Oraekei Daniel Ikechukwu1*, Okoye Odinachi Anthony2, Mba Ogbonnaya2, Abone Harrison Odera3, Obidiegwu Onyeka Chinwuba4

1Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Olivia University, Bujumbura, Burundi.
2Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, PMB 5025 Awka, Anambra State, Nigeria.
3Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
4Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
Daniel Ikechukwu Oraekei email: oraekeidanielikechukwu@gmail.com
Odinachi Anthony Okoye email: nachi.t.okoye@gmail.com
Ogbonnaya Mba email: mbabte@gmail.com
Harrison Odera Abone email: harrisonabone@gmail.com
Onyeka Chinwuba Obidiegwu email: oc.obidiegwu@unizik.edu.ng

*Corresponding author
Daniel Ikechukwu Oraekei,
1Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Olivia University, Bujumbura, Burundi.
Email: oraekeidanielikechukwu@gmail.com
Phone: +25771629919
ABSTRACT
Background: It is the function of the liver and kidneys to deal with processes concerning detoxification, metabolism, and the excretion of waste products. Aim: This study tested the liver and kidney protective effects of a combination of Z. officinale and A. sativum in Wister rats treated with doxorubicin. Methods: The qualitative phytochemical analysis and acute toxicity studies were carried out using standard methods. Bacterial lipopolysaccharide from Escherichia coli was used to induce systemic inflammatory and oxidative stress. The animals were pretreated for 14 days with the combined extracts of Z. officinale and A.sativum alone, the extracts with doxorubicin, and doxorubicin alone. LPS at 1 mg/kg intraperitoneally dissolved in normal saline was given daily to the animals along with the treatments for an additional 14 days. On the last day, the animals were anesthetized with ketamine and xylazine, and blood samples were withdrawn from the retro-orbital plexus of the animals into plain tubes. Serum alanine transaminase, Alkaline phosphatase, Serum creatinine, and blood urea nitrogen were estimated using standard methods. Results: among all tested phytochemicals, Z. officinale lacks tannins, steroids, Steroids and terpenoids, while A. sativum lacks saponins and glycosides. No mortality was observed after the acute toxicity study. Group 4 rats, which were treated with Z. officinae, A. sativun, and doxorubicin, showed lower serum levels of alanine aminotransferase, alkaline phosphatase, creatinine, and blood urea nitrogen than the control group. Conclusion: Z. officinale-A. sativum combination showed a favorable safety profile and also exhibited significant protective effects against chemotherapeutic liver and kidney toxicities.
Key words: Allium sativum, doxorubicin, kidney toxicity, liver toxicity, Zingiber officinale
INTRODUCTION
Background of the study
The liver and kidneys of the human body actively deal with processes concerning detoxification, metabolism, and the excretion of waste products. The protective role of natural products derived from plants against drug-induced damage to the organs has received significant attention. Zingiber officinale and Allium sativum are two widely used herbs in culinary and medicinal fields. They are well studied for their chemoprotective, anti-inflammatory, and antioxidant properties. (Oraekei et al., 2024). Z. officinale contains gingerol and shogaol, and A. sativum contains allicin and ajoene, and these are some of the active constituents that have properties to avert oxidative damage and enhance the functions of organs (Mao et al., 2019). Doxorubicin’s molecular composition leads to the production of free radicals and triggers oxidative stress, which is associated with cellular damage (Tacar et al., 2013). Doxorubicin is a chemotherapeutic agent that is effective in treating various cancers; it is known to induce oxidative stress, leading to hepatotoxicity and nephrotoxicity (Kciuk et al., 2023). Doxorubicin, also known as Adriamycin, is a widely used anthracycline antibiotic that’s actually derived from the bacterium Streptomyces peucetius. (Arcamone et al., 1969). Doxorubicin has been noted to have harmful effects on the liver (Abdulrhaman et al., 2025). It also decreases other protective components like cytochrome P-450 and glutathione in the rat’s liver (Timm etal., 2022). Notably, high glutathione levels have been shown to protect liver cells from Doxorubicin’s toxic effects (Deng et al., 2015). Doxorubicin’s long-term use is limited by severe side effects, including a potentially fatal heart condition that worsens with higher doses. (Belger et al., 2023). The combined use of Z. officinale and A. sativum may offer synergistic effects, potentially mitigating the toxic impact of chemotherapeutic agents like doxorubicin. This study aims to test the liver and kidney protective functions of a combination of Z. officinale and A. sativum in rats treated with doxorubicin. Through biochemical assays, the research seeks to determine whether this herbal blend can mitigate doxorubicin-induced toxicity and support liver and kidney health.
Aim of Study
The aim of the study is to test the liver and kidney protective effects of a combination of Z. officinale and A. sativum in Wister rats treated with doxorubicin.
Scope of Study
This study was narrowed to evaluate the biochemical changes in liver and kidney functions due to doxorubicin toxicity, assess key biomarkers like alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine and blood urea nitrogen (BUN).
Literature review
Studies have shown doxorubicin to be a highly chemotherapeutic agent used in treating various cancers. Its use is limited due to the toxic effect it causes on various organs, including the liver and kidneys (Alshabanah et al., 2010). Renal and Hepatic functions are assessed by testing biomarkers such as Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Creatinine levels, and Blood urea nitrogen (BUN) (Lala et al., 2023). Treatment with Z. officinale showed significant improvement in AST, ALT, and superoxide dismutase (SOD) activities (Abdel-Azeem et al., 2013). Z. officinale administered alone at 530 mg/kg body weight had a greater hepato-protective effect than when given in combination with A. sativum (Oraekei et al., 2024); and this study showed that Z. officinale significantly reduced liver and kidney damage, and the combination containing a higher proportion of Z. officinale was most protective than the other tested combinationse. High levels of ALP or BUN may indicate liver disease or a certain bone disorder or kidneys not functioning well (Lowe et al., 2023).
Herbal medicine combination in pharmacotherapy
When herbs are combined, a lot of interactions can occur, but the desirable interactions are those that can result in enhanced therapeutic benefit. The effects of herbal medicine combinations are usually variable. A herb can be used to potentiate the effect of another herb. An example is the combination of ginseng root and aconite daughter root in an anti-shock remedy (Che et al, 2013). In this combination, the aconite daughter root potentiated the effects of ginseng. Herbal drug combinations can also be antagonistic in their actions. An example is the interaction between turnip root and ginseng root, where ginseng is used as a tonic drug, but when used in the presence of turnip root, its effect will be reduced (Che et al, 2013).
Herb-Drug combination
The use of herb-drug combinations can lead to various clinical presentations, such as potentiation, as seen in the potentiation of the effect of oral corticosteroids by liquorice (Kahraman et al., 2021). The impact of herb-drug combinations can also provide effects that may be additive or antagonistic. Herb-drug combinations can lead to alterations in the gastrointestinal functions, which can affect drug absorption (Brantley et al., 2014). It can also cause induction and inhibition of metabolic enzymes and transport proteins (Fasinu et al., 2012). It can also lead to alteration of renal excretion of drugs and their metabolites (Dresser et al., 2002). Long-term use of St. John’s wort can lead to reduced clinical effectiveness of cytochrome P450 subtype CPY3A4 substrate drugs by CPY3A4 induction, which can cause rapid metabolism and a decrease in the dosage of the drugs (Markowitz et al., 2003).
Possible Herb-Drug Interactions
Herb-drug interactions can occur when herbal supplements are taken with prescription drugs and affect how the medications work in the body. There are so many herb-drug interactions like garlic increasing bleeding when taken with an anticoagulant (Hu et al., 2005). Ginseng interacts with anticoagulants and calcium channel blockers reducing their effects (Jiang et al., 2004). St John’s Wort poses high risks with drugs like cyclosporine, oral contraceptives, and indinavir (Roby et al., 2000).
Brief description of Zingiber officinale
Z. officinale is a rhizome that is widely used as a spice and a medicinal herb. It can be used fresh, dried, or in powdered form in the making of teas and cooking. It contains bioactive compounds like gingerol, which have anti-inflammatory or antioxidant properties. It is used in the treatment of nausea and for relief of cold (Mao et al., 2019).

Figure 1: Image of Z. officinale

Brief description of Allium Sativum
A. sativum is a bulbous plant in the onion family. It is widely used as a culinary spice and in traditional medicine. It has a pungent flavor, which comes from sulfur compounds like allicin. It enhances the cardiovascular system, supports the immune system, and has antimicrobial properties. (Ansary et al., 2020).
Figure 2: Image of A. Sativum.
Materials
Animals
Female Wister rats (230 – 240 g) were used for this study. All the animals were obtained from the animal house of the Department of Pharmacology and Toxicology, Enugu State University of Science and Technology, Enugu State, Nigeria. The animals were housed in standard laboratory conditions of 12 hours’ light, room temperature, 40-60% relative humidity, and fed with rodent feed (Guinea Feeds Nigeria Ltd). They were allowed free access to food and water. All animal experiments were conducted in compliance with the NIH guide for care and use of laboratory animals (National Institute of Health (NIH), 2011) Pub No: 85-23), and animal protocol was approved by Animal care and ethics committee of Enugu State University of Science and Technology with approval number ESUT/2025/AEC/0962/AP 845.
Plant materials
Fresh Z. officinale rhizome and A. sativum bulb were purchased from Ogbete main market in Enugu state, Nigeria.
Drug
Doxorubicin was used for this research.
Equipment
Glass column, flasks, beakers, test tubes, surgical blade, measuring cylinder, forceps, scissors, white transparent paper, Analytical Weighing balance(Metler H30, Switzerland), Electric oven, Water bath (Gallenkamp, England) Water bath, disposable pipette tips (Labcompare USA), intubation tubes, stop watch (Avi Scientific India), BUN and creatinine test kits (Teco Diagnostics, USA), precision pipettes (25, 50, 100, and 300 μl, 1,000 µL) (Labcompare USA), AST test kit (Span Diagnostics Ltd., India), UV-VIS spectrophotometer (Model 752, China), distilled or deionized water (SnowPure Water Technologies USA), micropipette (Finnipipette® Labsystems, Finland), disposable hand gloves (Supermax Malaysia), National Blender (Japan), ALP test kit (Span Diagnostics Ltd., India), ALT test kit (Span Diagnostics Ltd., India), plethysmometer (Biodevices, New Delhi, India).
Methods
Phytochemical analysis
The qualitative phytochemical analysis of the extracts was carried out using standard methods described by Odoh et al. (2019).
Test for alkaloids: The plant extracts (0.2 g) were heated in 20 mL of 2% acid solution (HCL) individually in a water bath for about 2 minutes. The resulting solutions were allowed to cool and then filtered, and then 5 mL of the filtrate was used for Hager’s test. The samples (5 mL) were placed in labeled test tubes, and a few drops of Hager’s reagent (saturated picric acid solution) were added. Formation of a yellow precipitate indicated the presence of alkaloids.
Test for glycosides
The samples were extracted with 1% H2SO4 solution in a hot water bath for about 2 minutes. The resulting solution was filtered and made distinctly alkaline by adding 4 drops of 20% KOH (confirmed with litmus paper). One milliliter of Fehling’s solution (equal volume of A and B) was added to the filtrates and heated on a hot water bath for 2 minutes. Brick red precipitate indicated the presence of glycosides.
Test for saponins
The plant extracts (0.2 g) were dissolved in methanol individually, and the resulting solutions were used for Frothing test. The samples (5 mL) were placed in labeled test tubes, and 5 mL of distilled water was added and the mixtures were shaken vigorously. The test tubes were observed for the presence of persistent froth.
Test for tannins
The plant extracts (0.2 g) were dissolved in methanol individually, and the resulting solutions were used for the test. To 3 mL of each of the samples, a few drops of 1% Ferric chloride were added and observed for brownish green or a blue-black coloration.
Test for flavonoids
Using methanol, 0.2 g of the plant extracts and fractions were dissolved individually, and the resulting solutions were used for Ammonium hydroxide test. A quantity of 2 mL of 10% ammonia solution was added to a portion of each of the samples and allowed to stand for 2 minutes. Yellow coloration at the lower ammoniacal layer indicated the presence of a flavonoid.
Test for steroids and terpenoids
Salkowski test: The plant extracts were dissolved in methanol individually, and the resulting solutions were used for the test. A 5 mL of each of the samples was mixed with 2 mL of chloroform, and concentrated H2SO4 was carefully added to form a layer. A reddish-brown coloration at the interface indicated a positive test.
Acute toxicity studies
Acute oral toxicity of the combination of Z. officinale, A. sativum (6:4) and doxorubicin (318, 212, and 5 mg/kg respectively) was performed according to the Organization of Economic Cooperation and Development (OECD, 2021) guideline 425 for testing of chemicals (Up and down method). The single combination dose was administered to the animal based on their body weight. The animals were closely observed for the first 30 minutes, then for 4 hours. Food was provided after 2 hours of dosing. After the survival of the first treated animal, 4 more animals were treated with the same dose at an interval of 48 hours each. The control group of rats (n = 5) was administered with distilled water (vehicle used in preparing the herbal mixture) in the same volume as that of the treated group. All the groups were closely observed for 6 hours and then at regular intervals for 14 days. The animals were weighed and observed for mortality, salivation, diarrhea, asthenia, hypo-activity, hyperactivity, piloerection, hyperventilation, aggressiveness, yellowing or loss of hair fur, drowsiness, convulsion, tremor, dizziness, and other obvious signs of toxicity.
Experimental design
Bacterial lipopolysaccharide (LPS) from Escherichia coli, purchased from Sigma-Aldrich, was used to induce systemic inflammatory and oxidative stress states. The animals were pretreated for 14 days with the combined extracts of Z. officinale and A. sativum alone; the extracts with doxorubicin; and doxorubicin alone. LPS at 1 mg/kg intraperitoneal (I.P) dissolved in normal saline was given daily to the animals along with the treatments for an additional 14 days. Treatment was done 30 minutes before the LPS injection. On the last day, 2 hours after injection of LPS, the animals were anesthetized with ketamine and xylazine, and blood samples were withdrawn from the retro-orbital plexus of the animals into plain tubes.
Animal grouping (5 animals per group)
A total of 25 rats were allocated into five groups of five rats each. Group 1 were uninduced control (Naïve) and were treated with normal saline + 5 ml/kg distilled water via the oral route (p.o.). Group 2 were the negative control and was treated with LPS 1mg/kg i.p + 5 ml/kg distilled water p.o. Group 3 were treated with Z. officinale and A. sativum combination 6:4 (318:212 mg/kg p.o.) + LPS 1mg/kg i.p. Group 4 were treated with Z. officinale: A. sativum: doxorubicin combination (318:212: 5 mg/kg) + LPS 1 mg/kg i.p. While group 5 were treated with doxorubicin 5 mg/kg i. p.
Serum preparation
At the end of the study, blood samples were collected through retro-orbital plexus into a plain covered test tube. The blood samples were allowed to clot by leaving them undisturbed at room temperature for 30 minutes. The clots were removed by centrifuging at 2,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant (serum) was immediately transferred into a clean polypropylene tube using a Pasteur pipette. The samples were maintained at 2–8 °C while handling and apportioned into 0.5 ml aliquots.
Hepatic function tests.
Quantitative determination of alanine aminotransferase (ALT)
Serum alanine transaminase was estimated by the method described by Oraekei et al., (2024) using the ALT test kit (Span Diagnostics Ltd., India). A 0.25 ml of mixture of L-alanine (200 mmol/l), α-oxoglutarate (2.0 mmol/l), and phosphate buffer (100 mmol/l) was added to 0.5 ml of each sample and blank (containing distilled water). They were mixed and incubated at 37 °C for exactly 30 minutes in a water bath. A 0.25 ml of 2,4- dinitrophenylhydrazine was added to the sample and blank test tubes and incubated again at room temperature for 20 minutes. A 2.5 ml of sodium hydroxide (0.4 mol) was then added to all the test tubes, and the absorbance of the sample was read against the blank at 546 nm using a UV-VIS spectrophotometer (Model 752, China). The ALT concentration was extrapolated from a graph of concentration against wavelength absorbance of known ALT concentrations.
Quantitative determination of alkaline phosphatase (ALP)
Alkaline phosphatase was estimated by the method described by Colville (2002) using the ALP test kit (Span Diagnostics Ltd., India). A 0.5 ml of Alkaline Phosphatase substrate was placed in the sample and blank labeled test tubes and equilibrated to 37 °C for 3 minutes. At a timed interval, 0.05 ml each of standard, control (deionized water), and sample was added to its respective test tubes. The mixture was incubated for 10 minutes at 37 °C. A 2.5 ml of alkaline phosphatase color developer (0.1 M Sodium Hydroxide and 0.1 M sodium Carbonate) was added and properly mixed. The absorbance of the samples was read at 590 nm using a UV-VIS spectrophotometer (Model 752, China) and recorded. ALP concentration was calculated using the equation below;
Calculation of ALP concentration
ALP= (Abs of samples x value of standard (IU/L¬))/(Abs of standard)
Where Standard Value = 50 IU/L
Renal function tests
Serum creatinine and blood urea nitrogen (BUN) were estimated by the method described by Tietz (1976) and Heinegard and Tiderstrom (1973), respectively, using creatinine and BUN test kits (Teco Diagnostics, USA).
Quantitative determination of creatinine
Creatinine working reagent was prepared by combining equal volumes of 10 mM picric acid and Creatinine buffer reagent (10 mM sodium borate, 240 nM sodium hydroxide). Then 3.0 ml of this reagent was added to labelled tubes (test, blank, and standard) to which 100 µl of serum (test), 5 mg/dl of Creatinine (Standard), and distilled water (blank) were added and mixed in their designated test tubes. The tubes were incubated at 37 °C for 15 minutes, and the absorbance was measured spectrophotometrically at 520 nm against a test blank. The concentration of Creatinine (mg/dl) was calculated thus:
Creatinine= (Abs of Test)/(Abs of Std) ×Conc.of Std
Where Abs = Absorbance, Std = Standard
Quantitative determination of blood urea nitrogen
A 1.5 ml of BUN Enzyme reagent (containing 10,000 µ/l Urease, 6.0 mmol/l sodium salicylate, 3.2 mmol/l sodium nitroprusside) was added to 10 µl of Test (serum), Standard (20 mg/dl), and Blank (distilled water) followed by incubation for 5 minutes at 37 °C. At a timed interval, 1.5 ml of BUN color developer (6 mmol/L of sodium Hypochlorite and 130 mmol/l sodium hydroxide) was added to each of the labelled tubes and were incubated for another 5 minutes at 37 °C. The absorbance of the tests and standards was measured spectrophotometrically at 630 nm against a blank. Urea nitrogen concentration (mg/dl) was calculated thus:
BUN= (Abs of Test)/(Abs of Std) ×Conc.of Std
Where Abs = Absorbance, Std = Standard
Results
Table 1: Phytochemical analysis of Z. officinale and A.sativum
Phytocompounds Zingiber officinale Allium sativum
Alkaloids + +
Saponins + –
Tannins – +
Flavonoids + +
Steroids and terpenoids – +
Glycosides + –
Yield 44.8 g (11.2%) 62.4 g (15.6%)
Key: + = Present; – = Absent
Acute toxicity study
No mortality was observed throughout the observational period. Reduced physical activities were observed after drug administration, but normalcy was restored 30 minutes later. Other observations were similar to those of the control group that received the vehicle. Delayed signs of toxicity were not recorded within the 14-day observational periods.
Liver and kidney function tests
Figure 1: Serum level of alanine aminotransferase (ALT)
Figure 2: Serum level of alkaline phosphatase (ALP)

Figure 3: Serum level of creatinine
Figure 4: Serum level of blood urea nitrogen (BUN)
Discussion
In the present study, the phytochemical composition and protective effects of a combined extract of Z. officinale and A. sativum against doxorubicin-induced toxicity were investigated, with a particular focus on liver and kidney function biomarkers. The phytochemical analysis revealed that both Z. officinale and A. sativum contain bioactive compounds such as alkaloids and flavonoids, known for their antioxidant, anti-inflammatory, and hepatoprotective properties. Z. officinale showed the presence of saponins and glycosides, which were absent in A. sativum, while A. Sativum uniquely contained tannins and a combination of steroids and terpenoids, which were absent in Z. officinale. These differences suggest that the combination of both plants could offer a wider spectrum of protective phytochemicals than each of the herbs alone. A study conducted by Mao et al., (2019) confirmed the presence of bioactive compounds like flavonoids and gingerols in Z. officinale, which exhibited antioxidant and anti-inflammatory properties.
The acute toxicity assessment showed no mortality or significant adverse effects in the treated animals over a 14-day observation period. Although a temporary reduction in physical activity was observed shortly after extract administration, the animals recovered within 30 minutes. This rapid return to normal behavior, coupled with the absence of delayed toxicity signs, suggests that the herbal combination is safe at the administered dosage. Z. officicinale was shown to be safe when administered in rats at doses up to 2000 mg/kg. (Rong et al., 2009)
Biochemical analyses further supported the extract’s protective effects. Doxorubicin, known for its potent chemotherapeutic activity as well as its hepatotoxic and nephrotoxic side effects, significantly elevated serum markers of liver and kidney injury. Alanine aminotransferase (ALT), a key indicator of liver cell damage, increased significantly following doxorubicin administration. However, animals pre-treated with the Z. officinale-A. sativum combination exhibited a significant reduction of ALT levels compared to the doxorubicin-only group, indicating a strong hepatoprotective effect of the extracts. Similarly, levels of alkaline phosphatase (ALP), another marker of hepatic function, were elevated by doxorubicin treatment but attenuated in animals co-treated with the extracts. The extract alone maintained ALP and ALT levels close to those of the healthy control group, suggesting it has no intrinsic hepatotoxicity and may even support liver health under normal conditions.
Renal functions, assessed via serum creatinine and blood urea nitrogen (BUN), also deteriorated significantly in response to doxorubicin. However, treatment with the Z. officinale-A. sativum combination weakened these effects. Although creatinine and BUN levels remained higher than those of untreated controls, they were significantly lower than in the doxorubicin-only group, indicating nephroprotection. The extract alone maintained creatinine and BUN levels within normal ranges, again reinforcing its safety and potential therapeutic value.
Overall, the results demonstrated that the combined extract of Z. officinale and A. sativum can effectively reduce biochemical signs of liver and kidney toxicity induced by doxorubicin. This protective effect is likely due to the synergistic action of the various phytochemicals present in both plants. Alkaloids, flavonoids, saponins, glycosides, tannins, and terpenoids are all known to contribute to antioxidant defense mechanisms and membrane stabilization, which may account for the observed mitigation of organ damage.
Conclusion
From this study, the Z. officinale-A. sativum combination not only showed a favorable safety profile but also exhibited significant protective effects against chemotherapeutic toxicity. These findings suggest that such a combination could serve as a promising adjunct therapy to reduce organ damage in patients undergoing doxorubicin treatment.

Acknowledgement
I am thankful to God for his unwavering support throughout this study. My appreciation also goes to Dr. Ajaghaku Lotenna Daniel and the laboratory technologists of the Pharmacology and Toxicology department, Enugu State University of Science and Technology, for their expertise that enabled the smooth completion of this study.
Disclosure of conflict of interest
Daniel Ikechukwu Oraekei declared no conflict of interest
Odinachi Anthony Okoye declared no conflict of interest
Ogbonnaya Mba declared no conflict of interest
Harrison Odera Abone declared no conflict of interest
Onyeka Chinwuba Obidiegwu declared no conflict of interest
Statement of ethical approval
Maintenance and care of all animals were carried out in accordance with EU Directive 2010/63/EU for animal experiments. Guide for the care and use of Laboratory Animals, DHHS Publ. # (NIH 86-123) were strictly adhered to. Animal protocol was approved by the Animal Care and Ethics Committee of Enugu State University of Science and Technology with approval number ESUT/2025/AEC/0962/AP 845. There was additional approval by the Nnamdi Azikiwe University’s Ethical Committee for the use of Laboratory Animals for Research Purposes (Approval number is NAU/AREC/2025/0077).

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