic extract.

Group 18: 2900 mg/kg clove and ginger (50% each) ethanolic extract.

Group 19: 5000 mg/kg clove and ginger (50% each) ethanolic extract

The LD₅₀ was determined by the graphical method.

Statistical Analysis

A completely randomized design was used for this experiment, and results obtained were analyzed statistically using one-way analysis of variance (ANOVA), which was carried out on the Statistical Package for Social Sciences (SPSS) version 22. The means were separated using Duncan’s Multiple Range and compared for significance at p < 0.05. The group results were presented as means ± standard deviation.

Evaluation of the Lipid Profile of Ginger and Clove Ethanol Extracts

The lipid profile parameters, which include total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TA), and low-density lipoprotein cholesterol (LDL-C). And Very Low-Density Lipoproteins Cholesterol (Low-Density Lipoproteins Cholesterol (LDL-C). LDL-C) were determined using a commercial test kit (Random Laboratory, United Kingdom). The spectrophotometric procedures as outlined by the kit manufacturer were strictly followed to arrive at results.

LD₅₀ of Ginger

Graded doses up to 6000 mg/kg of the extract produced no mortality in rats. The treated rats also did not show signs of severe toxicity like tremor, convulsions, writhing reflexes, and agitations but remained active and physically stable throughout the 24-hour period and a further 7 days of observation. However, 33.33 percent mortality was observed in the third phase, and the group administered 8000 mg/kg body weight, while all mice in the 10,000 mg/kg treated group died, representing 100 percent mortality. The application of Lorke’s formula therefore yielded an LD50 value of 7745.97 mg/kg body weight for the aqueous extract in rats. Results are presented in tables 1-4 below.

Table 1: Percentage Yield of Extracts.

Table 2: Stage 1: Acute Toxicity (LD₅₀) Evaluation of Ginger Extract in Rats.

Table 3: Stage 2: Acute Toxicity (LD₅₀) Evaluation of Ginger Extract in Rats.

Table 4: Stage 3: Acute Toxicity (LD50) Evaluation of Ginger Extract in Rats.

LD₅₀ = (D₀ x D₁₀₀)^1/2

Where:

D₀: Highest dose that gave no mortality.

D₁₀₀: Lowest dose that produced mortality

LD₅₀ = (6000 x 10000)^1/2

LD₅₀ = 7745.97 mg/kg body weight

LD₅₀ of Clove

Graded doses up to 10,000 mg/kg of the extract produced no mortality in rats throughout the 24-hour period of the study and a further 7 days. The animals instead remained active and physically stable throughout the 3 phases of the study period. Other signs of toxicity, including agitation, convulsions, calmness, restlessness, roughness of hairs, writhing reflexes, and uncoordinated movements, were also not observed. The LD₅₀ value for the extract was therefore established to be greater than 10,000 mg/kg body weight. Results are presented in tables 5-7 below.

Table 5: Stage 1: Acute Toxicity (LD₅₀) Evaluation of Clove Extract.

Table 6: Stage 2: Acute Toxicity (LD₅₀) Evaluation of Clove Extract.

Table 7: Stage 3: Acute Toxicity (LD₅₀) Evaluation of Clove Extract.

LD₅₀ >10,000 mg/kg body weight.

Result of the Effects of the Extracts on the Lipid Profile

The result of the effects of the extracts on the lipid profile, as shown in Table 8, reveals that the treatments with ginger and clove ethanolic extracts significantly influenced all lipid parameters compared to the control group. Total cholesterol levels were significantly reduced in all treated groups, with the lowest level observed in the group treated with 800 mg/kg of the combined ginger and clove ethanolic extracts (94.90±1.39), compared to the control group (117.62±1.45). HDL-C levels were higher in the treated groups compared to the control (66.66±0.56), with the highest value recorded in the group treated with 800 mg/kg of ginger extract (69.50±1.38). TAG levels were significantly lower in the treated groups compared to the control (79.82±1.15), with the lowest value observed in the group treated with 800 mg/kg of ginger extract (71.24±1.70). LDL-C levels were markedly reduced in all treated groups, with the most significant reduction in the group treated with 800 mg/kg of the combined ginger and clove extracts (13.69±2.17) compared to the control (35.00±1.70). VLDL-C levels also decreased significantly in all treated groups compared to the control (15.96±0.23), with the lowest value observed in the group treated with 800 mg/kg of ginger extract (14.25±0.34).

Table 8: Results of Anti-Inflammatory Study.

Values are presented as mean ± standard deviation. Means with different letter superscripts are significantly (P < 0.05) different within the column.

The significant reduction recorded in the LDL-C, VLD-L and TAG shows that both the low doses (400 mg/kg) and the high doses (800 mg/kg) of the ginger and clove and the combined extract of ginger and clove have the potential of improving the lipid profile of mice and, as such, can be used as supplements in proving the lipid profile of humans.

The percentage yield reported in this study shows a higher ethanolic yield in clove when compared with ginger. The subchronic and acute toxicity studies also demonstrated the safe doses of clove and ginger extract. However, higher doses above 5000 mg/kg and 6000 mg/kg body weight in ginger and clove, respectively, may result in some side effects such as weakness, depression, and low activity level as observed in the albino rats. Oral administration of ginger above 6000 mg/kg body weight resulted in 33% mortality, which may also result in death or other adverse effects in humans.

Hyperlipidemia, a disease condition, is marked by increasing levels of TG (triglyceride), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) in peripheral blood, as well as a drop in HDL level [6,7]. This disease condition results in the deposition of excess fat or lipid in the blood vessels, thus hindering the free flow of blood. High levels of low-density lipoproteins have been associated with cardiovascular diseases [8]. This increases the risk of heart disease and stroke as a result of the inability of food to flow through the arteries. Some authors have reported hypolipidemic activities of ginger and clove in combination with other spices and plant extracts. Khattab et al. [9] also demonstrated the effectiveness of a combined mixture of ginger and curcumin in significant reduction of hyperglycemia and hyperlipidemia in rats. Salih et al. [10] also documented the hypolipidemic effect of combined ginger and thyme extracts on low-density lipoproteins in rats. A combined therapy of clove and cinnamon extract on zebrafish also showed very strong hypolipidemic activity [11]. This trend is similar to the trend observed in this study. Combined clove and ginger extracts showed a significantly higher reduction in the total cholesterol and low-density lipoproteins when compared with either clove or ginger extract. Low-density lipoprotein (LDL) is the main cholesterol carrier in the bloodstream, and the concentration of LDL-cholesterol is directly correlated with the prevalence of coronary heart disease [11].

However, Fuhrman et al. [12] reported that 250 mg/kg of ginger extract administered to mice for 10 weeks reduced their LDL. Furhman et al. [12] and Thomson et al. [13] also recorded reductions in the TAG, total cholesterol (TC), and low-density lipoproteins (LDL) after administration of different doses of ginger ethanolic extract on mice. A significant reduction in the TC level of mice treated with combined therapy of 12.5% of clove ethanol extract and 12.5% of fermented ethanolic extract of ginger was reported by Abubakar et al. [14]. However, in this study, 400 mg/kg and 800 mg/kg of both the ginger and clove extract significantly increased the high-density lipoproteins in the albino rats. This means that oral administration of ginger and clove extract may reduce the incidence of hyperlipidemia in humans. The desirable limits for cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol are less than 200 mg/dl, less than 140 mg/dl, 60 mg/dl, and 60-130 mg/dl, respectively, while the borderline for cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol are 200-239 mg/dl, 150-199 mg/dl, 40-50 mg/dl, and 130-159 mg/dl, respectively [15].

The study shows that oral administration of extracts from ginger, clove, and combined extracts of clove and ginger are safe to a certain level (doses) and possess hypolipidemic activities such as lowering of triglycerides, total cholesterol, low-density lipoproteins, and very low-density lipoproteins and an increase in the level of high-density lipoproteins.

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