https://orcid.org/0000-0002-6911-8299
Figures
Abstract
Charmagaz seed oil (CSO), derived from Cucurbitaceae family, and is a traditional mix of four different seeds: pumpkin, cucumber, watermelon, and musk melon. Widely utilized in Asia, this blend is considered as a brain tonic and a nutritional powerhouse. Despite its significant ethno medicinal value, the potential toxicity and safety of this combination oil have not been scientifically documented. Therefore, the current study was conducted to assess the sub-acute toxicity of Charmagaz seed oil in rat model for the assessment of its safety profile. For acute oral toxicity CSO was given at doses of 50, 500, and 5000 mg/kg orally over 28 days in rats. No evidence of toxicity was observed in animals when acutely exposed to CSO, implying that the LD50 is higher than 5000 mg/kg body weight. Cell viability assay revealed that Charmagaz oil is relatively non-toxic, showing an inhibition rate below 50% following 24 and 48-hour exposures. In the Brine Shrimp bioassay, oil demonstrated no cytotoxic effects, unlike the standard cytotoxic drug Etoposide, which resulted in 70% mortality at a 7.5 μg/ml concentration. No treatment-related toxicity or death was seen in any of the animals receiving dosages of 500 and 5000 mg/kg during the course of the 28-day study period, according to sub-acute oral toxicity tests. Additionally, no significant alterations (p > 0.05) were detected in hematological or biochemical parameters across the dosed groups. The administration of Charmagaz seed oil led to a modest elevation in high-density lipoprotein cholesterol, likely due to its polyunsaturated fatty acids content. Since the liver is a major organ involved in lipid metabolism hence, histopathological analysis was conducted to determine the effects of Charmagaz oil on hepatic tissues. The analysis revealed a positive correlation with biochemical results from liver function enzyme tests. Thus, this study provides evidence suggesting the safety of Charmagaz oil consumption at doses up to 5000 mg/kg.
Citation: Adil A, Khan SS, Naeem S, Alqahtani A, Alqahtani T, Asgher Shuja A, et al. (2025) Evaluation of Sub-acute toxicity and safety profile of Charmagaz seed oil in rats. PLoS One 20(7): e0327697. https://doi.org/10.1371/journal.pone.0327697
Editor: Ghulam Yaseen, University of Education, PAKISTAN
Received: September 23, 2024; Accepted: June 18, 2025; Published: July 11, 2025
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The minimal dataset supporting the findings of this study is available within the paper and its Supporting Information files. Additional data are available from the corresponding author upon request.
Funding: This work was supported by the Deanship of Scientific Research at King Khalid University through Large group Research Project (grant number RGP2/384/46. The funder played role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript, also drafting the work or revising it critically for important intellectual content. The names of the authros include Ali Alqahtani and Taha Alqahtani belonging to Department of Pharmacology, College of Pharmacy, King Khalid University, Guraiger, Abha 62529, Saudi Arabia.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Subacute toxicity testing is essential for evaluating the reversibility of adverse effects, establishing dose-response relationships, and identifying target organ toxicity following repeated exposure in test animals. With the increasing utilization of natural extracts as therapeutic and functional agents, concerns regarding their safety have emerged [1]. Despite their popularity due to accessibility and affordability [2], many plant-based treatments lack sufficient toxicity data. Rigorous studies are needed to build confidence in their safe use [3]. With the WHO establishing guidelines and 80% of the population relying on herbal medicine, understanding potential toxic effects and informing consumers about risks is crucial [4]. The rising prevalence of herbal medicine use, driven by accessibility, affordability, and perceived safety, necessitates a thorough evaluation of potential toxicities. While traditional use suggests safety, limited toxicity data and recent reports of adverse effects raise concerns. Contamination by heavy metals, aflatoxins, and pathogenic microbes during preparation, as well as metal accumulation from soil, are potential sources of toxicity. Consequently, toxicity studies are crucial to establish the safety profile of medicinal plants and enhance consumer confidence [5].
The Cucurbitaceae family is known as guard & vine family, and includes around one thousands of species in about one hundred genus of plants. In human food conception, this family occupies the highest rank among the plant families and the major crop species of the family are Cucumber (Cucumis sativus), Pumpkin (Cucurbita maxima), Watermelon (Citrullus lanatus) and Musk melon (Cucumis melo) [6]. Plants within this family possess a long-standing history of ethno pharmacological and traditional medicinal use, supported by extensive documentation in traditional literature. The demonstrated phytochemical and pharmacological potential suggests these plants represent a promising source for the development of novel therapeutic compounds and functional food products [7]. The Cucurbitaceae family is established as a significant source of secondary metabolites exhibiting diverse biological activities, as evidenced by in vitro, in vivo, and clinical trial investigations [8].
Cucurbit seeds are significant in Islamic and Quranic contexts and are known for their traditional uses in Ayurveda and Chinese medicine [9]. Ethno medicinal literature indicates that these seeds are used to treat various health issues, including stomach disorders, intestinal parasites, dizziness, kidney inflammation, and depression [10]. Members of cucurbitaceae family have broad range of pharmacological activities including antioxidant [11,12], antitumor [13], antidiabetic [14], hepatoprotective [15], anti-obesity [16] and neurodegenerative diseases [17]. Numerous in-vitro and in-vivo investigations have linked the Cucurbita genus to these health-promoting properties. Clinical research results indicate that people with diabetes may benefit from cucurbita [18], patients with benign prostate hyperplasia, infertile women [19], postmenopausal women [20], and stress urinary incontinence in women [21,22].
Cucurbitaceae species are rich in diverse phytochemicals, including cucurbitacins, saponins, carotenoids, phytosterols, polyphenols, flavonoids, alkaloids, triterpenoids, and fatty acids, which contribute to their potential medicinal properties [23]. Seeds further contain cucurbitosides and lutein [23,24], and are a valuable source of lipids, proteins, carbohydrates, minerals (calcium, phosphorus, iron), and vitamin A [9]. The abundance of these bioactive compounds positions Cucurbitaceae as a promising source for novel natural therapeutics. Notably, Cucurbita species are abundant in natural antioxidants, such as polyphenols, terpenoids, and polyunsaturated fatty acids, which play a significant role in disease prevention. Phenolic compounds exhibit antioxidant activity through various mechanisms, including free radical scavenging, metal chelation, and modulation of enzyme activity, and may also enhance the action of other antioxidants [25].
Charmagaz is a traditional blend of four cucurbit seeds which are pumpkin (C. maxima), cucumber (C. sativus), watermelon (C. lanatus), and musk melon (C. melo). In literal terms, ‘char’ means four and magaz signify ‘brains’, i.e., four seeds that enhance cognitive abilities and increase memory. This combination is extensively used in Asia as a brain tonic and super food. The application of Cucurbitaceae members in public health, as nutraceuticals is associated with good ethno pharmacological profile and easy availability [10]. To the best of our knowledge, no published article is found in literature search concerning the possible toxic effect of the combination of four cucurbits cold-pressed seed oil. Therefore, the present study was designed to evaluate the sub-acute toxicity and safety of Charmagaz seed oil in rat models.
2. Materials and methods
2.1. Ethical statement
All animal experiments were conducted in accordance with the National Institute of Health’s (NIH) guide for the care and use of laboratory animals. The protocol was reviewed and approved by the Institutional Bioethical Committee of University of Karachi under approval number (IBC KU-413/2024) and the Advanced Studies and Research Board (Reference No. 05918/Pharm). All efforts were made to minimize animal suffering and to reduce the number of animals used.
All staff received specialised training in animal care and handling to ensure compliance with NIH regulations.
2.2. Collection of the plant material
Cucurbit fruits were bought from the nearby local market from which the seeds were removed, shade dried and were deposited at Herbarium, Centre for Plant Conservation, University of Karachi. The taxonomist identified and issued the specimen number 94589 to Cucumis sativus, 9501 to Cucurbita maxima, 94494 to Cucumis melo and 9462 to Citrullus lanatus for reference.
2.3. Extraction of Charmagaz seed oil
Charmagaz seed oil was received as an extract of four cucurbit (Cucumis sativus, Cucurbita maxima, Citrullus lanatus, Cucumis melo) seeds by cold pressing using vegetable oil screw press model Komet DD 85 G (IBG MonfortsOekotec GmbH & Co. KG, Monchengladbach, Germany), Cold pressing is done three times with directly pressing the raw/dried seed by continuous screw press at low temperature. This technique entails an extraction of a solid sample oil contained within a plant hopper in a cold process. Oil extraction was performed to get the Cucurbitaceae seeds (of 0. 25 kg each of the studied varieties altogether 1 kg) ground and subjected to pressure exerted with the help of the conical screw rotation. The pressure was applied on the oil and it was compelled to enter into the perforated tube. After that at the end of the shaft, the prescribed quantity of meal was ejected by calibrated orifice which generally behaves as a barrier to the flow of meal that may contain residual fat or nutrition value. The remaining oil transacts to the centrifuge for 15 minutes in order to filter the oil with plant material particles. This is then followed by filtration [26]. After further weighing the extracted oils, the following formula was used to calculate the yield percentage:
2.4. Drugs and chemicals
Sigma Aldrich (MO, USA) provided the dimethyl sulfoxide (DMSO), methylthiazolyldiphenyl-tetrazolium bromide (MTT), and 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The ATCC® CRL 1658 was used to obtain the viability of the NIH/3T3 cell line. Brine shrimp eggs (Artemia salina) were purchased from Ocean Nutrition CA, United States.
2.5. Determination of fatty acid composition by Gas chromatography flame ionization detection (GC-FID)
The analysis of fatty acid content was done in an Agilent 7890A gas chromatography equipped with a flame ionization detector (GC-FID) (Agilent Technologies, USA). The Charmagaz oil sample’s fatty acid methyl esters (FAMEs) were made using a 2N potassium hydroxide solution in methanol, following the procedure outlined by Akin et al. (2018). Briefly, 10 milliliters of hexane were used to dissolve 0.1 grams of Charmagaz seed oil that had been weighed into a centrifuge tube. A vigorous 60-second shaking was then applied to the tube along with 0.1 mL of 2N potassium hydroxide solution. Next, for five minutes at 2000 rpm, the mixture was centrifuged. Finally, the top layer was transferred to a vial for examination. We followed Gu et al. (2011) guidelines for operating conditions for FAME analysis, with a few minor adjustments [27]. Agilent’s HP-5MS (30m × 0.25 mm × 0.25µm) capillary column was used for the separations. Using a split ratio of 100:1, the injection volume was 1 µL. The temperature at the inflow was 230 °C. The temperature schedule for the oven was set to start at 60 °C and grow linearly to 175 °C (15 °C/min) and 240 °C (2 °C/min), maintaining these levels for ten minutes. Under the suggested procedure conditions, fatty acids were determined as g/100 g of total fatty acids by measuring the retention periods of a FAME mix C14:0–C22:0 standard that was administered. Using an Agilent ChemStation 2001–2010 data processor, the data was processed [27].
2.6. Antioxidant assay by DPPH method
A sample of Charmagaz oil was subjected for its capacity to scavenge the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) compound. For this test, one milliliter of a reagent solution of 0. 5 grams of the oil dissolved in 5 mL of ethyl acetate was mixed vigorously with 4 mL of DPPH solution (prepared and diluted to 10 − 4 M ethyl acetate) in a screw capped test tube with about 10 mL capacity. The reaction mixture then stood for 10 seconds to allow for the formation of the emulsion using the Vortex apparatus. The tube was then left for 30 minutes in complete darkness as the system began to achieve steady state dark adaptation. Subsequently, the amount of the mixture was determined by absorbance at 515 nm against a reagent blank. At the same time, a control sample, which was constituted by combining 1 milliliters of ethyl acetate and 4 milliliters of DPPH solution, was prepared and measured as well. Percentage inhibition of DPPH radical formation (I %) was determined using the following formula and Methanol was used pertaining to this test as the control group [28].
2.7. MTT assay using NIH/3T3 cell line
The MTT (Methylthiazolyldiphenyl-tetrazolium bromide) test was used to examine the cytotoxic potential of Charmagaz seed oil using the NIH/3T3 (ATCC® CRL 1658) cell line. The cells were cultured at 37°C in an incubator with 5 percent carbon-dioxide and maintained in DMEM supplemented with 10 percent fetal bovine serum. Every 24 hours, the medium was added in order to obtain 70–80% confluency. 6000 cells per well were collected and placed in 96-well plates with 200 µL of growth media in each well. A medium containing the test chemical at a concentration of 25–75 µM for Charmagaz seed oil was added after the 24-hour incubation period. Thus the untreated wells functioned as a control of positive growth. Each well was filled with 5 mg/ml MTT dye and incubated for an additional 3 hours. After removing the supernatant, 100 microliter of Dimethyl sulfoxide was introduced to each well in order to dissolve the formazan crystals. After 15 seconds of shaking the 96-well plate, the absorbance at 550 nanometer was measured using a spectrophotometer (Multiskan GO, Thermo Scientific). The following formula was used to calculate the percentage of cytotoxicity [29];
2.8. Brine shrimp bioassay
Charmagaz seed oil was evaluated for its acute toxicity using a bioassay that is known as Brine shrimp (Artemia salina) method. Cold pressed seed oil was diluted in Dimethyl sulfoxide (DMSO) and the volume was made up to 5mL with sea water of different concentrations, i.e., 10, 100 and 1000 µg/mL. The test was done in replicates and control consisting only of Dimethyl sulfoxide in seawater. Eggs were sown in natural sea water and nauplii were obtained after 48h and used to inoculate the test samples [30]. After 24h of incubation at room temperature in the light, the number of survivors in each test tube was counted and the percentage mortality [31] was calculated by following formula:
2.9. Sub-acute oral toxicity
In the present study, adult healthy Sprague Dawley rats (180–200 g, both sex) were purchased from the breeding unit of HEJ Institute of Chemistry, University of Karachi, Pakistan. The animals were housed in individual cages and were kept in standard settings, which included a 12:12 h light/dark cycle, a regulated room temperature of 23 ± 2 °C, stress-free, unlimited access to water, standard diets, and odor-free surroundings. The rats were given a week to become used to their new environment in order to minimize stress before the experiment began. We took all the necessary steps to reduce the number of animals used and their suffering.
2.9.1. Experimental groups and treatment.
The rats were divided up into four groups, each with 10 animals (five males and five females). Group I served as vehicle control and was given purified water orally. The other three groups, referred to as groups II, III, and IV, were given oral doses of Charmagaz seed oil for a duration of 28 days, at dosages of 50, 500, and 5000 mg/kg respectively. After receiving Charmagaz seed oil, each animal was observed every day to look for any changes in their overall behavior or other physiological functions. Every animal was euthanized using ketamine at a dose of 100 mg/kg, followed by cervical dislocation to ensure death [32,33].
2.9.2. Animal monitoring and welfare considerations.
Throughout the study, each animal was observed daily for any changes in overall behavior, physical appearance, and physiological functions, including mobility, grooming, feeding and excretory patterns. Monitoring was conducted at least twice daily to assess animal health, detect early signs of distress and identify severe adverse effects. Body weights were recorded weekly, and any signs of toxicity, such as lethargy, dehydration, abnormal breathing or convulsions were noted.
2.9.3. Humane endpoints and euthanasia criteria.
Specific human endpoints were established to ensure that animals were euthanized if they exhibit signs of severe distress or if their condition was deteriorating beyond recovery. The humane endpoints included:
- Rapid weight loss exceeding 20% body weight
- Inability to eat or drink
- Persistent labored breathing or unresponsive behaviors
- Signs of severe pain or distress that could not be alleviated
Animals that reached any of these human endpoints were immediately euthanized using ketamine at a dose of 100 mg/kg followed by cervical dislocation to ensure death.
2.9.4. Number of animals used and outcome.
A total of 40 animals were used in the study, with 10 animals in each group. No animals were found dead during the study period prior to euthanasia. All animals were euthanized at the end of the 28-day experimental period.
2.9.5. Efforts to minimize suffering.
Throughout the experiment, every effort was made to minimize animals suffering. Animals were housed in comfortable, odor-free surroundings with minimal environmental stress. Additionally animals that displayed any signs of pain or distress were monitored closely, and any adverse symptoms were managed as per protocol. Analgesics or other supportive measures were considered, if necessary, though no analgesics were required during the course of this study.
2.9.6. Duration of the experiment.
The experiment was conducted over 28 days, with daily observation and recording of health and behavior.
2.9.7. Determination of hematological parameters.
White Blood Cells (WBCs), Red Blood Cells (RBCs), hemoglobin (Hb) and platelets (PLT) counts were assessed in blood samples using hematology analyzer (Siemens AG, Berlin, Germany).
2.9.8. Determination of biochemical parameters.
High density lipoprotein cholesterol (HDL), total cholesterol (TC), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Creatinine (Cr) were performed by a local laboratory.
2.9.9. Histopathological examination.
Livers were obtained and brought into the laboratory for immediate fixation in 10% phosphate buffered formalin, processed for paraffin embedding and 5 micrometer sections were made along the longitudinal axis. The sections were stained with hematoxylin and eosin (H&E) to observe under the microscope [34].
3. Results
3.1. Extraction and evaluation of lipid content
The cold-pressed process yielded a total percentage of 57.5% for the lipid content of Charmagaz seed oil. Cucurbitaceae seeds have a high total lipid content, which may make them economically advantageous for industrial extraction. This is especially true when compared to other oilseed crops like soybean and corn, which have lipid concentrations of 18–20% and 3.1–5.7%, respectively [35].
3.2. Charmagaz seed oil analysis by gas Chromatography Fame Ionization Detection (GC-FID)
An overview of Charmagaz seed oil’s fatty acid composition is shown in Table 1 (S1 Fig). Due to the inclusion of key fatty acids including oleic, palmitic, stearic, and linoleic, this oil falls within the category of oleic-linoleic acid [36]. Due to their high proportion of unsaturated fatty acids (UFAs, 76.662%) and low percentage of saturated fatty acids (SFAs, 23.338%), these fatty acid compositions are thought to be perfect for edible oils. The results showed that the polyunsaturated fatty acids (PUFAs) were 41.754% and the monounsaturated fatty acids (MUFAs) were 34.908%.
3.3. Antioxidant Activity by DPPH Assay
Charmagaz seed oil, after 30 min of incubation quenched 31.1% of DPPH radicals which is comparable to that reported by Rezig et al., (2012) [28]. This indicates that cucurbit seed oil can neutralize free radicals, meaning they contain substances that can directly interact with and neutralize DPPH.
3.4. MTT assay using NIH/3T3 cell line
The cytotoxic potential of Charmagaz seed oil dissolved in DMSO) was observed using the NIH/3T3 cell line. This experiment was executed to screen for toxicity (if any) of Charmagaz seed oil on normal cells. The cytotoxicity profile was determined in terms of percent growth inhibition using MTT assay. Charmagaz oil was found considerably safe and showed less than 50% threshold of percentage inhibition after 24 h and 48 h treatment as shown in Fig 1. At the tested concentration of 25 µM and 50 µM, Charmagaz oil did not show any significant cytotoxic effect as all the cells were found viable. After 48 h, Charmagaz oil at concentration of 25 µM showed 22% killing. While other doses of Charmagaz oil after 24 h and 48 h showed less than 20% of growth inhibition. Thus, suggesting that our test oil is safe and harmless on cellular assay. The images are showing the very low toxic effects of Charmagaz oil (***p < 0.001) at 25 µM and 50 µM on NIH/3T3 cells after 24 h and 48 h treatment. Arrows are indicating the places where the cells are present with the normal morphology even after the treatment with Charmagaz oil as shown in Fig 2. However, after 48 h treatment with the test oil cells showing detachment but overall architecture of fibroblast cells were intact.
Three experiments were run and the data was represented as mean ± S.E.M. One-way ANOVA was used for data analysis. The significant values are labelled as ***p < 0.001, **p ≤ 0.01 and *p ≤ 0.05.
The images demonstrate the effects of Charmagaz oil on fibroblast cells (NIH/3T3 cells). The arrows are used to show the effect produced by Charmagaz oil on 3T3 cells.
3.5. Brine Shrimp Bioassay
As displayed in Fig 3 (S1 Table), Charmagaz seed oil exhibited no cytotoxic activity in relation to the standard drug Etoposide (70% mortality at 7.5 μg/ml concentration).
3.6. Acute oral toxicity
Oral administration of Charmagaz seed oil, at doses from 50 to 5000 mg/kg, did not produce any significant changes in behavioral responses (vocalization, self-trauma, isolation from cage mates, aggressiveness, ataxia, restlessness, grooming, hunched posture, porphyrin staining, soiled anogenital area etc.) and physiological responses (changes in body weight, water intake, food intake, body temperature, activity level, vital signs, respiratory distress etc.). During the 28-day experimental period, no deaths occurred in any of the groups. These results indicate that LD50 of Charmagaz seed oil is above 5000 mg per kg and the compound exerted substantial safety margin and seemed to be free from acute toxicity in rats.
3.7. Effect of Charmagaz seed oil on hematological parameters
As demonstrated in Table 2 (S2 Table), there was not a statistically significant difference in the relative blood parameters, such as RBCs and WBCs, between the Charmagaz seed oil-treated rats and the control group. Even at high dosages, Charmagaz seed oil does not seem to interfere with hematopoiesis, as shown by the lack of a discernible change in blood index levels in relation to the normal control group.
3.8. Effect of Charmagaz seed oil on biochemical parameters
The serum concentrations of Creatinine, AST and ALT were not affected significantly after the treatment with Charmagaz seed oil indicating no change in the normal level of these serum markers as shown in Table 3 (S3 Table), Furthermore, the value of the Total cholesterol and HDL was not reduced in the treated groups compared to the control group.
3.9. Effect of Charmagaz seed oil on histopathology
According to Fig 4, histological analysis of the liver tissue in the normal control group (A) demonstrated normal parenchyma with intact hepatic architecture. Hepatic tissue of 50 mg/kg group (B) of Charmagaz seed oil revealed normal parenchyma with intact architecture. Hepatic tissue of animals at the doses of 500 mg/kg (C1, C2) displayed an intact cellular architecture. Likewise, photomicrographs of hepatic tissue treated with 5000 mg/kg (D1, D2) revealed intact architecture devoid of any signs of necrosis, edema, or degenerative alterations.
Histopathological assessment of liver of Charmagaz seed oil treated groups showed no pathological changes after 28 days treatment as compared to control group.
4. Discussion
Plant-based herbal medicines have minimal adverse effects making them typically safe and effective. The rural populace may have used herbal medicines responsibly because they were thought to be safe. In developing countries with large rural populations, these preparations are often taken orally for extended periods without proper consultation or dosage control, which can lead to a lack of awareness about potential toxic effects. Consequently, there is a need for scientifically validated information on the oral toxicity of herbal medicines. Such data would not only assist in identifying safe dosages for future use but also reveal various clinical signs and symptoms related to the herbal agent being studied. While the pharmacological effects of all the four members from Cucurbitaceae family have been established, comprehensive knowledge about the toxicity of combination preparation is still missing. Therefore, this research study was conducted to assess the safety data of Charmagaz seed oil [37,38].
Toxicological characteristics of the plant extract or isolated components are examined as the first stage in the pharmacological activity screening process. When studying new compounds in animal research, the acute toxicity profile offers information that is helpful for labeling, classifying, and determining the dosage. No treatment-related fatalities occurred over the duration of the present investigation, indicating that Charmagaz seed oil at a high dosage of 5000 mg/kg BW was neither toxic nor deadly. As every animal used in the research lived until they were compelled to be put to death, a lethal dosage (LD50) of more than 5000 mg/kg BW of Charmagaz seed oil was discovered in rats. To ensure that there were no observable adverse effect levels (NOAELs) in rats, three distinct doses—50, 500, and 5000 mg/kgwere chosen for the 28-day repeated dose toxicity trial, which was designed based on the LD50 dosage. The preclinical research on the acute toxicity of Charmagaz seed oil for clinical medication safety has not yet been found, as far as we could find. Because of this, we carried out a thorough pre-study for the present study in order to evaluate the safety and harmful effects of Charmagaz seed oil on rats. In another study, Atmane et al. (2022) investigated the safety profile of cold-pressed oil extracted from Pinus halepensis Mill. Seeds (COPHS) in mice. Their findings demonstrated that oral administration of COPHS at doses up to 5000 mg/kg body weight did not result in mortality or any observable signs of toxicity. This study suggests a large safety margin (>5000 mg/kg) for cold-pressed seed oils, providing some context for the potential safety of Charmagaz cold-pressed seed oil, though further research is certainly necessary [39]. It is important to note that while this provides a relevant comparison, it does not directly address the safety of Charmagaz oil, and our research aims to specifically address this gap. Therefore, we have retained and emphasized the importance of future toxicological studies on Charmagaz seed oil.
GCFID analysis of the seed oil were carried out using standard biochemical procedures that reveals the seed oils of Cucurbitaceae contain palmitic acid, Linoleic acid, Oleic acid, myristic acid and stearic acid that are 16–18 carbon fatty acids requires for normal growth, development and life processes.. Our findings are in accordance with Rezig et al., 2021 that reported Cucurbitaceae seeds presented an alternative source of plant oil as rich in omega 3 and 6 fatty acids which may serve as raw material for food applications and provide health benefits [40].
Cucurbita species plants are abundant in phytochemicals and serve as a rich supply of antioxidants. The phytochemicals Cucurbitacins, saponins, carotenoids, phytosterols, and polyphenols are the most significant ones found in cucurbits. Phenolic chemicals function in various ways, such as “scavenging” free radicals, and are members of a large class of antioxidants. The human body has several mechanisms to counteract oxidative stress by using antioxidants, which are either naturally produced in situ, or externally supplied through food and/or supplements. Endogenous and exogenous antioxidants interfere with the oxidation process by reacting with free radicals, chelating free catalytic metals, and acting as oxygen scavengers. Therefore, the consumption of cucurbits, which contain polyphenols and terpenoids in a large amount, plays an important antioxidant role in prophylaxis against oxidative stress [41].
The DPPH test shows that Charmagaz seed oil has antioxidant activity. As reported by several studies, seeds concentrated a higher proportion of vegetable constituents like poly unsaturated fatty acids (PUFAs) which are both of nutritional or medicinal interest [41–43].
Similar to the findings reported by Rezig et al. (2012), 31.1% of DPPH radicals were quenched by cucurbit seed oil after 30 minutes of incubation [28]. Strong naturally occurring antioxidants including ascorbic acid, β-carotene, lycopene, and tocopherols are responsible for the free radical scavenging action. Our findings suggest that Charmagaz seed oil’s strong antioxidant levels may be a factor in its therapeutic potential because they may provide protection against oxidative damage brought on by free radical scavenging.
In the MTT assay, Charmagaz oil was determined to be relatively safe, demonstrating less than a 50% inhibition rate after treatments of 24 and 48 hours, as shown in Fig 1. Similarly, in Brine Shrimp (Artemia salina) bioassay, Charmagaz seed oil did not exhibit cytotoxic effects when compared to the standard drug Etoposide, which caused 70% mortality at a concentration level of 7.5 microgram per ml as shown in Fig 3. Acute toxicity research further supported the safety of cucurbit seed oil. Acute oral toxicity tests identify obvious unfavorable effects that may impair the lifetime of the experimental animals and give crucial information on dosing schedules and target organ toxicity. In the present investigation, the effect of Charmagaz seed oil was evaluated in rats at doses of 50, 500 and 5000 mg/kg for 28 days. It has been observed that acute administration of Charmagaz seed oil did not produce any significant changes in behavioral and physiological responses. During the 28-day experimental period, no deaths occurred in any of the groups. Our findings agree with the previously reported safety profile of Cucurbita maxima and Cucumis sativus by Wahid et al., (2020) and Cruz et al., 2006 respectively [44,45]. The acute oral toxicity study showed that the LD50 of Charmagaz seed oil exceeds 5000 mg/kg. This suggests a significant safety margin, as they appear to be free from sub-acute toxicity in rats.
The hematopoietic system is the most susceptible system for toxic substances and becomes a useful marker of physiological and pathological state in animals and humans. Being the major medium for transporting nutrient and other foreign substances, the hematological parameter can be useful in evaluating the degree of harm caused by foreign compounds. In the present research study, all these hematological parameters were found to be within reference range of rat, indicating that Charmagaz seed oil does not contain any toxic compounds which could cause any variation in normal physiology. Charmagaz seed oil showed no significant changes on hematological variables recorded between the rats in the experimental group and the control group. The outcomes have been presented in tabular form in Table 2. The seed oil was also found to be inactive at a dose of 5000 mg per kg on synthesis and proliferation of blood cells or destruction at any stage.
Biochemical parameters are very essential in safety evaluation as they act as indicators in response to signs and symptoms elicited by toxicants. Therefore, to evaluate the toxic effects attained by seed oil, determination of liver and kidney function is mandatory. It is important to comprehend that the liver is the second largest internal organ in the human body. Extremely it can be damaged by several toxic molecules and medicinal agents taken in overdoses. This is explained by the fact that damage to the liver cell’s plasma membrane releases elevated amounts of liver function enzymes into the circulation. Aspartate transaminase and Alanine aminotransferase are predominantly located in the hepatic cells, and rise in their concentrations reflect the injury to the said cells. As a result, studying serum concentrations of ALT and AST can be regarded as the most informative markers of hepatocyte damage [46]. In the current study, no changes were found in the levels of Alanine aminotransferase and Aspartate aminotransferase thus implying that oil has hepatoprotective action as depicted in Table 3 [47]. In a similar way, the obtained result support the study carried out by Abou Seif (2014) and Bazabang et al. (2018) [48,49] which revealed that Cucurbita maxima seed oil and Citrullus lanatus seed extract possess hepatoprotective effect in alcohol induced hepatotoxicity and oxidative stress. Furthermore, no histological alterations of the liver cells were evidenced in the treated animals at all the doses (the low, middle, and high doses), proposing the hepatoprotective effect of cucurbit oil on hepatic tissues. In this study, we have got a positive correlation between liver function test and histological architecture of liver tissues.
Liver is a crucial organ for lipid metabolism as it secretes bile which helps in the absorption of fats and fat-soluble vitamins. The effect of Charmagaz seed oil on lipid metabolism is an important parameter to determine its safety as the oil itself is metabolized by liver. Charmagaz seed oil belong to the oleic-linoleic acid type of oil owing to the presence of major fatty acids such as Linoleic, oleic, palmitic, and stearic (Table 1). These fatty acids are regarded as the first choice of edible oils because they contain a high percentage of unsaturated fatty acids and low percentage of saturated fatty acids [36]. Dyslipidemia is characterized by an abnormal blood lipid profile, is usually diagnosed from Triglycerides (TGs), Total Cholesterol (TC), Low density lipoprotein cholesterol (LDL-C), and High-density lipoprotein cholesterol (HDL-C) levels [50]. Interestingly, Charmagaz seed oil decreased total cholesterol and increased the levels of High-density lipoprotein cholesterol indicating its usefulness in the management of hypercholesterolemia (Table 3). Moreover, cucurbit oil at any of the dose did not derange the values of liver enzymes and lipid profile as compared to normal control indicating hepatoprotective activity which is due to the presence of PUFAs such as palmitic acid, linoleic acid, oleic acid, and stearic acid [51]. Moreover, Poly unsaturated fatty acid (PUFAs) including linoleic acid (LA) and alpha-linolenic acid (ALA) have shown antioxidant property by preventing lipid peroxidation and raising the levels of many cellular antioxidants including ascorbic acid, α-tocopherol and glutathione [52].
Serum creatinine concentration is the surrogate test used to determine renal function. Importantly, Serum creatinine is acknowledged as an inadequate gold standard for several reasons. Besides Liver, Kidney is also a very important organ in the body that helps to regulate the whole body by throwing out metabolism waste materials. Long term consumption of oil might harm the renal tubules of kidneys and thus produce nephrotoxicity. In order to evaluate normal kidney function, we assessed the degree of normal kidney function through serum creatinine levels after administering oil for 28 days. Serum creatinine concentration which tends to increase in cases of impaired kidney function was unaltered in the treated rats in relation to normal control as shown in (Table 3).
Along with the predominant PUFAs, cucurbitaceae seeds also possess high content of polyphenols (protocatechuic, p-coumaric, vanillic), squalene, tocopherols, phytosterols, and carotenoids (lycopene, lutein, and zeaxanthin). These phytoconstituents show a positive correlation with the plant’s nephron-protective activity [53,54].
Histopathological examination of hepatic tissues in the control group as shown in Fig 4 (A) demonstrates typical architecture of hepatocytes indicated by centrally & rounded nuclear membrane with clear cellular boundary. While evaluation of liver tissues of treated groups showed that Charmagaz seed oil did not affect the normal architecture compared to normal control. Hepatic tissue of animals at the doses of 500 mg/kg (C1, C2) and 5000 mg/kg (D1, D2) showed intact architecture. Also, no evidence of degenerative changes, edema or necrosis was noted. Our findings are in support of Belemkar and Shendge, 2021 who reported the protective effect of Citrullus lanatus seed extract on histopathology of liver tissues in rats. Moreover, the biochemical results (ALT, AST) further strengthened the histopathological findings as they interpret the normal liver function [55].
While traditional medicine systems and modern pharmaceuticals offer valuable approaches to healthcare, incorporating nutrient-rich plant-based foods, like cucurbit seeds, can play a vital role in disease prevention and health promotion [56]. Cucurbitaceae seeds are a promising source of bioactive compounds and can be utilized in functional foods [57]. These seeds are rich in beneficial compounds [58] and offer a sustainable dietary option due to their adaptability [57]. However, despite their nutritional value, cucurbit seeds, particularly from pumpkin and watermelon, are often underutilized and wasted [59]. Given the increasing focus on plant-based diets [58] and the challenges of food security [59], maximizing the use of these nutrient-dense seeds in diverse food applications, including nutraceuticals, represents a significant opportunity to improve community health and potentially lessen reliance on pharmaceutical interventions.
5. Conclusion
The current research study indicates that an oral dosage of up to 5000 mg/kg of Charmagaz seed oil is considered safe, as it did not exhibit any signs of sub-acute toxicity or cause mortality in rats. Furthermore, the oil did not result in any negative effects on the normal behavior of treated animals. During a 28-day period of repeated administration, the biochemical, hematological, behavioral, and histological indicators remained within the safe range for dosages of 50, 500, and 5000 mg/kg, in comparison to the untreated control group. These findings suggest that Charmagaz seed oil can be safely utilized in the evaluation of pharmacological and therapeutic effects. Moreover, these preclinical results also provide a basis to further explore the efficacy of Charmagaz seed oil in clinical practice. While the demonstrated benefits of cucurbit seeds are promising, further research, including detailed chronic and sub-chronic toxicity studies, is crucial to definitively establish their safety for widespread use. Additional investigation into the medicinal and therapeutic efficacy is also warranted to fully understand and leverage the potential health benefits of Cucurbitaceae seed oils.
Supporting information
S1 Fig. Fatty Acid Profile of Charmagaz Seed Oil by GC-FID.
https://doi.org/10.1371/journal.pone.0327697.s001
(PDF)
S1 Table. Percentage mortality of brine shrimps by Charmagaz seed oil.
https://doi.org/10.1371/journal.pone.0327697.s002
(DOCX)
S2 Table. Effect of Charmagaz seed oil on Hematological Parameters.
https://doi.org/10.1371/journal.pone.0327697.s003
(XLSX)
S3 Table. Effect of Charmagaz seed oil on Biochemical Parameters.
https://doi.org/10.1371/journal.pone.0327697.s004
(XLSX)
Acknowledgments
Authors are grateful to the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi for the support provided to conduct this experimental study.
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