Polyherbal extracts and their influence on antioxidant levels: a gender-based study in ratsABSTRACT
The study explores the effects of polyherbal extracts on antioxidant levels in male and female rats, focusing on catalase, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) peroxidase activities. Polyherbal formulations, which combine multiple herbs, are noted for their synergistic effects and superior antioxidant properties. This research investigates the differential antioxidant responses between male and female rats, an area of significant interest due to hormonal and metabolic differences influencing antioxidant capacity. Eighteen healthy rats, equally divided between males and females, were administered polyherbal extracts at doses of 100mg and 250mg. The study measured antioxidant levels using various assays, including colorimetric methods for GSH and TBARS assays for MDA. Results showed a dose-dependent increase in catalase and SOD activities in both sexes, with males exhibiting slightly higher activities. MDA levels, a marker of lipid peroxidation, decreased significantly with polyherbal treatment, with males showing a greater reduction. GSH levels increased dose-dependently, with males again showing higher levels than females. These findings indicate a more robust response to polyherbal extracts in males, underscoring the importance of considering sex differences in antioxidant research and therapeutic applications.
Keywords:
polyherbal extracts; oxidative stress; gender differences; rats
RESUMO
O estudo explora os efeitos dos extratos poli-herbais sobre os níveis de antioxidantes em ratos machos e fêmeas, com foco nas atividades da catalase, superóxido dismutase (SOD), malondialdeído (MDA) e glutationa (GSH) peroxidase. As formulações poli-herbais, que combinam várias ervas, são conhecidas por seus efeitos sinérgicos e propriedades antioxidantes superiores. Esta pesquisa investiga as respostas antioxidantes diferenciais entre ratos machos e fêmeas, uma área de grande interesse devido às diferenças hormonais e metabólicas que influenciam a capacidade antioxidante. Dezoito ratos saudáveis, divididos igualmente entre machos e fêmeas, receberam extratos poli-herbais em doses de 100mg e 250mg. O estudo mediu os níveis de antioxidantes usando vários ensaios, incluindo métodos colorimétricos para GSH e ensaios TBARS para MDA. Os resultados mostraram um aumento dependente da dose nas atividades de catalase e SOD em ambos os sexos, com os homens apresentando atividades ligeiramente mais altas. Os níveis de MDA, um marcador de peroxidação lipídica, diminuíram significativamente com o tratamento com ervas, sendo que os homens apresentaram uma redução maior. Os níveis de GSH aumentaram de forma dependente da dose, com os homens novamente apresentando níveis mais altos do que as mulheres. Esses resultados indicam uma resposta mais robusta aos extratos de ervas medicinais em homens, ressaltando a importância de considerar as diferenças de sexo na pesquisa de antioxidantes e aplicações terapêuticas.
Palavras-chave:
extratos de ervas medicinais; estresse oxidativo; diferenças de gênero; ratos
INTRODUCTION
Plants are known as rich sources of bioactive metabolite. Combination of plants as polyherbal mixture or formulation is a practice dated from the ancient time and in recent time gaining research attention. Polyherbal formulation usually involves harnessing the synergistic potentials of the various constituent phytochemicals to enhance their biological activities.
Phytochemicals of antioxidants activities have been reported from various plants, combining plants that are known to have potent antioxidants activities can enhance their ability to neutralize free radicals and reduce oxidative stress. By targeting multiple biochemical pathways, polyherbal mixtures often outperform single-herb treatments in scavenging reactive oxygen species (ROS), reducing lipid peroxidation, and stabilizing cell membranes, thus protecting against cellular damage. Recent studies have demonstrated that polyherbal extracts exhibit significant in vitro antioxidant activities via the DPPH and ABTS assays.
Polyherbal formulations were reported to effectively scavenge free radicals and reduce oxidative stress (Ghasemzadeh et al., 2010). Similarly, in-vivo studies have also indicated that polyherbal formulations can modulate oxidative status and improve overall health outcomes in animal models (Sabir et al., 2003).
Polyherbal formulations play a crucial role in mitigating oxidative stress which is a key factor in the development of various chronic diseases (Kumar et al., 2021; Katiyar et al., 2015; Parasuraman et al., 2014). Typically, oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize these harmful compounds through antioxidants. Antioxidants are crucial in preventing cellular damage, thereby reducing the risk of diseases such as diabetes, cardiovascular disorders, and cancer (Adam et al., 2016; Ozougwu,2016; Adwas et al., 2019; Galli et al., 2005). Polyherbal formulations offer superior antioxidant protection compared to single-herb preparations due to the diverse range of bioactive compounds they contain, which can act synergistically to enhance their therapeutic efficacy (Mapeka ,2023; Rajini and Muralidhara ,2023; Mishra & Singh, 2009). However, the level of response to antioxidants has been reported to be gender-specific due to hormonal variations and metabolic differences. For instance, female rats often exhibit higher levels of certain antioxidants, such as vitamin E, in comparison to males, which could influence their response to polyherbal treatments (Stankovic et al., 2011). Understanding these gender-specific responses is crucial for optimizing the therapeutic use of polyherbal formulations. This study aims to investigate the influence of polyherbal extracts on antioxidant levels in male and female rats, highlighting potential gender-specific responses. We hope to elucidate the potential gender-based variations in antioxidant response and provide insights into the development of more effective polyherbal therapies tailored to individual needs.
MATERIAL AND METHODS
The polyherbal mixture was formulated by combining equal portions of each plant in the mixture. These plants namely, Ziziphus spina-christi, Trigonella foenum-graecum, and Nigella sativa (ZTN) were obtained from various locations, each collected in quantities of five hundred grams. Following plant sample collection, the plants were ground to powdered and subjected to extraction in water as described in our previous study (Ammari et al., 2024; Alhimaidi et al., 2021). The resulting herbal mixture was prepared in two different concentrations levels (100 mg and 250mg) and kept at 20C for further studies.
The experimental design involved eighteen viable rats, equally distributed into nine females and nine males. The weight of the rats ranged between 200 and 230 grams and their age was between 12 to 14 weeks old. These animals were obtained from the animal facility at the Zoology Department of the Science College, King Saud University (KSU). The animals were acclimatized to a well-ventilated setting at a stable temperature of 25±2ºC, following a 12-hour light and dark cycle. Standard diet and water were supplied to the animal ad libitum. All experimental protocols were conducted in accordance with the guidelines set by the ethics committee and Institutional Animal Care at KSU (Approval no: KSU-SE-24-3). The rats were categorized into three groups, each comprising three females and three males. The control group receives water while the two other groups were administered with 100 mg/kg and 250 mg/kg of the ZTN polyherbal formulation, respectively. The treatment lasted for 14 days before the animals were sacrificed. Glutathione (GSH) Assay: The levels of reduced glutathione (GSH) in the plasma were measured using a colorimetric assay kit obtained from Cayman Chemical. The assay was performed according to the manufacturer’s instructions. Basically, a yellow-colored product was formed upon the reaction of plasma with DTNB (5,5'-dithiobis-2-nitrobenzoic acid), thereafter, the absorb absorbance was measured at 405 nm, and the GSH concentration was calculated using a standardized curve.
Malondialdehyde (MDA) Assay: The concentration of malondialdehyde (MDA), a marker of lipid peroxidation, was determined using a Thio barbituric acid reactive substances (TBARS) assay kit obtained from Sigma-Aldrich as TBARS Assay Kit. The assay involved mixing the plasma with TBA reagent, incubating the mixture at 95°C for 60 minutes, cooling it on ice, and then measuring the absorbance at 532nm. The MDA levels were quantified using a standardized curve generated from known MDA standards. Other Antioxidants: Additional antioxidants, such as superoxide dismutase (SOD) and catalase (CAT) activities were measured using specific assay kits available as., Sigma-Aldrich SOD and Catalase Assay Kits. These assays involved reactions that produce measurable color changes, with absorbance read at specific wavelengths as per the kit instructions (Szczeklik et al., 2018).
The antioxidant levels were expressed as means ± standard deviation (SD). Statistical analysis was performed using appropriate software (e.g., SPSS or GraphPad Prism). Comparisons between separate groups were made using one-way ANOVA followed by post hoc tests to determine significant differences.
RESULTS
The catalase activity in ZTN treated rats demonstrates a dose dependent response in both male and female rats. Figure 1 shows that the control group demonstrates a baseline level of catalase activity as the male and female rats showed the lowest catalase activity. The 100mg/kg ZTN treated group showed a significant increase in catalase activity in both male and female rats when compared with the control. Meanwhile, male rats exhibited a slightly higher catalase activity than female rats at 100 mg/kg. At the highest ZTN dosage (250 mg/kg), catalase activity was observed to be at highest level in both male and female rats. Male rats again showed a slightly higher catalase activity compared to female rats at this higher dosage.
ZTN polyherbal formulation enhanced antioxidant enzyme activities in both male and female rats, with males showing a marginally higher response. Figure 1 shows that both male and female rats exhibited a dose-dependent increase in SOD activities in relation to the increase in the ZTN polyherbal formulation doses. Across all treatment groups, male rats consistently demonstrated slightly higher SOD activities compared to female rats.
The study analyzed the effects of ZTN polyherbal formulation on malondialdehyde (MDA) levels, a marker of oxidative stress. Figure shows that both male and female rats displayed a dose-dependent decrease in MDA levels at higher dosage of the ZTN polyherbal extract. Male rats consistently showed a slightly greater reduction in MDA levels compared to female rats across all treatment groups. ZTN polyherbal extract effectively reduces oxidative stress, as indicated by lower MDA levels, in both male and female rats. However, male rats demonstrated a marginally better response to the treatment. These findings underscore the importance of considering sex differences in antioxidant research, which could inform more tailored therapeutic strategies.
In the control group, both male and female rats exhibited the lowest baseline levels of glutathione, indicating minimal oxidative stress resistance without any treatment. In 100mg/kg ZTN polyherbal administered group, a marked increase in glutathione levels was observed in both sexes. Male rats demonstrated a more pronounced increase compared to their female counterparts, suggesting a higher sensitivity or responsiveness to the polyherbal treatment at this dosage. At 250mg/kg, the glutathione levels increased significantly in both male and female rats, show casing a dose-dependent enhancement in antioxidant defense. Male rats again exhibited higher glutathione levels than female rats at this higher dosage, indicating a consistent trend of greater responsiveness to the polyherbal extract.
Figure 1 shows a clear dose-dependent increase in glutathione levels at higher doses of the polyherbal extract in both male and female rats. However, male rats consistently displayed higher glutathione levels across all treatment groups compared to female rats. This suggests that male rats may have a slightly more robust response to the antioxidant effects of the polyherbal therapy. The administration of ZTN polyherbal extracts significantly enhanced glutathione levels in both male and female rats, with males exhibiting a marginally better response. These findings underscore the importance of considering sex differences in antioxidant research and potential therapeutic applications, as males and females may respond differently to treatment.
The effects of ZTN polyherbal formulation on antioxidant levels in rats was assessed. Specifically, figure - shows the glutathione (GSH) peroxidase activity in male and female rats. There was a significant difference in the level of the enzyme in both sexes. The control group exhibited the lowest levels of glutathione peroxidase in both male and female rats, establishing a baseline for comparison. Following the administration of 100mg of the ZTN polyherbal formulation, a significant increase in glutathione peroxidase activity was observed in both sexes. Male rats exhibited a slightly higher increase compared to female rats, indicating a more pronounced response to the treatment at this dosage. At 250mg/kg, the glutathione peroxidase activity was more significant in both male and female rats. Despite both sexes showing substantial increases, male rats consistently had higher levels of this antioxidant enzyme compared to female rats at the same dosage. while both male and female rats responded positively to ZTN polyherbal formulation with increased glutathione peroxidase activity, male rats demonstrated a slightly more robust response. These findings highlight the importance of considering sex differences in antioxidant research.
DISCUSSION
This study investigates the effects of ZTN polyherbal formulation on antioxidant levels in male and female rats, revealing significant sex-based differences in response. The results are in tandem with recent studies that highlight the therapeutic potential of polyherbal formulations, and their differential impacts based on sex. Both male and female rats exhibited dose-dependent increase in catalase activity following ZTN treatment, while males consistently showing higher activity levels. Polyherbal formulations rich in phenolics and flavonoids significantly boost catalase activity (Ghasemzadeh et al. 2010). The slightly higher response in males could be attributed to metabolic differences affecting antioxidant enzyme activity. Similarly, antioxidant enzyme Superoxide Dismutase (SOD) activity was increase in both sexes, with a marginally higher increase in males. Polyherbal extracts have been reported to enhance SOD activity, providing robust protection against oxidative stress (Adam et al., 2016). The sex-specific difference suggests that male rats may have a more efficient mechanism for upregulating SOD in response to polyherbal treatments. MDA, a marker of lipid peroxidation, was found to be decreased significantly in both male and female rats after treatment with ZTN polyherbal formulation and males having a slightly greater reduction. A report by (Mishra and Singh, 2009) indicates that polyherbal formulations effectively reduce MDA levels, thereby mitigating oxidative damage. The slightly better response in males could be linked to their higher baseline oxidative stress, making the reduction more pronounced.
In addition, the dose-dependent increase in glutathione levels in both sexes while males showing slightly higher levels can be attributed to the presence of a critical component of the antioxidant defense system in the polyherbal formulation (Stankovic et al., 2011). The observed sex difference might be due to variations in glutathione metabolism or synthesis rates between males and females. Similarly, both male and female rats exhibited significant increases in glutathione peroxidase activity after polyherbal extract treatment, with males showing a slightly more robust response. Sabir et al. (2003) reported that enhanced glutathione peroxidase activity following polyherbal treatment highlights its role in reducing oxidative stress. The marginally higher activity in males suggests that sex-specific factors may influence the enzyme's regulation and activity.
CONCLUSION
The study confirms that ZTN polyherbal extracts significantly enhance antioxidant defenses in both male and female rats, while males showing a slightly more robust response across all measured parameters. These sex-specific differences underscore the necessity of considering gender as a biological variable in antioxidant research and therapeutic applications. Future studies should aim to elucidate the underlying mechanisms driving these differences and optimize polyherbal formulations accordingly.
ACKNOWLEDGMENTS
The authors sincerely acknowledge the Researcher Support Project (RSP-2024R112) for funding this work at King Saud University, Riyadh, Saudi Arabia.
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