Preclinical Safety Assessment and Serum Lipid Profiles of Wister Rats Following Acute and Subchronic Exposure to Water-Soluble Curcuminoid-Rich Extracts

Abstract

The present study aimed to establish water-soluble curcuminoid-rich extracts and assess their acute and subchronic toxicities, following the OECD guidelines. Water-soluble curcuminoid-rich extracts, namely, CRE-Ter [ternary complex of curcuminoid-rich extract (CRE), hydroxypropyl-β-cyclodextrin, and polyvinylpyrrolidone K30] and CRE-SD (CRE in a solid dispersion form with polyvinylpyrrolidone K30) were produced via green technology, and their curcuminoid content was subsequently quantified using a high-performance liquid chromatographic (HPLC) method. CRE-Ter and CRE-SD contained 17.8% (w/w) total curcuminoids (12.7% (w/w) of curcumin, 3.2% (w/w) of demethoxycurcumin, and 1.9% (w/w) of bisdemethoxycurcumin) and 7.5% (w/w) total curcuminoids (5.2% (w/w) of curcumin, 1.4% (w/w) of demethoxycurcumin, and 0.9% (w/w) of bisdemethoxycurcumin), respectively. The acute and subchronic toxicities of the extracts were investigated in both male and female rats. The limit test of acute toxicity revealed that the oral LD₅₀ of both CRE-Ter and CRE-SD was found to be greater than 2000 mg/kg, with no signs of acute toxicity or mortality during a single dose treatment of 2000 mg/kg. Similarly, regular oral administration of 0, 10, 30, and 300 mg/kg/day of CRE-Ter or CRE-SD for 90 days did not induce any significant toxicological effects on the clinical signs, body weights, food consumption, or water intake of both male and female rats. Moreover, no adverse effects were noted on the hematological or serum biochemical parameters. The gross appearance and histopathological analysis of the major visceral organs in treated groups were comparable to those of the control group. Interestingly, both CRE-Ter and CRE-SD significantly decreased the levels of lipid profiles and fasting blood glucose. These results clearly highlight the excellent safety profiles of CRE-Ter and CRE-SD when administered orally, paving the way for future drug development.

Research Highlights

• 1.Based on GHS, CRE-SD and CRE-Ter are classified as Category 5, LD₅₀ > 2000 mg/kg.
• 2.Both extracts did not result in any toxicologically significant in a subchronic toxicity test.
• 3.No adverse effects were found on the hematology or serum biochemistry parameters for both extracts.
• 4.The lipid profiles and fasting blood glucose levels were significantly decreased by both extracts.
• 5.These results support the highly safe and very low toxicity of both extracts.

1. Introduction

Curcuma longa L., commonly known as turmeric, is a widely used herb with both medicinal and culinary applications. It is a perennial plant from the Zingiberaceae family, exhibiting a rich history of therapeutic use very well documented in Indian and Chinese Medicine Systems. The major curcuminoids, namely, curcumin, demethoxycurcumin, and bisdemethoxycurcumin, obtained from turmeric are bioactive compounds that advocate potential health benefits. Over the past half-century, comprehensive research has demonstrated many health advantages of curcumin,¹ widely recognized for its ability to exert numerous pharmacological effects, making it a molecule of interest in health and medical research. Its health benefits largely stem from its potent antioxidant and anti-inflammatory attributes.² In addition, curcumin has been reported to showcase a potent and a broad-spectrum antiviral activity, effectively disrupting viral replication at multiple stages of infection.^1,3

Unfortunately, a number of preclinical and clinical studies have revealed that curcuminoids exhibited a low bioavailability.⁴ This might result from either low water solubility or rapid metabolism, leading to quick excretion from the body. To increase the bioavailability of curcuminoids, researchers have discovered multiple strategies, such as the use of nanoparticles or complexation with other compounds, that can improve their absorption and subsequent retention in the body.

Recently, two water-soluble curcuminoid-rich formulations, namely, CRE-Ter and CRE-SD, were developed via a green method to address the bioavailability challenges of curcuminoids.⁵⁻⁷ Herein, CRE-Ter is a ternary complex of curcuminoid-rich extract (CRE) with hydroxypropyl-β-cyclodextrin/HPβCD and polyvinylpyrrolidone-K30/PVP-K30. In contrast, CRE-SD is a solid dispersion form of CRE combined with PVP-K30. Based on the results of HPLC analysis, CRE-Ter and CRE-SD were found to contain total curcuminoid contents of 14% and 7% w/w, respectively. Thereby, the improved water solubility of CRE-Ter paved the way for the enhanced anticancer potential of curcuminoids against human lung adenocarcinoma (A-549), human cervical adenocarcinoma (HeLa), and human colon adenocarcinoma (HT-29) cell lines.⁶ Meanwhile, both CRE-SD and CRE-Ter also showed increased antiosteoclastogenesis activity of curcuminoids,^5,8 highlighting their potential therapeutic effects against cancer as well as osteoporosis.

It is a universal truth that plant-based biomolecules are safe only if used within an appropriate dosage range. Therefore, it must be acknowledged that not all plant-based products are risk-free; uncontrolled or empirical consumption can lead to unwanted health implications.⁹ Previous safety evaluation studies revealed that curcumin was well tolerated at a very high dose without any toxic effects. Furthermore, some clinical trials that also investigated safety parameters suggested that curcumin given at doses as high as 12 g/day orally for 90 days did not cause any adverse effects and was well tolerated.¹⁰⁻¹² The water-soluble forms of CREs are known for their high bioavailability due to efficient gastrointestinal absorption, leading to their enhanced effectiveness. Despite this advantage, there has been no in-depth safety evaluation of these water-soluble CREs. Given their increased absorption and potential for future use in dietary supplements or nutraceuticals, it is crucial to understand their toxicological profile. This study therefore focuses on assessing the safety of CRE-Ter and CRE-SD via acute and subchronic toxicities in both male and female rats, following OECD guidelines. These evaluations would pave the way for the clinical assessment of water-soluble CREs, underscoring their potential role as therapeutic agents.

2. Materials and Methods

2.1. Preparation and Standardization of CRE-SD and CRE-Ter

CRE was prepared using an eco-friendly microwave extraction technique, followed by fractionation on a Diaion HP-20 column.⁷ The solvent was then evaporated at 45 °C under reduced pressure, yielding a CRE with 89.5% w/w of total curcuminoids, composed of 72.8% curcumin, 12.5% demethoxycurcumin, and 4.2% bisdemethoxycurcumin. Subsequently, CRE-SD was produced by dispersing CRE in an ethanolic solution of PVP-K30 at a mass ratio of 1:10 (CRE to PVP-K30). As the solvent was evaporated, it yielded a yellow powder with a total curcuminoid content of 7.5 ± 0.26% w/w total curcuminoids (5.2 ± 0.26% w/w of curcumin, 1.4 ± 0.01% w/w of demethoxycurcumin, and 0.9 ± 0.01% w/w of bisdemethoxycurcumin).⁵ CRE-Ter was prepared via an already established method.⁶ Following this method, a yellow powder of CRE-Ter was produced that comprised a total curcuminoids of 17.8 ± 0.47% w/w total curcuminoids (12.7 ± 0.37% w/w of curcumin, 3.2 ± 0.07% w/w of demethoxycurcumin, and 1.9 ± 0.04% w/w of bisdemethoxycurcumin). Finally, the water solubility of both products, CRE-SD and CRE-Ter, was determined to be 28.0 and 70.3 μg/mL, respectively. Figure 1 shows the HPLC chromatograms of CRE-Ter and CRE-SD.

Figure 1.

HPLC chromatograms of (A) CRE-Ter and (B) CRE-SD.

2.2. Experimental Animals

Albino Wistar rats (8–10 weeks old), weighing approximately 200–250 g for females and 300–350 g for males, were utilized for acute and subchronic oral toxicity studies. All of the animals were obtained from Nomura Siam International (Bangkok, Thailand) and kept in an animal house (Prince of Songkla University, Hat-Yai, Thailand). Before commencing the experiment, the animals were allowed approximately 1 week to acclimatize to the laboratory conditions. The animals were provided with unrestricted access to irradiation-sterilized pellet food (no. CP082, Perfect Companion Group, Bangkok, Thailand) and distilled water under standard environmental conditions at a room temperature of 24 ± 2 °C, a relative humidity of 55 ± 10%, 12 h of light, and 12 h of darkness. The experimental protocols were ethically approved by the Institutional Animal Care and Use Committee (MHESI 6800.11/124).

2.3. Acute Oral Toxicity Study

Acute oral toxicity study of CRE-SD and CRE-Ter was performed in accordance with the OECD guidelines (test no. 420).¹³ This stepwise approach involves starting with a small group of three animals at each step and adjusting the procedure based on the observed outcomes, such as mortality and/or morbidity. This structured method helps determining the toxic dose range while allowing for data accuracy with fewer animals, making it a more ethical and efficient approach than other toxicity testing methods. In the current study, a single high dose of 2000 mg/kg was administered orally (po) to initially assess the potential toxicity of CRE-SD and CRE-Ter. This dosage helped establishing a baseline for categorizing and classifying substances according to the Globally Harmonized System (GHS) for chemical safety.

Herein, three male and three female rats were randomly assigned to independent groups for each extract, ensuring that both genders were represented. Following administration, the rats were carefully monitored for signs of toxicity in stages: first, continuously during the first 30 min, an early critical period, then at intervals up to 4 h, and finally every 24 h over a 14 day period. Food was withheld initially to avoid any interference with absorption and potential toxic effects, and it was provided approximately 1–2 h after dosing. If the initial group of rats survived and showed no signs of severe toxicity, three additional rats were given the same dose under identical conditions to confirm the findings. This follow-up step was crucial for validating that the tested dose was within a safe range and to rule out any unexpected adverse effects.

Throughout the 14 day observation period following treatment, each animal was closely monitored daily for any clinical signs of toxicity, as well as for changes in body weight and food and water intake. At the conclusion of the study, each animal was weighed again to assess any cumulative impact on body mass, which is a key indicator of general health and wellness. For comprehensive hematological and biochemical analyses, blood samples were collected via cardiac puncture under anesthesia using an overdose (100 mg/kg) of thiopental sodium (Scott-Edil Pharmacia, Chandigarh, India). Additionally, the major organs were carefully removed for necropsy to assess any visible pathological changes. The organs were weighed to determine their relative weights and subsequently preserved in 10% buffered formalin for histopathological evaluation.

2.4. Subchronic Oral Toxicity Study

A subchronic oral toxicity study of CRE-SD and CRE-Ter was performed in accordance with the OECD guidelines (test no. 408):¹⁴ repeated dose, 90 days, oral toxicity study in rodents, with a few modifications. A total of 80 Wister rats, comprising 40 males and 40 females, each weighing between 200 and 350 g, were randomly assigned to two experimental groups, CRE-SD and CRE-Ter. Animals were subsequently divided into four subgroups (10 males and 10 females per group) and received either distilled water or three doses, that is, 10 (low), 30 (medium), and 300 (high), of CRE-SD and CRE-Ter, respectively. Both CRE-SD and CRE-Ter were suspended in distilled water (1 mL/kg body weight) and orally administered by oral gavage at 1 mL/kg body weight daily for 90 days. Rats in control groups were administered orally with the same volume of distilled water (vehicle). All animals were observed once daily for clinical signs of toxicity and twice daily for mortality. Changes in behavior, food and water intake, and body weight were also recorded. Bodyweight, food, and water consumption were determined every week for 90 days. At the end of the 90 day period, the animals were sacrificed under anesthesia, and blood samples were collected for the measurement of hematological and biochemical parameters. After euthanasia, the rats were sacrificed and organs were removed for necropsy, organ weight measurement, and histopathological examination.

2.4.1. Determination of the Toxicity Signs, Body Weight, Food Consumption, and Water Intake

Throughout the study, each animal was closely observed once daily for any clinical signs that might indicate toxicity, following the guidelines set forth in OECD 407.¹⁴ Observations included detailed assessments of general behaviors, respiratory patterns, motor activities (including gait and coordination), reflex responses, and changes in skin and fur conditions. Any abnormalities were carefully documented, including the type of abnormality, the date on which it was first noticed, and its severity. In addition, to monitor body weight changes, each animal was weighed at the beginning of the study (day 1, prior to treatment) and then approximately every week (at intervals of 7 ± 1 days). Food intake was also measured daily to calculate feed efficiency, which was expressed in grams per animal per day. This was determined by recording the difference between the initial amount of food provided and the food remaining after 24 h, helping to assess any changes in consumption patterns over time. Before euthanasia, all of the animals were fasted overnight to standardize the conditions and reduce the potential variability in hematological and biochemical measurements.

2.4.2. Determination of the Hematological and Biochemical Indices

Clinical pathology assessments, including blood chemistry and hematology, were conducted on all animals designated for terminal sacrifice to evaluate their overall health and physiological status. The animals were humanely euthanized with an intraperitoneal injection of sodium thiopental at a dose of 100 mg/kg. Blood samples were collected via cardiac puncture to ensure adequate and uncontaminated specimens. The collected blood was divided into two types of tubes: EDTA-containing tubes, used for hematological analysis to evaluate parameters like complete blood count, and nonheparinized tubes, used for biochemical analysis of serum to assess markers of liver and kidney function, glucose, and cholesterol levels. This process provided comprehensive data on the potential toxicological effects of the tested compound.

Hematological assessments were conducted using the ADVA 2120i Hematology system, covering various parameters such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin level (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), and differential counts of leukocytes, including lymphocytes, neutrophils, and monocytes. Additionally, blood smear analysis was performed to examine the morphology of blood cells, focusing on abnormalities like atypical lymphocytes, anisocytosis, hypochromia, microcytes, poikilocytosis, ovalocytosis, and platelet characteristics.

For the analysis of serum biochemical parameters, blood samples collected in nonheparinized tubes were processed to separate the serum. The samples were first centrifuged at 3000 rpm for 10 min at a temperature of 5 °C to ensure the efficient separation of the serum from the blood cells. The resulting serum was then subjected to analysis using an automated biochemistry analyzer, specifically the Cobas 6000 analyzer (Roche Diagnostics, Penzburg, Germany). A comprehensive panel of biochemical markers was assessed to evaluate organ function, including serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), which are enzymes used to evaluate liver function and potential hepatocellular damage; blood urea nitrogen (BUN), a key marker of renal function; and creatinine, which helps in assessing kidney health and filtration efficiency.

2.5. Determination of the Gross Necroscopy and Relative Weights of the Major Organs

A comprehensive gross necropsy was conducted on all of the rats to assess any potential pathological changes. This procedure involved a thorough examination of both the thoracic organs (e.g., heart and lungs) and the entire abdominal cavity, including all internal organs, to detect any visible abnormalities such as discoloration, swelling, or lesions. To further investigate potential microscopic changes, we carefully examined the major vital organs under a light microscope to identify any structural abnormalities. Following the examination, the major organs, including the heart, liver, kidneys, lungs, spleen, adrenal glands, thymus, and reproductive organs (testes, uterus, and ovaries), were surgically removed for further analysis. Each organ was meticulously washed with ice-cold saline solution to remove blood and debris, placed on absorbent paper to remove excess moisture, and then weighed to determine the absolute organ weight by the formula given below:

2.6. Histopathological Examination of the Major Organs

For histopathological examinations, major organs (lung, heart, liver, kidney, and spleen) and reproductive organs (testis and ovary) were removed, weighed, and preserved in 10% buffered formalin (pH 7.4). Following fixation, tissue specimens were dehydrated in a graded series of ethanol (70–100%), cleared in xylene, and finally enclosed in paraffin. A 3–4 μm thin section was prepared by using a microtome and stained with hematoxylin and eosin. Under a light microscope, the sections were examined at ×40. Microscopic observations were performed by an initial unblinded comparison of all of the control and treated groups. Blind and semiquantitative scoring was applied when changes were suggested by the initial inspection.

2.7. Statistical Analysis

Data are presented as mean ± SEM. Body weights, food consumption, relative organ weights, and hematological and biochemical parameters were assumed to follow a normal distribution. Statistical differences between groups were analyzed using one-way ANOVA. If ANOVA indicated significant differences, Duncan’s posthoc test was applied to identify specific pairs of groups with statistically significant differences. Statistical significance was set at p < 0.05.

3. Results

3.1. Acute Oral Toxicity

The acute toxicity evaluation of CRE-SD and CRE-Ter demonstrated that a single oral dose of 2000 mg/kg could not induce any mortality or observable toxicity in male or female rats during a 14 day observation period. No significant alterations were detected in body weight, food and water intake, or clinical behavior of the treated groups compared to the control group. Comprehensive analyses of clinical pathology revealed no significant deviations in hematological or biochemical parameters, and there were no notable changes in relative organ weights or histopathological findings. These findings indicated that both CRE-SD and CRE-Ter were well-tolerated at doses of up to 2000 mg/kg. Consequently, based on the Globally Harmonized Classification System (GHS), both the extracts were categorized as Category 5 substances (LD₅₀ > 2000 mg/kg), indicating that they belong to the least toxic class and pose minimal risk under the tested conditions.

3.2. Subchronic Oral Toxicity

3.2.1. Effects of CRE-SD and CRE-Ter on Mortality and Physical and Behavioral Observations

According to the results of the subchronic oral toxicity study, the treated groups showed no adverse effect or toxicity over the course of the 90 days (Table 1). Observations performed on a routine basis concluded no mortality/morbidity, revealing no clinical sign of tremors, convulsions, hair loss, abnormal gait/posture, unusual fecal consistency, reduced defecation, or distress during handling. Furthermore, no behavioral abnormalities or visible indications of toxicity were evident by the end of the study period, suggesting that the administration of the extracts was well-tolerated without causing noticeable harm or discomfort to the animal models.

Table 1. Subchronic Oral Toxicity Effects of CRE-SD and CRE-Ter on Physical and Behavioral Observations of Male Rats for 90 Days^a.

	CRE-SD (mg/kg)								CRE-Ter (mg/kg)
	male				female				male				female
observations	C	10	30	300	C	10	30	300	C	10	30	300	C	10	30	300
fur appearance	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
feces (bolus)	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
salivation	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
eyes	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
mucous membrane	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
lethargy	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF
convulsion	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF
tremors	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF
twitches	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF
vocalization	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
gait	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
body posture	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N	N
mortality	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF	NF

^aC: control, 10: 10 mg/kg, 30: 30 mg/kg; 300: 300 mg/kg, N: normal, NF: not found

During the course of the subchronic oral toxicity test, none of the treated groups died or showed obvious clinical signs (Table 1), such as tremors or convulsions, hair loss, abnormal gait and body posture, soft or mucoid feces, decreased defecation or feces that were smaller than usual, or vocalization when handled. No behavioral changes or visual symptoms of toxicity related to the administration of the extracts were observed at the conclusion of the study period.

3.2.2. Effects of CRE-SD and CRE-Ter on Bodyweight, Food Consumption, and Water Intake

The body weight data for the control group and those treated with CRE-SD and CRE-Ter are shown in Figure 2, illustrating consistent trends across the study period. Both male and female rats in the treated groups exhibited mean weekly body weights that closely matched those of the control group, with no statistically significant differences observed between the groups. This indicated that the administration of CRE-SD and CRE-Ter did not adversely affect the growth or physical development of the animals. Similarly, assessments of food and water intake revealed no significant deviations (p > 0.05) between the treated and control groups in either gender throughout the study. These findings suggested that the extracts did not interfere with normal metabolic functions or feeding behavior in the tested rats, further supporting their safety under the experimental conditions.

Figure 2.

Changes in the body weights of male and female rats treated with CRE-SD and CRE-Ter in the subchronic toxicity study. Each point represents the mean ± SEM (n = 10).

3.2.3. Effects of CRE-SD and CRE-Ter on Hematological Parameters

The hematological effects resulting from oral administration of CRE-SD and CRE-Ter at doses of 10, 30, and 300 mg/kg for a period of 90 days were evaluated in male and female rats, with the findings summarized in Tables 2 and 3, respectively. The results demonstrated that key hematological parameters, such as hemoglobin levels and total WBC counts, remained within the normal physiological range for all treated groups. Statistical analysis revealed no significant differences (p > 0.05) in these or other hematological parameters between the treated groups and the control group. While some parameters exhibited minor variations, these changes were not biologically significant and remained within the expected range for healthy animals. These findings suggested that long-term oral administration of CRE-SD and CRE-Ter did not adversely affect the hematological profile of rats, further supporting their safety at the tested doses.

Table 2. Subchronic Oral Toxicity Effects of CRE-SD and CRE-Ter on the Hematological Parameters of Male Rats^a.

		CRE-SD (mg/kg)				CRE-TER (mg/kg)
parameters	control	10 mg/kg	30 mg/kg	300 mg/kg	control	10 mg/kg	30 mg/kg	300 mg/kg
WBC (103/μL)	2.25 ± 0.56	2.93 ± 0.09	2.84 ± 0.39	2.98 ± 0.37	2.40 ± 0.60	2.60 ± 0.37	2.03 ± 0.47	3.18 ± 0.63
lymph (%)	2.85 ± 0.59	2.76 ± 0.69	2.84 ± 0.39	2.62 ± 0.47	1.70 ± 0.40	1.85 ± 0.29	1.40 ± 0.33	2.30 ± 0.46
Mon (%)	0.10 ± 0.04	0.08 ± 0.02	0.08 ± 0.02	0.08 ± 0.04	0.04 ± 0.04	0.08 ± 0.02	0.08 ± 0.04	0.08 ± 0.02
Gran (%)	0.80 ± 0.19	0.98 ± 0.22	0.62 ± 0.10	0.08 ± 0.14	0.66 ± 0.17	0.78 ± 0.15	0.78 ± 0.23	0.86 ± 0.16
RBC (106/μL)	9.69 ± 0.66	11.14 ± 0.87	9.83 ± 0.69	10.91 ± 0.79	10.00 ± 0.90	8.99 ± 0.35	8.94 ± 0.13	9.36 ± 0.47
HGB (g/dL)	17.04 ± 1.27	19.18 ± 1.66	17.74 ± 1.34	18.95 ± 1.58	17.05 ± 1.44	16.12 ± 0.70	15.86 ± 0.39	17.00 ± 0.78
HCT (%)	53.16 ± 3.71	62.44 ± 5.22	54.98 ± 3.86	61.60 ± 4.78	52.88 ± 4.43	49.84 ± 2.09	49.06 ± 1.10	52.62 ± 2.23
MCV (fL)	54.92 ± 0.28	55.98 ± 0.62	55.98 ± 0.32	56.40 ± 0.52	56.32 ± 0.65	55.46 ± 0.45	54.92 ± 0.67	54.46 ± 0.46
MCH (pg)	17.52 ± 0.15	17.98 ± 0.26	17.98 ± 0.12	18.04 ± 0.24	18.10 ± 0.20	17.84 ± 0.14	17.68 ± 0.22	18.14 ± 0.15
MCHC (g/dL)	31.96 ± 0.26	32.18 ± 0.14	32.18 ± 0.18	32.04 ± 0.19	32.20 ± 0.10	32.30 ± 0.14	32.28 ± 0.13	32.24 ± 0.19
RDW (%)	13.66 ± 0.45	14.60 ± 0.41	13.60 ± 0.44	14.24 ± 0.62	14.34 ± 0.32	13.04 ± 0.30	13.74 ± 0.31	13.42 ± 0.32
PLT (104/μL)	114.5 ± 6.85	101.2 ± 6.96	100.4 ± 9.36	102.1 ± 5.85	102.6 ± 10.57	117.1 ± 3.62	107.0 ± 8.48	101.9 ± 5.79
MPV (fL)	6.40 ± 0.10	6.34 ± 0.02	6.56 ± 0.20	6.52 ± 0.24	6.18 ± 0.13	6.40 ± 0.12	6.40 ± 0.13	6.50 ± 0.18
PDW	16.12 ± 0.07	16.08 ± 0.05	16.08 ± 0.05	16.08 ± 0.06	16.34 ± 0.09	16.28 ± 0.07	16.12 ± 0.10	16.18 ± 0.10

^aValues are the mean ± SEM for six rats in each group. WBC: white blood cell count, lymph: leukocyte differential count, Mon: monocytes, Gran: granulocyte, RBC: red blood cell count, HGB: hemoglobin concentration, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: MCH concentration, RDW: red blood cell distribution width, PLT: platelet count, MPV: mean platelet volume, and PDW: platelet distribution width.

Table 3. Subchronic Oral Toxicity Effects of CRE-SD and CRE-Ter on the Hematological Parameters of Female Rats^a.

		CRE-SD (mg/kg)				CRE-TER (mg/kg)
parameters	control	10 mg/kg	30 mg/kg	300 mg/kg	control	10 mg/kg	30 mg/kg	300 mg/kg
WBC (103/μL)	1.06 ± 0.40	1.72 ± 0.28	1.30 ± 0.31	1.33 ± 0.10	1.92 ± 0.65	1.28 ± 0.45	2.20 ± 0.35	1.32 ± 0.48
lymph (%)	1.17 ± 0.26	1.36 ± 0.22	1.84 ± 0.80	1.58 ± 0.56	1.88 ± 0.41	1.20 ± 0.29	1.58 ± 0.24	1.25 ± 0.38
Mon (%)	0.03 ± 0.03	0.04 ± 0.02	0.04 ± 0.04	0.04 ± 0.02	0.03 ± 0.02	0.08 ± 0.02	0.08 ± 0.04	0.03 ± 0.02
Gran (%)	0.37 ± 0.07	0.32 ± 0.04	0.40 ± 0.18	0.40 ± 0.13	0.48 ± 0.11	0.43 ± 0.08	0.58 ± 0.12	0.33 ± 0.07
RBC (106/μL)	10.12 ± 0.40	9.76 ± 0.42	8.83 ± 0.56	9.20 ± 0.49	9.33 ± 0.52	8.66 ± 0.35	10.02 ± 0.52	10.38 ± 0.52
HGB (g/dL)	19.88 ± 1.02	19.22 ± 1.11	17.24 ± 1.16	17.50 ± 0.86	18.50 ± 1.18	17.46 ± 0.87	19.86 ± 1.11	20.20 ± 1.02
HCT (%)	61.04 ± 2.83	59.74 ± 3.28	54.00 ± 3.36	54.42 ± 2.40	54.35 ± 2.33	52.15 ± 1.89	59.74 ± 3.05	59.78 ± 2.70
MCV (fL)	60.28 ± 0.85	61.14 ± 0.94	61.22 ± 0.26	59.34 ± 0.74	61.06 ± 0.90	62.28 ± 0.78	59.70 ± 0.68	59.60 ± 0.43
MCH (pg)	19.56 ± 0.29	19.62 ± 0.32	19.44 ± 0.19	19.02 ± 0.21	19.78 ± 0.38	20.06 ± 0.24	19.76 ± 0.29	19.04 ± 0.09
MCHC (g/dL)	32.48 ± 0.21	32.10 ± 0.17	31.84 ± 0.24	32.10 ± 0.30	32.42 ± 0.22	32.32 ± 0.29	33.16 ± 0.19	32.62 ± 0.33
RDW (%)	13.64 ± 0.34	12.40 ± 0.30	12.28 ± 0.62	12.06 ± 0.33	12.46 ± 0.55	12.58 ± 0.34	12.66 ± 0.15	12.40 ± 0.13
PLT (104/μL)	74.5 ± 12.10	82.6 ± 5.96	93.3 ± 7.52	97.5 ± 8.32	84.3 ± 5.30	87.3 ± 4.26	66.7 ± 12.87	70.2 ± 3.60
MPV (fL)	6.38 ± 0.04	6.74 ± 0.14	6.56 ± 0.25	6.60 ± 0.21	6.26 ± 0.16	6.44 ± 0.17	6.56 ± 0.14	6.50 ± 0.10
PDW	16.20 ± 0.10	16.42 ± 0.17	16.28 ± 0.10	16.26 ± 0.07	16.24 ± 0.09s	16.26 ± 0.10	16.28 ± 0.09	16.34 ± 0.05

^aValues are the mean ± SEM for 6 rats in each group. WBC: white blood cell count, lymph: leukocyte differential count, Mon: monocytes, Gran: granulocyte, RBC: red blood cell count, HGB: hemoglobin concentration, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: MCH concentration, RDW: red blood cell distribution width, PLT: platelet count, MPV: mean platelet volume, and PDW: platelet distribution width.

3.2.4. Effects of CRE-SD and CRE-Ter on Serum Biochemical Indices

The serum biochemical indices of male and female rats after treatment with CRE-SD and CRE-Ter are summarized in Tables 4 and 5, respectively. According to the biochemical results, the levels of kidney function markers (BUN and CRE) remained unchanged in all treated groups comparable to the control ones, indicating no adverse effects on kidney health. Similarly, the levels of liver function indicators (ALP, SGOT, SGPT, and TB) did not show any significant changes (p > 0.05), instead very minor and statistically insignificant fluctuations were observed in their levels.

Table 4. Subchronic Oral Toxicity Effects of CRE-SD and CRE-Ter on the Serum Biochemical Indices of Male Rats^a.

		CRE-SD (mg/kg)				CRE-TER (mg/kg)
parameters	control	10 mg/kg	30 mg/kg	300 mg/kg	control	10 mg/kg	30 mg/kg	300 mg/kg
BUN (mg%)	17.45 ± 0.27	17.77 ± 0.58	19.16 ± 0.60	19.15 ± 0.66	18.54 ± 1.10	19.28 ± 1.17	16.83 ± 0.68	19.91 ± 1.01
CRE (mg%)	0.70 ± 0.02	0.68 ± 0.03	0.65 ± 0.01	0.66 ± 0.01	0.79 ± 0.05	0.73 ± 0.02	0.74 ± 0.03	0.73 ± 0.02
ALP (mg%)	76.40 ± 6.79	77.80 ± 8.92	78.60 ± 7.57	87.00 ± 3.35	66.60 ± 8.12	78.60 ± 5.26	76.40 ± 6.52	76.80 ± 3.93
SGOT (U/L)	120.0 ± 6.72	124.2 ± 6.28	132.8 ± 3.51	131.4 ± 10.90	148.0 ± 7.57	142.2 ± 5.67	177.2 ± 8.86	151.8 ± 6.69
SGPT (U/L)	42.60 ± 2.40	40.50 ± 3.15	39.25 ± 4.49	40.00 ± 2.37	42.00 ± 4.46	35.80 ± 1.53	58.80 ± 8.55	53.20 ± 14.71
TB (mg%)	0.04 ± 0.00	0.03 ± 0.01	0.05 ± 0.00	0.04 ± 0.00	0.04 ± 0.00	0.04 ± 0.00	0.04 ± 0.00	0.04 ± 0.01
CLT (mg%)	75.60 ± 5.81	62.00 ± 4.24	69.60 ± 5.22	54.40 ± 3.78b	60.80 ± 5.56	68.00 ± 6.01	57.20 ± 5.11	55.60 ± 10.14
TG (mg%)	147.2 ± 14.80	125.8 ± 18.56	97.2 ± 16.59	72.8 ± 9.53b	76.20 ± 11.02	94.40 ± 12.14	77.80 ± 7.54	62.00 ± 23.95
HDL-C (mg%)	50.20 ± 3.97	40.80 ± 3.77	48.40 ± 2.94	43.40 ± 3.49	36.60 ± 3.89	43.40 ± 3.82	36.40 ± 3.20	33.00 ± 5.75
LDL-C (mg%)	19.42 ± 0.86	13.65 ± 1.60b	13.55 ± 0.97b	15.36 ± 1.32b	17.68 ± 2.93	17.08 ± 1.49	12.28 ± 1.71	14.68 ± 0.78
VLDL-C (mg%)	25.84 ± 4.43	28.24 ± 4.46	22.60 ± 4.26	17.32 ± 3.22	15.24 ± 2.20	18.88 ± 2.43	15.56 ± 1.51	12.40 ± 4.79

^aBUN: blood urea nitrogen, CRE: blood creatinine, ALP: alkaline phosphatase, SGOT: serum glutamic-oxaloacetic transferase, SGPT: serum glutamic pyruvate transaminase, TB: total bilirubin, CLT: cholesterol, TG: triglyceride, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, and VLDL-C: very low-density lipoprotein cholesterol.

^bThe values were significantly different (p < 0.05) when compared to the control group.

Table 5. Subchronic Oral Toxicity Effects of CRE-SD and CRE-Ter on the Serum Biochemical Indices of Female Rats^a.

		CRE-SD (mg/kg)				CRE-TER (mg/kg)
parameters	control	10 mg/kg	30 mg/kg	300 mg/kg	control	10 mg/kg	30 mg/kg	300 mg/kg
BUN (mg%)	15.77 ± 0.94	18.29 ± 0.65	18.92 ± 1.01	18.67 ± 1.23	16.95 ± 1.36	15.12 ± 0.59	17.02 ± 0.38	17.55 ± 1.02
CRE (mg%)	0.63 ± 0.02	0.63 ± 0.01	0.64 ± 0.02	0.69 ± 0.01	0.65 ± 0.02	0.71 ± 0.02	0.67 ± 0.03	0.65 ± 0.02
ALP (mg%)	31.80 ± 3.12	28.20 ± 1.28	28.40 ± 2.25	27.60 ± 2.58	27.20 ± 1.16	29.40 ± 1.91	26.00 ± 2.21	26.40 ± 3.23
SGOT (U/L)	122.6 ± 7.98	132.0 ± 25.17	134.0 ± 6.48	110.0 ± 7.94	135.0 ± 6.01	152.0 ± 7.35	126.4 ± 12.53	168.8 ± 15.26
SGPT (U/L)	38.20 ± 3.12	55.75 ± 12.88	43.75 ± 5.46	28.25 ± 1.48	35.20 ± 1.46	36.00 ± 4.68	35.60 ± 6.56	104.00 ± 42.40
TB (mg%)	0.08 ± 0.01	0.08 ± 0.01	0.06 ± 0.00	0.06 ± 0.01	0.07 ± 0.00	0.09 ± 0.02	0.08 ± 0.01	0.08 ± 0.01
CLT (mg%)	54.60 ± 3.54	61.00 ± 4.49	52.20 ± 2.99	36.00 ± 4.64b	42.60 ± 4.82	53.80 ± 5.73	50.40 ± 6.62	49.60 ± 6.38
TG (mg%)	44.00 ± 3.97	42.75 ± 3.06	37.75 ± 1.61	36.40 ± 4.88	31.60 ± 1.29	30.60 ± 2.16	40.80 ± 7.14	34.20 ± 2.67
HDL-C (mg%)	37.80 ± 2.52	37.75 ± 2.17	32.60 ± 1.81	32.50 ± 5.51	29.20 ± 2.85	36.60 ± 4.34	37.20 ± 3.29	35.60 ± 5.09
LDL-C (mg%)	9.80 ± 1.02	11.58 ± 1.72	9.90 ± 1.31	11.12 ± 1.21	10.72 ± 0.74	12.26 ± 0.50	10.42 ± 1.05	9.26 ± 1.19
VLDL-C (mg%)	8.80 ± 0.79	8.55 ± 0.61	7.55 ± 0.32	7.28 ± 0.98	6.32 ± 0.26	6.12 ± 0.43	8.16 ± 1.43	6.84 ± 0.53

^aBUN: blood urea nitrogen, CRE: blood creatinine, ALP: alkaline phosphatase, SGOT: serum glutamic-oxaloacetic transferase, SGPT: serum glutamic pyruvate transaminase, TB: total bilirubin, CLT: cholesterol, TG: triglyceride, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, and VLDL-C: very low-density lipoprotein cholesterol.

^bThe values were significantly different (p < 0.05) when compared to the control group.

In the case of male rats, a repeated oral administration of CRE-SD regularly for 90 days, at all its dosages, produced a dose-dependent reduction in low-density lipoprotein cholesterol (LDL-C) levels. However, it could induce a notable decline in total cholesterol (CLT) and triglyceride (TG) levels only at a dosage of 300 mg/kg. Conversely, for female rats, a significant reduction in CLT was observed only at 300 mg/kg. However, no significant changes were observed in the other lipid profile parameters. Unlike CRE-SD, treatment with CRE-Ter could not produce any of the substantial changes (p > 0.05) in lipid profile indices in either of the sex, suggesting that it is minimal or no impact on the given markers.

Interestingly, a dramatic fall was observed in fasting blood glucose (FBG) levels following a 90 day repeated administration of CRE-SD at doses of 30 and 300 mg/kg in female rats (Figure 3B). Thus, a substantial fall was seen in FBG levels of female rats across all the tested doses of CRE-Ter (Figure 3C).

Figure 3.

Fasting blood glucose levels of male and female rats treated with CRE-SD (A: male and B: female) and CRE-Ter (C: male and D: female) in the subchronic toxicity study. Each point represents the mean ± SEM (n = 10).

3.2.5. Effects of CRE-SD and CRE-Ter on Gross Necropsy and Relative Weight of the Major Organs

A detailed gross necropsy was conducted on the major organs (liver, kidneys, heart, lungs, spleen, testis, ovaries, and uterus) of rats to investigate the potential damage brought about by the administration of the respective curcuminoid extracts. This analysis involved the treated group of rats to be compared to that of the control group to assess any morphological abnormality or deviation. The findings revealed no observable pathological changes or abnormalities in any of the organs examined, suggesting that the administration of the curcuminoid extracts was not associated with any adverse effects on organ morphology.

Apart from this, at the point of necropsy, the ROW of these organs was recorded to comprehend potential alterations caused by the administration of curcuminoid extracts for a longer period of time. The data, summarized in Table 6, illustrated no significant differences (p > 0.05) in ROW between the control and the treatment groups for male as well as female rats. The consistent results regarding the ROW for both sexes vividly concluded that SD and CRE-Ter could not inflict any of the untoward effects on the internal organs by keeping their weights within a normal range. These results clearly advocated for the complete safety of the extracts with respect to the integrity and function of internal organs.

Table 6. Subchronic Oral Toxicity Effects of CRE-SD and CRE-Ter on the Relative Weight of the Major Organs.

		CRE-SD (mg/kg)				CRE-TER (mg/kg)
parameters	control	10 mg/kg	30 mg/kg	300 mg/kg	control	10 mg/kg	30 mg/kg	300 mg/kg
male
liver	2.4 ± 0.25	2.60 ± 0.13	2.42 ± 0.21	2.56 ± 0.23	2.51 ± 0.05	2.55 ± 0.27	2.60 ± 0.17	2.63 ± 0.35
kidney	0.53 ± 0.01	0.56 ± 0.03	0.57 ± 0.01	0.61 ± 0.02	0.56 ± 0.01	0.55 ± 0.02	0.56 ± 0.02	0.58 ± 0.01
heart	0.21 ± 0.05	0.23 ± 0.03	0.24 ± 0.03	0.23 ± 0.01	0.23 ± 0.02	0.21 ± 0.02	0.23 ± 0.03	0.24 ± 0.04
lung	0.29 ± 0.05	0.29 ± 0.04	0.31 ± 0.05	0.31 ± 0.05	0.30 ± 0.04	0.29 ± 0.05	0.29 ± 0.04	0.30 ± 0.04
spleen	0.14 ± 0.02	0.13 ± 0.02	0.14 ± 0.01	0.15 ± 0.01	0.15 ± 0.02	0.12 ± 0.01	0.14 ± 0.02	0.14 ± 0.02
testis	0.69 ± 0.10	0.75 ± 0.07	0.78 ± 0.08	0.79 ± 0.05	0.77 ± 0.05	0.80 ± 0.09	0.75 ± 0.02	0.81 ± 0.03
female
liver	2.73 ± 0.10	2.44 ± 0.05	2.46 ± 0.17	2.45 ± 0.16	2.46 ± 0.40	2.40 ± 0.12	2.56 ± 0.18	2.88 ± 0.32
kidney	0.29 ± 0.03	0.26 ± 0.02	0.28 ± 0.03	0.29 ± 0.03	0.25 ± 0.02	0.26 ± 0.02	0.29 ± 0.02	0.30 ± 0.02
heart	0.41 ± 0.03	0.39 ± 0.01	0.45 ± 0.03	0.44 ± 0.04	0.38 ± 0.04	0.40 ± 0.04	0.44 ± 0.03	0.45 ± 0.03
lung	0.64 ± 0.05	0.58 ± 0.02	0.62 ± 0.05	0.60 ± 0.03	0.61 ± 0.04	0.61 ± 0.03	0.60 ± 0.03	0.65 ± 0.05
spleen	0.19 ± 0.03	0.18 ± 0.02	0.18 ± 0.02	0.18 ± 0.02	0.19 ± 0.02	0.16 ± 0.02	0.18 ± 0.02	0.20 ± 0.02
ovary/uterus	0.26 ± 0.02	0.31 ± 0.07	0.27 ± 0.04	0.27 ± 0.04	0.19 ± 0.03	0.23 ± 0.02	0.25 ± 0.05	0.33 ± 0.12

3.2.6. Effects of CRE-SD and CRE-Ter on the Histopathology of the Major Organs

A comprehensive histopathological evaluation was performed to assess the hazardous impact of curcuminoid extracts on the liver and kidney, which are the key metabolic and excretory organs. Microscopic examinations of these organs revealed normal cellular architecture, with no observable alterations in tissue morphology across all of the groups, showing striking histopathological uniformities between the control group and the groups treated with CRE-SD or CRE-Ter. Representative photomicrographs revealing the histological architecture are presented in Figures 4 and 5, further illustrating the normal cellular appearances in these organs.

Figure 4.

Photomicrographs of hematoxylin and eosin-stained sections of liver and kidney in control and experimental groups treated with CRE-SD daily for 90 days. No significant alteration was observed in all treatment groups (×200 magnification).

Figure 5.

Photomicrographs of hematoxylin and eosin-stained sections of liver and kidney in control and experimental groups treated with CRE-Ter daily for 90 days. No significant alteration was observed in all treatment groups (×200 magnification).

Besides, the histopathological investigation was further extended to the heart, lungs, spleen, testis (in males), ovaries, and uterus (in females) in order to comprehend a detailed evaluation of potential systemic effects. This was done to thoroughly assess whether the treatment caused any harmful effects throughout the body. The analysis revealed no signs of damage, abnormalities, or structural changes in these organs, indicating that the treatment did not cause any adverse effects in the animals. Although detailed data for these specific organs have not been presented in the figures, the findings authenticated that CRE-SD/CRE-Ter induced no damage to the microscopic structure or integrity of these organs. Thereby, the given curcuminoid extracts were found to be completely safe to maintain the tissue integrity of visceral and reproductive organs in rodent models.

4. Discussion

This study presents detailed safety profiles of two water-soluble CRE formulations, namely, CRE-SD and CRE-Ter. Herein, both male and female Wisterr rats were used to assess acute and subchronic oral toxicities, following the stringent guidelines set by the OECD. This adherence ensures that the findings are scientifically robust and reliable. While previous studies have consistently shown that turmeric extracts and curcumin are well-tolerated, even at exceptionally high doses without significant toxic effects,^15,16 the current investigation specifically focuses on the safety of these water-soluble formulations, offering new insights into their tolerability. The evaluation of CRE-SD and CRE-Ter is essential due to their enhanced bioavailability compared with traditional turmeric extracts or curcumin, which could potentially influence their pharmacokinetic and safety parameters. While the improved bioavailability enhances therapeutic effectiveness, it also underscores the importance of thorough toxicity testing to confirm their safety for use as nutraceutical or phytopharmaceutical products. This study addresses a significant gap in safety data, providing essential evidence to support the development and broader application of these formulations in healthcare and medicine.

The acute toxicity study assessed the safety of CRE-SD and CRE-Ter by administering a single oral dose of 2000 mg/kg to rats, following fixed-dose guidelines for acute toxicity testing. Throughout the 14 day observation period, neither formulation resulted in any visible signs of toxicity or mortality in the treated animals. The rats exhibited normal food and water intake with no significant differences in body weight compared to the control groups, indicating that the formulations did not negatively impact growth or overall health. Comprehensive clinical pathology analyses further confirmed the absence of toxicity, as hematological and serum biochemical parameters remained within normal limits, showing no evidence of systemic or organ-specific dysfunction. Additionally, gross necropsy and histopathological evaluations of the major organs, including the liver, kidneys, heart, lungs, spleen, and reproductive organs (testes in males and ovaries and uterus in females), revealed no abnormalities or treatment-related damage. These results clearly demonstrated that a single oral dose of 2000 mg/kg of CRE-SD or CRE-Ter was well-tolerated, with no adverse effects on the physiological, biochemical, or histological parameters in male or female rats.

Fixed-dose acute toxicity testing guidelines for rodents specify a dosage range of 2000 to 5000 mg/kg of body weight. However, if no deaths or significant toxic effects are observed at the 2000 mg/kg dose, further increasing the dose is deemed unnecessary. Administering higher doses would not only lack scientific justification but also be less cost-effective and conflict with the principles of the 3Rs (Replacement, Reduction, and Refinement), which aim to minimize animal use and suffering in research. Based on these considerations, this study capped the maximum dose at 2000 mg/kg. This approach reflects an ethical commitment to responsible testing practices, while ensuring compliance with regulatory standards and optimal resource utilization.

Under the Globally Harmonized System (GHS), substances with an LD₅₀ between 2000 and 5000 mg/kg are classified as Category 5, indicating an extremely low risk of oral toxicity. This study showed that both CRE-SD and CRE-Ter had an LD₅₀ exceeding 2000 mg/kg, placing them within this low-toxicity category.¹⁵ This outcome unveils the protective effects of CRE-SD and CRE-Ter on animal and human health at the higher dosage levels, supporting their potential for safe application as nutraceuticals or phytotherapeutic agents.

Acute toxicity studies, which assess the effects of a single high dose of a drug, have limited clinical relevance since medications are typically administered in smaller, repeated doses over extended periods. Prolonged exposure to a xenobiotic (drug), even at meager doses, could unveil subacute or subchronic toxic effects that are not evident in acute testing. Thereby, the present study not only focuses on acute toxicity assessment but also extends to a 90 day repeated oral toxicity evaluation of CRE-SD and CRE-Ter in male as well as female rats. According to the results, repeated oral administration of 10, 30, and 300 mg/kg of CRE-SD or CRE-Ter for the period of 90 days could not inflict any morbidity or mortality in either sex of the rats.

Appetite is a fundamental biological instinct essential for regulating the body weight. As a result, tracking food and water intake along with body weight changes is a routine procedure to evaluate the overall health and well-being of animals in toxicity studies. In this study, the control group and those treated with CRE-SD and CRE-Ter exhibited steady weight gain throughout the period of 90 days. These observations clearly demonstrated that administering CRE-SD and CRE-Ter at doses as high as 300 mg/kg could not interfere with the normal metabolic processes of rodent models.

The hematopoiesis is an integral component of the biological system exhibiting higher sensitivity against the toxic effects of drugs or herbal substances in both humans and animals.¹⁶ In the present study, hematological parameters were evaluated to observe any changes resulting from the 90 day subchronic toxicity testing of CRE-SD and CRE-Ter. According to the results, repeated oral administration of these substances could not inflict any of the untoward effects on the production of blood cells. There were no significant differences in the hematological values of treated groups compared with that of the control group. Interestingly, the observed changes in hematological parameters of treated rats in our study remained within the normal physiological range, and similar results were also reported previously in a couple of studies.^17,18 These findings authenticated that CRE-SD and CRE-Ter, even after prolonged exposure at tested doses, exhibited no toxic effects on the hematopoietic system, further supporting their safety profile.

Evaluating liver and kidney function is a vital part of toxicity studies because these organs play a central role in detoxifying the body, metabolizing the substances, and excreting the waste. This makes them highly susceptible to toxic damage. For the kidneys, specific markers, such as BUN and creatinine levels, are routinely measured. These markers provide crucial insights into renal health, where elevated levels indicate impaired kidney function.¹⁹ This study monitored the levels of BUN and creatinine to evaluate kidney function after 90 days of repeated oral administration of CRE-SD and CRE-Ter. The results showed no significant differences in these markers between the treated groups and the control group, indicating that neither formulation caused any renal toxicity. Liver function was assessed by measuring key biomarkers, such as ALP, AST/SGOT, ALT/SGPT, and TB. Elevated ALT and AST levels typically indicate acute liver damage, while prolonged injury can lead to reduced enzyme levels due to extensive liver cell loss. In this study, no significant changes in these liver enzymes or other markers were observed in the treated groups compared with the controls. These findings demonstrated that repeated oral doses as high as 300 mg/kg of CRE-SD and CRE-Ter could not induce any hepatic or renal toxicity, supporting the safety of these formulations for the tested doses and duration.

Serum lipid profiles were also analyzed following the subchronic administration of CRE-SD and CRE-Ter. Notably, the high dose of CRE-SD (300 mg/kg) led to a significant reduction (p < 0.05) in certain lipid parameters, including CLT and TGs. Additionally, LDL-C levels were significantly lowered at all tested doses of CRE-SD (10, 30, and 300 mg/kg). Interestingly, these lipid-lowering effects were observed exclusively in male rats treated with CRE-SD, with no similar changes noted in the groups receiving CRE-Ter. Previous research regarding the effects of curcumin on lipid profiles has shown inconsistent results in animals as well as humans. For instance, a previous study examining the impact of a 6 month curcumin supplementation in healthy human participants found no significant changes in serum lipid parameters, including TG, CLT, LDL-C, or HDL-C. This suggested that curcumin might not consistently influence the lipid metabolism in humans, particularly under normal physiological conditions.²⁰ Another study conducted on patients with metabolic syndrome and related conditions reported a notable decline in FBG, TG, and CLT levels. However, no significant effects were observed on LDL-C or HDL-C levels.²¹ Additionally, the use of a high-bioavailability form of curcumin was found to enhance its lipid-lowering efficacy in animal studies.

This study evaluated the effects of 90 day subchronic administration of CRE-SD and CRE-Ter on vital organs, including the liver, kidney, heart, lung, spleen, and sexual organs, using gross examination, ROW, and histopathology. Gross examination assessed visible anatomical changes, ROW was used to detect organ size changes indicative of hypertrophy or atrophy,²² and histopathological analysis provided microscopic insights into structural and cellular alterations. Together, these methods allowed for a comprehensive assessment of drug-induced toxicity and anatomical changes in the targeted organs. A histopathological analysis was performed to validate the biochemical results and detect any structural abnormalities. Given that the liver, as the primary organ of metabolism, and the kidney, as the main organ responsible for elimination, are most susceptible to damage from drug exposure or extracted substances, the histological examination revealed no abnormalities in these organs across all dose levels of CRE-SD and CRE-Ter in both male and female rats. These findings aligned with the serum biochemical evaluations, which showed normal liver and kidney function parameters. Additionally, no notable changes were observed in other organs, including the heart, lungs, spleen, and reproductive organs.

5. Conclusions

Based on the GHS classification, CRE-SD and CRE-Ter were categorized as Category 5, reflecting minimal to no toxicity potential. Overall, the findings of this study unequivocally demonstrated the absence of toxicity associated with acute and subchronic oral administration of CRE-SD and CRE-Ter, with LD₅₀ values surpassing 2000 mg/kg of body weight. Neither compound caused lethality nor elicited any adverse effects on hematological, biochemical, or histopathological parameters. These results provided compelling evidence of the safety profile of CRE-SD and CRE-Ter, underscoring their significant potential for pharmaceutical development. However, rigorous preclinical and clinical trials remain essential to validate their safety and efficacy before approval for human consumption, paving the way for their integration into therapeutic applications.

Data Availability Statement

All the data generated or analyzed during this study are included in this published article

Author Contributions

P.P., D.C., M.K., and A.I. conceived and designed the research study. A.I. and P.P. conducted all the experiments. All the authors analyzed the data, discussed the findings, and prepared the manuscript.

The authors declare no competing financial interest.

Notes

All applicable guidelines for the care and use of animals were followed. All experiments were approved by the Prince of Songkla University Ethical Committee (reference number: MHESI 6800.11/124). This article does not contain any studies performed with human participants.