Crude and Nanosynthesized Selected Medicinal Plants Demonstrate Low Acute Toxicity and Hematopoietic Efficacy in Wistar Rats

*Corresponding author: Babayemi Olawale Oladejo Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Crude and Nanosynthesized Selected Medicinal Plants Demonstrate Low Acute Toxicity and Hematopoietic Efficacy in Wistar Rats Muftau Kolawole Oladunmoye 1, MacDonald Idu 2, Isiaka Adekunle Amoo 3, Babayemi Olawale Oladejo 1, *, Ruth Ebunoluwa Bodunrinde 4 and Elizabeth Tomilayo Akinterinwa 1 1 Department of Microbiology, Federal University of Technology Akure, Nigeria. 2 Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria. 3 Department of Chemistry, Federal University of Technology Akure, Nigeria. 4 Department of Microbiology, Joseph Sarwuan Tarka University, Makurdi, Nigeria. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 Publication history: Received on 26 September 2025; revised on 09 November 2025; accepted on 12 November 2025 Article DOI: https://doi.org/10.30574/gscbps.2025.33.2.0431 Abstract This study evaluated the in vivo biosafety profile of extracts from Hibiscus sabdariffa, Justicia carnea, Zingiber officinale and honey alongside their nanosynthesized extracts. Acute (single-dose) and 28-day sub-chronic (100, 200, and 400 mg/kg/day) oral toxicity studies were conducted in Wistar rats following OECD guidelines. Assessments included behavioral observations, body temperature and weight changes, hematological parameters and liver markers including Alanine transaminase (ALT), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) using standard laboratory techniques. Acute toxicity test revealed no mortality or significant adverse behavioral changes up to 400 mg/kg. In the 28-day toxicity study, all formulations were generally well-tolerated. The administration of plants at doses of 100, 200, and 400 mg/kg and NPs does not appear to have a biologically significant effect on the body temperature of albino rats over a 28-day period. Also, the acute toxicity of the plant extracts at 400 mg/kg and the NPs at E9 +NPs1 body weight had no observable deleterious effects on the rat’s health, temperature and behavioral features. Hematological analyses indicated significant modulatory effects as extracts administration at 28 days, markedly increasing platelet counts; 1128.50 × 10⁹/L at 200 mg/kg and RBC counts 7.30 × 10¹²/L at 100 mg/kg). This showed that these plants could possess pharmacokinetic potentials which could modulate immune response and serve as potential alternative therapy against infection disease agents. Keywords: Hibiscus sabdariffa; Justicia carnea; Liver markers; Nanoparticles; Toxicity; Zingiber officinale 1. Introduction The escalating crisis of antimicrobial resistance (AMR) represents one of the most significant threats to modern healthcare, rendering conventional antibiotics progressively ineffective against common pathogens [1]. The antimicrobial activity of plant extracts has garnered much attention in the biomedical research field due to the growing threat over antibiotic resistance and the need for alternative therapeutic agents. Plants are known to produce various array of secondary metabolites, many of which have antimicrobial properties. Many of these compounds have been shown to exhibit antimicrobial activity against a wide range of pathogens, including bacteria, fungi, and viruses. Several studies have demonstrated that combinations of plant compounds can exhibit synergistic or additive effects, where the antimicrobial activity of the mixture is greater than the sum of the activities of its individual components. Plant-derived compounds often act on several targets within microbial cells, thereby making it difficult for pathogens to develop resistance [1,2]. The failure of empirical antibiotic therapy due to resistance necessitates an urgent paradigm shift toward novel therapeutic strategies. One promising avenue is the exploration of agents that, rather than directly GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 220 targeting the microbe, bolster the host's own defense mechanisms—a strategy known as immunomodulation [3]. Concurrently, strengthening the host's hematopoietic (blood-forming) system is vital for maintaining physiological resilience and ensuring a robust supply of immune cells during infection. Medicinal plants remain a vast reservoir of complex phytochemicals with diverse pharmacological activities. Zingiber officinale (ginger) is widely recognized for its potent anti-inflammatory and immunomodulatory properties, primarily attributed to compounds like gingerols, which modulate critical signaling pathways such as NF-κB [4]. Hibiscus sabdariffa (roselle) has a long history in traditional medicine for treating UTIs, with modern studies corroborating its antimicrobial, antioxidant, and immunomodulatory effects [5]. Similarly, Justicia carnea is extensively used in African ethnomedicine, lauded for its remarkable hematopoietic ("blood-boosting") properties. Recent in vivo studies have validated this traditional use, demonstrating its capacity to significantly increase red blood cell counts, hemoglobin, and packed cell volume, alongside exhibiting anti-inflammatory activity [6]. Despite this therapeutic potential, the clinical application of crude herbal extracts is often hampered by poor phytochemical stability, low bioavailability, and rapid metabolic degradation [7]. Nanotechnology, specifically the green synthesis of nanoparticles using plant extracts, offers a transformative solution. This method uses the plant's own phytochemicals as reducing and capping agents, creating nanosynthesized formulations that enhance stability, improve delivery, and potentiate biological efficacy [8]. Recent studies confirm that nanoparticles synthesized from Z. officinale and H. sabdariffa exhibit superior antibacterial activity against Escherichia coli and Staphylococcus aureus compared to crude extracts [9,10]. A significant gap persists between the in vitro potentials of these herbal extracts and the requisite in vivo data for therapeutic development. While the immunomodulatory and hematopoietic properties are documented, there is a lack of rigorous, comparative in vivo studies evaluating their safety and efficacy, especially for their nanosynthesized counterparts, in the context of an active infection. Furthermore, the toxicity profile of these specific formulations, as mandated by OECD guidelines, has not been thoroughly established. Therefore, the aim of this study was to evaluate and compare the acute and sub-chronic toxicity profiles, as well as the hematopoietic and immunomodulatory efficacy, of crude and nanosynthesized extracts from H. sabdariffa, J. carnea, and Z. officinale in honey. 2. Methods 2.1. Plant Collection and Authentication The plants used in this study were H. sabdariffa L. (calyces), Z. officinale L. (rhizomes), and J. carnea L. (leaves), identified with voucher numbers UIH-23344, UIH-23356 and UIH-23346 respectively. Honey was obtained from the School of Agriculture and Agricultural Technology (FUTA), Nigeria and proximate analysis was conducted on it [11,12]. 2.2. Extraction of Plant Materials using Different Solvents Ethanol, chloroform and hot water extracts were derived from H. sabdariffa calyx, J. carnea leaves, and Z. officinale rhizomes. The plant materials were air-dried at room temperature for 7–10 days to constant weight, then ground into fine powder using a mechanical blender. The dried, powdered material (100 g) was mixed with the respective solvents at a 1:10 ratio (100 g in 1000 mL of solvent) for each solvent and soaked for 72 hours with intermittent agitation to facilitate extraction. The mixture was then filtrated/sieved using muslin cloth followed by Whatman No. 1 filter paper to remove solid residues [13,14]. The filtrates were concentrated under vacuum using a rotary evaporator (RE-52A, Union Laboratories, England) at 37°C to obtain the crude extracts. The 100% stock concentrates of the extracts were obtained and stored in a refrigerator at 4°C in sterile universal bottles until further use. 2.3. Green Nano-synthesis and Physical Characterization Silver nanoparticle (AgNP) synthesis involved preparing an aqueous solution (1 mM) of silver nitrate (AgNO₃) in 250 mL Erlenmeyer flasks, to which plant extract was added for reduction into Ag⁺ ions for each type of plant extract. The composite mixture was kept on a magnetic hot plate or microwave oven for complete bioreduction at a power of 300 W for 4 minutes discontinuously to prevent an increase of pressure. In the meantime, the colour change of the mixture from faint light to yellowish brown to reddish brown to colloidal brown was monitored periodically (time and colour change were recorded along with periodic sampling and scanning by UV-visible spectrophotometry) for a maximum of 30 minutes. The reactions were carried out in dark (to avoid photoactivation of AgNO₃) at room temperature. Complete reduction of AgNO₃ to Ag⁺ ions was confirmed by the change in colour from colourless to colloidal brown. After irradiation, the dilute colloidal solution was cooled to room temperature and kept aside for 24 hrs for complete bioreduction and saturation. Then, the colloidal mixture was sealed and stored properly for further use. The formation GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 221 of AgNPs was furthermore confirmed by spectrophotometric analysis [15,16]. To isolate the nanoparticles, the colloidal solution was centrifuged at 4000 rpm for 15 min to remove large aggregates, followed by ultracentrifugation at 25,900 rpm for 30 min to pellet the nanoparticles. The pellets were washed thrice with deionized water to remove unbound phytochemicals and redispersed in sterile water for characterization. 2.4. Animal Handling Ethical approval for the use and handling of animals was obtained from the Center for Research and Development (CERAD) of the Federal University of Technology Akure (FUTA) Ethical Committee for the use and care of laboratory animals. One hundred and twenty (120) Wistar rats (weighing 18–24 g) were used for this study and were purchased at the breeding colony of the Department of Animal Production and Health, Federal University of Technology Akure. The rats were maintained at 25°C on a 12-hour light/dark cycle with access to food and water for two weeks before the commencement of the experiment. Animal handling was in accordance with the rules and regulations prepared by the Washington Institute for Laboratory Animal Research [17]. 2.5. Acute Toxicity Assay Healthy female Wistar rats were used in this study according to the instructions of the Organization for Economic Cooperation and Development (OECD) for acute oral toxicity tests [18]. All animals were fasted overnight but had free access to water and were weighed before administration of the extracts. The animals were randomly divided into five groups (n = 4 per group) for each extract. Group 1 (Control) received distilled water orally; groups 2, 3 and 4 (Acute toxicity) received 100 mg/kg, 200 mg/kg and 400 mg/kg respectively, while group 5 (nanoparticles) received corresponding plant-based nanosynthesized extract [19,20]. The five groups were also reproduced for the plants used: H. sabdariffa, J. carnea and Z. officinale. The animals were then observed for mortality, signs of acute toxicity and behavioural changes (aggression, unusual vocalization, agitation, sedation and somnolence, convulsions, tremors, ataxia, catatonia, paralysis, fasciculation, prostration and unusual locomotion and asphyxia) for the first 30 mins, 1 hr, 5 hrs and finally periodically up to 48 hrs. All experimental animals were individually observed daily for general behaviour and body weight changes, dangerous symptoms and mortality [21,22]. 2.6. Sub-Chronic Toxicity Assay The experiment was conducted according to the protocols described by OECD Guideline 407 [19,23,24] with minor modifications. This was a 28-day sub-chronic toxicity test and a continuation from the acute testing. Prior to treatment, rats were handled individually and carefully examined for abnormal behaviour and appearance. The plant extracts were dissolved in distilled water, and respective nanoparticles were administered orally once a day for 28 consecutive days. Group 1 (Control) received distilled water orally; groups 2, 3 and 4 (Sub-chronic toxicity) received 100, 200 and 400 mg/mL respectively, while group 5 (Nanoparticles) received corresponding plant-based nanosynthesized extract. The five groups were also reproduced for the plants used: H. sabdariffa, J. carnea and Z. officinale. The animals were observed daily during the experimental period for mortality or morbidity, changes in posture, changes in the fur, skin, eyes, mucous membranes and behaviours. At the end of the first 7 days, the first set of animals, at 14 days another set, and at 21 days a different set were bled to assess internal organs; blood samples were taken by retro-orbital puncture using capillary tubes for hematological and liver biochemical studies [25]. 2.7. Hematological and Liver Biochemical Test Blood samples taken in EDTA tubes were used for hematological treatment using an automated hematology analyzer (ABX Pentra XL 80, France). The differential count of leukocytes was performed with light microscopy after haematological staining (fixation with May Grunewald and staining with Giemsa stain. In each case, 100 cells were counted. Blood samples taken in anticoagulant-free tubes were used for biochemical analysis and were centrifuged at 3000 rpm for 10 min. The sera were separated, stored at -20°C and used for evaluation. Liver biochemical parameters such as; Alanine transaminase (ALT), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) were estimated using URIT 8021A automated analyzer (URIT Medical Electronic Group Co., Ltd.) [26]. 2.8. Statistical analysis All experiments were performed in triplicate, data were expressed as mean ± standard error (SE). Differences were analyzed using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test at p < 0.05 using SPSS version 25. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 222 3. Results 3.1. Nanosynthesized Crude Plant Extracts The green synthesis of AgNPs was initially confirmed by visual color changes in the reaction mixtures which confirm the green synthesis of silver nanoparticles (AgNPs). This color shift is a key indicator that silver ions (Ag+) have been reduced to silver atoms (Ag0), forming the nanoparticles (Table 1). Table 1 Visual Confirmation of Silver Nanoparticle (AgNP) Synthesis Plant Extract Color Before Reaction Color After Reaction (AgNPs formed) Justicia carnea Light Green Reddish-Brown Zingiber officinale Light Yellow Dark Brown Hibiscus sabdariffa Light Purple Dark Brown 3.2. Behavioural Observations of Oral Acute Toxicity in Rats administered with Nanosynthesized Crude Extracts The appearance and behavioral observations of the oral administration of H. sabdariffa ethanol J. carnea hot water, Z. officinale hot water extract and their nanoparticles (NPs) in rats is shown in Table 2. There was no observable changes in the body weight, temperature, eye colour and rate of respiration. In addition, there was no lethality and unconsciousness record in any of the experiment groups. However, sedation, slight reduction in food intake and raised fur of rats were observed only immediately after administration which normalizes within 1-2 hours. 3.3. Effects of Administration of H. sabdariffa Ethanol Extract on Body Temperature of Albino Rats Throughout the study, the temperature ranged between 36°C and 39°C across all groups, which is within the normal range for rats and there were no clear dose-dependent trend in temperature changes. Both 200 and 400 mg/kg groups showed significantly higher temperatures than 100 mg/kg and control groups on the last day of the experiment. By day 28, the higher doses (200 and 400 mg/kg) resulted in elevated temperature ranges compared to the lower dose and control as shown in Table 3. 3.4. Effects of Administration of J. carnea hot water Extract on Body Temperature of Albino Rats All groups, including the control, showed some fluctuations in temperature ranges over the 28-day period. These fluctuations appeared to be within a relatively narrow range of 36.5°C to 38.5°C. There was no consistent pattern of temperature increase or decrease over time in any of the treatment groups compared to the control, suggesting that the J. carnea hot water extract does not have a pronounced long-term effect on body temperature as shown in Table 4. 3.5. Effects of Administration of Z. officinale hot Water Extract on Body Temperature of Albino Rats There was no clear dose-dependent effect on temperature ranges across the different dosage groups (100 mg/kg, 200 mg/kg and 400 mg/kg) compared to the control. There are no consistent patterns of temperature increase or decrease over time in any of the treatment groups compared to the control. The fluctuations appear random and similar across all groups as shown in Table 5. 3.6. Effects of Administration of Nanoparticles of H. sabdariffa, J. carnea and Z. officinale Extracts on Body Temperature of Albino Rats There was no significant differences (P<0.05) between treatments and control for most days. No consistent pattern of temperature increase or decrease over time for any treatment. Fluctuations appeared random and mostly within the range of control group variations. Treatment group nHSE showed the highest temperature value (38.15°C on Day 7) while nJCH, was generally similar to control, with a slight decrease on day 22 (36.20°C). Group nZOH was closest to the control values throughout the study as shown in Table 6. GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 223 Table 2 General Appearance and Behavioural Observations of Oral Acute Toxicity in Rats administered with Plant Extracts and Nanoparticles Observations Control Experimental Groups (HSE) Experimental Groups (JCH) Experimental Groups (ZOH) Experimental Groups (NPs) 100 mg/kg 200 mg/kg 400 mg/kg 100 mg/kg 200 mg/kg 400 mg/kg 100 mg/kg 200 mg/kg 400 mg/kg HSE JCH ZOH Body weight N C C C C C C C C C C C C Temperature N N N N N N N N N N N N N Food/water intake N N N R N N N N N R N R R Rate of Respiration N NE NE NE NE NE NE NE NE NE NE NE NE Foot thickness N NE NE NE NE NE NE NE NE NE NE NE NE Change in skin N NE NE NE NE NE NE NE NE NE NE NE NE Eye colour N NE NE NE NE NE NE NE NE NE NE NE NE Raised fur N NP P P NP P P NP P P NP NP NP Drowsiness NP NP NP NP NP NP NP NP NP NP NP NP NP Coma NP NP NP NP NP NP NP NP NP NP NP NP NP Death A A A A A A A A A A A A A Keys: HSE= H. sabdariffa Ethanol Extract, JCH= J. carnea Hot Water Extract, ZOH= Z. officinale Hot Water Extract, NPs= Nanoparticles, A= Alive, C= Change, NC= Not change, NE= No effect, NP= Not present, R= Reduce, P= Present Table 3 Effects of Administration of H. sabdariffa Ethanol Extract on Body Temperature of Albino Rats Days 100 mg/kg 200 mg/kg 400 mg/kg Control 1 38.20±0.35a 37.98±0.83a 38.30±0.79a 36.85±0.05a 4 36.68±0.14a 36.95±0.30a 38.23±0.17b 36.15±0.15a 7 37.48±0.58a 37.60±0.29a 37.30±0.47a 36.35±0.35a 10 37.53±0.29ab 37.00±0.06a 38.23±0.23b 37.10±0.10a 13 37.20±0.42a 37.67±0.27a 37.60±0.60a 37.80±0.10a 16 37.65±1.05a 37.45±0.25a 37.25±0.65a 36.40±0.40a 19 37.60±1.20a 37.50±0.30a 37.15±0.75a 37.05±0.05a 22 37.10±0.10b 36.25±0.25ab 36.00±0.00a 37.25±0.25b 25 37.25±0.25a 37.40±0.10a 37.25±0.25a 36.85±0.05a 28 37.50±0.00b 39.10±0.10c 39.10±0.10c 36.50±0.00a Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 224 Table 4 Effects of Administration of J. carnea hot water Extract on Body Temperature of Albino Rats Days 100 mg/kg 200 mg/kg 400 mg/kg Control 1 38.53±0.34a 37.20±0.70a 37.15±0.70a 36.85±0.05a 4 36.58±0.08a 37.53±0.54a 37.33±0.33a 36.15±0.15a 7 36.43±0.30a 37.50±0.11b 37.10±0.17bc 36.35±0.35a 10 37.40±0.12a 36.70±0.70a 37.27±0.23a 37.10±0.10a 13 37.17±0.28a 38.04±0.43a 37.37±0.22a 37.80±0.10a 16 37.80±0.10a 37.45±0.35a 37.35±0.35a 36.40±0.40a 19 37.15±0.05a 37.20±0.60a 37.10±0.00a 37.05±0.05a 22 37.75±0.05b 36.80±0.10a 37.80±0.00b 37.25±0.25ab 25 37.65±0.05b 37.10±0.10a 37.80±0.00b 36.85±0.05a 28 37.05±0.05b 36.80±0.10ab 38.20±0.00c 36.50±0.00a Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference Table 5 Effects of Administration of Z. officinale hot Water Extract on Body Temperature of Albino Rats Days 100 mg/kg 200 mg/kg 400 mg/kg Control 1 37.58±0.55a 37.33±0.74a 36.88±0.05a 36.85±0.05a 4 37.33±0.74a 36.88±0.56a 37.58±0.55a 36.15±0.15a 7 36.88±0.56a 36.88±0.56a 37.03±0.19a 36.35±0.35a 10 37.57±0.07a 37.73±0.38a 37.13±0.43a 37.10±0.10a 13 37.17±0.28a 36.70±0.69a 37.27±0.09a 37.80±0.10a 16 37.80±0.10a 37.65±0.15a 37.15±0.95a 36.40±0.40a 19 37.00±0.10a 37.75±0.35a 37.65±1.45a 37.05±0.05a 22 37.15±0.15a 37.35±0.35a 37.15±0.15a 37.25±0.25a 25 37.20±0.20a 37.35±0.35a 37.40±0.00a 36.85±0.05a 28 36.35±0.35a 37.45±0.45a 36.70±0.00a 36.50±0.00a Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference Table 6 Effects of Administration of Nanoparticles of H. sabdariffa, J. carnea and Z. officinale Extracts on Body Temperature of Albino Rats Days nHSE nJCH nZOH Control 1 37.58±0.83a 37.50±0.97a 37.73±0.70a 36.85±0.05a 4 37.18±0.35a 37.08±0.15a 37.50±0.97a 36.15±0.15a 7 38.15±0.60a 37.73±0.70a 37.03±0.47a 36.35±0.35a 10 37.53±0.38a 37.73±0.23a 37.43±0.40a 37.10±0.10a 13 37.37±0.23a 36.78±0.37a 36.87±0.75a 37.80±0.10a 16 37.45±0.05a 37.15±0.05a 36.75±0.05a 36.40±0.40a GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 225 19 37.50±0.60a 37.05±0.15a 36.45±0.65a 37.05±0.05a 22 37.05±0.50ab 36.20±0.20a 37.00±0.00ab 37.25±0.25b 25 37.30±0.00a 36.45±0.45a 37.55±0.05a 36.85±0.05a 28 36.80±0.00b 37.10±0.10c 36.50±0.00a 36.50±0.00a Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference. Keys: nHSE – nanosynthesized H. sabdarifa Ethanol, nJCH – nanosynthesized J. carnea Hot water, nZOH – nanosynthesized Z. officinale Hot water 3.7. Effects of Administration of H. sabdariffa Ethanol Extract on Body Weight of Albino Rats There was no consistent dose-dependent trend across all time points. However, the 400 mg/kg dose generally showed lower weight gain compared to other groups. All groups revealed an overall increase in weight over the 28-day period. The 100 mg/kg group exhibited the most pronounced weight gain, surpassing the control group from Day 13 onward. The 400 mg/kg group consistently showed the least weight from Day 7 onward as shown in Table 7. 3.8. Effects of Administration of J. carnea hot water Extract on Body Weight of Albino Rats The result in Table 8, revealed that there was no consistent dose-dependent trend across all time points. However, the 200 mg/kg dose generally showed higher weight gain, especially in the latter half of the study. The extract appears to affect weight gain differently at various doses. The 200 mg/kg dose promotes weight gain more than the control, which could be beneficial or concerning depending on the context. 3.9. Effects of Administration of Z. officinale hot Water Extract on Body Weight of Albino Rats All groups, including the control, showed an overall increase in body weight over the 28-day period, indicating normal growth. The 100 mg/kg group showed the least weight gain, often lower than the control. The 200 mg/kg group consistently showed the highest weight gain among treatment groups. The control group showed steady weight gain, ending with the second-highest weight by Day 28 as shown in Table 9. 3.10. Effects of Administration of Nanoparticles of H. sabdariffa, J. carnea and Z. officinale Extracts on Body Weight of Albino Rats All nanoparticle extract administered groups showed higher body weights compared to the control. Treatment group nHSE group showed the highest final body weight, followed closely by nJCH and nZOH. For the first 19 days, there were no statistically significant differences between groups. Group nHSE appeared to have the most pronounced effect on weight gain, especially in the later stages of the study as shown in Table 10. Table 7 Effects of Administration of H. sabdariffa Ethanol Extract on Body Weight of Wistar Rats Days 100 mg/kg 200 mg/kg 400 mg/kg Control 1 93.00±1.22a 119.25±3.20b 118.25±4.97b 98.33±0.33a 4 98.75±0.48a 113.25±1.11b 109.75±4.50b 94.67±0.33a 7 101.50±1.55a 123.25±1.25b 108.25±4.57a 100.00±0.00a 10 129.33±10.35a 125.00±3.61a 108.67±6.44a 112.33±0.33a 13 137.00±9.61b 108.67±6.44ab 102.67±7.54a 116.33±0.33ab 16 135.50±0.50c 125.00±2.00b 112.00±1.00a 120.33±0.33b 19 134.00±1.00b 133.50±8.50b 114.00±0.00a 125.00±0.58ab 22 148.00±0.00d 126.50±0.50b 111.50±0.50a 130.33±0.33c 25 141.50±0.50c 130.50±0.50b 110.50±0.50a 132.00±0.00b 28 142.50±0.50c 141.50±0.50c 121.50±0.50a 132.00±0.00b Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 226 Table 8 Effects of Administration of J. carnea hot water Extract on Body Weight of Wistar Rats Days 100 mg/kg 200 mg/kg 400 mg/kg Control 1 107.75±4.60a 101.75±5.50a 101.25±5.42a 98.50±0.50a 4 96.25±2.10a 96.25±1.93a 104.00±1.47a 95.50±0.50a 7 105.75±5.38a 105.25±5.96a 113.50±2.75a 100.50±0.50a 10 114.00±7.51a 110.00±7.57a 116.67±3.38a 111.50±0.50a 13 116.00±9.50a 121.33±5.33a 116.67±2.96a 116.00±0.00a 16 112.00±14.00a 136.00±5.00a 115.50±0.50a 120.50±0.50a 19 123.00±11.00a 136.00±2.00a 119.00±1.00a 126.50±0.50a 22 118.50±0.50a 138.50±0.50c 117.50±0.50a 130.00±0.00b 25 121.50±0.50b 141.50±0.50d 112.50±0.50a 131.50±0.50c 28 129.00±0.00b 150.50±0.50d 123.00±0.00a 132.00±0.00c Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s Honestly Significant Difference Table 9 Effects of Administration of Z. officinale hot water Extract on Body Weight of Wistar Rats Days 100 mg/kg 200 mg/kg 400 mg/kg Control Day 1 108.25±4.87a 116.00±7.45a 107.50±3.52a 98.50±0.50a Day 4 97.75±6.38a 114.25±6.54a 106.25±4.25a 95.50±0.50a Day 7 96.50±7.41a 116.25±6.02a 108.00±2.89a 100.50±0.50a Day 10 108.00±4.73a 120.33±7.06a 113.33±2.60a 111.50±0.50a Day 13 115.67±3.48a 126.33±8.84a 118.00±2.52a 116.00±0.00a Day 16 115.00±6.00a 135.50±2.50b 114.50±2.50a 120.50±0.50ab Day 19 118.00±7.00a 137.00±2.00a 115.50±3.50a 126.25±3.53a Day 22 118.50±0.50a 148.50±0.50c 118.00±0.00a 130.50±0.50b Day 25 124.50±0.50b 160.50±0.50d 120.50±0.50a 132.00±0.00c Day 28 128.50±0.50a 168.00±0.00d 124.50±0.50a 131.50±0.50c Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference Table 10 Effects of Administration of Nanoparticles of H. sabdariffa, J. carnea and Z. officinale Extracts on Body Weight of Wistar Rats Days nHSE nJCH nZOH Control Day 1 102.75±5.39a 115.00±2.68a 117.25±3.90a 98.50±0.50a Day 4 100.50±5.11a 107.75±4.19a 113.25±3.20a 95.50±0.50a Day 7 100.50±3.30a 114.25±2.53a 111.00±9.89a 100.50±0.50a Day 10 108.00±2.00a 117.00±3.21a 115.67±13.86a 112.50±0.50a GSC Biological and Pharmaceutical Sciences, 2025, 33(02), 219-233 227 Day 13 109.00±6.43a 126.33±3.93a 130.00±8.54a 116.00±0.00a Day 16 118.50±1.50a 122.50±1.50a 122.00±2.00a 120.00±0.00a Day 19 126.00±1.00a 129.50±0.50a 126.00±1.00a 125.50±0.50a Day 22 137.00±0.00b 136.50±0.50b 130.50±0.50a 130.00±0.00a Day 25 141.50±0.50d 139.00±0.00c 136.50±0.50b 132.00±0.00a Day 28 143.50±0.50c 141.00±0.00b 141.50±0.50bc 132.00±0.00a Data are presented as Mean ± S.E (n=3). Values with the same superscript letter(s) along the same rows are not significantly different (p<0.05) according to Tukey’s honestly significant difference. Keys: nHSE – nanosynthesized H. sabdarifa Ethanol, nJCH – nanosynthesized J. carnea Hot water, nZOH – nanosynthesized Z. officinale Hot water 3.11. Effects of Administration of H. sabdariffa Ethanol Extract on Liver Biomarkers of Wistar Rats The effects of H. sabdariffa ethanol extract on liver function tests in albino rats over a 28-day period is shown in Figure 1. The liver enzymes measured were AST, ALT and ALP at days 7, 14, 21, and 28. Higher doses (200 mg/kg and 400 mg/kg) generally resulted in more pronounced changes in enzyme levels compared to the 100 mg/kg dose and control. AST levels elevated at all doses compared to control, particularly at days 7 and 14. The 200 mg/kg dose showed the highest AST levels at day 7. ALT levels showed significant elevations, particularly at day 21 for all doses while, the 400 mg/kg dose resulted in the highest ALT levels at day 21. 3.12. Effects of Administration of J. carnea hot water Extract on Liver Biomarkers of Wistar Rats The 200 mg/kg and 400 mg/kg doses showed the highest AST levels, suggesting potential hepatocellular damage at higher concentrations. ALT levels also showed significant elevations, particularly at day 14 for the 100 mg/kg dose and day 7 for the 400 mg/kg dose. The control group maintained relatively stable enzyme levels throughout the study period as shown in Figure 2. 3.13. Effects of Administration of Z. officinale hot Water Extract on Liver Biomarkers of Wistar Rats The 200 mg/kg dose often showed the most pronounced changes, particularly for ALT and AST. Enzyme levels fluctuated over time, with different patterns for each enzyme, dose, and time point. AST levels was generally elevated across all doses compared to control, with the 200 mg/kg dose showing the highest levels, particularly at day 21. ALT levels showed significant elevations, particularly for the 200 mg/kg dose at day 7. The 400 mg/kg dose also showed elevated ALT at day 7, but to a lesser extent than the 200 mg/kg dose as shown in Figure 3. 3.14. Effects of Administration of Nanoparticles of H. sabdariffa, J. carnea and Z. officinale Extracts on Liver Biomarkers of Wistar Rats Higher doses of 100 mg/kg and 400 mg/kg resulted in more pronounced changes in enzyme levels compared to the 100 mg/kg dose and control. AST was elevated at all doses compared to control, particularly at days 7 and 14. The 200 mg/kg dose showed the highest AST levels, especially at day 7. ALT showed significant elevations, particularly at day 21 for all doses while, 400 mg/kg dose resulted in the highest ALT levels at day 21. ALP demonstrated less changes compared to AST and ALT. Some elevation were observed in the enzyme levels at day 14 for the 100 mg/kg and 200 mg/kg doses with little to no changes in the control group as shown in Figure 4.