https://orcid.org/0009-0008-0388-3482
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Abstract
Liver fibrosis, a consequence of chronic liver injury, is characterized by excessive accumulation of the extracellular matrix, oxidative stress, and activation of hepatic stellate cells. This study evaluated the effectiveness of Ziziphus jujuba (Z. jujuba) seed powder and seed oil, both separately and in combination, in alleviating CCl4-induced liver fibrosis, and linked the in vivo effects to their phytochemical profiles. The seed powder extracts, using acetone and ethanol, showed significant antioxidant activity, with DPPH values reaching up to 90.13%, and exhibited a high phenolic content of 79.57 mg GAE/g in the ethanol extract. Gas chromatography–mass spectrometry (GC–MS) analysis identified several bioactive compounds, including derivatives of 1,4-benzenedicarboxylic acid (30.46% in powder, 20.43% in oil), hexanedioic acid bis(2-ethylhexyl) ester (25.31% in powder, 20.78% in oil), and oleic acid (10.61% in powder, 16.06% in oil. In vivo, carbon tetrachloride (CCl4) administration led to elevated levels of ALT, AST, ALP, and bilirubin, causing disruptions in oxidative, lipid, inflammatory, hematological, and nutritional parameters. All treatment groups showed improvements in these parameters, with Group 4 (G4) exhibiting the most pronounced hepatoprotective effects, including reductions in ALT (75.56 U/L), AST (125.76 U/L), ALP (125.43 U/L), and bilirubin (0.40 mg/dL) levels. Oxidative stress markers were reduced, with MDA at 5.47 nmol/g protein and NOx at 46.81 nmol/g protein, whereas antioxidant defenses were enhanced, as evidenced by SOD activity at 71.69 U/mg and catalase restoration. Lipid profiles were normalized, with triglycerides (TG) at 80.01 mg/dL, HDL at 34.60 mg/dL, and LDL at 22.30 mg/dL. Additionally, cytokine levels, specifically TNF-α and IL-6, decreased, red and white blood cell differentials were restored, and feed and water intakes and serum protein levels improved. These findings highlight the synergistic anti-fibrotic and hepatoprotective properties of Z. jujuba seed powder and oil, likely facilitated by the bioactive compounds in both the polar and lipid phases. However, further research into histopathology and confirmation of molecular pathways is crucial for clinical application.
Citation: Tanvir L, Afzal K, Abbas A, Amjad A, Iqbal TB, Sarwar N, et al. (2026) Integrating phytochemical analysis and experimental validation of Ziziphus jujuba seed powder and oil to ameliorate CCl4-induced liver fibrosis in sprague dawley rats. PLoS One 21(2): e0343428. https://doi.org/10.1371/journal.pone.0343428
Editor: Chun-Hua Wang, Foshan University, CHINA
Received: October 4, 2025; Accepted: February 4, 2026; Published: February 20, 2026
Copyright: © 2026 Tanvir 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 relevant data are within the paper.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors declare that no competing interests exist.
Abbreviations: Z. jujuba, Ziziphus jujuba; CCl₄, Carbon Tetrachloride; GC-MS, Gas Chromatography-Mass Spectrometry; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; ALP, Alkaline Phosphatase; MDA, Malondialdehyde; NOx, Nitric Oxide Metabolites; CAT, Catalase; HDL, High-Density Lipoprotein; LDL, Low-Density Lipoprotein; VLDL, Very Low-Density Lipoprotein; WBC, White Blood Cell; RBC, Red Blood Cell; HCT, Hematocrit; TBARS, Thiobarbituric Acid Reactive Substances; TGF-β, Transforming Growth Factor Beta; NF-κB, Nuclear Factor Kappa B
Introduction
Liver fibrosis is a complex pathological condition that arises from persistent and chronic liver injury. It is characterized by an excessive buildup of extracellular matrix (ECM) proteins, such as collagen, which disrupts the normal architecture of the liver and impairs its functionality [1,2]. This condition is widely considered an intermediate stage in the progression toward cirrhosis, liver failure, and hepatocellular carcinoma, all of which are associated with significant morbidity and mortality globally [3]. Unlike acute liver injury, fibrosis is a dynamic process that involves a continual wound healing response that fails to resolve, resulting in pathological scarring [4]. The growing incidence of liver fibrosis is strongly associated with the increasing prevalence of metabolic diseases, viral infections, and alcohol-related liver disease, particularly in low- and middle-income countries. This trend highlights the urgent need for effective therapeutic interventions to address this escalating health challenge [5].
Hepatic stellate cell (HSCs) activation is a key pathological feature of liver fibrosis. Under normal conditions, these cells remain quiescent and primarily function as vitamin A storage cells. However, upon liver injury, HSCs transform into myofibroblast-like cells that produce fibrillar collagens and other ECM components, thereby exacerbating tissue scarring and disrupting the hepatic microarchitecture [6,7]. The molecular and cellular mechanisms underlying HSC activation involve inflammatory signaling pathways, including cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), as well as oxidative stress mediated by reactive oxygen species (ROS). Additionally, immune cell recruitment contributes to an inflammatory environment that perpetuates fibrosis [8,9]. The progression of liver fibrosis also involves dysregulated signaling pathways, such as TGF-β, platelet-derived growth factor (PDGF), and the gut–liver axis, which promote fibrogenic responses and ECM remodeling [10–12]. Despite significant progress in understanding these underlying mechanisms, effective anti-fibrotic therapies remain elusive. Current therapeutic strategies primarily focus on addressing the root cause of liver fibrosis, rather than directly reversing the condition.
Historically, medicinal plants have played a pivotal role in managing liver diseases owing to their bioactive compounds, which possess antioxidant, anti-inflammatory, and immunomodulatory properties [13–15]. Among these plants, Z. jujuba (common name: Unaab) has attracted significant attention because of its rich phytochemical composition and wide range of pharmacological activities. This deciduous tree, native to Asia and cultivated across subtropical regions, produces seeds that are abundant in flavonoids, saponins, triterpenic acids, and unsaturated fatty acids, including oleic and linoleic acids. These compounds contribute to the antioxidant and hepatoprotective effects of the plant [16,17]. Research has shown that compounds extracted from Z. jujuba seeds, such as jujubosides, modulate key inflammatory and fibrogenic pathways, including TLR4-MyD88 and NF-κB signaling. This modulation leads to a reduction in hepatic damage and fibrosis in experimental models, supporting the plant’s potential as a therapeutic agent for liver disease [18].
Despite extensive research on the antioxidant and anti-inflammatory benefits of Z. jujuba seed derivatives, there are limited comparative studies on the therapeutic potential of its seed oil versus seed powder in the context of liver fibrosis. Seed oil, which is rich in lipid-soluble antioxidants and essential fatty acids, may exert cellular effects compared to the complex phytochemical matrix found in seed powder [19,20]. Given the global increase in liver fibrosis, there is an urgent need for novel therapeutic options, such as Z. jujuba seed powder and oil, which have shown promise in preclinical studies. This rise in liver disease underscores the importance of natural food products, especially those enriched with phytochemicals, in reducing the global burden of liver disease. Understanding the differential effects of Z. jujuba seed oil and powder is crucial for optimizing their use as nutraceuticals or adjunctive therapies for chronic liver diseases. The present study aimed to evaluate the anti-fibrotic potential of Z. jujuba seed powder and oil in a rat model of CCl₄-induced liver fibrosis. Phytochemical analysis was conducted using GC–MS, and the therapeutic efficacy was assessed using biochemical, oxidative stress, and histopathological parameters (Fig 1).
Materials and methods
Raw material procurement
Seeds of Z. jujuba Mill. were procured locally from Multan, Pakistan. Their authenticity was confirmed by experts from the Department of Food Science and Nutrition, Bahauddin Zakariya University, Multan. The seeds were washed several times with distilled water to remove any impurities. The samples were then dried in a hot air oven (Model BJ-9176) at 40°C for 48 h until the moisture was removed. Dried seeds were ground to a coarse powder using a mechanical grinder, sieved to obtain a uniform particle size, and stored in airtight containers at 4°C until use. Seed oil was extracted via cold pressing using a mechanical oil press (Model GD-HOPM-150) at ambient temperature to preserve the bioactive compounds. The extracted oil was filtered and stored in amber bottles at 4°C.
Phytochemicals analysis
To evaluate the antioxidative potential of Z. jujuba seed powder and oil, a series of in vitro assays was conducted. For the extraction process, 10 g of seed powder was added to 100 mL of solvent in a 1:10 (w/v) ratio. The solvents used were ethanol, hexane, acetone, and distilled water. Each mixture was placed in a conical flask and agitated continuously on an orbital shaker for 36 h to ensure the maximum extraction of antioxidant constituents. For the oil samples, 1 mL of oil and 5 mL of either ethanol or methanol were mixed and vortexed thoroughly to ensure complete homogenization. After extraction, all samples were filtered using filter paper (Whatman No. 4), and the resulting filtrates were concentrated using a rotary evaporator (Model: RE202).
DPPH for free radical scavenging activity
The antioxidant properties of Z. jujuba seed powder and oil were evaluated using the DPPH radical-scavenging technique [21]. A 0.5 mL sample of Z. jujuba seed powder extract and Z. jujuba seed oil was mixed with 3 mL of DPPH solution and then diluted with 2 mL of methanol. These mixtures were kept in the dark for a set period, after which their absorbance was recorded at 517 nm using a UV–visible spectrophotometer (Model no: 823−0210 P-2-R) to assess their free radical scavenging ability.
Ferric reducing antioxidant power (FRAP)
The antioxidant capacity of Z. jujuba seeds and their oils was evaluated using the ferric reducing antioxidant power (FRAP) assay [22]. To conduct the test, FRAP reagents, including FeCl₃·6H₂O, acetate buffer, and TPTZ solution, were combined with the Z. jujuba seed powder and oil. The mixture was then incubated at 37 °C in the dark for 30 min. The absorbance of each sample was recorded at 593 nm using a spectrophotometer (Model 823−0210 P-2-R).
Total phenolic content (TPC)
The total phenolic content of Z. jujuba seed powder and seed oil extracts was determined using the Folin–Ciocalteu method, and the results were expressed in mg GAE/g [23]. All mixtures were kept in a dark room for 1 h, and the absorbance of both the seed powder and seed oil extracts was determined using a spectrophotometer at 760 nm (Model no: 823−0210 P-2-R).
Quantification of phytochemicals of seed powder and seed oil of Z. jujuba by GC/MS
The phytochemical components of the methanolic extract of Z. jujuba seeds and the hexane extract of its oil were analyzed using gas chromatography-mass spectrometry (GC-MS) (model number: Agilent 5977 B GC/MSD) with a thermal desorption system [24]. The gas chromatography column used had dimensions of 30 m length, 0.25 mm internal diameter, and 0.25 µm film thickness. The initial temperature of the column was set at 100 °C and increased by 5 °C per minute until it reached 280 °C, where it was maintained for 3 min. Subsequently, the temperature was increased to 280 °C at a rate of 15 °C/min, with a 35-minute isothermal hold. Helium was used as the inert carrier gas at a flow rate of 1.41 mL/min. Samples diluted to 1% v/v were introduced with an injection volume of 1 µL in splitless mode. The mass spectrometer was operated at an ion source temperature of 230 °C and an interface temperature of 270 °C. Data collection was conducted in full-scan mode over a mass-to-charge (m/z) range of 40–650. The volatile compounds from the Z. jujuba seed powder and seed oil were identified by matching mass fragmentation data (MS), Kovats indices (KI), and chromatographic peaks with reference spectra from the NIST 20 (National Institute of Standards and Technology) Mass Spectral Library, RTLPEST 3 libraries, and the 4th edition of Adams [59] and by comparison with the previously published literature [60,61].
Study animal, experimental design, and dose selection
Forty male Sprague-Dawley rats, weighing–180–220 g and aged 8–10 weeks were obtained from the animal facility at the Department of Pharmaceutical Sciences, BZU, Multan. The rats were kept in polypropylene cages, with eight rats per cage, under controlled environmental conditions: a temperature of 22 ± 2°C, relative humidity of 50–60%, and a 12-hour light/dark cycle. Throughout the study, the animals had unrestricted access to standard basal diet and water. The experimental design included five groups and eight male Sprague-Dawley rats in each group: normal control (G0) and disease control (G1), while the intervention groups received a standard basal diet supplemented with Z. jujuba seed powder at 5 g/kg (G2), seed oil at 5 mL/kg (G3), or their combination at 5 g/kg + 5 mL/kg (G4). Z. jujuba seed powder and seed oil was administered daily via oral gavage tube mixed with standard basal diet during the experimental period in the experimental animals. Z. jujuba seed powder and seed oil were dissolved in 0.5% (w/v) sodium carboxymethyl cellulose, which was used as a vehicle during the administration to–G2-G4 and orally administered at a dose of 10mL/kg for the study period. However, for the G0 and G1 groups, only 0.5% (w/v) sodium carboxymethyl cellulose was administered orally at a dose of 10mL/kg body weight of experimental animal. The dosages of Z. jujuba seed powder and seed oil were established based on the existing literature on the clinical practices of Chinese medicine practitioners [62]. The human-equivalent lower dose is 0.81 g/kg, which corresponds to 3.2 times the clinical dosage, whereas the human-equivalent higher dose is 3.23 g/kg, equating to 12.9 times the clinical dosage [63,64] (Table 1).
Induction of liver fibrosis
Liver fibrosis was induced by administering carbon tetrachloride (CCl₄) via intraperitoneal injection at a dosage of 1 mL/kg, mixed in a 1:1 ratio with olive oil. This was performed twice a week for 6 weeks, totaling 12 injections [25]. The treatments were initiated simultaneously with CCl₄ injections.
Ethical statements, sample collection, and Humane endpoint
The Institutional Review Committee approved the experimental protocol (Approval No: BZU/IAEC/2024/032) used in this study involving experimental animals. Before the experiments began, all animals were randomly assigned to different groups. This research strictly followed the National Institutes of Health (NIH) regulations and guidelines [58]. Efforts were made to minimize animal distress in line with the principles of the National Center for the Replacement, Refinement, and Reduction of Animal Research (NC3Rs), as detailed in the Animal Research Reporting of In vivo Experiments (ARRIVE) guidelines (S1 File). Following the treatment phase, the rats were deprived of food overnight and anesthetized with a ketamine/xylazine mixture (80/10 mg/kg). Blood was drawn via cardiac puncture using sterile syringes, left to clot, and centrifuged at 3000 rpm for 10 min to obtain the serum. The serum samples were stored at −20°C for later biochemical analysis [26]. Animals were euthanized by cervical dislocation, and their livers were quickly removed, washed with ice-cold saline, weighed, and divided for biochemical testing.
Body weight measurement and food consumption
The body weights of the rats were recorded weekly and at the end of the study. Relative liver weight was calculated as the percentage of liver weight to final body weight. The food intake of the animals in the different groups was observed weekly. The per capita water and food consumption for each week was calculated using the following formula:
Oxidative stress markers in liver tissue
Liver tissue samples (0.25 g) from each experimental group were homogenized in 1 mL of ice-cold phosphate-buffered saline (PBS) with 0.1 M EDTA at pH 7.4. The homogenized mixtures were centrifuged at 12,000 × g for 20 min at 4 °C. The obtained supernatant was used for biochemical analysis. The protein concentration in the tissue homogenates was determined using the Bradford assay with bovine serum albumin as the standard [27].
Malondialdehyde (MDA) determination
The levels of MDA, which serves as a marker for lipid peroxidation, were assessed in 10% liver homogenates prepared using a 0.9% NaCl solution through a thiobarbituric acid reactive substances (TBARS) assay [28].
Catalase activity
Catalase activity was evaluated by observing the rate of hydrogen peroxide (H₂O₂) breakdown. A small amount of liver supernatant (1 μL) was mixed with 1 mL of 2 mM H₂O₂ in a 0.1 M PBS solution at pH 7.0. The reduction in absorbance at 240 nm was measured every 30 s for 2-minute period. Catalase activity was determined using a molar extinction coefficient of 43.6 M ⁻ ¹cm ⁻ ¹ and reported as units (U) per milligram of protein [29].
Nitric Oxide (NOx) level assay
To indirectly estimate NO levels, total nitrate and nitrite concentrations, which are stable metabolites of NO, were measured. The assay mixture comprised 100 µL of liver homogenate, 400 µL of Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid), and 500 µL of distilled water. The samples were incubated at room temperature for 10 min to facilitate color development. The absorbance was immediately measured at 540 nm using a spectrophotometer. The concentration of nitric oxide was determined from a sodium nitrite standard curve and expressed in µmol per mg of protein [30].
Superoxide Dismutase (SOD)
Superoxide dismutase activity was evaluated by measuring its ability to decrease nitrotetrazolium chloride photochemical reduction under a controlled environment. The superoxide anions produced by riboflavin-mediated illumination react with SOD and decrease formazan product formation. The assay utilized 10 μL of liver tissue homogenate mixed with 641 μL of 0.067 M phosphate-buffered saline (pH 7.0), 40 μL of 0.1 M EDTA, 20 μL of 1.5 mM nitro blue tetrazolium (NBT), and 9 μL of 0.1 mM riboflavin. The mixture was stirred gently and illuminated with a 40-W lamp at a distance of 15 cm for 15 min. The absorbance was measured immediately at 560 nm using a spectrophotometer. One SOD unit represented the enzyme quantity required to inhibit 50% of NBT reduction within 1 min at 25°C, expressed as U/mg protein [68].
Liver function tests and inflammatory markers
Blood samples were obtained in vials coated with EDTA for hematological testing and gel vials without anticoagulants for serum biochemical analysis [31]. To separate the serum, clotted blood was centrifuged at 3500 rpm for 10 min using a centrifuge (Model # Z326K), and the serum was then stored at −20 °C until needed. Hematological parameters, such as total white blood cell (WBC) count and its differentials, were assessed using an automated hematology analyzer (Cobas 6000). Serum biochemical markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, urea, uric acid, and creatinine, were evaluated using automated clinical chemistry analyzers (Lisa 300 Hycel automation). Moreover, inflammatory markers (TNF-α and IL-6) were measured using an enzyme-linked immunosorbent assay (ELISA) kit using a microtiter plate reader. The absorbance was measured at 450 nm, and the concentration of these anti-fibrotic cytokines was expressed as pg/mL [71].
Lipid and lipid profile
The lipid profile, which encompasses high-density lipoprotein (HDL), total cholesterol (TC), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and triglycerides (TG), was assessed using standard enzymatic techniques and calculation methods widely recognized in clinical settings. Total protein concentration was determined using a colorimetric assay. These measurements provide vital insights into cardiovascular risks and metabolic health [32].
Statistical analysis
Each test was conducted three times, and the results are presented as the mean ± standard deviation (SD). To compare the groups statistically, a one-way analysis of variance (ANOVA) was utilized, followed by the Least Significant Difference (LSD) post-hoc test, using SPSS software version 16. A p-value of less than 0.05 was considered statistically significant.
Results
Key findings of the study investigating the therapeutic effects of Ziziphus jujuba seed powder and seed oil on CCl₄-induced liver fibrosis in rats. This includes the analysis of multiple biochemical, hematological, and physiological parameters, emphasizing antioxidant activity, liver function, hematology, and metabolic profiles, with data derived directly from group-wise experimental measurements. Additionally, water and feed intakes were observed throughout the 4-week trial.
Antioxidant activity of Z. jujuba seed powder and oil
The antioxidant capacity of Z. jujuba seeds was confirmed using multiple assays, including DPPH radical scavenging, TPC, and FRAP (Table 2). Seed extracts, particularly those prepared with acetone and ethanol, exhibited higher radical scavenging activity (up to 90.13%) than seed oil extracts, demonstrating the abundance of polar antioxidant phytochemicals, such as flavonoids and phenolics, in the seed matrix. Hexane extracts exhibited lower antioxidant activity, reflecting the limited capacity of the solvent to solubilize polar compounds.
TPC analysis indicated that the ethanol-extracted seed powder possessed the highest phenolic content (79.57 mg GAE/g), which correlated with its strong antioxidant activity. Seed oil phenolics were present at lower levels but contributed significantly to the overall antioxidant potential. FRAP assays supported these findings, with the acetone seed extract showing a superior reducing power.
Phytochemical compounds identification of Z. jujuba seed powder by gas chromatography-mass spectrometry (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) analysis of the methanolic extract of Ziziphus jujuba seed powder identified several bioactive compounds with varying abundances. The extract was dominated by 1, 4-Benzenedicarboxylic acid derivatives (30.46%) and Hexanedioic acid, bis(2-ethylhex) (25.31%), followed by Bis(2-ethylhexyl) phthalate and related phthalates (17.59%), and Oleic acid (10.61%). These major constituents suggest the presence of fatty acids, esters, and phthalate derivatives, which are often associated with antioxidant, antimicrobial, and plasticizer properties. Minor compounds, such as n-hexadecanoic acid (2.63%), octadecanoic acid (2.38%), and various unsaturated fatty acids (linoleic and linolenic acid derivatives, 3.62%), contribute to the nutritional and pharmacological potentials of the extract. The relatively high proportion of fatty acid esters and phthalate derivatives indicates that the seed extract is rich in lipid-soluble bioactive molecules, which may play a role in its traditional medicinal use (Fig 2; Table 3).
Phytochemical compounds identification of Z. jujuba seed oil by gas chromatography-mass spectrometry (GC-MS)
GC–MS analysis of the hexane extract of Z. jujuba seed oil revealed the presence of diverse fatty acids, esters and phthalate derivatives. The extract was primarily dominated by hexanedioic acid and bis(2-ethylhexyl) (20.78%) and 1,4-Benzenedicarboxylic acid derivatives (20.43%), which together accounted for over 40% of the total composition. Another major component was Oleic acid (16.06%), a monounsaturated fatty acid known for its nutritional and therapeutic value. Moderate proportions of phthalate derivatives (7.81%), methyl esters of linoleic and linolenic acids (4.59–4.85%), and n-hexadecanoic acid (4.23%) were also identified. Several minor constituents, including methyl stearate derivatives (1.34%) and octadecatrienoic acid methyl esters (<1%), contributed to the lipid profile (Table 4). Z. jujuba seed oil is rich in unsaturated fatty acids (oleic, linoleic, and linolenic acids), which are nutritionally beneficial, while the presence of phthalate-related compounds suggests possible environmental or solvent-related contamination (Fig 3; Table 4).
Feed and water intake of experimental animals
The results indicate that CCl₄-induced liver fibrosis negatively impacted feed and water intake in rats, with the Disease control group (G1) consistently having the lowest consumption throughout the experimental period. Treatment with Z. jujuba seed powder (G2) and seed oil (G3) moderately improved intake, whereas the combined treatment group (G4) exhibited the greatest restoration of both feed and water consumption, almost reaching or exceeding the levels of the normal control group (G0). These findings suggest that the treatments enhance physiological recovery and alleviate disease-induced anorexia and dehydration, promoting a superior nutritional status and possibly accelerating the healing processes (Fig 4 and Fig 5).
Effect of Z. jujuba powder and oil on Liver function biomarkers
As indicated in Table 5, G1 rats exhibited a marked increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin levels, confirming hepatocellular damage and cholestasis. The administration of Z. jujuba seed powder (G2), seed oil (G3), and their combination (G4) ameliorated these biochemical indicators, with the combined treatment showing the most effective reduction in ALT (75.56 U/L), ALP (125.43 U/L), AST (125.76 U/L), and bilirubin (0.40 mg/dL) levels. These results illustrate the potent hepatoprotective effects of Z. jujuba formulations in mitigating liver dysfunction and cholestasis caused by fibrosis (Table 5; Fig 6).
Oxidative stress and fibrosis biomarkers
CCl₄ administration significantly increased malondialdehyde (MDA) levels to 9.15 nmol/g protein in the disease control group (G1) compared to 7.31 nmol/g protein in the normal control (G0), indicating heightened lipid peroxidation and oxidative damage. Nitric oxide (NO) levels, also indicative of oxidative stress, were elevated to 61.34 nmol/g protein in G1 from 28.89 nmol/g protein in G0, while SOD levels were decreased in G1 56.51 U/mg from 78.37 U/mg in G0. Treatment with Z. jujuba seed powder (G2), seed oil (G3), or a combination of both (G4) progressively reduced these markers. G4 showed the greatest reduction in MDA (5.47 nmol/g protein) and NOx (46.81 nmol/g protein) levels and an elevation in SOD (71.69 U/mg), demonstrating effective antioxidant defense. Catalase (CAT) enzyme activity, which was suppressed in G1 (5.51 U/mg protein) relative to G0 (8.37 U/mg protein), was restored by treatments, with G4 achieving 8.69 U/mg protein, was restored by treatments, with G4 achieving 2.67 U/mg protein, suggesting improved enzymatic scavenging of reactive oxygen species, as shown in Fig 7 and Table 6.
Effect of Z. jujuba seed powder and oil on lipid profile and glucose levels
As shown in Table 7, exposure to CCl- resulted in an increase in serum triglyceride (TG), very low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) levels, whereas high-density lipoprotein (HDL) levels were reduced. Treatment with Z. jujuba seed powder (G2) and seed oil (G3) partially reversed these effects, with decreased TG (109.92 and 95.23 mg/dL, respectively) and increased HDL (25.54 and 32.00 mg/dL, respectively). The combined treatment (G4) yielded the most significant improvements, normalizing TG and HDL levels to 80.01 mg/dL and 34.60 mg/dL, respectively, which were closer to the control values. LDL levels were also substantially lower in G4 (22.30 mg/dL) than in G1 (41.09 mg/dL).
Fasting glucose showed minor, statistically nonsignificant variations between groups, with G1 slightly higher (114.23 mg/dL) than G0 (100.00 mg/dL), and G4 nearly restored to normal (98.92 mg/dL). These data suggest that Z. jujuba treatment effectively ameliorates lipid abnormalities induced by fibrotic liver damage with a limited impact on glucose homeostasis (Table 7).
Effect of Z. jujuba seed powder and oil on inflammatory markers
CCL4 administration significantly increased the levels of TNF-α to 17.90 pg/mL in the disease control group (G1) compared to 8.03 pg/mL in the normal control (G0), indicating an active immune response, stellate cell activation, and progression toward scar tissue accumulation. IL-6 levels were also slightly elevated to 28.38 pg/mL in G1, from 21.53 pg/mL. Treatment with Z. jujuba seed powder (G2), seed oil (G3), or a combination of both (G4) progressively reduced these markers. G2 showed the greatest reduction in TNF-α (11.21 pg/mL), followed by G4 (12.49 pg/mL). IL-6 levels were also restored by the treatments, with G4 achieving 18.81 pg/mL, suggesting reduced inflammatory signaling and stellate cell activation, as shown in Table 8.
Effect of Z. jujuba seed powder and oil on Serum protein profile
Serum total protein concentration decreased in G1 (5.87 g/dL) versus G0 (6.80 g/dL), reflecting liver synthetic dysfunction. Supplementation with Z. jujuba improved protein levels, with G4 reaching 5.98 g/dL, indicating the partial restoration of hepatic biosynthesis. Globulin levels remained relatively unchanged across the groups (Fig 8).
Effect of Z. jujuba seed powder and oil on hematological profile
The hematological data showed a significant reduction in the RBC count in the disease control group (G1:6.19 ± 0.33 × 10⁶/μL) compared to the normal control group (G0:7.80 ± 0.35 × 10⁶/μL), indicating anemia induced by CCl₄ toxicity. Hemoglobin levels followed a similar decline in G1 (10.23 ± 0.49 g/dL) compared to G0 (12.50 ± 0.66 g/dL). The hematocrit percentage also significantly decreased in G1 (33.33 ± 1.32%) compared to G0 (43.50 ± 1.93%). Treatment with seed powder (G2), seed oil (G3), and their combination (G4) improved these indices, with G4 nearing normal values (RBC: 7.39 ± 0.59 × 10⁶/μL; Hb: 13.81 ± 0.77 g/dL; HCT: 41.50 ± 1.21%). The mean corpuscular volume, hemoglobin, and hemoglobin concentrations were similar across the groups. These findings reflect the protective effects of Z. jujuba interventions on erythropoiesis and overall hematological health in rats with fibrosis (Fig 9).
Effect of Z. jujuba seed powder and oil on differential White blood cell (WBC) count
Liver fibrosis induced by CCl₄ resulted in a significant increase in the total WBC count to 14.22 × 10³/µL in the disease group (G1) compared to 8.32 × 10³/µL in the normal control group (G0). This finding indicates an activated inflammatory response in the cells. Lymphocytes decreased to 45.23% in G1, whereas neutrophils increased to 47.27%, indicating a shift toward neutrophil inflammation. The monocyte count increased to 6.50%. The treatment groups showed improvement with combined treatment (G4); nearly normalizing values WBC count was 8.80 × 10³/µL, lymphocytes 67.98%, neutrophils 26.72%, and monocytes 4.20%. Eosinophil and basophil counts remained unchanged and within the normal range. These changes demonstrate that Z. jujuba treatment effectively mitigates the fibrotic inflammatory profile (Fig 10).
Discussion
This study explored the potential therapeutic effects of Z. jujuba seed powder and oil on liver fibrosis in rats induced by carbon tetrachloride (CCl₄). The adverse effects of CCl₄ on feed and water intake, hematological parameters, liver function, oxidative stress, and fibrosis markers were significantly ameliorated by the treatments, highlighting the hepatoprotective and antifibrotic capabilities of Z. jujuba. This discussion integrates these findings with the current literature and elaborates on the mechanistic insights.
The acetone and ethanol extracts of the seed powder exhibited the highest antioxidant activity in both DPPH and FRAP assays. Interestingly, Zheng et al. (2022) reported that flavonoid-rich seed extracts attenuate oxidative injury in vivo via Nrf2 activation and suppression of NF-κB signaling [33]. Seed oil ethanolic extracts also demonstrated notable FRAP values despite lower phenolic content, consistent with previous findings, which attributed oil antioxidant effects to tocopherols, phytosterols, and unsaturated fatty acids, similar to our study [34]. Furthermore, a previous study found that lipid-soluble constituents from Z. jujuba seeds can enhance antioxidant defenses in vivo, complementing phenolic-driven radical scavenging from the powder [35].
GC-MS analysis of Z. jujuba seed powder and seed oil identified several bioactive compounds with potential anti-fibrotic effects relevant to liver fibrosis [36]. Oleic acid, the predominant monounsaturated fatty acid in both extracts, ameliorates liver fibrosis by modulating the NF-κB signaling pathway, which is critically involved in hepatic inflammation and fibrogenesis [37]. NF-κB inhibition leads to reduced activation of HSCs, the key effectors of fibrosis, thereby decreasing extracellular matrix deposition and inflammatory cytokine production [6,7]. Linoleic acid, another significant unsaturated fatty acid, has been demonstrated to interfere with the transforming growth factor-beta (TGF-β) and Smad signaling cascades, which are central pathways in promoting HSC activation and fibrotic progression in the liver [38]. Through the downregulation of TGF-β1 expression, linoleic acid reduces collagen synthesis and fibrosis advancement. Moreover, n-hexadecanoic acid (palmitic acid) may exert anti-inflammatory effects by repressing profibrotic gene expression, although some studies suggest that it can also promote fibrosis depending on the context, highlighting its complex role [36]. Since, Oleic acid’s high presence in the extract may explain the observed reduction in NF-κB signaling, as evidenced by previous studies [36,38].
GC-MS analysis identified bioactive compounds such as oleic acid, linoleic acid, and phthalate derivatives, including bis (2-ethylhexyl) phthalate and hexanedioic acid Bis(2-ethylhexyl) ester. While these compounds are acknowledged for their therapeutic properties, the presence of phthalates raises concerns regarding potential contamination from extraction materials, as phthalates are not naturally found in plants [65]. Phthalates, including Bis (2-ethylhexyl) phthalate, are recognized as environmental contaminants and endocrine disruptors that may interfere with the observed biological effects. These compounds have been shown to affect macrophage activity and fibrogenic signaling, potentially resulting in liver damage. Therefore, it is essential to distinguish the effects of Z. jujuba from the potential impact of phthalate contamination to accurately interpret the results [65–67]. To minimize contamination, future research should employ glassware and solvents that are less susceptible to phthalate introduction and ensure meticulous management of the extraction process. Phthalate derivatives, such as Hexanedioic acid, Bis(2-ethylhexyl) ester in seed oil, have been reported to modulate macrophage activity and fibrogenic signaling in experimental models [39]. These findings warrant a careful evaluation of the therapeutic applications and extraction purity. Overall, the significant active compounds in Z. jujuba seed extracts potentially confer hepatoprotective effects by targeting key fibrotic pathways, such as NF-κB and TGF-β/Smad, which aligns with multiple studies investigating natural product interventions in liver fibrosis [37,38,40]. The promising effects of Z. jujuba seed powder and oil in Sprague Dawley rats highlight the therapeutic potential of the current study, which significantly improved antifibrotic biomarkers, especially hydroxyproline and oxidative stress markers.
The decline in feed and water intake observed in the fibrotic group (G1) reflects systemic toxicity and hepatic dysfunction resulting from the oxidative damage and inflammation. The improvement in intake in the treatment groups, particularly in the combined seed powder and oil group (G4), suggests the mitigation of metabolic stress and restoration of gastrointestinal function. Our observations are consistent with those of Begum et al. (2022), who reported enhanced appetite and hydration in rodents treated with hepatoprotective phytoconstituents [41].
In rats, liver fibrosis caused by CCl4 led to a marked increase in liver enzymes such as ALT, AST, ALP, and bilirubin. However, the administration of Z. jujuba seed powder and oil significantly improved the levels of these enzymes, indicating strong hepatoprotective properties. Comparable findings have been reported in other interventional studies involving Z. jujuba and natural products in liver fibrosis animal models. Li et al. (2023) demonstrated that flavonoids extracted from Z. jujuba protected hepatocytes against acetaminophen-induced liver injury by significantly reducing ALT and AST levels through antioxidant and anti-inflammatory mechanisms [42]. Similarly, Liu et al. (2015) showed that polysaccharides isolated from Z. jujuba markedly suppressed increases in ALT, AST, and ALP in CCl4-injured mice, indicating the mitigation of hepatic damage via the enhancement of antioxidant enzymes and reduction of lipid peroxidation [43]. Furthermore, Z. jujuba seed powder and oil have been shown to significantly downregulate elevated liver enzymes, such as ALT and AST, in vivo, primarily by modulating key signaling pathways, including NF-κB and TGF-β/Smad, which reduce hepatic inflammation and fibrosis progression [44]. This hepatoprotective effect is coupled with antioxidant and anti-inflammatory actions, which collectively improve liver function and structure in fibrosis models. These studies collectively endorse the hepatoprotective and anti-fibrotic potential of Z. jujuba components, consistent with our biochemical findings of decreased serum liver enzyme activity and bilirubin levels in the present study.
Z. jujuba seed powder and oil contain significant bioactive compounds, such as oleic acid, linoleic acid, and methyl esters, which play crucial roles in modulating oxidative stress markers, including MDA, NOx, SOD, and CAT, in liver fibrosis. Oleic acid reduces hepatic oxidative stress by activating Nrf2-dependent antioxidant pathways, thereby decreasing MDA levels and restoring SOD and CAT activity [45]. Similarly, linoleic acid supplementation attenuates lipid peroxidation and inflammatory responses by promoting Nrf2 signaling and scavenging reactive oxygen species, consequently lowering MDA and NOx levels and enhancing antioxidant enzyme defense [46]. These actions collectively suppress the oxidative damage implicated in hepatic stellate cell activation and fibrosis progression [47,48]. Thus, the antioxidant properties of Z. jujuba constituents likely contribute to the observed normalization of oxidative stress markers in liver fibrosis models, highlighting their therapeutic potential in targeting oxidative stress-linked pathways.
Moreover, the bioactive compounds in Z. jujuba seed powder and oil not only play role in modulate oxidative stress markers but also inflammatory markers (TNF-α, IL-6) by reducing hepatic stellate cell activation and ECM deposition. Elevated TNF-α and IL-6 levels in liver fibrosis activate the NF-kB and JAK/STAT pathways, promoting the trans-differentiation of HSCs into myofibroblasts that secrete ECM via TGF-β and JNK signaling [70]. Thus, the anti-inflammatory and hepatoprotective properties of Z. jujuba help restore the inflammatory markers to their normal values in a liver fibrosis model [69].
Furthermore, Z. jujuba seed powder and oil had a positive impact on the lipid profile by reducing total cholesterol, LDL, and triglyceride levels while improving HDL. These changes likely stem from bioactive compounds such as oleic acid and linoleic acid, which regulate lipid metabolism by modulating the PPARα and AMP-activated protein kinase (AMPK) pathways, thus improving lipid clearance and reducing hepatic steatosis [49]. This lipid-lowering effect has been observed in multiple intervention studies, where natural products rich in unsaturated fatty acids significantly alleviated dyslipidemia associated with liver fibrosis and metabolic dysfunction [36,50].
Regarding hematological parameters, treatment with Z. jujuba formulations improved RBC and WBC counts, which are typically suppressed under fibrotic conditions owing to systemic inflammation and oxidative stress. These effects can be attributed to the antioxidant and immunomodulatory compounds in the seed extracts, which enhance hematopoiesis and resolve inflammation by regulating cytokine signaling and oxidative stress pathways [48,51]. Similar improvements in blood cell indices have been documented in preclinical models treated with flavonoid-rich botanicals, reinforcing the role of Z. jujuba in restoring hematological homeostasis during liver injury [52–54].
Mechanistically, Z. jujuba seed constituents likely interfere with pro-fibrogenic signaling pathways, including TGF-β and hepatic stellate cell activation pathways. The synergism between the water-soluble polyphenols in the seed powder and the lipid-phase antioxidants in the seed oil may account for the enhanced therapeutic efficacy observed with the combination treatment. This integrative phytochemical approach offers comprehensive modulation of fibrosis pathophysiology beyond what single extracts achieve, consistent with the multi-target potential of medicinal plants [55–57].
The primary limitation of this study is the absence of a liver histopathological analysis of the Z. jujuba seed powder and seed oil treatment groups, in comparison with a normal control and a disease control group, to accurately assess liver architecture and hepatocyte condition. Additionally, this study did not explore molecular pathways, such as TGF-β and NF-κB protein expression, or clinical validation. The findings are preliminary and based only on a CCl4-induced animal model. However, these preclinical findings provide compelling evidence supporting Z. jujuba seed powder and oil as promising candidates for the adjunctive management of liver fibrosis, especially in high-burden, resource-limited settings, such as Pakistan. Moreover, emerging nanotechnologies, such as nano-emulsions, nanoparticle-based delivery, and nano-encapsulation, represent promising strategies to enhance the bioavailability and targeted delivery of Z. jujuba bioactive compounds to hepatic cells. Nano-encapsulation holds great potential for improving specific liver cell targeting, maximizing therapeutic efficacy, and minimizing systemic side effects. Future research should focus on developing and optimizing nanocarrier systems for Z. jujuba formulations to realize their full clinical potential in treating liver fibrosis.
Conclusion
This study revealed that Z. jujuba seed powder and oil exhibit hepatoprotective and antifibrotic properties in a rat model of CCl₄-induced liver fibrosis. Gas chromatography-mass spectrometry (GC-MS) analysis identified bioactive compounds such as oleic acid, linoleic acid, and various fatty acid methyl esters, which likely contribute to the observed hepatoprotective effects. The combination of seed powder and oil significantly normalized the liver function enzymes (ALT, AST, and ALP) and bilirubin levels, effectively reversing hepatocellular injury and cholestasis. Moreover, Z. jujuba treatment improved oxidative stress markers (MDA, NOx, SOD, and CAT activity), suggesting an antioxidant defense mechanism through the modulation of pathways involved in oxidative damage and fibrogenesis. Restoration of the lipid profiles and hematological parameters (RBC and WBC) further underscores the therapeutic potential of the extract in mitigating liver damage and fibrosis. Additionally, Z. jujuba reduced TNF-α and IL-6 levels, indicating decreased inflammatory signaling and activation of stellate cells. Future research should explore the molecular mechanisms underlying these effects and confirm their safety and efficacy in larger animal models or clinical settings to validate Z. jujuba as a standardized treatment for liver fibrosis and related conditions.
Supporting information
S1 File. Details animals based experiments was reporting as per Animal Research Reporting of In vivo Experiments (ARRIVE) guidelines.
https://doi.org/10.1371/journal.pone.0343428.s001
(DOCX)
References
- 1. Mayorca-Guiliani AE, Leeming DJ, Henriksen K, Mortensen JH, Nielsen SH, Anstee QM, et al. ECM formation and degradation during fibrosis, repair, and regeneration. NPJ Metab Health Dis. 2025;3(1):25. pmid:40604328
- 2. Tsomidis I, Voumvouraki A, Kouroumalis E. Immune checkpoints and the immunology of liver fibrosis. Livers. 2025;5(1):5.
- 3. Lai JC-T, Liang LY, Wong GL-H. Noninvasive tests for liver fibrosis in 2024: are there different scales for different diseases? Gastroenterol Rep (Oxf). 2024;12:goae024. pmid:38605932
- 4. Hong Y-K, Chang Y-H, Lin Y-C, Chen B, Guevara BEK, Hsu C-K. Inflammation in wound healing and pathological scarring. Advances in Wound Care. 2023;12(5):288–300.
- 5. Kim HY, Yu JH, Chon YE, Kim SU, Kim MN, Han JW, et al. Prevalence of clinically significant liver fibrosis in the general population: A systematic review and meta-analysis. Clin Mol Hepatol. 2024;30(Suppl):S199–213. pmid:39074982
- 6. Chen L, Guo W, Mao C, Shen J, Wan M. Liver fibrosis: pathological features, clinical treatment and application of therapeutic nanoagents. J Mater Chem B. 2024;12(6):1446–66. pmid:38265305
- 7. Kamm DR, McCommis KS. Hepatic stellate cells in physiology and pathology. J Physiol. 2022;600(8):1825–37. pmid:35307840
- 8. Zhang Y, Ren L, Tian Y, Guo X, Wei F, Zhang Y. Signaling pathways that activate hepatic stellate cells during liver fibrosis. Front Med (Lausanne). 2024;11:1454980. pmid:39359922
- 9. Wiering L, Subramanian P, Hammerich L. Hepatic stellate cells: dictating outcome in nonalcoholic fatty liver disease. Cell Mol Gastroenterol Hepatol. 2023;15(6):1277–92. pmid:36828280
- 10. Sultana M, Islam MA, Khairnar R, Kumar S. A guide to pathophysiology, signaling pathways, and preclinical models of liver fibrosis. Mol Cell Endocrinol. 2025;598:112448. pmid:39755140
- 11. Wang L, Zhao W, Xia C, Li Z, Zhao W, Xu K, et al. TRIB3 mediates fibroblast activation and fibrosis though interaction with ATF4 in IPF. Int J Mol Sci. 2022;23(24):15705. pmid:36555349
- 12. Tan Z, Sun H, Xue T, Gan C, Liu H, Xie Y, et al. Liver fibrosis: therapeutic targets and advances in drug therapy. Front Cell Dev Biol. 2021;9:730176. pmid:34621747
- 13. Han W, Li H, Jiang H, Xu H, Lin Y, Chen J, et al. Progress in the mechanism of autophagy and traditional Chinese medicine herb involved in alcohol-related liver disease. PeerJ. 2023;11:e15977. pmid:37727691
- 14. Kamelnia E, Mohebbati R, Kamelnia R, El-Seedi HR, Boskabady MH. Anti-inflammatory, immunomodulatory and anti-oxidant effects of Ocimum basilicum L. and its main constituents: A review. Iran J Basic Med Sci. 2023;26(6):617–27. pmid:37275758
- 15. Fu K, Wang C, Ma C, Zhou H, Li Y. The potential application of chinese medicine in liver diseases: a new opportunity. Front Pharmacol. 2021;12:771459. pmid:34803712
- 16. Popstoyanova D, Gerasimova A, Gentscheva G, Nikolova S, Gavrilova A, Nikolova K. Ziziphus jujuba: applications in the pharmacy and food industry. Plants (Basel). 2024;13(19):2724. pmid:39409594
- 17. Khabbazeh S, Al-Masoudi N, Al-Masri R. The benefits of jujube: Extracting phenols and saponins for medicinal and nutritional uses. Asian Journal of Pharmaceutical Sciences. 2024;19(4):235–44.
- 18. Liang Q, Liu X, Peng X, Luo T, Su Y, Xu X, et al. Salvianolic acid B in fibrosis treatment: a comprehensive review. Front Pharmacol. 2024;15:1442181. pmid:39139645
- 19. Yang F, Bai L, Tao Y-Q. Study on antioxidant activity of Jujube seed oil. Journal of Food Bioactives. 2023;:27–35.
- 20. Zazouli S, Chigr M, Ramos PAB, Rosa D, Castro MM, Jouaiti A, et al. Chemical profile of lipophilic fractions of different parts of Zizyphus lotus L. by GC-MS and evaluation of their antiproliferative and antibacterial activities. Molecules. 2022;27(2):483.
- 21. Ji X, Hou C, Yan Y, Shi M, Liu Y. Comparison of structural characterization and antioxidant activity of polysaccharides from jujube (Ziziphus jujuba Mill.) fruit. Int J Biol Macromol. 2020;149:1008–18. pmid:32032709
- 22. Shi Q, Zhang Z, Su J, Zhou J, Li X. Comparative analysis of pigments, phenolics, and antioxidant activity of Chinese Jujube (Ziziphus jujuba Mill.) during fruit development. Molecules. 2018;23(8):1917. pmid:30071615
- 23. Wei X, Zhang R, Yang D, Zhao Z, Zhang X, Wu S, et al. Paclobutrazol induces changes in transcriptomic and endogenous hormone profiles in yellow camellia for reproductive phase transition. Plant Growth Regul. 2025;105(4):1029–41.
- 24. Patil T. Phytochemical screening and determination of bioactive constituents of methanolic leaf extract of Ziziphus jujuba (Mill.). Ind J Pure App Biosci. 2021;9(2):34–40.
- 25. Zhang L, Liu C, Yin L, Huang C, Fan S. Mangiferin relieves CCl4-induced liver fibrosis in mice. Sci Rep. 2023;13(1):4172. pmid:36914687
- 26. Akter MA, Yesmi̇n N, Talukder MBA, Alam MM. Evaluation of anaesthesia with xylazine-ketamine and xylazine-fentanyl-ketamine in rabbits: A comparative study. Journal of Advances in VetBio Science and Techniques. 2023;8(1):38–46.
- 27. Cheng H-Y, Chao J, Chiu C-S, Hsieh I-C, Huang H-C, Wu L-Y, et al. Hepatoprotective and antioxidant effects of Wu-Zi-Yuan-Chung-Wan against CCl4-induced oxidative damage in rats. Eur J Inflamm. 2021;19.
- 28. Hamam H, Demi̇r H, Aydin M, Demi̇r C. Determination of some Antioxidant Activities (Superoxide Dismutase, Catalase, Reduced Glutathione) and Oxidative Stress Level (Malondialdehyde Acid) in Cirrhotic Liver Patients. Middle Black Sea Journal of Health Science. 2022;8(4):506–14.
- 29. Abdelghffar EA, Obaid WA, Alamoudi MO, Mohammedsaleh ZM, Annaz H, Abdelfattah MAO, et al. Thymus fontanesii attenuates CCl4-induced oxidative stress and inflammation in mild liver fibrosis. Biomed Pharmacother. 2022;148:112738. pmid:35202909
- 30. Brizzolari A, Dei Cas M, Cialoni D, Marroni A, Morano C, Samaja M, et al. High-Throughput Griess Assay of Nitrite and Nitrate in Plasma and Red Blood Cells for Human Physiology Studies under Extreme Conditions. Molecules. 2021;26(15):4569. pmid:34361720
- 31. Li Z, LoBue A, Heuser SK, Li J, Engelhardt E, Papapetropoulos A, et al. Best practices for blood collection and anaesthesia in mice: Selection, application and reporting. Br J Pharmacol. 2025;182(11):2337–53. pmid:40234101
- 32. Pancholia AK, Kabra NK, Gupta R. Laboratory evaluation of lipid parameters in clinical practice. Indian Heart J. 2024;76 Suppl 1(Suppl 1):S29–32. pmid:38431087
- 33. Zheng Q, Li Q, Yang T, Qi W, Zhang Y, Wang L, et al. Flavonoid Glucosides from Ziziphus jujuba Seeds Improve Learning and Memory in Mice. Rev Bras Farmacogn. 2022;32(1):99–110.
- 34. Arab R, Casal S, Pinho T, Cruz R, Freidja ML, Lorenzo JM, et al. Effects of seed roasting temperature on sesame oil fatty acid composition, lignan, sterol and tocopherol contents, oxidative stability and antioxidant potential for food applications. Molecules. 2022;27(14):4508. pmid:35889377
- 35. Rajaei A, Salarbashi D, Asrari N, Fazly Bazzaz BS, Aboutorabzade SM, Shaddel R. Antioxidant, antimicrobial, and cytotoxic activities of extracts from the seed and pulp of Jujube (Ziziphus jujuba) grown in Iran. Food Sci Nutr. 2020;9(2):682–91. pmid:33598153
- 36. Fridén M, Rosqvist F, Ahlström H, Niessen HG, Schultheis C, Hockings P, et al. Hepatic Unsaturated Fatty Acids Are Linked to Lower Degree of Fibrosis in Non-alcoholic Fatty Liver Disease. Front Med (Lausanne). 2022;8:814951. pmid:35083257
- 37. Luedde T, Schwabe RF. NF-κB in the liver--linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2011;8(2):108–18. pmid:21293511
- 38. Kasahara N, Imi Y, Amano R, Shinohara M, Okada K, Hosokawa Y, et al. A gut microbial metabolite of linoleic acid ameliorates liver fibrosis by inhibiting TGF-β signaling in hepatic stellate cells. Sci Rep. 2023;13(1):18983. pmid:37923895
- 39. Lu Z, Li Q, Dai Y, Pan X, Luo X, Peng R, et al. Association of co-exposure to polycyclic aromatic hydrocarbons and phthalates with oxidative stress and inflammation. Sci Total Environ. 2024;912:169513. pmid:38154630
- 40. Huang W, Wang Y, Jiang X, Sun Y, Zhao Z, Li S. Protective effect of flavonoids from Ziziphus jujuba cv. Jinsixiaozao against Acetaminophen-Induced Liver Injury by Inhibiting Oxidative Stress and Inflammation in Mice. Molecules. 2017;22(10):1781. pmid:29053632
- 41. Begum R, Papia SA, Begum MM, Wang H, Karim R, Sultana R, et al. Evaluation of hepatoprotective potential of polyherbal preparations in CCl4-Induced Hepatotoxicity in Mice. Adv Pharmacol Pharm Sci. 2022;2022:3169500. pmid:35265845
- 42. Li X, Lao R, Lei J, Chen Y, Zhou Q, Wang T, et al. Natural Products for Acetaminophen-Induced Acute Liver Injury: A Review. Molecules. 2023;28(23):7901. pmid:38067630
- 43. Liu G, Liu X, Zhang Y, Zhang F, Wei T, Yang M, et al. Hepatoprotective effects of polysaccharides extracted from Zizyphus jujube cv. Huanghetanzao. Int J Biol Macromol. 2015;76:169–75. pmid:25709018
- 44. Li X, Li S, Li N. Research progress on natural products alleviating liver inflammation and fibrosis via NF-κB Pathway. Chem Biodivers. 2025;22(4):e202402248. pmid:39576739
- 45. Giudetti AM, Vergara D, Longo S, Friuli M, Eramo B, Tacconi S, et al. Oleoylethanolamide reduces hepatic oxidative stress and endoplasmic reticulum stress in high-fat diet-fed rats. Antioxidants (Basel). 2021;10(8):1289. pmid:34439537
- 46. Zhang Q, Jiang Y, Qin Y, Liu J, Xie Y, Zhang L, et al. Linoleic Acid Alleviates Lipopolysaccharide Induced Acute Liver Injury via Activation of Nrf2. Physiol Res. 2024;73(3):381–91. pmid:39027955
- 47. Thiruvengadam M, Venkidasamy B, Subramanian U, Samynathan R, Ali Shariati M, Rebezov M, et al. Bioactive compounds in oxidative stress-mediated diseases: targeting the NRF2/ARE signaling pathway and epigenetic regulation. Antioxidants (Basel). 2021;10(12):1859. pmid:34942962
- 48. Pan X, Ma X, Jiang Y, Wen J, Yang L, Chen D, et al. A comprehensive review of natural products against liver fibrosis: flavonoids, quinones, lignans, phenols, and acids. Evid Based Complement Alternat Med. 2020;2020:7171498. pmid:33082829
- 49. Ahmadi M, Shirafkan H, Mozaffarpur SA, Rezghi M. Impact of jujube fruit on serum lipid profile, glycemic index, and liver function: a systematic review and meta-analysis. Nutr Diabetes. 2025;15(1):22. pmid:40399274
- 50. Aziz T, Niraj MK, Kumar S, Kumar R, Parveen H. Effectiveness of Omega-3 polyunsaturated fatty acids in non-alcoholic fatty liver disease: a systematic review and meta-analysis. Cureus. 2024;16(8):e68002. pmid:39347373
- 51. Yi Y-L, Li Y, Guo S, Yan H, Ma X-F, Tao W-W, et al. Elucidation of the reinforcing spleen effect of jujube fruits based on metabolomics and intestinal flora analysis. Front Cell Infect Microbiol. 2022;12:847828. pmid:35402299
- 52. Kandimalla R, Dash S, Kalita S, Choudhury B, Malampati S, Kalita K, et al. Protective effect of bioactivity guided fractions of Ziziphus jujuba Mill. Root bark against hepatic injury and chronic inflammation via inhibiting inflammatory markers and oxidative stress. Front Pharmacol. 2016;7:298. pmid:27656145
- 53. Wan L, Jiang J-G. Protective effects of plant-derived flavonoids on hepatic injury. Journal of Functional Foods. 2018;44:283–91.
- 54. Pandey B, Thapa S, Kaundinnyayana A, Panta S. Hepatoprotective effects of Juglans regia on carbon tetrachloride-induced hepatotoxicity: In silico/in vivo approach. Food Sci Nutr. 2024;12(9):6482–97. pmid:39554326
- 55. Song Z, Bu S, Sang S, Li J, Zhang X, Song X, et al. The active components of traditional chinese medicines regulate the multi-target signaling pathways of metabolic dysfunction-associated fatty liver disease. Drug Des Devel Ther. 2025;19:2693–715. pmid:40231197
- 56. Pandey B, Baral R, Kaundinnyayana A, Panta S. Promising hepatoprotective agents from the natural sources: a study of scientific evidence. Egypt Liver Journal. 2023;13(1).
- 57. Pandey B, Thapa S, Biradar MS, Singh B, Ghale JB, Kharel P, et al. LC-MS profiling and cytotoxic activity of Angiopteris helferiana against HepG2 cell line: Molecular insight to investigate anticancer agent. PLoS One. 2024;19(12):e0309797. pmid:39739862
- 58.
Institute of Laboratory Animal Resources (US), Committee on Care, Use of Laboratory Animals. Guide for the care and use of laboratory animals. 86. US Department of Health and Human Services, Public Health Service, National Institutes of Health; 1986.
- 59. Waterman PG. Identification of essential oil components by gas chromatography/mass spectroscopy. Biochemical Systematics and Ecology. 1996;24(6):594.
- 60. Al-Reza SM, Bajpai VK, Kang SC. Antioxidant and antilisterial effect of seed essential oil and organic extracts from Zizyphus jujuba. Food Chem Toxicol. 2009;47(9):2374–80. pmid:19563858
- 61. Luo H, Sun S-J, Wang Y, Wang Y-L. Revealing the sedative-hypnotic effect of the extracts of herb pair Semen Ziziphi spinosae and Radix Polygalae and related mechanisms through experiments and metabolomics approach. BMC Complement Med Ther. 2020;20(1):206. pmid:32615973
- 62. Du C, Pei X, Zhang M, Yan Y, Qin X. 1 H-NMR based metabolomic study of sedative and hypnotic effect of ziziphi spinosae semen in rats. Chin Traditional Herbal Drugs. 2019;50(10):2405–12.
- 63.
Food and Drug Administration. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. 2005. https://www.fda.gov/media/72309
- 64. Hua Y, Guo S, Xie H, Zhu Y, Yan H, Tao W-W, et al. Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou Seed Ameliorates Insomnia in Rats by Regulating Metabolomics and Intestinal Flora Composition. Front Pharmacol. 2021;12:653767. pmid:34220499
- 65. Manayi A, Saeidnia S. A concern on phthalate pollution of herbal extracts/medicines and detection methods. Research Journal of Pharmacognosy. 2015;2(3):49–54.
- 66. Omidpanah S, Saeidnia S, Saeedi M, Hadjiakhondi A, Manayi A. Phthalate contamination of some plants and herbal products. Boletín latinoamericano y del caribe de plantas medicinales y aromáticas. 2018;17(1).
- 67. Zhang M, Hou J. Phthalate contamination in food: occurrence, health risks, biomarkers for detection, and mitigation strategies to enhance food safety. J Agric Food Chem. 2025;73(22):13178–94. pmid:40417958
- 68. Shukla S, Chopra D, Patel SK, Negi S, Srivastav AK, Ch R, et al. Superoxide anion radical induced phototoxicity of 2,4,5,6-Tetraminopyrimidine sulfate via mitochondrial-mediated apoptosis in human skin keratinocytes at ambient UVR exposure. Food Chem Toxicol. 2022;164:112990. pmid:35398180
- 69. Hong S, Kim Y, Sung J, Lee H, Heo H, Jeong HS, et al. Jujube (Ziziphus jujuba Mill.) Protects Hepatocytes against Alcohol-Induced Damage through Nrf2 Activation. Evid Based Complement Alternat Med. 2020;2020:6684331. pmid:33424992
- 70. Kuchay MS, Martínez-Montoro JI, Kaur P, Fernández-García JC, Ramos-Molina B. Non-alcoholic fatty liver disease-related fibrosis and sarcopenia: An altered liver-muscle crosstalk leading to increased mortality risk. Ageing Res Rev. 2022;80:101696. pmid:35843589
- 71. Abdel-Moneim AM, Al-Kahtani MA, El-Kersh MA, Al-Omair MA. Free Radical-Scavenging, Anti-Inflammatory/Anti-Fibrotic and Hepatoprotective Actions of Taurine and Silymarin against CCl4 Induced Rat Liver Damage. PLoS One. 2015;10(12):e0144509. pmid:26659465