Acute Toxicity, Anti-oxidative, and Anti-inflammatory Activities of a Combination of Zingiber officinale, Allium sativum, and Doxorubicin in Wister Rats Model

Acute Toxicity, Anti-oxidative, and Anti-inflammatory Activities of a Combination of Zingiber officinale, Allium sativum, and Doxorubicin in Wister Rats Model

Oraekei Daniel Ikechukwu^1,2*, Okonkwo Chukwuemeka Micheal¹, Mba Ogbonnaya¹, Abone Harrison Odera³, Obidiegwu Onyeka Chinwuba⁴, Chukwuka Benjamin Uzodinma¹.

¹Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, PMB 5025 Awka, Anambra State, Nigeria.

²Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences. Oliva University, Number 4 & 5 Mayugi Avenue, Mukaza, Bujumbura, Burundi.

³Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, PMB 5025 Awka, Anambra State, Nigeria.

⁴Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, PMB 5025 Awka, Anambra State, Nigeria. 

Oraekei Daniel Ikechukwu email: di.oraekei@unizik.edu.ng

Okonkwo Chukwuemeka Micheal email: Michaelfact91@gmail.com

Mba Ogbonnaya email: mbabte@gmail.com

Abone Harrison Odera email: harrisonabone@gmail.com

Obidiegwu Onyeka Chinwuba email: oc.obidiegwu@unizik.edu.ng

Chukwuka Benjamin Uzodinma email: cb.uzodimma@unizik.edu.ng

 

*Corresponding author

Dr. Oraekei Daniel Ikechukwu,

Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Oliva University, Number 4 & 5 Mayugi Avenue, Mukaza, Bujumbura, Burundi.

Email: di.oraekei@unizik.edu.ng

Phone: +2348061629459; +25771629919

ABSTRACT

Background: Herb-drug combination therapy involves using herbal remedies together with conventional drugs. This can lead to beneficial or harmful interactions. These interactions can affect drug metabolism, efficacy, and toxicity, and can alter the way the body processes and responds to drugs, leading to enhanced effectiveness or causing toxicity. Objectives: This study aimed to evaluate the safety of a blend of Zingiber officinale, Allium sativum, and doxorubicin for a possible drug-herb combination against breast cancer. Methods: Z. officinale, and A. sativum were extracted with 95% methanol, and the extracts were subjected to phytochemical analysis, acute toxicity study, anti-oxidant, and anti-inflammatory tests were performed using standard methods. Results: Alkaloids, Saponins, Flavonoids, and glycosides were present in Z. officinale, and Tannins, Steroids, and terpenoids were absent. In A. sativum, alkaloids, flavonoids, glycosides, and tannins were present, while saponins, steroids, and terpenoids were absent. No mortality was observed throughout the observational period. In group 5, doxorubicin alone had a reduction of superoxide dismutase (1.298 ± 0.03 mg/ml) that was even worse than the reduction caused by lipopolysaccharide. This was ameliorated by a 6:4 blend of Z. officinale: A. sativum, which, when given together with doxorubicin, raised the superoxide dismutase to 2.944 ± 0.11 mg/ml. This herbal blend also decreased nuclear factor kappa β from 9.364 ± 0.19 ng/ml in the doxorubicin-only group to 6.324 ± 0.22 ng/ml when the herbs were blended with doxorubicin. Conclusion: The 6:4 blend of Z. officinale: A. sativum may be useful in preventing the side effects of doxorubicin, especially those resulting from oxidative stress and inflammation.

 

Keywords: Acute toxicity, Allium sativum, Anti-inflammatory, Anti-oxidant, Zingiber officinale.

 

INTRODUCTION

Background of the study

Herb-drug combination therapy can lead to either beneficial effects or toxic interactions (Bukowska et al., 2025). These interactions can affect drug metabolism and efficacy, and can alter the body's responses to drugs, leading to enhanced effectiveness or adverse reactions (Gamil et al., 2025). Some herbs may enhance the effects of orthodox drugs and may require a reduction of the dose of the orthodox drug (Ameade et al., 2018). This may reduce the adverse effects and improve treatment outcomes. On the other hand, certain combinations can lead to treatment failure and adverse effects such as increased risk of bleeding, serotonin syndrome, or liver damage (Czigle et al., 2023). Evidence has shown that incorporating Chinese herbal medicines into synthetic drug-based therapies delivers benefits in the treatment of many multifactorial diseases (Li et al., 2022). Herb-drug interactions may be due to the interactions between the phytochemicals in the herbal medicines and active ingredients of prescription drugs (Borse et al., 2019). This can occur through several mechanisms that can be classified as either pharmacokinetic or pharmacodynamic interactions (Gouws & Hamman, 2020). The concurrent use of herbal medicines and conventional drugs is of important concern in breast cancer treatment (Al-Masri et al., 2025). It is recommended that oncologists and clinical pharmacologists should take into account the challenges associated with herb-drug interactions in their routine practices, particularly during chemotherapy among cancer patients (Bazrafshani et al., 2023). Z. officinale and A. sativum combination had anti-breast cancer activities and decreased breast tumor volume, tumor weight, and cancer antigen 15-3 (CA 15-3) (Ihekwereme et al., 2024). Consequently, a combination of these two herbs and doxorubicin is expected to interact synergistically to treat breast cancer, especially when the breast cancer is diagnosed early.

Statement of the problem

Treatment of breast cancer and cancer in general with Pharmaceutical/orthodox drugs was known to be followed by a lot of adverse effects. This may be due to the mechanisms through which orthodox anti-breast cancer drugs perform their action. Doxorubicin acts by generating free radicals that kill cancerous cells. But these free radicals can also affect body organs and tissues. This study therefore verified whether blending doxorubicin with herbs with proven anti-oxidative and anti-inflammatory activities will work synergistically in breast cancer treatment.

Justification

Z. officinale and A. sativum had good anti-breast cancer activities comparable to that of doxorubicin at the ratio of 6:4 (318:212 mg/kg body weight). (Ihekwereme et al., 2024). It was recommended that these two herbs, blended with doxorubicin, should be studied further to find out if such a blend will synergistically bring about a radical cure for breast cancer. This present study evaluated the safety of such a blend as a stepping stone to considering such a drug-herb blend.

Aim

This study aimed to evaluate the safetyof a blend of Z. officinale, A. sativum, and doxorubicin for a possible drug-herb combination against breast cancer.

Specific objectives

·        To carry out an acute toxicity study of a blend ofZ. officinale, A. sativum, and doxorubicin.

·        To evaluate the anti-oxidative effects of a blend ofZ. officinale, A. sativum, and doxorubicin.

·        To estimate the anti-inflammatory activities of a blend of Z. officinale, A. sativum, and doxorubicin.

Scope of the study

This study was narrowed down to investigating the drug-herb interaction between Z. officinale, A. sativum, and doxorubicin. A blend of these three ingredients was prepared and subjected to acute toxicity testing as to ascertain the safety of such a combination. Superoxide dismutase (SOD) assay was carried out to indicate the antioxidant potential of the blend. The combination of Z. officinale, A. sativum methanol extracts, and doxorubicin was tested for its effectiveness in inhibiting inflammation caused by carrageenan-induced paw edema.

 

Expected outcome

Because Z. officinale, A. sativum, and doxorubicin had been individually confirmed to be safe, it is expected that a blend of the three drugs will also be safe. Doxorubicin works by generating free radicals that destroy breast cancer cells, while the two herbs, Z. officinale and A. sativum, were confirmed in several previous studies to have remarkable antioxidant and anti-inflammatory activities. It is therefore expected that a blend of these three drugs will have antagonistic effects with respect to oxidative stress and inflammation responses. However, this is desirable in breast cancer treatment, whereby the herbs will ameliorate the oxidative stress caused by treatment with doxorubicin.

Literature review

Herbal drugs have a long history of use in traditional medicine, and they are increasingly attracting attention and being integrated into modern pharmacotherapy (Yuan et al., 2016). Even though some herbal medicines have shown effectiveness, they should be used with caution, and healthcare providers should be consulted, especially while using them together with synthetic drugs. The practice of herbal drug use dates back to ancient civilizations, where plants were revered for their healing properties (Petrovska et al., 2012). Traditional medical systems like Ayurveda, Traditional Chinese Medicine (TCM), and Native American remedies all bear witness to the historical significance of herbal medicine (Coopoosamy et al., 2023). Approximately 80% of the global population is estimated to use herbal medicinal products for their therapeutic benefits (Balkrishna et al., 2024). Plants' different natural products have inspired the design, discovery, and development of new drugs (Chaachauay et al., 2024). Currently, there are worldwide initiatives targeted at finding herbal treatments in plants and promoting them using a reliable drug delivery system for people, based on the fact that each disease’s cure can be found in nature (Hardeep SG, 2023).

Some research documents on herb-drug combinations

The phenomenon of herb-drug interactions is of significant importance in clinical practice, particularly when conventional medications and herbal remedies are used concomitantly. These interactions can result in a range of effects, including increased bleeding risk, serotonin syndrome, and decreased drug bioavailability, among other adverse effects (Fugh-Berman, 2000). Therefore, healthcare professionals need to be aware of potential herb-drug interactions. They should also contribute to providing solutions for the risks. Some of these solutions include rendering patient education and monitoring the patients. However, some herb-drug interactions can be synergistic, whereby the effects of the blend are greater than either of the herb or drug given alone (Zhou et al., 2016). The possibility of herbal medicines and conventional drugs being concurrently used is high (Pan et al., 2022). There is a need to improve phytovigilance of the potential harms and benefits as a result of the combined use of herbal medications with pharmaceutical drugs (Babos et al., 2021). Natural products, including crude extracts, herbal formulas, and bioactive compounds from plants, hold great potential to prevent and treat cancers (Wang et al., 2012). More importantly, some herbal drugs can reduce the incidence of chemotherapy-induced toxicity, including oral mucositis, gastrointestinal toxicity, and hepatotoxicity (Fu et al., 2018). This confers on herbal drugs the great potential as adjuvant therapy for the prevention and treatment of chemotherapy-induced side effects (Okem et al., 2023). Herb-drug interaction could remain unidentified in the case where there is limited knowledge of the effects of herbal medicines and their safety (Amaeze et al., 2020).

Pharmacological uses of Z. officinale and A. sativum

Ginger's pharmacological uses are diverse, encompassing anti-inflammatory, anti-nausea, and antioxidant properties, among others. It is used for conditions like arthritis, nausea, and vomiting, and some studies suggested potential benefits in managing heart disease, diabetes, and cancer. Z. officinale is one of the most widely used natural products consumed as a spice and medicine for treating diabetes, flatulent intestinal colic, indigestion, infertility, inflammation, insomnia, a memory booster, nausea, rheumatism, stomach ache, and urinary tract infections (Unuofin et al., 2021). A. sativum, on the other hand, has a range of pharmacological uses, including its potential to manage blood pressure, support cardiovascular health, enhance immunity, and potentially protect against cancer. It is also known for its antimicrobial, anti-inflammatory, and antioxidant properties. A certain study reported that A. sativum is an aromatic herbaceous plant that is consumed worldwide as food and a traditional remedy for various diseases. It has been reported to possess several biological properties, including anticarcinogenic, antioxidant, antidiabetic, renoprotective, anti-atherosclerotic, antibacterial, antifungal, and antihypertensive activities in traditional medicines (El-Saber et al., 2020). A. sativum has a high medicinal value and is used to cure a variety of human diseases. It has anti-inflammatory, rheumatological, ulcer inhibiting, anticholinergic, analgesic, antimicrobial, antistress, antidiabetes, anticancer, liver protection, anthelmintics, antioxidants, antifungal, and wound healing properties, as well as properties that help with asthma, arthritis, chronic fever, tuberculosis, runny nose, malaria, leprosy, skin discoloration, and itching, indigestion, colic, enlarged spleen, hemorrhoids, fistula, bone fracture, gout, urinary tract disease, diabetes, kidney stones, anemia, jaundice, epilepsy, cataract, and night blindness (Tesfaye, 2021). Based on the fact that a blend of Z. officinale and A. sativum at a ratio of 6:4 which is equivalent to 318:212 mg/kg body weight, had remarkable anti-breast cancer activities and decreased breast tumor volume, tumor weight and cancer antigen 15-3 (CA 15-3) in a manner that is comparable to a standard anti-breast cancer drug, doxorubicin (Ihekwereme et al., 2024); this current study evaluated the safety of combining the blend of these two herbs with doxorubicin for possible application in breast cancer treatment.

 

Materials and methods

Animals

Swiss female Albino rats (230 – 240 g) were employed for the study. All the animals were obtained from the animal house of the Department of Pharmacology, Enugu State University of Science and Technology, Agbani. 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). 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).

 

Drugs

Doxorubicin was the only pharmaceutical drug used for this study.

Plant material

Fresh Zingiber officinale rhizome and Allium sativum bulb were purchased from Ogbete main market in Enugu state, Nigeria.

 

Chemicals and Reagents

Wash Buffer Concentrate (Sigma Aldrich Germany), Assay Buffer (Alpco USA), TMB Substrate (Cayman Chemical USA), Stop Solution (Cayman Chemical USA), Hydrochloric acid (Prime laboratories, India); Dragendoff reagent (Sigma Aldrich, United States of America); Ammonia (Shackti Industrial Gases, India), sodium hydroxide (Treveni Chemical Pvt., India); Ferric chloride (AkashPurochem. Pvt., India); Fehling’s solution (Lab care Diagnostics, India); Million reagent (Interlab Chemical Pvt., India): Ethanol (TAJ Pharmaceutical Ltd., India); Acetic anhydride (Ashok Organics Industries, India); Concentrated sulfuric acid (Navin Chemical Pvt., India), Acetic acid (Kayla Africa Suppliers, South Africa); Molisch reagent (Interlab Chemical Pvt., India); alcoholic alpha naphatol (Prat Industry Corcopation, India).

 

Equipment

Glass column, flasks, beakers, test tubes, measuring cylinders, surgical blade, forceps, scissors, graph paper, white transparent paper, rotary evaporator, Analytical Weighing Balance (Metler H30, Switzerland), Electric Oven (Gallenkamp, England), Spectrophotometer (B. Bran Scientific &Instrument Company, England), Water Bath (Techmel & Techmel, Texas, USA), National Blender (Japan), Micropippete (Finnipipette® Labsystems, Finland), Plethysmometer (Biodevices, New Delhi, India) and Intubation tubes. Precision pipettes (25, 50, 100 and 300 μl, 1,000 µL) (Labcompare USA); Disposable pipette tips (Labcompare USA); Distilled or deionized water (SnowPure Water Technologies USA); Plate shaker (Biocompare USA); Microwell plate reader (BioTek India); Centrifuge (Sharplex Filters Pvt., India); Vortex mixer (Bionics Scientific Technologies (P) LTD, India); Graduated cylinder for 500 ml (Boenmed Healthcare Co. Ltd, Hong Kong); Stop watch (Avi Scientific India); EDTA containers (Sure Care Corporation), heparinized capillary tube (Thomas Scientific, USA), disposable hand gloves (Supermax Malaysia), toilet tissue.

Extraction

The Z. officinale and A. sativum were scraped, sliced, and pulverized to a powdered paste using an electronic blender and kept in clean, airtight amber colored bottles separately. Then, 400 g of each of the powdered plant material was cold macerated in 95% methanol. The mixture was allowed to stand for 3 days (72 hours) with intermittent agitation. It was filtered, and the filtrate was concentrated first using a vacuum rotary evaporator at 50ºC and then with a freeze dryer at -55ºC. The extracts were stored in a refrigerator until used.

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, filtered, and then 5 mL of the filtrate was used for the Dragendorff’s test.  To each labeled test tube, 5 mL of the sample was added, followed by1 mL of Dragendorff’s reagent. Formation of orange or red precipitates indicated the presence of an alkaloid.

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 and fractions (0.2 g) were dissolved in methanol individually, and the resulting solutions were used for the Frothing test: The samples (5 mL) were placed in labeled test tubes, and 5 mL of distilled water was added; 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 was dissolved individually, and the resulting solutions were used for the 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 study

Acute oral toxicity of the combination of Z. officinale, A. sativum (6:4), and doxorubicin at 318, 212, and 5 mg/kg body weight, respectively, was performed according to the Organization of Economic Cooperation and Development (OECD) 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, hypoactivity, hyperactivity, piloerection, hyperventilation, aggressiveness, yellowing or loss of hair fur, drowsiness, convulsion.

 

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, A. sativum alone, the combination of the extracts with doxorubicin, and doxorubicin alone. LPS at 1 mg/kg 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 grouped into five with five rats in each group. Group 1 were un-induced control/naïve and were treated with normal saline injection + 5 ml/kg distilled water orally (p.o.). Group 2 were the negative control which were treated with LPS 1 mg/kg intraperitoneal (i.p.) + 5 ml/kg distilled water p.o.). Group 3 rats were treated with Z. officinale and A. sativun combination 6:4 (318:212 mg/kg p.o.) + LPS 1mg/kg i.p. Group 4 were treated with Z. officinale: A. sativun: (318:212: 5 mg/kg) and doxorubicin blend + 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. After collection of the whole blood, 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.

 

Antioxidant Activity

Serum Superoxide Dismutase (SOD) Assay Procedure

Assay of SOD was performed using the ElabScience kit (USA) following the manufacturer's instructions. The ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in the kit has been pre-coated with Rat SOD1. First wells for diluted standard, blank, and sample were determined. Then, 50 µl each of the dilutions of standard, blank, and samples were added into the appropriate wells. Thereafter, 50 µl of Biotinylated Detection Ab working solution was added to each well immediately. The plate was covered with the sealer provided in the kit and incubated for 45 min at 37 ℃. Solution from each well was decanted, followed by the addition of 350 µl of wash buffer to each well. Excess conjugate and unbound sample or standard are washed from the plate three times, and 100 µl Avidin conjugated to Horseradish Peroxidase (HRP) were added to each microplate well and incubated for 30 minutes at 37 °C. The solution from each well was decanted and the washing process repeated for 5 times. Then 90 µl of Substrate Reagent was added to each well, covered, and incubated for 15 min at 37 °C in the dark. The enzyme-substrate reaction was terminated by the addition of 50 µl of Stop Solution to each well, and the color change was measured by spectrophotometry at a wavelength of 450 nm. The concentration of Rat SOD1 in the samples was then determined by comparing the OD of the samples to the standard curve.

 

Determination of anti-inflammatory activity

Nuclear factor kappa B (NF-kβ) assay

Serum NF-kβ activity was estimated by quantitative sandwich enzyme immunoassay (ELISA) technique as described by Jin et al. (2005) using rat-specific NF-kβ assay kit (Elabscience Biotechnology Co., Ltd., China). One hundred microliters of the serum samples and standards were placed in duplicates in their designated wells. The wells were covered with a sealer and incubated at 37 °C for 90 minutes. After incubation, the fluids in the wells were emptied (without washing), followed immediately by the addition of 100 µl of dilute biotinylated detection antibody. The wells were covered again with a sealer, the contents gently mixed and incubated for 60 minutes at 37 °C. After incubation, the contents of the wells were emptied by decanting, and then the wells were washed by adding 350 µl of dilute wash buffer to each well. This was allowed to soak for 2 minutes and then the solution was decanted and the plate was patted dry with a clean absorbent paper. The washing was repeated 3 times. Then 100 µl of dilute horseradish peroxidase (HRP) conjugate working solution was added to each well, the plate/wells covered with a plate sealer and incubated at 37 °C for 30 minutes. After incubation, the contents of the wells were decanted, and the plate was patted dry on absorbent paper. The wash process was repeated again 5 times, this time with 350 µl of dilute wash buffer, which was allowed to soak for 1 minute during each wash. The plate was also emptied and patted dry during each wash (5 times). After washing, 90 µl of substrate reagent was added to each well, and the plate was covered with a new plate sealer. The plate was incubated for 15 minutes at 37°Cwith the plate/wells well protected from light. After incubation, 50 µl of stop solution was added to each well, and the absorbance was read immediately at 450 nm.

 

Statistical analysis

Results were presented as Mean ± Standard Error of Mean (S.E.M). Means were analyzed using one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test for multiple comparisons. P < 0.05 was set to be statistically significant. Results analysis was conducted using the Statistical Package for the Social Sciences, SPSS version 20.

Results and Discussion

Results

Table 1: Phytochemical analysis of Zingiber officinale and Allium sativum ethanol extracts

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 the control group that received the vehicle. Delayed signs of toxicity were not recorded within the 14 days’ observational periods.

Figure 1: Serum concentration of superoxide dismutase (SOD)

 

Figure 2: Serum concentration of nuclear factor kapper β (NF-kβ)

 

Discussion

Phytochemical analysis

The phytochemicals present in Z. officinal were alkaloids, saponins, flavonoids, and glycosides, while tannins, steroids, and terpenoids were absent. The phytochemicals present in A. sativum were alkaloids, tannins, flavonoids, and steroids and terpenoids, while saponins and glycosideswere absent. Phytochemicals, which are bioactive compounds found in plants, have been reported to be responsible for the pharmacological activities of herbs (Kumar et al., 2023). These compounds contribute to the medicinal properties of plants and are the basis for many traditional and modern herbal remedies. Indeed, phytochemical analysis and pharmacological studies of traditional medicinal plants are integral to validating their therapeutic potential (Jordan, 2024). Understanding the phytochemical and therapeutic properties of medicinal plants is a key factor for developing effective medicines (Rabizadeh et al., 2022).

Acute toxicity study

No mortality was observed throughout the observational period. This indicated that the blend of Z. officinale, A. sativum, and doxorubicin was safe at the administered doses of 318, 212, and 5 mg/kg body weight, respectively. This outcome was exceptionally interesting because a blend of herbal and pharmaceutical drugs is not necessarily safe for humans and can be risky. While some herbal remedies may be beneficial, they can also interact negatively with pharmaceutical drugs, potentially leading to harmful side effects or reduced effectiveness of either the herb or the medication. But in this case, the reverse was the case. According to an earlier study, the use of herbal medicines is not without risk, and when they are administered together with allopathic medicines, they can lead to herb-drug interactions, the outcome of which ranges from negligible to life-threatening situations (Gouws et al., 2020). The experience of the use of herbal medicines and preparations and the risks of interactions between herbal and conventional medicines was investigated, and a total of 38.3% found the use of herbal remedies safe and harmless, while 57.3% of respondents regarded the combination of herbal and regular drugs as unsafe (Sile et al., 2023). This again buttressed the fact that the finding that a blend of Z. officinale, A. sativum, and doxorubicin was safe at the administered doses of 318, 212, and 5 mg/kg body weight, respectively, was exciting and promising.

 

Antioxidant activity

In the antioxidant assay, group 1 rats, which were treated with 5 ml/kg body weight of distilled water and served as the overall control group, had a serum superoxide dismutase (SOD) concentration of 3.520 ± 0.15 ng/ml, which indicated the natural level of SOD in the rats. In group 2, bacterial lipopolysaccharide was used to induce oxidative stress, which led to the rats having reduced serum concentration of SOD (1.552 ± 0.14 ng/ml). SOD is an antioxidant enzyme, and its decrease in the body results in oxidative stress. SOD is a crucial enzyme that helps neutralize harmful superoxide radicals, which are a type of reactive oxygen species (ROS). When SOD levels are reduced, these ROS can accumulate, causing damage to cells and tissues, a state known as oxidative stress (Chen et al., 2023). Group 3 rats were treated with a blend of Z. officinale and A. sativum in a proportion of 6:4 (318:212 mg/kg body weight). This proportion was confirmed in a preceding research to have the best antioxidant effects when oxidative stress was induced in rats with 7,12-dimethylbenz(a)anthracene (Oraekei et al., 2024). Expectedly, this combination ameliorated the decrease in SOD caused by LPS and raised the SOD level to 2.944 ± 0.11 mg/ml, which was significant when compared with group 2, which had an SOD level of 1.552 ± 0.14 mg/ml (p=1.99 x 10-11). When doxorubicin was added to the blend in group 4, there was yet an increment in SOD level, which was significant when compared with group 2. In group 5, doxorubicin alone had a reduction of SOD (1.298 ± 0.03 mg/ml) that was even worse than the reduction caused by LPS. This showed the extent to which the blend of Z. officinale and A. sativum in a proportion of 6:4 (318:212 mg/kg body weight was able to remedy the oxidative stress caused by doxorubicin when group 4 raised the SOD level to 2.090 ± 0.16 mg/ml (p = 8.25 x 10-12).

 

Anti-inflammatory activity

In the test for inflammatory activities of this blend of Z. officinale and A. sativum in a proportion of 6:4 (318:212 mg/kg body weight with doxorubicin, similar trend was observed in which LPS increased the inflammatory marker, nuclear factor kappa β (NF-kβ) from 2.464 ± 0.11 mg/ml to 8.128 ± 0.20 mg/ml which was significant (p = 6.00 x 10-9). Doxorubicin in group 5 increased NF-kβ even more (9.364 ± 0.19 mg/ml) than the increment in group 2. Elevated levels of nuclear factor kappa B (NF-kβ) in the nucleus of a cell are indeed a strong indicator of inflammation. NF-kβ is a protein complex that acts as a transcription factor, meaning it controls which genes are turned on or off in a cell. When the body experiences inflammation, NF-kβ is activated and moves into the cell's nucleus, where it triggers the expression of genes that promote inflammation. In addition to being a possible transcription factor, it plays a very significant role in the establishment of inflammation-associated immunological responses in host cells (Mukherjee et al., 2024). Again, both the blend of Z. officinale and A. sativum in a proportion of 6:4 (318:212 mg/kg body weight in group 3 and such a blend with doxorubicin in group 4 reduced NF-kβ level when compared with LPS group 2, as well as doxorubicin only group 5.

 

Conclusion

The blend of Z. officinale and A. sativum in a proportion of 6:4 (318:212 mg/kg body weight) was confirmed to possess the potential to reduce the oxidative stress and inflammation caused by doxorubicin. As such, this blend of Z. officinale and A. sativum in a proportion of 6:4 (318:21 2 mg/kg body weight) can be used to counter the adverse effects of orthodox drugs that cause their toxicity via an increase in reactive oxygen species and activation of inflammatory mediators.

 

Acknowledgement

I am thankful to God for his unwavering support throughout this study. My appreciation also goes to the head, staff, and laboratory technologists of the Pharmacology and Toxicology department for providing the necessary facilities that enabled the smooth completion of this study

 

Disclosure of conflict of interest

Oraekei Daniel Ikechukwu declared no conflict of interest

Okonkwo Chukwuemeka Micheal declared no conflict of interest

Mba Ogbonnaya declared no conflict of interest

Abone Harrison Odera declared no conflict of interest

Obidiegwu Onyeka Chinwuba declared no conflict of interest

Chukwuka Benjamin Uzodinma 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).

 

 

References

·       Alexander SK, Chrisanty C, Lumbwe K, Ian B, Patience C, Majorie M. (2016). Vitamin C Levels in Lemons (Citrus Limon) Grown in Zambia; an Opportunity for Utilisation in Fish Feeds. Journal of Agriculture and Life Sciences; Vol. 3, No. 1. Accessed July, 2025.

 

·       Al-Masri, M., Darwish, R., Safi, Y., Mustafa, A., Alzyoud, D., Almasri, M., Kahhaleh, B., & Khader, M. (2025). Herbal and alternative medicine use: a cross sectional study to evaluate the prevalence and predictors of use in cancer patients. Frontiers in pharmacology, 16, 1535795. https://doi.org/10.3389/fphar.2025.1535795.

 

·       Amaeze O. U.*, olugbakeef O. A., lawal M. (2020). Knowledge of Herbal Medicines and Herb-drug Interaction Among Medical and Pharmacy Students of the University of Lagos, Nigeria. Nig. J. Pharm. Res, 16 (1) pp 61-70. https://doi.org/10.4314/njpr.v16i1.7.

 

·       Ameade, E. P. K., Ibrahim, M., Ibrahim, H. S., Habib, R. H., & Gbedema, S. Y. (2018). Concurrent Use of Herbal and Orthodox Medicines among Residents of Tamale, Northern Ghana, Who Patronize Hospitals and Herbal Clinics. Evidence-based complementary and alternative medicine: eCAM, 2018, 1289125. https://doi.org/10.1155/2018/1289125.

 

·       Antonio DM, Giuseppe DRS, Aurora C, Maria AR, Claudio D. (2021). Morphological characteristics, ascorbic acid, and antioxidant activity during fruit ripening of four lemons (Citrus limon (L.) Burm. F.) cultivars. Scientia Horticulturae; Volume 276, 109741. https://doi.org/10.1016/j.scienta.2020.109741.

 

·       Babos, M. B., Heinan, M., Redmond, L., Moiz, F., Souza-Peres, J. V., Samuels, V., Masimukku, T., Hamilton, D., Khalid, M., & Herscu, P. (2021). Herb–Drug Interactions: Worlds Intersect with the Patient at the Center.Medicines,8(8), 44. https://doi.org/10.3390/medicines8080044.

 

·       Balkrishna A, Sharma N, Srivastava D, Kukreti A, Srivastava S, Arya V. (2024). Exploring the Safety, Efficacy, and Bioactivity of Herbal Medicines: Bridging Traditional Wisdom and Modern Science in Healthcare. Future Integrative Medicine. 3(1):35-49. doi: 10.14218/FIM.2023.00086.

 

·       Banerjee, S., Chanda, A., Adhikari, A., Das, A., & Biswas, S. (2014). Evaluation of Phytochemical Screening and Anti-Inflammatory Activity of Leaves and Stem of Mikania scandens (L.) Wild.Annals of medical and health sciences research,4(4), 532–536. https://doi.org/10.4103/2141-9248.139302.

 

·       Bazrafshani, M.S., Pardakhty, A., Kalantari, K B, Haleh T, Sadra G P, SaraH, ShivaA, SoheilM, AminB, A., & HamidS. (2023). The prevalence and predictors of herb-drug interactions among Iranian cancer patients during chemotherapy courses.BMC Complement Med Ther23, 41. https://doi.org/10.1186/s12906-023-03869-1.

 

·       Borse, S. P., Singh, D. P., & Nivsarkar, M. (2019). Understanding the relevance of herb-drug interaction studies with special focus on interplays: a prerequisite for integrative medicine. Porto biomedical journal, 4(2), e15. https://doi.org/10.1016/j.pbj.0000000000000015.

 

·       Bukowska, B., Grzegorowska, A., Szczerkowska-Majchrzak, E., Bukowski, K., Kadac-Czapska, K., Grembecka, M., & Broncel, M. (2025). Hazardous Interactions Between Food, Herbs, and Drugs in the First Stage of Biotransformation: Case Reports of Adverse Drug Interactions in Humans. International Journal of Molecular Sciences, 26(11), 5188. https://doi.org/10.3390/ijms26115188.

 

·       Chaachouay, N., & Zidane, L. (2024). Plant-Derived Natural Products: A Source for Drug Discovery and Development.Drugs and Drug Candidates.3(1), 184-207. https://doi.org/10.3390/ddc3010011.

 

·       Chaughule, R.S., Barve, R.S. (2023). Role of herbal medicines in the treatment of infectious diseases. Vegetos 2023. https://doi.org/10.1007/s42535-022-00549-2. Accessed July, 2025.

 

·       Chen, L., Liu, Y., Zhang, Y., Zhang, Y., Wang, W., Han, H., Yang, C., & Dong, X. (2023). Superoxide dismutase ameliorates oxidative stress and regulates liver transcriptomics to provide therapeutic benefits in hepatic inflammation. Peer J, 11, e15829. https://doi.org/10.7717/peerj.15829.

 

·       Coopoosamy, R., Singh, K., Naidoo, K. & Nadasan, D.S. (2023). ‘The role of phytomedicine: Bridging the gap between the past, present, and future’, Journal of Medicinal Plants for Economic Development 7(1), a197. https://doi.org/10.4102/jomped.v7i1.197.

 

·       Cotoraci C, Ciceu A, Sasu A, Hermenean A. (2021). Natural Antioxidants in Anemia Treatment.International Journal of Molecular Sciences; 22(4):1883. https://doi.org/10.3390/ijms22041883.

 

·       Czigle, S., Nagy, M., Mladěnka, P., Tóth, J., & OEMONOM. (2023). Pharmacokinetic and pharmacodynamic herb-drug interactions-part I. Herbal medicines of the central nervous system. PeerJ, 11, e16149. https://doi.org/10.7717/peerj.16149.

 

·       El-Saber Batiha, G., Magdy Beshbishy, A., G Wasef, L., Elewa, Y. H. A., A Al-Sagan, A., Abd El-Hack, M. E., Taha, A. E., M Abd-Elhakim, Y., & Prasad Devkota, H. (2020). Chemical Constituents and Pharmacological Activities of Garlic (Allium sativum L.): A Review.Nutrients,12(3), 872. https://doi.org/10.3390/nu12030872.

 

·       Emanuel L. P, Susan F. R, Kijakazi O. M, Hamisi M. M. (2014). Ethno-medicinal knowledge and plants traditionally used to treat anemia in Tanzania: A cross-sectional survey. Journal of Ethnopharmacology; Volume 154, Issue 3, Pages 767-773. https://doi.org/10.1016/j.jep.2014.05.002.

 

·       Fugh-Berman A. (2000). Herb-drug interactions. Lancet (London, England), 355(9198), 134–138. https://doi.org/10.1016/S0140-6736(99)06457-0.

 

·       Emire A S. and Sudip K R. (2005). Proximate composition and physico-chemical properties of improved dry bean (Phaseolus vulgaris L.) varieties grown in Ethiopia. LWT - Food Science and Technology; Volume 38, Issue 4, Pages 331-338, https://doi.org/10.1016/j.lwt.2004.07.002.

 

·       Fu, B., Wang, N., Tan, H. Y., Li, S., Cheung, F., & Feng, Y. (2018). Multi-Component Herbal Products in the Prevention and Treatment of Chemotherapy-Associated Toxicity and Side Effects: A Review on Experimental and Clinical Evidences. Frontiers in pharmacology, 9, 1394. https://doi.org/10.3389/fphar.2018.01394.

 

·       Gamil, N.M, Elsayed, H.A, Hamed, R.M,  Salah E.T, Ahmed A.M, Mostafa H.A, El-Shiekh R.A,  & Abou-Hussein (2025).   Insights from herb interactions studies: a foundational report for integrative medicine. Futur J Pharm Sci 11, 46. https://doi.org/10.1186/s43094-025-00794-7.

 

·       Gouws, C., & Hamman, J. H. (2020). What are the dangers of drug interactions with herbal medicines? Expert Opinion on Drug Metabolism & Toxicology,16(3), 165–167. https://doi.org/10.1080/17425255.2020.1733969.

 

·       Hardeep SG. (2023). Recent development of novel drug delivery of herbal drugs, RPS Pharmacy and Pharmacology Reports, Volume 2, Issue 4, October 2023, rqad028,https://doi.org/10.1093/rpsppr/rqad028. Accessed: April 28, 2025.

 

·       Ihekwereme PC, Oraekei DI*, Asomugha RN, Abone HO, Ezugwu MI and Nnamani ME. (2024). Anti-breast cancer activities of Zingiber officinale and Allium sativum ethanol extracts in 7,12-dimethylbenz[a]anthracene induced female Wister rats. GSCBPS. 29(01), 224–232 DOI: https://doi.org/10.30574/gscbps.2024.29.1.0304.

 

·       Jin, S., Lu, D., Ye, S., Ye, H., Zhu, L., Feng, Z., Liu, S., Wang, D., & Hu, Q. (2005). A simplified probe preparation for ELISA-based NF-kappaB activity assay.Journal of biochemical and biophysical methods,65(1), 20–29. https://doi.org/10.1016/j.jbbm.2005.08.006.

 

·       Jordan L. (2024). Phytochemical Analysis and Pharmacological Activities of Traditional Medicinal Plants. J. Chem. Pharm. Res. 16:198. DOI:10.37532/0975-7384.2024.16(9).198.

 

·       Kumar, A., P, N., Kumar, M., Jose, A., Tomer, V., Oz, E., Proestos, C., Zeng, M., Elobeid, T., K, S., & Oz, F. (2023). Major Phytochemicals: Recent Advances in Health Benefits and Extraction Method. Molecules (Basel, Switzerland), 28(2), 887. https://doi.org/10.3390/molecules28020887.

 

·       Li, C., Jia, Ww, Yang, Jl. et al., (2022). Multi-compound and drug-combination pharmacokinetic research on Chinese herbal medicines.Acta Pharmacol Sin 43, 3080–3095 (2022). https://doi.org/10.1038/s41401-022-00983-7.

 

·       Lorke D (1983). A new approach to practical acute toxicity testing.Arch Toxicol. 54(4):275-87.

 

·       Mukherjee S, Dutta S, Pati S, Sarkar T, Paul S, Chakraborty S, Basak U, Dhar S, Das T, Sa G. (2024). Nuclear Factor Kappa-B: The El Dorado of inflammatory immune response. J Cell Mol Immunol. 2024; 3(1):6-19. Accessed July, 2025.

 

·       Msomi Z.N and Simelane M.B.C. (2019). Herbal Medicine [Internet]. Herbal Medicine. IntechOpen; 2019. Available from: http://dx.doi.org/10.5772/intechopen.72816.

 

·       Odoh, U. E., Obi, P. E., Ezea, C. C., Anwuchaepe, A. U. (2019a). Phytochemical methods in plant analysis. 1st Ed. Pascal Communications, Nsukka, Enugu, Nigeria. 47 p.

 

·       Okem, A., Henstra, C., Lambert, M., & Hayeshi, R. (2023). A review of the pharmacodynamic effect of chemo-herbal drug combinations therapy for cancer treatment.Medicine in Drug Discovery,17, Article 100147.https://doi.org/10.1016/j.medidd.2022.100147. Accessed: April 28, 2025.

 

·       Oluyomi S A and Bukola T O (2014). "Lipid Profile and Oxidative Stress Markers in Wistar Rats following Oral and Repeated Exposure to Fijk Herbal Mixture", Journal of Toxicology, vol. 2014, 7 pages. https://doi.org/10.1155/2014/876035.

 

·       Oraekei D I, *. Peter C I, Asomugha R N, Abone H O, Ezugwu M I, and Adione N M. (2024). Potentials of Zingiber officinale and Allium sativum ethanol extracts to inhibit oxidative stress and inflammation resulting from cancer induction with 7,12-dimethylbenz[a]anthracene. World Journal of Biology Pharmacy and Health Sciences, 2024, 19(02), 340–350 Article DOI: https://doi.org/10.30574/wjbphs.2024.19.2.0540.

 

·       Pan, H. Y., Wu, L. W., Wang, P. C., Chiu, P. H., & Wang, M. T. (2022). Real-world Evidence of the Herb-drug Interactions.Journal of food and drug analysis,30(3), 316–330. https://doi.org/10.38212/2224-6614.3428.

 

·       Petrovska B. B. (2012). Historical review of medicinal plants' usage. Pharmacognosy reviews, 6(11), 1–5. https://doi.org/10.4103/0973-7847.95849.

 

·       Rabizadeh, F., Mirian, M. S., Doosti, R., Kiani-Anbouhi, R., & Eftekhari, E. (2022). Phytochemical Classification of Medicinal Plants Used in the Treatment of Kidney Disease Based on Traditional Persian Medicine. Evidence-based complementary and alternative medicine: eCAM,2022, 8022599. https://doi.org/10.1155/2022/8022599.

 

·       Ranasinghe, P., Ranasinghe, P., Abeysekera, W. P., Premakumara, G. A., Perera, Y. S., Gurugama, P., & Gunatilake, S. B. (2012). In vitro erythrocyte membrane stabilization properties of Carica papaya L. leaf extracts.Pharmacognosy research, 4(4), 196–202. https://doi.org/10.4103/0974-8490.102261.

 

·       Sammán, N., Maldonado, S., Alfaro, M. E., Farfán, N., & Gutierrez, J. (1999). Composition of different bean varieties (Phaseolus vulgaris) of Northwestern Argentina (Region NOA): cultivation zone influence.Journal of agricultural and food chemistry; 47(7), 2685–2689. https://doi.org/10.1021/jf970967v.

 

·       Sile, I., Teterovska, R., Onzevs, O., & Ardava, E. (2023). Safety Concerns Related to the Simultaneous Use of Prescription or Over-the-Counter Medications and Herbal Medicinal Products: Survey Results among Latvian Citizens.International Journal of Environmental Research and Public Health,20(16), 6551. https://doi.org/10.3390/ijerph20166551.

 

·       Tesfaye A. (2021). Revealing the Therapeutic Uses of Garlic (Allium sativum) and Its Potential for Drug Discovery.The Scientific World Journal,2021, 8817288. https://doi.org/10.1155/2021/8817288. Accessed: April 28, 2025.

 

·       Unuofin J O*Nelisiwe P M, Oluwatomiwa K P, Sogolo L L. (2021). Ginger from Farmyard to Town: Nutritional and Pharmacological Applications. Front. Pharmacol., Sec. Ethnopharmacology. Volume 12 – 2021.https://doi.org/10.3389/fphar.2021.779352. Accessed: April 28, 2025.

 

·       Wang, H., Khor, T. O., Shu, L., Su, Z. Y., Fuentes, F., Lee, J. H., & Kong, A. N. (2012). Plants vs. cancer: a review on natural phytochemicals in preventing and treating cancers and their druggability. Anti-cancer agents in medicinal chemistry, 12(10), 1281–1305. https://doi.org/10.2174/187152012803833026.

 

·       Yuan, H., Ma, Q., Ye, L., & Piao, G. (2016). The Traditional Medicine and Modern Medicine from Natural Products. Molecules (Basel, Switzerland), 21(5), 559. https://doi.org/10.3390/molecules21050559.

 

·       Zhou X, Seto SW, Chang D, Kiat H, Razmovski-Naumovski V, Chan K and Bensoussan A (2016) Synergistic Effects of Chinese Herbal Medicine: A Comprehensive Review of Methodology and Current Research. Front. Pharmacol. 7:201. doi: 10.3389/fphar.2016.00201.