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AMELIORATIVE  EFFECT  OF  NATURAL  SPICES  MIXTURE (ginger, garlic, and 

turmeric) ON CADMIUM  INDUCED  NEPHROTOXICITY

1 Mba Eunice Azuka, 2 Ogugua Victor Nwadiogbu, 3 Emmanuel Ike Ugwuja, 4 Aniagolu 

Miriam Obiageli.

1DEPARTMENT OF MEDICAL BIOCHEMISTRY, COLLEGE OF MEDICINE ENUGU 

STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY NIGERIA

2DEPARTMENT OF BIOCHEMISTRYUNIVERSITY OF NIGERIA NSUKKA 

3DEPARTMENT OF  BIOCHEMISTRY,  EBONYI STATE STATE UNIVERSITY NIGERIA

4DEPARTMENT OF MEDICAL MEDICAL LABORATORY SCIENCE, COLLEGE OF 

MEDICINE ENUGU STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY NIGERIA

ABSTRACT

Cadmium  (Cd),  a  known  ubiquitous  naturally  occurring  toxic  element  has  been  recognized  to 

have deleterious effect in biological systems, affecting mostly the testis, liver, pancreas, kidneys,

and bone. Cd accumulates mainly in the kidney and liver which are critical targets for its acute 

toxicity. Evaluation of toxic potentials of this metal is important for the risk assessment of those 

ordinarily  exposed  to  it.  This  study  investigated  the  effects  of  natural  spices  mixture  on  renal 

toxicity  in  rats  exposed  to  cadmium.  Graded  doses  (200  mg,  300  mg,  and  400  mg/Kg  body 

weight)  of  natural  spices  mixture  were  administered  orally  to  the  rats.  After  forty  two  days,

significant  increases  in  serum  urea,  and  cratinine  concentrations  were  observed  in cadmium 

exposed rats compared to control group (P < 0.05). Natural spices mixture treatment of cadmium 

exposed rats significantly loweredurea and creatinine concentrations, compared to cadmiumalone 

group (P < 0.05).Natural spices mixture had significantly decreased all renal function parameters 

and  protected  kidney  cells  against  Cd  induced  toxicity. The  histology  evaluation  of  kidney 

tissues  of  the  experimental  rats  showed  that  the  induction  of  Cd  toxicity  without  treatment 

caused  significant  glomerular  necrosis,  while  the  administration  of  natural  spices  mixture 

protected  the  kidney  tissues  from  necrosis  and  other  pathological  changes. The  results  of  this 

study supported the potential ameliorative effects of natural spices mixture on rat kidney tissues 

against cadmium induced nephrotoxicity.

KEY WORDS: Cadmium chloride; natural spices mixture (ginger, garlic, and turmeric); kidney;

urea; creatinine; rat.



 

 

INTRODUCTION 

Background of study 

In the present era, environmental contamination is one of the significant constraints of modern 

human society (Ali and Khan 2019; Afzal et al., 2019). Among environmental contaminants, 

heavy metals (HMs) are the most toxic due to their persistent and bioaccumulative nature; thus, 

creating a deleterious risk to biological substances (Ali et al., 2008). Globally, the rate of HM 

mobilization and transport in the ecological system has been extraordinarily expanded since the 

1940s due to rapid growth rate of industrialization (Anyanwu et al., 2018). Although, these 

metals naturally exist in the soil in a very minute concentration through characteristic lithogenic 

as well as pedogenic means (Wuana and Okieimen, 2011). However, various anthropogenic 

practices including mining, improper industrial as well as urban waste disposal, combustion of 

non-renewable energy sources, metallurgical industries, chemical fertilizers, and improper 

handling of industrial effluents are fundamental contributors to aggregate these metals in soil 

(Tchounwou et al., 2012; Yuan et al., 2019). Non-essential HMs including lead (Pb), cadmium 

(Cd), and silver (Ag) have no or very little biological activity and their exposure above 

permissible limit poses a hazard to biological systems by interfering with their physiological and 

metabolic processes, contaminating food chain, causing ecological imbalances and resulting in 

lethal health issues due to their toxic nature (Haider et al., 2021). Cadmium is a highly noxious 

HM that is deleterious for biological systems through its uptake and accumulation in phototrophs 

and consequent trophic transportation. Cadmium is released into the environment via both 

natural and anthropogenic systems. Among natural systems, volcanic emissions, forest fires, 

weathering of Cd-containing rocks, and waste water are the principal means of mobilizing it 

from the lithosphere (Choppala et al., 2014). Anthropogenic activities such as application of 

phosphate-based fertilizers (Roberts 2014), manufacturing of Ni–Cd batteries, Zn mining, 

agricultural practices such as waste water irrigation, electroplating, application of urban compost 

as well as metal-based pesticides, and industrial emission as a byproduct (Zhao et al., 2015) are 

mainly responsible for Cd aggregation in soil. Human exposure to cadmium and cadmium 

compounds may occur in both occupational and environmental settings, the latter primarily via 

the diet and drinking water (ATSDR, 1989). A variety of industrial activities can lead to Cd 

exposure. Examples includes mining and metallurgical processing, combustion of  fossil  fuel 

(Genchi et al., 2020),  textile  printing, production of phosphate fertilizers,  recycling  of  ferrous  

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scrap, electronic waste, motor  oils, disposal  and  incineration  of  Cd-containing products.The 

increasing industrial uses of cadmium causes soil, air and water contaminations. However, 

human exposure to cadmium occurs chiefly through inhalation and ingestion of contaminated 

food and water (Ige et al., 2011). Tobacco in all its forms contains appreciable amounts of  Cd  

and  tobacco  smoke  is  one  of  the  largest  single sources  of  Cd  exposure  in  humans  (Ibiam 

and Uwakwe (2009). Foods derived from plants, depending on the level of soil contamination, 

generally contain higher concentrations of Cd than meat, egg, milk, and dairy products. Among 

these, rice and wheat, green leafy vegetables,potatoes, carrot, and celery can contain higher 

concentrations of the metal than other food from plants. Vegetarians and shellfish consumers 

may be exposed to a higher cadmium intake than omnivores (Sirot et al., 2008).  

 

Studies have proposed various mechanisms for Cd toxicity which include, modification of 

biomolecules, modulation of DNA repairs and genotoxic effects, antagonism and displacement 

of bivalent metal ions (zinc, selenium, calcium, and copper from their binding sites), induction of 

oxidative stress and impairment of p53 protein involved in suppression of cancer (Anetor, 2012; 

El-Boshy et al., 2015; Gong and Wang, 2019). Cadmium toxicity in liver seems to be mediated 

by the production of reactive oxygen spices (ROS) known to induce necrosis in various rat 

organs (Razinger et al., 2008, Hsu et al., 2007), lipids peroxidation (Borges et al., 2008) and 

decrease in antioxidant enzymes (El-Sharaky et al., 2007). 

 

Condiments such as ginger, garlic, and turmeric which have been generally used as spices, 

flavouring agent, food preservative and medicine, have been revealed to play important role in 

the amelioration of heavy metal toxicity. This may be due to the presence of biologically active 

polyphenols and antioxidants present in them (El-Sayed et al., 2015). For example, concurrent 

intake of garlic, ginger and nutmeg at culinary dose in diet has been found to have both 

therapeutic and prophylactic effect at mitigating Cd toxicity (Ugwuja et al., 2016). This 

reaffirmed the safety of spices combinations as being currently practiced. Some agents like 

curcumin, caffeic acid, phenylethyl ester found in spices like turmeric have been shown to be 

effective in preventing cadmium toxicity (Gong et al., 2012 Gong et al., 2014). Similarly, studies 

have shown that ginger (Zingiber officinale) has ameliorative effect against cadmium-induced 

liver and kidney injury in rat model (Egwurugu et al., 2007; Mohammad et al., 2013). Eteng et 

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al. (2014) have also demonstrated the reversal of cadmium induced toxicity after dietary 

supplementation with garlic, ginger and cabbage in male albino rats. 

Statement of problem 

Cadmium toxicity occurs even at a very low level in the blood and toxicity is usually without 

obvious signs and symptoms. With increase in industrialization/urbanization, heavy metals, 

including cadmium are released into the ecosystem, which will ultimately find its way into 

human systems. Accumulation of cadmium in human systems, if left untreated may lead to 

adverse health effects, especially in pregnant women and children, who have been found to be 

most vulnerable (Gehan and Ayman, 2010). Nanoparticle based antidote is very expensive and 

not readily accessible. Many chelating agents and antagonists have been found to reduce Cd 

toxicity, but part of them reveals undesirable side effects, intrinsic limitations and variability in 

the efficacy (Gonick, 2008; Jalilehvand et al., 2009). Reports have been inconsistent on the roles 

of spices in protecting against cadmium toxicity. Considering the rise in industrial activities with 

exponential emission of toxic metals, including cadmium, which has been classified as a 

category one carcinogen (IARC, 1994), coupled with reports of beneficial effects of nutrients 

and natural spices at ameliorating heavy metal toxicity, there is need to explore the potential 

beneficial effect of spices mixture at mitigating Cd toxicity.  

AIM AND OBJECTIVES 

This study was carried out to investigate the effects of natural spices mixture (ginger, garlic, and 

turmeric) on Cd-induced nephrotoxicity in male albino rats, and therefore sought to: 

1. Determine the levels of serum urea and creatinine in order to assess the effects of     

administration of natural spices mixture on renal function of cadmium-induced nephrotoxicity   

    in albino rats. 

2. Examine thekidney cell histology of the rats for morphological changes due to administration 

    of natural spices mixture on cadmium-induced nephrotoxicity in albino rats. 

3. Compare the levels of these chemical parameters, and histological changes in the treated rats  

    in relation to the control rats. 

 

 

 

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Significance of the study 

The data generated from this study may have policy relevance in designing mitigating strategy at 

reducing/preventing Cd toxicity, especially in cadmium polluted settings for the improvement of 

health and wellbeing of residents.  

 

Scope of the study 

This is a sub-acute study, and is limited to animal model – adult male albino rats of age 120 – 150 days. 

 

MATERIALS AND METHODS 

Subjects – rats 

Twenty adult male albino rats, weighing between 150 and 205g were used for this study.  The 

rats were obtained from the Animal House of Enugu State University of Science and 

Technology, College of Medicine (ESUCOM), Enugu. They were arranged into five groups (A, 

B, C, D, and E) of four rats each and were kept for five days to acclimatize before initiating the 

study. They were allowed free access to clean drinking water, and were fed on standard growers 

mash rat pellets (Grand Cereals LTD, Enugu) throughout the period of the study.  

 

The rats in group A were not induced with cadmium chloride, received no treatment with natural 

spices mixture, and served as normal control, while group B was administered 25 mg/kg body 

weight of cadmium chloride (CdCl2) without any treatment with natural spices mixture, and 

served as positive control. Those rats in groups C, D, and E were induced with 25 mg CdCl2/kg 

body weight and treated with graded concentrations 200 mg/kg, 300 mg/kg, and 400 mg/kg body 

weights respectively for forty two days. All administrations were through oral route. Two rats 

from each group were sacrificed at the end of the treatment period and their kidneys harvested. 

 

Reagents and chemicals 

Analytical grade of Cadmium Chloride (CdCl2) in the form of pure crystals were obtained from 

Riedel-De HaenAGSeelze-Hannover Germany through the Analar grade Laboratory reagents 

and chemical dealer at Ogbete main market, Enugu State. The reagents used for the biochemical 

assays were commercial test kits and products of Randox (USA). 

 

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Natural Spices Mixture  

Ginger rhizomes, garlic bulbs and turmeric rhizomes were purchased from Ogbete main market 

Enugu, Enugu State. The ginger, turmeric and garlic were peeled and all the three spices were 

washed with water. Exactly 5.0g of each spice were homogenized and soaked overnight in 

distilled water. It was filtered and the filtrate was concentrated at 600C using electric oven. It was 

carefully evapourated to dryness in water bath at 400C (Sofowora, 1977). 

 

 

Collection of blood samples and preparation of serum  

At the end of the experiment (42 days), the animals were fasted overnight for sample collection 

and harvest of organs. The specimens (blood samples) were collected through the retro-bulbar 

plexus of the nasal canthus, into plain bottles, for the estimation of the chemical parameters. 

Serum samples were prepared from the whole blood as follows:  

Three milliliters (3ml) of whole blood was collected into clean plain test tube. This was allowed 

to clot and retract, and then centrifuged at 3,000 rpm for five minutes. The supernatant (serum) 

was separated from the sediment (red cells) and transferred into another clean plain test tube, for 

the chemical analyses.  All serum samples were refrigerated (-4OC) until they were analysed. 

Two rats from each group were sacrificed by euthanasia, with chloroform, their kidney dissected 

out and fixed in 10% formol saline solution, for histological studies. 

 

Serum Urea and Creatinine Estimation 

Serum urea concentration was estimated by the modified diacetylmonoxime method of Wybenga 

et al., 1971; while serum creatinine concentration was determined using alkaline picrate method 

of Fabiny and Ertingshaysen, 1971. 

 

Principles 

In acid solution, diacetylmonoxime is hydrolysed to diacetyl, which reacts with urea to form a 

pink condensation product, which is measured at 480nm.  The concentration of urea in each 

sample was determined by interpolation from urea standard curve. Creatinine in the sample 

reacts with picrate in alkaline medium, forming a red-orange coloured complex.  The intensity of 

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the colour read at 500nm is directly proportional to the concentration of creatinine present in the 

sample.  The complex formation rate is measured in a short period to avoid interference from 

non-creatinine chromogens and creatinine present in the samples was interpolated from the 

creatinine standard curve. 

 

Histopathological examination 

The histological evaluation was carried out using the method described by Drury et al., (1967). 

Tissue sections of the kidney from all the experimental groups were fixed in 10% formol saline 

for a minimum of 48 hours. The tissues were subsequently processed in an automatic tissue 

processor (trimmed, dehydrated in graded concentrations of alcohol (70%, 80%, 90%, and 

absolute alcohol), cleared in three grades of xylene and embedded in molten paraffin wax). On 

solidifying, thin sections (about 4-5 microns thick) were cut using rotary microtome, and stained 

by heamatoxylin and eosin (H & E) method, permanently mounted on degreased glass slides 

using DPX mountant. The prepared slides were examined using a light Olympus microscope 

(magnification X100). The photomicrographs were taken using microscope camera at x400 

magnification. 

 

Staining Principle 

Haematoxylin was used as a nuclear stain preceding staining of cytoplasm and connective tissues 

with eosin.  Sections were stained with haematoxylin stain of sufficient power and for long 

enough to ensure some overstraining of nuclei, the superfluous and obscurative coloration of 

structures being removed by treatment with acid alcohol. Eosin stains connective tissues and 

cytoplasm in varying intensity and shades of primary colour giving a most useful differential 

stain with haematoxylin. 

 

 ANALYSIS OF RESLTS 

Statistical Analysis of results was carried out using Statistical Product and Service Solutions 

(SPSS) version 20. Results were expressed as mean ± standard error. Statistical differences were 

evaluated using One way Analysis of Variance (ANOVA), followed by Duncan's Multiple 

Range Test to detect the significant differences among the mean values of the different groups at 

p-values < 0.05. 

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RESULTS 

After forty two days, significant increase in serum urea and creatinine were observed in 

cadmium-exposed rats without treatment, compared to the normal control group (P< 0.05), while 

treatment with natural spices mixture had significant effects on these renal function parameters. 

Natural spices mixture treatment of cadmium exposed rats significantly lowered serum urea and 

creatinine concentrations compared to the cadmium alone group (P < 0.05). The concentrations 

of urea (2.48±0.10 mmol/L), and creatinine (50.25±1.18 µmol/L) in group D, which were treated 

with 300 mg/kg body weight of natural spices mixture were comparable with their levels in the 

normal control (2.74±0.05 mmol/L; 49.50±1.32 µmol/L), showing better ameliorative effect at 

mid dose. Group E rats which were treated with high dose of natural spices mixture recorded a 

non significant decrease (P > 0.05) when compared to cadmium alone group (Table 1). 

 

The histological evaluation of kidney tissues of the experimental rats showed that the normal 

control rats had normal histological architecture characterized by intact convoluted tubules (CT) 

and glomeruli (G) as shown in plate 1. However, the induction of Cd toxicity without treatment 

caused a significant glomerular necrosis (plate 2), while the administration of natural spices 

mixture 300 mg/kg, protected the kidney tissues from necrosis and other pathological changes 

(Plate 3). Morphological changes in those groups treated with low dose of SM (200 mg/kg) and 

high dose of same (400 mg/kg) manifested as mild capsular hypertrophy, brush border loss, and 

tubular atrophy (Plates 4, and 5). 

 

TABLE 1: Effect of spices mixture on the indices of renal function of cadmium induced 

toxicity in albino rats 

      

Parameters  Group A Group B Group C Group D Group E 

Urea (mmol/L) 2.74±0.05 5.88±0.14 3.80±0.08 2.48±0.10 5.63±0.15 

Creatinine (µmol/L)  49.50±1.32 106.75±1.18 65.50±1.32 50.25±1.18 81.75±3.93 

 

Results are expressed as Mean + Standard Error of Mean (n = 4). 

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Plate 1. Photomicrograph of normal control kidney. 

The kidney tissues showed intact convoluted tubules (CT) and glomeruli (G).  

H & E. magnification 100 X 

 

 

 

 

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Plate 2. Photomicrograph of cadmium exposed but untreated kidney. 

The kidney tissues showed glomerular necrosis (circled area), and hypertrophy of capsular space. 

H & E. magnification 100 X 

 

 

 

Plate 3. Photomicrograph of the group that received 300 mg SM/kg b.w. 

The kidney tissues appeared normal with intact convoluted tubules (CT) and glomeruli (G). 

H & E. magnification 100 X 

 

 

 

 

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Plate 4. Photomicrograph of the group that received 200 mg SM/kg b.w. 

The tissues showed mild tubular atrophy and loss of brush boarders 

H & E. magnification 100 X 

 

 

 

Plate 5. Photomicrograph of the group that received 400 mg SM/kg b.w. 

Tissue showed mild tubular atrophy and loss of brush borders 

H & E. magnification 100 X 

 

 

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DISCUSSIONS 

The diverse deleterious health effect upon exposure to heavy metals in the environment, 

especially Cadmium (Cd), a heavy metal of considerable toxicity with destructive impact on 

most organ system, is of serious concern and a global issue. Literature survey shows different 

treatment methods for Cd toxicity such asNanoparticle based treatments and use of chelating 

agents. The main disadvantage of synthetic chelators is that the chelation process removes vital 

nutrients along with toxic metals. However, natural spices mixture (ginger, garlic, and turmeric) 

could provide a credible alternative to the presently used drugs for the amelioration of cadmium 

toxicity to avoid long term adverse health effects. This study evaluated the ameliorative effects 

of natural spices mixtures on cadmium nephrotoxicity. 

 

In the present study, serum urea, and creatinine were estimated to assess renal glomerular 

function. The data obtained showed significant increase in renal function biomarkers; urea, and 

creatinine following six weeks of Cd exposure. This can be attributed to the impairment of the 

kidney, muscle wasting (weight loss), and dehydration (diarrhea) caused by the toxicant. The 

change in the levels of serum renal biomarkers represents renal damage caused by Cd as reported 

by Bekheet et al., (2011). These data were supported by others who reported that cadmium 

toxicity induced tubular necrosis or loss of the brush border and damage in the small tubules of 

kidney (Wang et al., 2011). Studies by Jacquillet et al., (2006) suggested that in chronic 

Cd2+ contamination, Cd2+ has an effect in the proximal as well as in distal tubule, leading to a 

pronounced renal defect. This can affect the filtration as well as absorption of these substances in 

the renal tubules.  

 
In addition to that, the elevated blood urea and creatinine concentrations, caused by cadmium 

toxicity may be related to disorder in protein catabolism as a result of increase in the synthesis of 

arginase enzyme involved in urea production (Tormanen, 2006), and glomerular functional 

disturbances such as lower filtration rate which results in poor creatinine clearance in cadmium 

intoxicated rats (L'Azou et al., 2002). 

 

The levels of renal function parameters, urea and creatinine, of group treated with 300 mg 

natural spices mixture/kg body weight, was comparable to those of normal control and achieved 

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717148/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717148/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717148/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717148/


 

 

a statistically significant decrease (P < 0.05) when compared with CdCl2 induced but untreated 

group. Groups treated with low dose (200 mg/kg), or medium dose (300 mg/kg) of natural spices 

mixture, were better protected from Cd nephrotoxicity. Treatment with high dose of natural 

spices mixture (400 mg/kg), caused a non significant (p > 0.05) decrease in the serum urea, and 

creatinine. The degree of increase in the levels of these renal parameters may be an indication of 

extent of renal tissue damage and impairment of kidney functions 

 

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