https://orcid.org/0000-0001-7220-6356
Figures
Abstract
Cadmium (Cd), a highly toxic heavy metal, is used in food and agricultural products while displaying nephrotoxicity to animals and humans. The genus Syzygium (Myrtle family) is rich in precious phenolic metabolites with various therapeutic values. This study investigated the phenolic content and the therapeutic potential of the defatted 80% aqueous methanol extract (DE) of S. malaccense and S. samarangense leaves against Cd-induced kidney injury in rats for the first time. High-performance liquid chromatography-Mass spectrometry (HPLC-MS), in addition to Folin-Ciocalteu and aluminium chloride colourimetric methods, depicted the phenolic metabolites, total phenolic content, and total flavonoid content, respectively. The nephroprotective effect was investigated using fifty-six female Sprague Dawley rats divided into eight groups: control group, CdCl2-treated group (3 mg/kg/i.p/7 days), and three groups of each species treated with the DE (250, 500, and 1000 mg/kg/o.p., respectively). The phytochemical analysis revealed the richness of S. samarangense DE by phenolic and flavonoid content over S. malaccense. The HPLC-MS showed the tentative identification of sixty-two compounds, in positive and negative ionization modes, belonging to phenolic acids (1−6), flavonoids (7−52), and miscellaneous compounds (53−62). Both extracts were considered safe up to 5 g/kg. At the maximum tested dose (100 mg/Kg), the DEs significantly (p < 0.001) boosted the levels of antioxidant markers by 3.3–6 fold, lessened the inflammatory indicators by 66.8%−75.1%, and reduced the apoptotic parameters by 45.4–73.3%, compared to the CdCl2-treated group. Additionally, the DEs maintained the mitochondrial function and inhibited autophagy via decreasing adenosine monophosphate-activated protein kinase by 49.2%−50.6%, and baclin-1 by 49.5%−56.1%. Additionally, the DEs increased the mammalian target of rapamycin (mTOR) by 4.7–4.9 fold. Additionally, the DE ameliorated CdCl2-induced elevations in serum ALT and AST, indicating a protective effect against systemic toxicity. Ultimately, the DE of S. malaccense and S. samarangense protect against Cd-induced nephrotoxicity that may be correlated to their abundant phenolic content. However, selecting suitable formulations and implementing clinical studies are among the future directions.
Citation: Moharram FA, Salem SS, Shabana S, El-Sayed EK, Mohamed SK, Khattab MA, et al. (2025) HPLC-MS profiling and protective potential of the defatted aqueous methanol extract of two Syzygium species against cadmium chloride-induced nephrotoxicity in rats. PLoS One 20(8): e0329586. https://doi.org/10.1371/journal.pone.0329586
Editor: Saikat Dewanjee, Jadavpur University, INDIA
Received: April 14, 2025; Accepted: July 16, 2025; Published: August 14, 2025
Copyright: © 2025 Moharram et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All available data are presented in the paper.
Funding: The grants from the National Science and Technology Council of Taiwan (MOST 111-2321-B-255-001 and MOST 111-2320-B-038-040-MY3) supported this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Cadmium (Cd) is a hazardous heavy metal renowned for its toxic potential to the kidneys [1]. Humans may be at risk from two types of Cd exposure. On top is the occupational Cd exposure through engaging with industrial activities [2] in addition to non-occupational exposure via contaminated food and water [3]. After Cd-exposure, multifactorial mechanisms are implicated in the pathogenesis of Cd-toxicity, such as oxidative stress, mitochondrial dysfunction, apoptosis, and inflammation [4–6]. Moreover, autophagy pathways are considered sensitive biomarkers for kidney damage upon Cd intoxication [7,8]. Currently, there is no standard therapy for Cd-induced nephrotoxicity, which underscores the urgent demand for the scientific community to discover a promising treatment, particularly given the growing global interest in the therapeutic values of antioxidants [9].
Phenolic compounds are a miscellaneous group of plant-derived metabolites that include different subclasses. They possess a unique chemical scaffold with potent antioxidant and anti-inflammatory activities [10–12]. Since oxidative stress and inflammation play essential roles in the prognosis of kidney diseases, phenolic metabolites offer potential as natural mediators that can modify these pathological processes [13–19]. Several plants are abundant in phenolics, including but not limited to Syzygium species (Myrtle family) [20,21]. Among the limitley explored species are Syzygium malaccense (L.) Merr. & L. M. Perry and Syzygium samarangense (Blume) Merr. & L. M. Perry. They are distributed in tropical and subtropical regions of Africa and Asia, and are renowned for their edible fruits [21–24]. In traditional medicine, S. malaccense was used as an anti-inflammatory, while the leaves and fruits were pharmacologically proven to possess anti-microbial and antioxidant activities, at least in part, due to their phenolic composition [25–31]. Extracts of S. samarangense displayed anti-inflammatory, analgesic, hepatoprotective, and diuretic effects [21,32]. These pharmacological activities are attributed to different classes of phenolic metabolites [21,33–35]. Although some biological activities were reported for S. malaccense and S. samarangense, their potential protective effect against Cd-induced nephrotoxicity has not been reported. Hence, encouraging our research team in the current study to investigate their nephroprotective potential.
Plant extracts represent a complex mixture of hundreds of diverse metabolites [36], making their analysis a challenging task. Although different chromatographic and spectroscopic approaches are available, high-performance liquid chromatography-mass spectrometry (HPLC-MS) permits comprehensive extract analysis on its own [37]. As a state-of-the-art analytical coupling approach, it merges the efficient separation aptitudes of HPLC with the supreme sensitivity and specificity of MS [37]. Hence, it provides valuable and prompt structural information on the metabolites detected without the need for isolation. Additionally, it is noticeable by its applicability to varied analyte types (ranging from small molecules to large polymers), providing higher resolution, greater sensitivity, and precision [36,37].
In the current study, we aimed to profile the phenolic content and composition of the defatted aqueous methanol extract of S. malaccense and S. samarangense using HPLC-MS. In addition, we evaluated their nephroprotective activity in a Cd-induced nephrotoxicity in vivo model.
Materials and methods
Plant material
Syzygium malaccense (L.) Merr. & L. M. Perry and Syzygium samarangense (Blume) Merr. & L. M. Perry leaves were supplied (June to August 2021) from Mazhar Botanical Garden, Cairo, Egypt. The plants were collected after the garden authorities’ permission, following the local garden`s guidelines for collection under the supervision of Dr. Trease Labib, a senior botanist at Mazhar Botanical Garden, who confirmed the species’ identity and the species’ names following the International Plant Name Index (IPNI). Samples from each species were kept in the Pharmacognosy Department, Faculty of Pharmacy, Helwan University under the numbers 01Sma, 2021, and 01Ssa, 2021. The plant material under study is endotoxin-free, and the study protocol was approved by the Ethics Committee of the Faculty of Pharmacy, Helwan University (Approval No: 11A2023).
Chemicals and reagents
Methanol, n-hexane, normal saline, buffered formalin, and phosphate buffer were supplied from El Nasr Pharmaceutical Chemicals Co. (Gesr El Suez, Cairo, Egypt). Folin-Ciocalteu reagent, gallic acid, catechin, AlCl3, anhydrous cadmium chloride (CdCl2), ethanol, sodium thiopental, and hematoxylin-eosin staining solution were obtained from Sigma-Aldrich Inc. (St. Louis, MO, United States).
Preparation of the defatted extract (DE)
S. malaccense (275 g) and S. samarangense (230 g) leaves were individually extracted with 80% aqueous methanol under reflux (3 x 4 L). The filtered, pooled extracts were evaporated under reduced pressure (60°C) to yield 35.0 g and 22.0 g of S. malaccense and S. samarangense dry extracts, respectively. The extracts were defatted by n-hexane under reflux (3 x 1 L) followed by solvent evaporation under vacuum using a rotary evaporator, followed by lyophilization to yield 10.0 g and 7.0 g (n-hexane) and 20.0 g and 10.0 g (defatted extract, DE) for S. malaccense and S. samarangense, respectively. The dried DE from each species was subjected to quantitative and qualitative phenolic profiling, and we investigated its protective potential in the Cd-induced nephrotoxicity in vivo model. The remainder of the extract was kept in a well-sealed container at −80°C.
Estimation of total phenolic content (TPC)
It was estimated following the Folin-Ciocalteu colorimetric method [38]. The absorbance was determined at 725 nm on a UV-visible spectrophotometer (Jasco V-730, Jasco Corporation, Japan). The results were calculated from the calibration curve of the standard (gallic acid 50–300 μg/mL) using the following equation:
The results were stated as gallic acid (mg) equivalent (mg GAE/mg) of dry weight.
Estimation of total flavonoid content (TFC)
It was measured colorimetrically using the AlCl3 [38]. The absorbance was measured at 510 nm on a UV-visible spectrophotometer (Jasco V-730, Jasco Corporation, Japan). The results were deduced from the equation of the calibration curve constructed from the standard (catechin, 50–300 μg/mL) as follows:
Results were expressed as milligrams of catechin equivalent (mg CE/mg of dry weight).
Determination of phenolic profile using HPLC-MS
The HPLC-MS analysis was performed on the XEVO® triple quadrupole (TQD) mass system (Waters™ Corporation, Milford, MA, United States) operated in negative and positive ionization modes. Chromatographic separation was carried out on the ACQUITY UPLCBEH C18 column (1.7 µm x 2.1 mm x 50 mm, 28.4 °C) using 0.1% formic acid in H2O as solvent A and MeOH as solvent B. Isocratic gradient elution was implemented using a flow rate of 0.2 mL/min for 32 min as follows: 10% B for 5 min, 30% B for 10 min, 70% B for 7 min, then 90% B for 10 min. The MS acquisition range was m/z 50–1000.
In vivo nephrotoxic activity induced by cadmium chloride (CdCl2)
Experimental animals.
Adult female Sprague-Dawley rats (9–10 weeks old, 180–200 g) were purchased from 223 VACSERA (Helwan, Cairo, Egypt). Rats were housed (3–4 rats/cage) at a controlled 224 temperature of 22°C ± 3°C with a 12-hour light/dark cycle. Standard rodent chow and water were provided ad libitum. All procedures were conducted following the ethical standards approved by the Ethics Committee of the Faculty of Pharmacy, Helwan University (Approval No: 11A2023). The study was conducted in compliance with the ARRIVE guidelines 2.0, the EU Directive 2010/63/EU for the protection of animals used for scientific purposes, and the NIH Guide for the Care and Use of Laboratory Animals (8th edition). The experimental design included the possibility of mortality due to the nature of the pharmacological intervention. Therefore, animals were monitored at least twice daily for clinical signs of pain, distress, or illness, including reduced mobility, abnormal posture, respiratory distress, piloerection, and failure to groom. If any animal exhibited signs of severe distress or met predetermined humane endpoints (e.g., > 20% weight loss, loss of appetite > 48 hours, or unresponsiveness), it was euthanized immediately via an overdose of sodium thiopental administered intraperitoneally (≥150 mg/kg). No animals died unexpectedly, and euthanasia was performed according to ethical guidelines to minimize suffering. All efforts were made to refine procedures and reduce the number of animals used. The Humane endpoints checklist is supplied as supporting material S1.
Acute toxicity study.
We used adult female rats, strain Sprague Dawley (180–200 g, n = 6), to establish the acute study. In a dose-dependent approach, the rats were administered S. malaccense DE and S. samarangense DE at 50 mg/kg, 200 mg/kg, 500 mg/kg, 1g/kg, 2.5 g/kg, and 5 g/kg. The rats were well observed for 24 h for any symptoms of toxicity, side effects, behavioral changes, movement patterns, diarrhea, and death.
Experimental design.
Fifty-six rats were randomly divided into eight groups (n = 7). The control group received 0.9% saline solution as a vehicle for seven days. In the CdCl2 group (group 2), rats were injected with CdCl2 (3 mg/kg/ intraperitoneal) for 7 days [39]. Animal groups 3–5 were treated for seven days with the DE of S. malaccense at 250, 500, and 1000 mg/kg orally, about one hour before the administration of CdCl2. On the other hand, groups 6–8 were treated for seven days with the DE of S. samarangense at 250, 500, and 1000 mg/kg doses, respectively, about one hour before the administration of CdCl2. On the 8th day, rats were intraperitoneally injected with sodium thiopental (40 mg/kg) [40] to induce anesthesia. Blood samples were collected from the retro-orbital plexus using non-heparinized tubes. Rats were euthanized by cervical dislocation, and the kidneys were isolated, washed with cold saline, and weighed. The right kidneys were homogenized in an ice-cold phosphate buffer (10 mM, pH 7.4) to prepare tissue homogenates of 10% w/v for biochemical parameters. The left kidneys were preserved in formal saline (10%) for histopathological analysis.
Measurements of biochemical markers
Serum urea and creatinine.
Urea and creatinine levels were determined using colorimetric kits (Catalog #K375-100 and K625-100, respectively) purchased from BioVision (Milpitas, United States). The colour intensity was measured on a Jenway™ 7315 spectrophotometer (Fisher Scientific, Leicestershire, UK).
Oxidative stress parameters.
The levels of GSH and SOD in the kidney tissue homogenates were quantified using the colorimetric kit (Catalog #K464-100 and K335-100 respectively) obtained from BioVision (Milpitas, United States). The colour intensity was measured on a Jenway™ 7315 spectrophotometer (Fisher Scientific, Leicestershire, UK).
Inflammatory parameters.
Using specific ELISA kits, the levels of TNF-α (Catalog # 438204, BioLegend, San Diego, United States), IL-1β (Catalog # E0119Ra, Bioassay Technology Laboratory, Shanghai, China), and NF-κB p65 (Catalog # ABIN1380657, Antibodies-online, Canada) were quantified in kidney tissue homogenates using NS 100 microplate reader (Heruvan Lab Systems, Selangor, Malaysia).
Apoptotic parameter.
The level of the apoptotic marker, caspase-3, was determined in the kidney tissue homogenates using an ELISA kit (Catalog # E4592-100, BioVision, Milpitas, United States) and NS 100 microplate reader (Heruvan Lab Systems, Selangor, Malaysia).
Mitochondrial dysfunction parameter.
The ATP level in kidney tissue homogenates was estimated using a colorimetric Kit (Catalog # ab83355, Abcam, Cambridge, United Kingdom). The colour intensity was measured on a Jenway™ 7315 spectrophotometer (Fisher Scientific, Leicestershire, UK).
Autophagic parameters.
For the determination of autophagic parameters, ELISA Kits for the phosphorylated mammalian target of rapamycin (p-mTOR) (Catalog # ab279869, Abcam, Cambridge, United Kingdom), Beclin-1 (Catalog# ER0416, FineTest, Wuhan, China), and phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) (Catalog # ER0730, FineTest, Wuhan, China) were used according to the manufacturer’s procedures. In brief, samples were added to their corresponding microtiter plate wells coated with the targeted monoclonal antibodies. Accordingly, any rat p-mTOR, beclin-1, or p-AMPK in the samples would bind to their corresponding immobilized antibodies. The wells were washed, and biotin-conjugated anti-rat p-mTOR, beclin-1, or p-AMPK antibodies (1:100) were added to their corresponding plates. The plates were washed, and avidin-horseradish peroxidase (avidin-HRP) was added. Then, the wells were washed, and tetramethylbenzidine (TMB) substrate solution was added to produce a blue color directly proportional to the amount of the detected proteins in the sample. The reaction was terminated by adding the stop solution, which changed the color from blue to yellow, and the intensity of the color was measured at 450 nm using an NS 100 microplate reader (Heruvan Lab Systems, Selangor, Malaysia).
Assessment of liver enzymes.
To evaluate the systemic toxicity of cadmium, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Biomatik ELISA kits (Cat no: EKU02211, EKE62019, Kitchener, Ontario, N2C 1N6, Canada, respectively). The procedures were carried out according to the manufacturer’s instructions
Histopathological analyses.
Kidney tissue samples from the investigated groups were fixed in 10% neutral buffered formalin for three days. The samples underwent a series of ethanol dehydration steps, followed by xylene clearing and embedding in Paraplast embedding media. Kidney tissue sections (4 µm) were cut using a rotary microtome and then fixed on glass slides for further examination. Finally, using hematoxylin and eosin, tissue slices were stained and inspected under a light microscope [41].
Statistical analysis
All experiments were implemented in triplicate, and values were represented as mean ± SEM. Results were analyzed using ANOVA followed by Tukey’s test, while the student’s t-test was implemented to analyze the TPC and TFC data. The Prism software, version 8, was used for the statistical analysis (GraphPad Software Inc., San Diego, United States). P-value, which is less than 0.001, is statistically significant and denoted with an asterisk or a letter, as indicated.
Results
Extraction, quantification, and identification of phenolic metabolites
Quantification of phenolic and flavonoid contents from two Syzygium species.
As stated earlier, the total phenolic and flavonoid contents in the DE of the two investigated species were measured using colourimetric assays. The results (Table 1) have shown that S. samarangense possesses a TPC that is 1.5-fold higher than S. malaccense, and the difference is statistically significant. Interestingly, this is the first study reporting the TPC for S. samarangense. However, it has been previously reported from S. malaccense by several researchers, notably ranging from 62.31 ± 8.32 to 88.73 ± 7.71 mg GAE/g [25,30]. On the other hand, S. samarangense showed significantly higher total flavonoid content than S. malaccense by 1.2-fold. This is the first report on the TFC of S. samarangense, although it has been quantitatively documented before (8.10 ± 46.75 CE/g) by Batista et al. [25]. The possible deviation in our data from those reported in the literature could be attributed to the geographical collection site’s impact and the variations in the extraction conditions (solvent, time, and temperature) [42].
HPLC-MS analysis for defatted extract (DE) of S. malaccense and S. samarangense leaves.
The HPLC-MS for the DE of the two species revealed the presence of sixty-two secondary metabolites belonging to various classes, which were tentatively identified in positive and negative modes (Table 2). Thirty-nine were detected in S. malaccense, while forty-six were in S. samarangense. The HPLC-MS chromatograms of the two species are displayed in Figs 1 and 2.
Phenolic acids are widely distributed in all native Australian flora, including Syzygium species [43]. Based on their chemical scaffold, they are sub-classified into hydroxybenzoic and hydroxycinnamic acid derivatives [44]. In the current study, we identified three hydroxybenzoic acid derivatives in the negative mode of S. malaccense (1, 2, 6) and five in S. samarangense (1–3, 5, 6). Additionally, one hydroxycinnamic acid derivative (4) was detected in the positive mode of S. malaccense. Compounds 1, 2, 3, and 5, were identified as gallic acid, methyl gallate, ellagic acid glucoside, and ellagic acid with m/z [M-H]-169.0373, 183.0698, 463.1513, and 301.0638, respectively. Compounds 4 and 6 were identified as hydroxycaffeic acid and gallic acid glucoside with m/z [M + H]+ 197.1579 and 333.1248, respectively. Compounds 1, 2, 5, and 6 were identified before from S. samarangense [32,45], while compound 1 was previously reported from S. malaccense [30,46]. The remaining compounds were identified for the first time.
Flavonoids are the most abundant bioactive metabolites widely distributed in medicinal plants. They are crucial metabolites with favorable therapeutic effects on multiple diseases [47]. Chemically, flavonoids comprise 15 carbon atoms in their basic frame, which are allocated as two six-membered rings and one three-carbon unit coupled to them as C6-C3-C6. Based on structural differences, they are classified into seven subclasses: flavonols, flavones, isoflavones, anthocyanidins, flavanones, flavanols, and chalcones [48]. In the current study, we annotated 46 flavonoids, including 15 flavonols, 7 flavanones, 3 flavones, 7 isoflavonoids, and 14 chalcones (Table 2).
Another notable subclass of flavonoids is the flavonols. Herein, eight flavonols (7, 8, 10, 13, 14, 17, 18, and 19) were identified from both species. In addition, four (12, 15, 16, 20) were identified from S. malaccense, and three from S. samarangense (9, 11, 21). Compounds 7 and 8 were identified as myricetin (7) and myricetin-O-rhamnoside (8) with m/z [M-H]- 317.1821 and 463.2279, respectively. Compounds 9, 10, 11, 13, 15, 16, 17, and 18 were identified in the negative mode as isoquercetin (m/z [M-H]- 463.1943), quercetin (m/z [M-H]- 301.0468, hyperin (m/z [M-H]- 463.1249), isorhamnetin-O-glucoside (m/z [M-H]- 477.1945), quercitrin (m/z [M-H]- 447.2192), kaempferol-O-glucoside (m/z [M-H]- 447.2192), quercetin 4′-O-glucuronide (m/z [M-H]- 477.2255), and mearnsitrin (m/z [M-H]- 477.2544). On the other side, compounds 12, 14, 19, 20 and 21 were identified in the positive mode as isorhamnetin-O-rhamnoside (m/z [M + H]+ 463.1954), mearnsetin (m/z [M + H]+ 333.11245), kaempferol (m/z [M + H]+ 287.1558), quercetin 3-O-acetyl-galactoside) -O-rhamnoside (m/z [M + H]+ 653.4450) and quercetin 3’-sulfate (m/z [M + H]+ 383.4042). Compounds 7 (myricetin), 8 (myricetin- O-rhamnoside), 10 (quercetin), 14 (mearnsetin), 15 (quercitrin), 18 (mearnsitrin), and 19 (kaempferol) were identified before from S. malaccense leaves grown in Brazil but not from the Egyptian species [25,46], while 13 (isorhamnetin-O-glucoside), 16 (kaempferol-O-glucoside were identified from its fruits [46]. Flavonols 12 (isorhamnetin-O-rhamnoside), 17 (quercetin 4′-O-glucuronide), and 20 (quercetin 3-O-acetyl-galactoside)-O-rhamnoside) were detected for the first time. Ultimately, compounds 8 (myricetin-O-rhamnoside), 9 (isoquercitrin), 10 (quercetin), and 11 (hyperin) were identified before from the leaves of S. samarangense grown in Egypt [32,45], compounds 8, 10, and 11 were identified from its fruits, while compounds 7 and 19 were detected in its stem bark [36]. Flavonols 7, 13, 14, 17, 18, 19, and 21 were tentatively identified for the first time from S. samarangense leaves. Additionally, two flavone glycosides were identified in the positive mode of S. malaccense as diosmin (23) (m/z [M + H]+ 609.4222) and apigenin-7-O-diglucuronide (24) (m/z [M + H]+ 623.4117), while 4’,7-tetrahydroxyflavone (22) (m/z [M-H]- 285.1601) was identified in the negative mode of S. samarangense. This is the first time to identify the forementioned flavone glycosides from both species, though 22 and 23 were previously detected in other Syzygium species [49]. Concerning the flavanone sub-class, eight of them were identified in the analyzed extracts. Pinocembrin 25 (m/z [M-H]- 255.1476/ [M + H]+ 257.1037) and cryptostrobin 26 (m/z [M-H]- 269.1739/ [M + H]+ 271.0981) were identified in both negative and positive modes of the two species. Naringenin 28 was identified in the positive mode of S. malaccense (m/z[M + H]+ 273.2100) while strobopinin 27 (m/z [M-H]- 269.1747), 7-hydroxy-5-methoxy-6,8-dimethylflavanone 30 (m/z [M-H]- 297.2050), and 8-prenylnaringenin 31 (m/z [M-H]- 339.3448) were identified in the negative mode of S. samarangense. Additionally, 8-methylpinocembrin 29 (m/z [M + H]+ 271.1037) and 6-geranyl naringenin 32 (m/z[M + H]+ 409.4701) were identified in its positive mode. This is the first time identifying the flavanones class of metabolites in S. malaccense, except naringenin 28, which was identified before in the Brazilian species [46]. 26 was previously detected in the leaf extract of S. samarangense cultivated in Egypt [32], while 25, 27, 29, and 30 were identified from its stem bark [35]. 31 and 32 were identified for the first time from the Egyptian species, while previously reported in other Myrtle species [49]. Skimming the presence of other flavonoid subclasses, three isoflavones were tentatively identified in S. malaccense. One of which, namely biochanin A 33 (m/z [M-H]- 283.1853/ [M + H]+ 285.1370), is detected in both negative and positive modes. On the other hand, 37 and 38 were identified as pseudobaptigenin (m/z [M + H]+ 269.1739) and glycitein m/z [M-H]- 283.1666), respectively. Moreover, 4’-O-methyl equol 33, dihydrodaidzein 35, glycitin-6’‘-O-acetate 36, and genistein 4’,7-O-diglucuronide 39 were identified in the negative mode of S. samarangense with m/z [M-H]- 255.1296, 255.1142, 487.4554, and 621.4432, respectively. Interestingly, this is the first report of isoflavones from the two species investigated, but they have been previously detected in other Myrtaceae species [49,50].
Chalcones and their derivatives represent an interesting category of flavonoids due to their versatility and effective bioactivities. They comprise two aromatic rings bridged by an α, β-unsaturated system of three carbons. In this regard, nine (41–43, 45–47, 49, 50, and 52) and twelve (40, 42–52) chalcones were identified from S. malaccense and S. samarangense, respectively. Chalcones 42, 43, 45, 46, 47, 49, 50, and 52 were detected in positive and negative modes of both species and elucidated as aurentiacin 42 (m/z [M-H]- 297.1133/ [M + H]+ 299.1569), cardamonin 43 (m/z [M-H]- 269.1631/ [M + H]+ 271.1143), 2’,4’-dihydroxy-6’-methoxy-3’-methyl chalcone 45 (m/z [M-H]- 283.1888/ [M + H]+ 285.2387), stercurensin 46 (m/z [M-H]- 283.1625/ [M + H]+ 285.1329), demethoxymatteucinol 47 (m/z [M-H]- 283.1846/ [M + H]+ 285.1332), 2’,4’-dihydroxy-6’-methoxy-3’-methyl dihydrochalcone 49 (m/z [M-H]- 285.2119/ [M + H]+ 287.1356), 2′,4′-dihydroxy-6′- methoxy3′,5′- dimethyl dihydrochalcone 50 (m/z [M-H]- 297.2200/ [M + H]+ 299.1672) and dimethyl cardamonin 52 (m/z [M-H]- 297.2204/ [M + H]+ 299.1618). On the other side, 6,8-di-C-methyl pinocembrin-5-methyl ether 41 was detected in the positive mode of S.malaccense (m/z [M + H]+ 299.1618), while phloretin 40, uvangoletin 44, 2’-hydroxy-4’,6’-dimethoxy chalcone 48 and 4’,6’-dihydroxy-3’,5’-dimethyl-2’-methoxy chalcone 51 were detected in the negative mode of S. samarangense with m/z [M-H]- 273.0029, 271.1406, 283.2011 and 297.1987, respectively. Interestingly, this is the first study to report the detection of chalcones in the leaves of S. malaccense, while chalcones have been reported before from S. samarangense. For instance, 45 was previously identified in S. samarangense leaves’ extract [51], compounds 42, 44, 46, 47, 49, 51, and 52 were detected in its stem bark [35], and compound 43 from the fruits [52] as well as from other Syzigium species [49,51].
Lignin is the most vital and profuse biopolymer that connects cellulose and hemicellulose fibers, providing a rigorous structure to the plant cell [53]. Herein, only one lignin, lariciresinol-sesquilignan 53, was identified for the first time from the negative mode of S. malaccense with (m/z [M-H]- 555.4398), but it was previously detected from other Myrtaceae species [49]. Two derivatives were identified for the first time from S. malaccense, one of which is 5-heptadecylresorcinol 54 and detected in both ionization modes (m/z [M-H]- 347.2201/ [M + H]+349.2050), while the other is adipostatin E 55 and detected in the negative mode only (m/z [M-H]- 255.1296). The two compounds were identified earlier from the leaves of S. samarangense grown in China [54].
Ultimately, three miscellaneous metabolites were detected for the first time in S. malaccense extract and elucidated as malic acid 58 (m/z [M-H]- 132.9923), quinic acid 59 (m/z [M-H]- 191.1055), and stigmastanol trans-ferulate 61 (m/z [M + H]+ 593.3953). To our knowledge, compounds 58 and 59 were previously identified from S. samarangense [32,46]. On the other side, bergapten 56 (m/z [M-H]- 215.1005), citric acid 57 (m/z [M-H]- 191.0651), 3,5-dimethyl-resveratrol 60 (m/z [M-H]- 255.1323), cyanidin 3-O-glucosyl-rutinoside 62 (m/z [M + H]+ 758.7891).
Acute toxicity study
The acute toxicity study was performed to ensure the safe dosing range of the extracts before proceeding to further pharmacological evaluations. Understanding the acute toxicity profile allows the selection of appropriate doses for efficacy studies while minimizing potential adverse effects. Herein, twenty-four hours post-extract administration, no changes were observed in the motor activity or the behavior of the tested animals, and no mortality was recorded. Therefore, both DEs were considered safe till 5 g/kg, and, consequently, we selected dose levels of 250, 500, and 1000 mg/kg to evaluate the extract’s nephroprotective effect.
In vivo nephroprotective activity
Effect of the DE of S. malaccense and S. samarangense on the levels of urea and creatinine.
As seen in Table 3, CdCl2 induced kidney tissue damage that affected the function of the glomeruli, evidenced by the significant (p < 0.001) increase in the levels of urea and creatinine in CdCl2-treated rats by 4.4-fold and 3.7-fold, respectively, compared to the control group. Both tested extracts preserved the renal function evidenced by the significant (p < 0.001) decrease in urea and creatinine levels as follows: DE of S. malaccense in doses 250, 500, and 1000 mg/kg significantly declined urea levels by 33.5%, 64.1%, and 69.2%, respectively; and decreased creatinine levels by 22.3%, 52.9%, and 60.6%, respectively, compared to the CdCl2-treated rats. Meanwhile, S. samarangense significantly (p < 0.001) decreased urea levels by 45.3%, 68.9%, and 70.2%, respectively, and decreased creatinine levels by 25.8%, 61.5%, and 65.4%, respectively, compared to the CdCl2-treated rats.
Effect of the DE of S. malaccense and S. samarangense leaves on oxidative stress parameters.
Oxidative damage induced by CdCl2 is evidenced by the marked (p < 0.001) decrease in GSH by 74.6% and SOD by 84.4%, compared to the control group. Doses of 250, 500, and 1000 mg/kg from the DE of S. malaccense significantly (p < 0.001) boosted the level of GSH by 1.7, 2.7, and 3.3 folds, respectively; and SOD by 2.4, 4.3, and 5 folds, respectively, compared to CdCl2-treated group. Meanwhile, doses of 250, 500, and 1000 mg/kg from S. samarangense significantly (p < 0.001) enhanced the level of GSH by 1.7, 2.7, and 3.3 folds, respectively. In addition, they potentiated the level of SOD by 2.6, 4.8, and 6.4 folds, respectively, compared to the CdCl2-treated group. These findings support their antioxidant property (Figs 3A and 3B).
Data represented as mean±SEM of n = 7. a: significant from the control group at p < 0.001, b: significant from the CdCl2 group at p < 0.001.
Effect of the DE of S. malaccense and S. samarangense leaves on inflammatory parameters.
Inflammation is involved in CdCl2-induced damage, as seen in CdCl2-treated rats. They manifested a marked (p < 0.001) elevation in TNF-α, IL-1β, and p-NF-κB p65, by 4.8, 4.0, and 3.5 folds, respectively, compared to the control rats. Treatment with S. malaccense DE (250, 500, and 1000 mg/kg) significantly (p < 0.001) reduced TNF-α by 37.4%, 57.9%, and 71.9%; decreased IL-1β by 35.6%, 62.4%, and 68.1%; and reduced p-NF-κB p65 by 27.3%, 60.9%, and 66.8%, respectively, compared to the CdCl2-treated rats. Additionally, treatment with S. samarangense (250, 500, and 1000 mg/kg) significantly (p < 0.001) decreased TNF-α by 41.3%, 57.9%, and 75.1%; and reduced IL-1β by 34.9%, 61.4%, and 71.9%; and decreased p-NF-κB p65 by 34.7.3%, 60.1%, and 65.4%, respectively, compared to the CdCl2-treated rats. These results indicate the promising anti-inflammatory activity of the tested extracts (Figs 3C-3E).
Effect of the DE of S. malaccense and S. samarangense leaves on the apoptotic parameter caspase-3.
Treatment with CdCl2 induced apoptosis of renal tissue as indicated by the marked (p < 0.001) increase in caspase-3 level in CdCl2-treated rats by 5.6-fold compared to the control rats. Meanwhile, administration of S. malaccense (250, 500, and 1000 mg/kg) significantly (p < 0.001) reduced the caspase-3 level by 21.2%, 38.2%, and 45.4%, respectively, compared to the CdCl2-treated rats. Moreover, treatment with S. samarangense (250, 500, and 1000 mg/kg) significantly (p < 0.001) decreased the caspase-3 level by 33.8%, 47.5%, and 73.3%, respectively, compared to the CdCl2-treated rats. These results suggest the anti-apoptotic and cytoprotective effect of S. malaccense and S. samarangense DEs (Fig 3F).
Effect of the DE of S. malaccense and S. samarangense leaves on the mitochondrial dysfunction parameters.
The CdCl2-treated group showed a significant (p < 0.001) reduction in ATP by 68.5% compared to the control rats. Groups treated with the DE of S. malaccense at doses 250, 500, and 1000 mg/kg showed a marked (p < 0.001) increase in ATP by 1.6, 2.7, and 3.2 folds, respectively, compared to the CdCl2-treated group. Additionally, groups administered the DE of S. samarangense at doses 250, 500, and 1000 mg/kg showed a significant (p < 0.001) increase in ATP by 1.6, 2.5, and 3.3 folds, respectively, compared to the CdCl2-treated rats (Fig 4A).
Data represented as mean±SEM of n = 7. a: significant from the control group at p < 0.001, b: significant from the CdCl2 group at p < 0.001.
Effect of the DE of S. malaccense and S. samarangense leaves on autophagy parameters.
Induction of autophagy was detected by measuring p-mTOR and beclin-1. As presented in Figs 4B and 4C, the CdCl2-treated group demonstrated a marked (p < 0.05) decrease in p-mTOR by 79.6% and a marked (p < 0.001) increase in beclin-1 by 2.4-fold compared to the control group. Meanwhile, groups administered with 250, 500, and 1000 mg/kg of S. malaccense DE significantly (p < 0.001) increased p-mTOR by 2, 3.5, and 4.7 folds; and decreased beclin-1 by 12.9%, 43.3%, and 49.5%, respectively, compared to CdCl2-treated groups. Groups treated with 250, 500, and 1000 mg/kg of S. samarangense DE significantly (p < 0.001) increased p-mTOR by 2.3, 3.9, and 4.9 folds; and decreased beclin-1 by 27.8%, 45.4%, and 56.1%, respectively, compared to CdCl2-treated groups. Regarding the level of p-AMPK, the CdCl2-treated group showed a marked (p < 0.001) elevation in p-AMPK by 2.1-fold compared to the control group. Groups treated with the DE of S. malaccense at doses 250, 500, and 1000 mg/kg showed a significant (p < 0.001) decrease in p-AMPK by 22.4%, 43.2%, and 49.2%, respectively, compared to the CdCl2-treated group. Additionally, groups administered the DE of S. samarangense at doses 250, 500, and 1000 mg/kg showed a marked (p < 0.001) decrease in p-AMPK by 21.1%, 41.2%, and 50.6%, respectively, compared to the CdCl2-treated group (Fig 4D).
Effect of the DE of S. malaccense and S. samarangense on serum ALT and AST levels.
As delineated in Table 4, CdCl2-treated rats exhibited a significant elevation in serum ALT and AST levels compared to the control group (p < 0.001) by 2.8 and 2.9 folds, respectively, indicating systemic hepatic toxicity. Treatment with the DE of S. malaccense at doses 250, 500, and 1000 mg/kg significantly decreased the serum level of ALT by 26.1%, 33.3%, and 57.5%, and AST by 34.9%, 53.4%, and 58.6%, respectively, compared to the CdCl2 group (p < 0.05). Meanwhile, treatment with the DE of S. samarangense at 250, 500, and 1000 mg/kg significantly decreased the serum level of ALT by 30.5%, 46.8%, and 58.6%, and AST by 35.4%, 56.1%, and 62.0%, respectively, compared to the CdCl2 group (p < 0.05). These results suggest a protective effect of the DEs against CdCl2-induced liver injury.
Effect of the DE of S. malaccense and S. samarangense leaves on histopathological examination of the kidney tissues.
As seen in Fig 5, normal kidney samples showed almost intact, well-organized morphological features of renal parenchyma. It showed apparent intact renal tubular segments and intact tubular epithelium (arrow), minimal records of degenerated tubular epithelial cells, intact renal corpuscles (star), and intact vasculatures. However, CdCl2-treated samples showed multiple focal records of periglomerular and perivascular mononuclear inflammatory cell infiltrates accompanied by higher fibroblastic activity (red arrow) with dilatation of renal vasculatures (red star). Both DEs of S. malaccense and S. samarangense (250 mg/kg) showed mild persistent records of perivascular and periglomerular inflammatory cell infiltrates (red arrow) with almost intact vasculatures and apparent intact nephronal segments. S. malaccense at a dose of 500 mg/kg showed higher protective efficacy with a few occasional records of interstitial inflammatory cell infiltrates (red arrow). In comparison, 1000 mg/kg demonstrated higher protective efficacy with almost intact renal parenchyma resembling normal control samples. The 500 mg/kg and 1000 mg/kg of S. samarangense showed higher protective efficacy with almost intact renal parenchyma resembling normal control samples.
Normal kidney samples showed intact renal parenchyma with renal tubular segments and intact tubular epithelium (arrow), intact renal corpuscles (star), and intact vasculature. CdCl2-treated samples showed multiple focal periglomerular and perivascular mononuclear inflammatory cell infiltrates accompanied by higher fibroblastic activity (red arrow) with dilatation of renal vasculatures (red star). DEs of S. malaccense and S. samarangense at 250 mg/kg doses showed mild perivascular and periglomerular inflammatory cell infiltrates (red arrow) with almost intact vasculatures and nephron segments. S. malaccense at a dose of 500 mg/kg showed higher protective efficacy with few occasional interstitial inflammatory cell infiltrates (red arrow). In comparison, the 1000 mg/kg showed higher protective efficacy with almost intact renal parenchyma. 500 mg/kg and 1000 mg/kg of S. samarangense showed a higher protective efficacy with almost intact renal parenchyma resembling normal control samples.
Discussion
The kidneys are among the most energy-intensive organs, crucial for maintaining water and salt equilibrium [69]. They receive nearly 25% of the cardiac output and are responsible for blood pressure management and continuous blood filtration [70]. Cadmium (Cd) pollution is escalating globally due to heightened industrial operations, which have augmented its availability and environmental persistence. Furthermore, it poses a significant public health hazard owing to its non-biodegradability and extended biological half-life (10–30 years) [71]. Environmental Cd may accumulate in different human organs, including the liver and lungs. However, the kidney is still the main target of its accumulation [72,73], making it mainly susceptible to Cd-mediated nephrotoxicity. Flavonoids are unique, natural bioactive secondary metabolites with a basic flavan skeleton. They comprise two phenyl rings connected to a central heterocyclic pyran scaffold, forming a C6-C3-C6 system with a distinguished polyphenolic feature. Flavonoids are allocated into six major groups, viz. flavanols, flavanones, flavonols, flavones, isoflavonoids, and anthocyanidins, in addition to other minor classes including chalcones, dihydrochalcones, and aurones. Flavonoids are found extensively in plant-based meals, so they are ingested through fruits and vegetables [74]. They exhibited beneficial pharmacological effects, including antioxidant, free radical scavenging, anti-inflammatory, immunomodulatory, and renal protective effects [75]. Given the global health burden of Cd toxicity on human health, and the potential therapeutic impacts of polyphenols, especially flavonoids, we investigated the protective potential of two phenolic-rich Syzygium species against cadmium chloride-induced nephrotoxicity in rats. The study showed high serum creatinine and urea levels in Cd-treated rats, indicating kidney injury. The levels of these parameters are usually used as biomarkers for assessing kidney function [76]. According to Gowda et al. [77], urea is a byproduct of the breakdown of proteins and amino acids, and an increase in its blood level is typically linked to renal failure or illness. Another biomarker is creatinine, which is mainly removed by glomerular filtration and created during muscle metabolism from creatine and phosphocreatine. In the current investigation, we reported that the DE of S. malaccense and S. samarangense reversed Cd-induced nephrotoxicity, evidenced by decreased blood creatinine and urea levels. This finding follows previous reports, which revealed that phenolic compounds improved the nephrotoxicity induced by heavy metals such as Cd [78–80].
Oxidative damage accompanied by free radical overproduction is one of the main processes that are activated upon Cd intoxication. It was established that Cd causes oxidative stress by influencing the pro-to-antioxidant ratio. Additionally, in the biological system, Cd does not undergo redox processes, however, it can generate oxidative stress by intracellular GSH reduction [81], since Cd can bind with the functional thiol (-SH) groups in both enzymatic and non-enzymatic cellular compounds [82]. Moreover, Cd can inhibit other antioxidant enzymes such as SOD which was attributed to the Cd interaction with the enzyme protein part causing a configurational change in the enzyme and results in depressing SOD catalytic activity [83]. The two examined extracts are rich mainly in flavonoids belonging to various classes, exemplified in flavonols (7−21), flavone (22−24), flavanones (25–32), isoflavones (33−39), and chalcones (40−52). Flavonoids exert their antioxidant effect via direct and indirect mechanisms. Directly by donating electrons that neutralize reactive oxygen species (ROS) [84] and free radicals such as peroxynitrite (ONOO−), hydroxyl radical (OH·), and peroxyl radical (ROO·), hence decreasing ROS and other free radicals levels in the body [85]. Though the sugar part of the flavonoid skeleton is critical for their antioxidant activity, the aglycones are more effective than their corresponding glycosides [86]. On the other hand, the indirect antioxidant effect of flavonoids is associated with inducing the production or activation of antioxidant enzymes and suppressing pro-oxidant enzymes. Flavonoids can activate the intracellular antioxidant signaling pathways to induce the production of intracellular antioxidant elements including GSH and SOD. We cannot neglect the effect of other phenolic classes detected in the two tested DE, such as phenolic acids (1−6), resveratrol derivatives (60), and lignans (53), which are all characterized by their potential antioxidant effect [49,87]. In all, our result revealed that the reduced GSH and SOD levels in the Cd-administrated rats were significantly restored by using the DE of S. malaccense and S. samarangense, especially at higher doses (500 and 1000 mg/kg). Moreover, several flavonoids and phenolic acids identified in this study were previously reported to have a strong antioxidant effect [33,47,49] and protect against kidney injury [75,87,88]. It is well-known that oxidative stress and inflammation are interrelated processes where elevated levels of ROS enhance NF-κB activation and release of inflammatory cytokines. NF-κB is an essential modulator of the immune system and several inflammatory disorders. Furthermore, it is essential to activate inflammatory cytokines including IL-1β and TNF-α [89]. Prior research has demonstrated that exposure to Cd in renal tissues can trigger the activation of NF-κB, leading to an increase in TNF-α, IL-1, and IL-6 [82,90,91]. Our study demonstrated that the examined extracts significantly decreased the levels of NF-κB, TNF-α, and IL-1β, especially at higher doses. These results might be attributed to flavonoids, confirmed by earlier reports that disclosed the anti-inflammatory potential of flavonoids and phenolic acids [33,47,49]. Through a variety of processes, including immune cell regulation and enzyme and transcription factor inhibition, flavonoids produce their anti-inflammatory effects. Prior research has demonstrated that flavonoids affect immune cell development, activation, and signaling transduction, which can interfere with cytokine and chemokine synthesis and secretion [47].
Cd-induced oxidative stress and inflammation may trigger apoptosis which has an essential role in Cd-caused nephrotoxicity [92]. Apoptosis results from an imbalance between apoptotic and anti-apoptotic molecules, which is caused by both dependent and independent mitochondrial pathways [5]. Because it can initiate the apoptotic pathway by inducing other caspase enzymes, caspase-3 is considered an essential apoptotic marker [93]. A previous report coincides with our finding that Cd exposure significantly elevates caspase-3 level in the rat’s renal tissues [6,94]. In the present study, treatment with our investigated extracts protected against Cd-induced renal apoptosis by inhibiting the activation of caspase-3, which indicates their anti-apoptotic potential against Cd exposure. This effect may be at least in part, due to the extracts’ flavonoid and phenolic acids content. Both are renowned for their valuable therapeutic activity on renal health due to their antioxidant, anti-inflammatory, and anti-apoptotic properties [75].
Mitochondria are referred to as the engine of the cell due to their capability to generate ATP via oxidative phosphorylation [95]. They are critical organelles in preserving cellular homeostasis, and therefore, their damage may result in tissue or cellular damage [96]. Renal mitochondrial dysfunction is associated with inflammation, apoptosis, and tissue damage, which raises the risk of death and morbidity [97]. It was documented that Cd exposure decreases the level of ATP because of mitochondrial damage [98], which is consistent with our result. Meanwhile, the level of ATP is increased upon the treatments with the two extracts investigated.
Autophagy is an evolutionary conserved catabolic process used by eukaryotic cells to break down damaged or unnecessary proteins and organelles [99]. It is crucial for the upkeep and survival of cells [100]. It participates in Cd-induced nephrotoxicity [101]. Furthermore, it has been reported to be the early response of a cell to Cd toxicity in a concentration- and time-dependent way [102]. During the early stage of exposure, low levels of Cd may trigger protective autophagy in renal tubular epithelial cells [103–105], but with continuous exposure, excessive autophagy ultimately fails to maintain cell viability and then triggers cell death [7]. Autophagy can result from the activation of many signaling pathways by cellular proteins and/or protein kinases [106]. Amongst them, the AMPK/mTOR is the most significant pathway in autophagy regulation [107]. AMPK usually motivates autophagy by impairing mTOR, which plays an important role in monitoring cell growth, proliferation, and autophagy [108]. Moreover, beclin-1 controls autophagy membrane biosynthesis as it is necessary to initiate autophagosome formation [109]. It was reported that Cd exposure significantly decreases mTOR levels, activates the AMPK, and increases protein levels of beclin-1 [110], which is consistent with our results. Meanwhile, our results showed that the administration of both DEs significantly suppressed the excessive autophagy process, evidenced by decreased levels of AMPK and beclin-1 and increased levels of mTOR. The suppressed autophagy effect of the extracts can be elucidated by their antioxidant, anti-inflammatory, antiapoptotic, and cytoprotective activities.
Histopathological analysis of the kidney tissues showed that Cd caused numerous pathological lesions characterized by inflammatory cell infiltration and vascular dilatation. Additionally, the usual histological features of the renal tubules were lost. Cd exposure results in multiple focal records of periglomerular and perivascular mononuclear inflammatory cell infiltrates accompanied by higher fibroblastic activity with dilatation of renal vasculatures. These histomorphological alterations may be due to the direct damage of the renal tissues and increased ROS generation brought on by exposure to Cd [111]. Hence, resulted in oxidative damage [112] and morphological changes in renal tissue. However, the S. malaccense and S. samarangense DEs, especially at higher doses, display potential protective effects on the kidney tissues, which may be related to the strong antioxidant potential of flavonoids. Flavonoids significantly attenuated the oxidative stress, causing a decrease in pathological changes [49,87].
Cd is well-documented to induce not only nephrotoxicity but also systemic toxicity, particularly hepatotoxicity, through oxidative stress and mitochondrial dysfunction [113,114]. Elevated ALT and AST levels have been observed in the CdCl2 group, confirming hepatic injury. The ability of the DE of S. malaccense and S. samarangense to reduce these liver enzyme levels suggests a broader systemic protective role, possibly via antioxidant or anti-inflammatory effects.
Conclusion
The current research reported that the defatted aqueous methanol extracts (DE) of S. malaccense and S. samarangense leaves are rich in various classes of flavonoids, such as flavonols, flavone, flavanones, isoflavones, and chalcones, in addition to other phenolic compounds. The administration of the two investigated extracts significantly attenuated Cd-induced renal toxicity by fighting renal oxidative stress, apoptosis, and inflammatory reactions. Moreover, the two extracts alleviated mitochondrial dysfunction and inhibited the autophagy level in a dose-dependent manner. The nephroprotective effects of the two Syzygium extracts against Cd-induced nephrotoxicity may be attributed to anti-inflammatory, antiapoptotic, and antioxidant activities of their constitutive phenolic metabolites. Moreover, the DE not only protects renal function but also mitigates systemic Cd-induced hepatic toxicity. However, implementing in vitro experiments using renal cell lines and setting quality control parameters for the HPLC-MS are among the study limitations. In all, the two Syzygium extracts can be used, at least in part, as a protective agent against environmental toxicity by heavy metals such as cadmium. Nevertheless, future clinical studies are required to test their toxicity and curing ability for Cd-induced renal dysfunctions in humans. Furthermore, formulating these extracts into a suitable pharmaceutical preparation will facilitate their usage as protective nutraceuticals for kidney nephrotoxicity.
Studies in animal statements
The Faculty of Pharmacy’s Ethical Animal Care and Use Committee approved the experimental protocol at Helwan University (approval number: 11A2023). The animal experiment complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Also, it followed the guidance on the operation of the Animals (Scientific Procedures) Act 1986, the association guidelines, EU directive 2010/63 for the protection of animals used for scientific purposes, the NIH (National Research Council) Guide for the Care and Use of Laboratory Animals (8th edition), and national guidelines for animal care (European Community Directive, 6/609/EEC).
Supporting information
S1 Checklist. Humane endpoints checklist.
This checklist details the implementation of humane endpoints in the animal experiment described in the study. It includes information on monitoring criteria, euthanasia timing, animal welfare considerations, and compliance with ethical standards, as per PLOS ONE and ARRIVE guidelines.
https://doi.org/10.1371/journal.pone.0329586.s001
(DOCX)
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