Pre- and post-treatment effects of date fruit phenolics in cisplatin-induced hepatotoxicity

Abstract

Cisplatin is a popular chemotherapy drug effective in treatment of many solid tumours, however, it is limited by its liver toxicity essentially induced by oxidative stress and inflammation. Meanwhile plant phenolics have a long history of antioxidative and anti-inflammatory capabilities, but, information on the ability of phenolics derived from Date (Phoenix dactylifera) Fruits to modulate this condition remains underexplored. This study investigates the effects of Date Fruit Phenolics (DFP) against cisplatin-induced hepatic damage in male Wistar rats. Wistar rats were administered DFP (200 mg/kg body weight) daily for 7 days, while treated with or without a single intraperitoneal dose of cisplatin (5 mg/kg body weight) either 2 hrs prior to DFP on day 1 (CisB+DFP) or 2 hrs prior to DFP on day 7 (CisE+DFP). All the animals were allowed to have access to water and feed ad-libitum. They were sacrificed 24 hrs after cisplatin administration for histological (Hematoxylin/Eosin, and Periodic Acid Schiff stains), biochemical, inflammatory and redox biomarker analyses. Chromatographic, spectroscopic, and in vitro antioxidative assays were performed to elucidate the phytochemical content, structural features, and antioxidative potentials of DFP. Cisplatin significantly increased hepatic injury markers, lipid peroxidation, and nitric oxide levels, while decreasing glutathione, glutathione peroxidase, catalase, and superoxide dismutase activities (p < 0.05). Meanwhile, DFP significantly modified these observations, with corroborative evidence from histological analysis. Additionally, the chemical constituents in DFP showed significant antioxidative and functional properties. The findings suggest that DFP could offer effective protection against cisplatin-induced hepatotoxicity which could be beneficial for combination therapy in cancer treatment.

Graphical Abstract

Highlights

• •Both Pre- and Post-treatment approaches with Date Fruit Phenolics (DFP) were used in this study.
• •DFP exerts significant free radical scavenging activity comparable to that of Vitamin C.
• •GCMS analysis of DFP identified many popular bioactive phenolic compounds.
• •Some functional groups linked to bioactivity were revealed via FTIR analysis DFP.
• •DFP exerted significant hepatoprotective effects against Cisplatin-induced hepatic injury.

1. Introduction

Cancer is a major worldwide cause of mortality and although over recent years there have been significant developments in cancer treatment strategies, chemotherapy is still the most often used treatment [16]. Preventing the spread and development of cancer cells depends mostly on chemotherapeutic medicines [32]. Among these drugs, cisplatin (Cis), a platinum-based medication formally called as cis-diammineplatinum (II) dichloride stays among the most effective anticancer treatments [32]. Often considered as the "penicillin of cancer," it has been used extensively in medical treatment worldwide and is the first major chemotherapy medicine the FDA licensed under the name Platinol for cancer treatment in 1978 [50]. Among the several human cancers treated with cisplatin are bladder, testicular, head and neck, ovarian, and lung tumours. Its efficacy spans a spectrum of cancer types including lymphomas, carcinomas, sarcomas, and germ cell tumours [4]. However, the use of cisplatin has sadly been linked to many side effects, including peripheral neuropathy, myelosuppression, gastrointestinal toxicity, nephrotoxicity, hepatotoxicity, and ototoxicity [19], [64]. The quality of life has suffered; this has resulted in dyspepsia (an upset stomach) marked by bloating, nausea, and burping. Though the renal excretion route is the primarily clearance route for cisplatin, it is also known to undergo partial metabolism in the liver, where the generation of reactive species contributes to hepatotoxicity. Reports suggest that hepatic mitochondrial damage contributes significantly to an early event in this process, this is explained by the high mitochondrial content in hepatocytes (Bademci et al., 2020). Being a vital organ for endogenous drugs and xenobiotics metabolism and detoxification, the liver becomes the focus of concentration for cisplatin accumulation, therefore causing hepatotoxicity [26]. Recent studies underline that the main processes causing cisplatin-induced hepatotoxicity are increased oxidative stress and inflammation [66]. Accumulation of cisplatin in the liver tissues considered a major consequence, raises a major risk of its associated hepatotoxicity, and even liver failure [37]. Cisplatin-induced cytotoxicity comprises of multiple interconnected mechanisms, including cell membrane peroxidation, mitochondrial malfunction, suppression of protein synthesis, oxidative stress, inflammation and DNA damage [24], [51]. Involvement of antioxidants has great potential to reduce cisplatin-induced toxicity [66], since oxidative stress is a fundamental mechanism driving hepatotoxicity by cisplatin. Various plants and natural chemicals have been investigated to address cisplatin-induced toxicity while keeping its anti-tumour activity, thereby showing their possibility in ameliorating side effects [18], [28], [37], [56]. Phoenix dactylifera L., also known as date palm, is a native of arid and semi-arid regions and it is extensively grown as an economic and food crop throughout the Middle East, Southern Europe, North Africa, South America, India, and Pakistan [30]. The several parts of the date palm—its fruits, seeds, pollen, and leaves—are linked to a wide range of health benefits. These benefits include: antioxidant, antidiabetic, anticancer, antimicrobial, antihyperlipidemic, hepatoprotective, nephroprotective, neuroprotective, gastrointestinal, and sexual improvement capabilities [2], [30], [46]. The significant pharmacological properties of date palm are attributed to its potent constituents, including flavonoids, phenolics, carotenoids, minerals, vitamins, amino acids, organic acids, and fatty acids [52], [8]. Among the listed phytoconstituents of date palm, its phenolics hold a critical medicinal position including anti-inflammatory, antioxidant, antimicrobial, and anticancer activities e.t.c. Hmidani et al. [39], [47], [58]. Given the important function of liver in drug metabolic and detoxification processes, P. dactylifera phenolics could provide encouraging prospects for therapeutic uses in diseases related to hepatoprotection. Therefore, the aim of this work is to assess the possible protective capacity of P. dactylifera water-soluble phenolics on cisplatin-induced hepatotoxicity. Present work uses a dual approach of pre- and post-administration of the P. dactylifera phenolics contents. This work could be important in the development of tailored cancer treatment plans to guarantee minimum organ toxicity increases the advantages of chemotherapy.

2. Material and methods

2.1. Date fruits and chemicals

Fresh Date (Phoenix dactylifera) fruits were bought from a popular fruit market at Oke Baale, Osogbo, Nigeria and identified at the Plant Biology Department, Osun State University, Osogbo, Nigeria by Associate Professor Maboob Adekilekun Jimoh, who was blind to the scheme of the study as Phoenix dactylifera. The fruits were additionally deposited at Obafemi Awolowo University Herbarium and given voucher number IFE-18183. Cisplatin with manufacturing license number, L/19/2292/KB was manufactured by Zuvius Lifesciences Limited, a WHO-GMP Certified Company, at Kharuni-Lodhimajra Road, VIL. Nandpur, Baddie, Distt Solan, Himachal Pradesh, India-173205. Glutathione (GSH), 5′,5′-dithiobis-2-nitrobenzene (DTNB), 2-thiobarbituric acid (TBA), Biuret and 1 chloro-2, 4-dinitrobenzene (CDNB) and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich (St Louis, MO, USA). All other reagents and chemicals used in this study were of analytical grade and water was glass distilled.

2.2. Preparation of water-soluble phenolics date palm Fruits

Water-soluble phenolics were extracted from the dried date palm Fruits by following the methods reported by [71] and [42] with slight modifications. Briefly, date palm fruits were processed to remove the attached specimens on the surface and were washed carefully in tap water. Seeds were removed from the processed fruits, and the seedless portions were dried at 50 °C in the oven and comminuted into powder. 100 g of the powdered fruits were defatted in 85 % ethanol (1:10) for 24 hrs in a water bath with a shaker at room temperature and were filtered. The residues were air-dried and then soaked in 10 volumes of distilled water at 97 °C under stirred conditions for 1 hr. Then the aqueous extracts were centrifuged at 3000 rpm for 15 min and the supernatant was concentrated under a vacuum to about one-quarter of its original volume. One volume of absolute ethanol was added and kept at 4 °C overnight. The mixture was centrifuged at 3000 rpm for 15 min and the pellet was freeze-dried to get the Date Fruit Phenolics (DFP) used for this study.

2.3. Fourier transform infrared spectroscopy analysis (FTIR)

The functional group of the Date Fruit Phenolics (DFP) was analysed using FTIR spectroscopic technique. Anhydrous potassium bromide was mixed with approximately 5 mg of the extract, and spectra were taken between the wavelengths of 500 and 4000 cm⁻¹.

2.4. Gas chromatography-mass spectrometry (GC-MS) analysis of water-soluble date fruit phenolics

The gas chromatography-mass spectrometry (GC-MS) analysis was carried out to determine the composition of the obtained Date Fruit Phenolics via GC-MS Model: QP2010 plus Shimadzu, Japan incorporating an AOC-20i auto-sampler and gas chromatograph interfaced to a mass spectrometer (GC-MS). The proportion of the sample analyte was indicated as a percentage through normalization of the peak area. Analysis of the mass spectrum was performed by referencing the National Institute of Standards and Technology (NIST) database. The patterns of fragmentation in the mass spectrum of the unidentified components were matched against those of identified compounds stored in the NIST library (NIST 11). The relative proportion of each plant-derived compound was determined by contrasting its average peak area against the entire area. Moreover, the identification of the components within the test materials included determining their names, molecular weights, and structures.

2.5. Determination of DFP content and in-vitro antioxidant activities

The phenolic content of DFP was determined by the Folin–Ciocâlteu technique as described by [43]. The 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of DFP was determined by following the method described by [14]. Nitric oxide scavenging assay was carried out by following the Griess protocol reported by [48] and [27] with little modifications. Hydroxyl radical scavenging activity was determined by following the method described by [44]. Hydrogen peroxide scavenging activity was carried out following the protocol reported by [13]. The free radical scavenging activity of DFP was compared with the radical scavenging activities of standard compounds (ascorbic acid and gallic acid).

2.6. Experimental animals and ethics approval

The animals used for this study were thirty-six (36) male rats of Wistar strain, weighing about 160–190 g which were obtained from the Central Animal House, College of Health Sciences, Osun State University, Osogbo. The animals were allowed 7 days for acclimatization in plastic cages in the animal house at an ambient temperature of 25 °C and a relative humidity of 45–55 %, with 12 h each of dark and light cycles. They were maintained on normal laboratory chow and fresh water ad libitum. The handling and use of the animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The ethical approval reference number: UNIOSUNHREC 2022/121B of the ethical committee of the Osun State University, College of Health Sciences Osogbo, Nigeria, was obtained for the research.

2.7. Experimental design

The experimental rats were randomly sorted into six sets (n = 6). The negative control set received only distilled water, two positive control sets (CisB and CisE) received single intra-peritoneal administration of 5 mg/kg body weight of cisplatin either on day 1 (beginning of the experiment) or day 7 (End of the experiment) respectively, DFP set received 200 mg/kg body weight of Date Fruit Phenolics orally. DFP + CisB set received 200 mg/kg body weight of Date Fruit Phenolics for 7 consecutive days orally, 2hrs after the administration of cisplatin (CisB) while the last set, DFP + CisE received 200 mg/kg body weight of Date Fruit Phenolics for 7 consecutive days orally, and a single administration of cisplatin (CisE) on day 7 (Table 1). This dose of cisplatin was chosen in line with several earlier reports that single intraperitoneal administration of 5 mg/kg is associated with toxicities in many important organs [38], [59]. The acute oral toxicity study (OECD) that was conducted on the extract to ascertain the maximum safe dose and preliminary investigations were the basis for the use of 200 mg/kg bwt of DFP in this investigation. Additionally, five doses of the extract, ranging from 75 to 1000 mg/kg bwt, were used in the early in vivo studies, and 200 mg/kg was selected for additional research based on its effectiveness. This dose is also supported by literature [33], [45]. The rats were euthanized 24 h after the last dose of treatment was administered with a 200 mg/kg sodium pentobarbital euthanasia solution.

Table 1.

Experimental design for the in vivo study.

Group	Treatment	Day(s) of Administration
Control	Distilled water (oral)	Days 1–7
CisB	Cisplatin (5 mg/kg, i.p.)	Day 1
CisE	Cisplatin (5 mg/kg, i.p.)	Day 7
DFP	DFP (200 mg/kg, oral)	Days 1–7
DFP + CisB	DFP (200 mg/kg, oral) + Cisplatin (5 mg/kg, i.p.)	DFP: Days 1–7; Cisplatin: Day 1 (2 h before DFP)
DFP + CisE	DFP (200 mg/kg, oral) + Cisplatin (5 mg/kg, i.p.)	DFP: Days 1–7; Cisplatin: Day 7

2.8. Tissue collection preparation

Blood was collected from the rats into non-heparinized bottles through the retro-orbital plexus and then centrifuged at 3500 rpm for 15 min at room temperature to obtain the serum. The serum obtained was then used for biochemical analyses. The solid tissues of interest i.e. the liver tissues were excised and rinsed in an ice-cold solution of 1.15 % KCl, a portion was kept in 10 % formalin for histological examination and the remaining were kept at –20°C for biochemical analyses. The tissues were homogenized in 100 mM potassium phosphate buffer, pH 7.5. The homogenates were centrifuged at 10,000 g for 20 min at 4 °C, and the supernatant was kept at −20 °C to be used for subsequent biochemical assays.

2.9. Determination of serum hepatic function biomarkers

The estimation of serum hepatic function biomarkers was carried out in accordance with the manufacturer’s guide using commercial kits (RANDOX Laboratories Ltd, Crumlin, County Antrim, UK) using the following principles: Serum hepatic function biomarkers, Alkaline phosphatases (ALP) activity and bilirubin were determined following the method described by [34]. Acid phosphatase (ACP) activity was determined following the instructional manual.

2.10. Estimation of antioxidant and Lipid peroxidation status

The activities and concentration of antioxidant enzymes and molecules in the liver tissues of the experimental animals were estimated according to the following principles: Catalase (CAT) activity was measured using hydrogen peroxide as the substrate according to the method previously described by [17]. Superoxide dismutase (SOD) activity was determined by measuring the inhibition of autoxidation of epinephrine at pH 10.2 and 30 °C according to [53]. Reduced glutathione (GSH) was determined according to the method of [12]. Activity of Glutathione peroxidase (GPx) was estimated following the method reported by [63]. The lipid peroxidation status of the treated samples was determined as the formation of thiobarbituric acid reactive substances (Malondialdehyde (MDA)) during an acid heating reaction, according to [57]. Nitric Oxide (NO) level was determined by the method of [35].

2.11. Histological analysis and special stains

2.11.1. Tissue preparation and staining

Fresh liver tissue specimens were collected from the experimental animals and promptly fixed in 10 % neutral buffered formalin to preserve the tissue histoarchitectural profile. After fixation, tissues were processed for paraffin embedding. Sections of about 4–5 µm were sectioned using a rotary microtome, floated on a water bath, and transferred onto glass slides. Slides were then deparaffinized and rehydrated for staining.

2.11.2. Histological staining and evaluation

For general histological analysis, Hematoxylin and Eosin (H&E) stain technique was employed. A semi-quantitative scoring scale (0–4) was used to describe the level of hepatic injury in each group (Table 2). Additionally, Periodic Acid-Schiff (PAS) histochemical stain was used to demonstrate glycogen content in the tissue. It involved treating sections with Periodic Acid and Schiff's reagent. Stained sections were examined under a light microscope (X400 magnification) for glycogen deposition and representative fields were photographed for documentation. A semi-quantitative scoring scale (0–5) was used to describe the level of glycogen deposition in each group (see Table 3)

Table 2.

Semi-quantitative scoring system for hepatic injury in the experimental rats.

Score	Histopathological Features (Liver)
0	Normal hepatic architecture
0.5	Focal injured areas (e.g., single-cell changes without structural impact)
1	Less than 10 % of hepatic tissue showing injury (mild hepatocyte changes, focal vacuolation)
2	10–25 % of hepatic tissue affected (moderate hepatocyte degeneration, sinusoidal changes)
3	25–75 % of hepatic tissue involved (marked fragmentation, perinuclear spacing, vein atrophy)
4	More than 75 % of hepatic tissue affected (widespread necrosis, severe sinusoidal occlusion, vein collapse)

Table 3.

Semi-quantitative PAS scoring system for hepatic glycogen deposition.

Score	Description
0	No staining
1	Very poor glycogen deposition
2	Poor
3	Moderate
4	Strong
5	Very strong glycogen staining

2.12. Statistical analysis

The data were expressed as mean ± standard deviation (SD) after analysis by one-way analysis of variance (ANOVA) with the aid of Graph Pad Prism software, version 8.0.2 for Windows (GraphPad Software, San Diego, CA), followed by Post hoc Bonferroni comparative test. Differences between the mean values of different groups were considered statistically significant at P < 0.05.

3. Results

3.1. FTIR spectra of functional groups in Date Fruit Phenolics (DFP)

Fourier-transform infrared (FT-IR) spectrum of DFP is shown in Fig. 1. Typical absorption peaks at, 1650.03 cm⁻¹ correlating with conjugated C O group, 1510.61 cm⁻¹ correlating with aromatic C C hydrocarbon group, and 1050.55 cm⁻¹ correlating with O-H bending vibration, (hydroxyl group) were shown by the extract. All these functional groups are known to be characteristics of phenolics [31].

Fig. 1.

a.) FT-IR spectra of DFP b.) Functional groups and vibration types identified in DFP.

3.2. Gas chromatography/mass spectrometric components in Date Fruit Phenolics (DFP)

The GC-MS analysis of DFP revealed very important phenolic compounds through NIST 11 at different retention times as shown in Fig. 2. These phenolic compounds previously reported to be crucial bioactive principles having antioxidant, and cytoprotective properties. These are highly valuable for the healing of oxidative damage and other various medicinal activities.

Fig. 2.

a.) GCMS chromatogram of DFP b.) Compounds identified from DFP.

3.3. Free radical scavenging activities of Date Fruit Phenolics (DFP)

The free radical scavenging activity of DFP as shown in Fig. 3(a-d) is in comparison with the radical scavenging activities of Ascorbic Acid and Gallic acid which are known radical scavengers. The date fruit phenolics exhibited significantly lower free radical (DPPH-(Fig. 3a), Nitric Oxide-(Fig. 3b), Hydroxyl radical-(Fig. 3c), and Hydrogen peroxide (Fig. 3d), scavenging activities compared to the pure antioxidant standards (ascorbic acid and gallic acid at p < 0.05). However, its activity is still considerable and within a comparable range, since the DFP is in its crude form, this indicates that the extract possesses strong free radical scavenging potential despite the complexity of its phytochemical composition.

Fig. 3.

Free radical scavenging activities of DFP, Ascorbic Acid and Gallic Acid. a) DPPH Radical Scavenging Activity b) Nitric Oxide (NO) radical scavenging activity c) Hydroxyl Radical Scavenging Activity d) Hydrogen Peroxide (H₂O₂) scavenging activity. The values are expressed as mean ± SD for n = 3. * shows significant difference at P < 0.05 as compared to Ascorbic acid: ^# shows significant difference at P < 0.05 as compared to gallic acid: ^** shows significant difference at P < 0.05 as compared to both Ascorbic acid and gallic acid.

3.4. Serum hepatic function biomarkers

Cisplatin caused significant increase in hepatic activities of alkaline phosphatase (Fig. 4a) and acid phosphatase (Fig. 4b) with increase in bilirubin concentration (Fig. 4c) relative to control, reflecting hepatic dysfunction (P < 0.05). There was no significant difference between the two time points for single cisplatin administration (day 1 and day 7). In contrast, treatment with DFP alone or in combination with cisplatin (DFPCisB and DFPCisE) significantly moderated these elevations.

Fig. 4.

Liver function biomarkers (a) ALP (b) ACP (c) Bilirubin. The values are expressed as mean ± SD for n = 6. ^a P < 0.05 level of significant as compared to the control: ^b P < 0.05 level of significant as compared to CisB: ^c P < 0.05 level of significance as compared to CisE: ^dP < 0.05 level of significance as compared to DFPCisB: ^e P < 0.05 level of significance as compared to DFPCisE.

This suggests that DFP confers a hepatoprotective effect by stabilizing liver function markers even under cisplatin-induced stress (Fig. 4a–c). The lack of significant difference between the two cisplatin time-points indicates that cisplatin’s hepatotoxicity is consistently manifested across the treatment duration, reinforcing the reliability of this dose as a model of hepatic injury.

3.5. Hepatic antioxidant enzyme status

Single administration of cisplatin at both time-points caused a marked suppression of the hepatic antioxidant defence system when compared with the control group. The activities of superoxide dismutase (SOD) (Fig. 5a), catalase (CAT) (Fig. 5b), and glutathione peroxidase (GPx) (Fig. 5d), as well as the level of the non-enzymatic antioxidant glutathione (GSH) (Fig. 5c), were all significantly reduced (P < 0.05) following cisplatin administration. This reduction is a reflection of the oxidative stress burden and depletion of endogenous antioxidants induced by

Fig. 5.

level of antioxidant molecule and antioxidant enzyme activities in liver: (a) superoxide dismutase (SOD), (b) Catalase (CAT), (c) Reduced Glutathione (GSH) and (d) Glutathione peroxidase (GPX). The values are expressed as mean ± SD for n = 6. a P < 0.05 level of significant as compared to the control: b P < 0.05 level of significant as compared to CisB: c P < 0.05 level of significance as compared to CisE: ^dP < 0.05 level of significance as compared to DFPCisB.

cisplatin.

However, combination of cisplatin treatment with DFP or DFP alone (DFPCisB, DFPCisE, and DFP groups) significantly enhanced the hepatic antioxidant status. The activities of SOD, CAT, and GPx, and GSH levels were elevated toward control values. This suggests that DFP offers partial protection against cisplatin hepatotoxicity (Fig. 5a–d).

3.6. Liver inflammatory and lipid peroxidation markers

Single treatment of animals with cisplatin at both time-points resulted in a significant elevation of the hepatic nitric oxide (Fig. 6a), and malondialdehyde) levels (Fig. 6b) when compared with the control group. This elevation is an implication of induced inflammation and lipid peroxidation by cisplatin toxicity. Conversely, treatment with DFP alone or in combination with cisplatin (DFPCisB, DFPCisE, and DFP groups) significantly suppressed these molecules relative to the cisplatin-only groups. This suggests that DFP offers anti-inflammatory protection and partial protection from cisplatin- induced lipid peroxidation (Fig. 6a–b).

Fig. 6.

Inflammatory and Lipid peroxidation markers concentration in the liver. (a) NO: Nitric Oxide Concentration (b) Malondialdehyde (MDA) level. The values are expressed as mean ± SD for n = 6. ^a P < 0.05 level of significance as compared to the control: ^b P < 0.05 level of significance as compared to CisB: ^c P < 0.05 level of significance as compared to CisE: ^d P < 0.05 level of significance as compared to DFPCisB: ^e P < 0.05 level of significance as compared to DFPCisE.

.

3.7. Photomicrograph of hematoxylin and eosin section of the experimental rat liver

H&E-stained liver sections are shown in Fig. 7A–F. The control group (Fig. 7A) revealed well-preserved hepatocytes (yellow arrows), sinusoid-lining cells (red arrows), and patent sinusoids (white arrows). The Cisplatin-treated groups (Figs. 7B, 7C) exhibited significant histopathological damage, including fragmented and vacuolated hepatocytes (yellow arrowheads), occluded sinusoids (red arrowheads), and hypertrophied central portal vein (CpV). Administration of DFP alone (Fig. 7D) showed that the normal hepatic morphology is preserved, this indicates no observable deviations. In the DFPCisB and DFPCisE groups (Fig. 7E–F), hepatocytes showed only mild deviations, including occasional fragmented hepatocytes (yellow arrowheads) and partially occluded sinusoids (black arrows). This indicates that the morphology of the hepatocytes were relatively preserved. Semi-quantitative liver injury scores across the six experimental groups. Scores were derived from H&E-stained sections based on the extent of hepatocyte fragmentation, vacuolation, sinusoidal occlusion, and overall tissue integrity (Fig. 7G).

Fig. 7.

H&E-stained sections of the liver from the experimental rats (X400) Semi-quantitative hepatitis injury scores. (a) Control: Liver section shows well-preserved architecture with normal hepatocytes (yellow arrows), sinusoid-lining cells (red arrows), and intact sinusoids (white arrows). (b) CisB: Histoarchitecture of the liver is compromised, showing fragmentation of hepatocytes (yellow arrowheads), occluded sinusoids (red arrowheads), and hypertrophied central portal vein. (c) CisE: Hepatocytes appear fragmented, vacuolized, and display perinuclear spacing (yellow arrowheads). The central portal vein is morphologically distorted (double asterisks). (d) DFP: Sections are preserved, showing no observable deviations from normal architecture. (e) DFPCisB: Largely preserved histoarchitecture with minor deviations, including occluded sinusoids (black arrows) and fragmented hepatocytes (yellow arrowheads). (f) DFPCisE: Similar preservation is observed, with only few histological deviations (occluded sinusoids and fragmented hepatocytes). The yellow arrows are showing normal hepatocytes; The CpV connotes the central portal vein. (g) Semi-quantitative hepatitis injury scores across the six experimental groups.

3.8. Photomicrograph of Periodic Acid Schiff (PAS) staining section of the experimental rat liver

Treatment with cisplatin caused significant histological deviations and energy loss in the functional integrity of the hepatic cells with significantly reduced or poor glycogen staining, indicative of energy loss and impaired functional integrity of the liver cells. While DFP caused no visible lesion, it suppressed histopathological deviations caused by cisplatin at both time points. (Fig. 8 (A-F)). These observations were further supported by the semi-quantitative glycogen deposition scores where control and DFP groups showed the highest scores, cisplatin groups the lowest, and the co-treatment groups intermediate values (Fig. 8G).

Fig. 8.

PAS-stained sections of the liver from experimental rats (×400) and semi-quantitative glycogen deposition scores. (a) Control: Liver sections well preserved with abundant glycogen deposits in hepatocytes (scores 4–5). (b) CisB: The liver sections were marked with histological deviations ranging from occluded sinusoids, atrophied hepatocyte vacuolation, and absence of visible sinusoid-lining cells. The glycogen reaction is poor suggestive of energy loss and showing the functional integrity of the liver (scores 1–2). (c) CisE: The liver sections were marked with histological deviations ranging from occluded sinusoids, atrophied hepatocyte vacuolation, and absence of visible sinusoid-lining cells. The glycogen reaction is poor suggestive of energy loss and showing the functional integrity of the liver (scores 1–2). (d) DFP: Preserved liver structure with strong glycogen staining (scores 4–5), comparable to control. (e) DFPCisB: The histological reaction of the PAS is slightly active as evidence of glycogen deposits (scores 2–3) are seen around the hepatocytes. The yellow arrows show intact cuboidal hepatocytes surrounded by sinusoidal channels. (f) DFPCisE: The histological reaction of the PAS is slightly active as evidence of glycogen deposits (scores 2–3) are seen around the hepatocytes. The yellow arrows show intact cuboidal hepatocytes surrounded by sinusoidal channels. (g) Semi-quantitative glycogen deposition scores; Graphical representation of glycogen staining intensities across experimental groups. Yellow arrows: Intact cuboidal hepatocytes surrounded by sinusoidal channels.

4. Discussion

The possible preventive properties of water-soluble Date Fruit Phenolics (DFP) against cisplatin-induced liver damage in rats are investigated in this work. Using dual (pre- and post-treatment) strategies, the study aims to find whether DFP can reduce cisplatin-induced hepatotoxicity, a major consequence compromising its therapeutic efficacy. Along with related death, structural and functional abnormalities in the liver, cisplatin is accompanied by significant nephrotoxic and hepatotoxic side effects involving inflammatory and oxidative stress pathways notwithstanding its efficacy as an anticancer drug [11].

To understand the possible mechanistic basis of the protective effects of DFP, the extract was chemically characterized. The Gas Chromatography-Mass Spectrometry (GC–MS) investigation of DFP turned up a wide range of phenolic chemicals, which have different health effects [69]. These isolated phenolic components from DFP include Naringenin, Apigenin, Epicatechin, Rosmarinic acid, Kaempferol, Quercetin, Rutin, and Myricetin. Particularly, these compounds have been extensively investigated for their varied biological and pharmacological activities and could explain their contribution to the possible therapeutic properties of DFP (Hu et al., 2022; [68]). For instance, Quercetin, Rutin, Naringenin, Apigenin, Rosmarinic acid, and Myricetin are extensively investigated phenolics known for many recorded medicinal and pharmacological activity including antimicrobial, antioxidant, anti-inflammatory, and antitumour effects [65], [68]. The special combination of these components in DFP implies that this extract has great pharmacological potential and provides a multifarious way to support health. These compounds likely act synergistically to provide the observed hepatoprotection. The FTIR spectrum of DFP in this study revealed distinct absorption bands attributable to phenolic hydroxyl groups [61], [67].

Antioxidant properties of phenolics are crucially related to their several health advantages [20]. Recent scientific studies have focused on the contribution of phenolic compounds free radical scavenging especially in date fruits [65]. The results of this study coincide with studies of Djaoudene et al. suggesting that the total phenolic content of DFP is closely connected with its capacity to remove free radicals. This is relevant because an increase of reactive oxygen species (ROS) eventually leads to oxidative stress, an important mechanism in cisplatin-induced hepatotoxicity. The liver is particularly high in polyunsaturated fatty acids [60] making it highly susceptible to lipid peroxidation, where ROS covalently bond to cell membrane macromolecules to cause severe peroxidative destruction of membrane lipids, mitochondria, lysosomes, and endoplasmic reticulum. Apart from using GC-MS and FTIR to identify several phenolic compounds and functional groups in DFP, we performed experiments for free radical scavenging activities to evaluate its antioxidant ability. The antioxidant capacity of DFP was assessed by means of scavenging activity against free radicals including DPPH, nitric oxide, hydroxyl, and hydrogen peroxide. Since DPPH• is a persistent free radical, it is often employed to evaluate the efficiency of natural antioxidants in scavenging free radicals in vitro [36]. In this study, the DPPH radical scavenging activity of DFP was observed, this is consistent with earlier report which proved that date phenolics showed significant DPPH radicals (Sumaira et al., 2017). Because of its special chemical characteristics, nitric oxide, a free radical molecule created endogenously in the human body, is essential in controlling many physiological processes including circulatory, immunological, neurological, and antioxidant reactions [49]. Its broad participation in biological systems means that both underproduction and overproduction can cause health problems [21]. The nitric oxide-radical scavenging capacity of date phenolics has been widely established in literature [39] which is in agreement with the results obtained in this study. Identified as the most reactive free radical, the hydroxyl ion readily permeates cell membranes and reacts with important biomolecules including lipids, proteins, and DNA, therefore causing cell damage and finally cell death [42]. DFP showed a remarkable hydroxyl radical scavenging activity in the present study, this is in agreement with previous study [73]. Many plant extracts have their hydrogen-donating ability evaluated using the scavenging activity of hydrogen peroxide since these radicals function as hydrogen-acceptor molecules [3], [7]. DFP showed remarkable antioxidant qualities comparable to those of ascorbic acid and gallic acid in the in-vitro antioxidant assay reported in the present work. This implies that DFP may act as a donor of free protons or hydrogen ions and help to stabilize damaging free radicals in cells. The free-radical scavenging capacity of date phenolics has been widely established in literature [5], [39], [45], [54].

Despite its efficacy against solid tumours, the clinical utility of cisplatin is constrained by its tendency to induce toxicity in various tissues. Particularly cisplatin-induced hepatotoxicity is linked to multiple processes starting with the too high formation of reactive oxygen species, which causes oxidative stress, inflammation, DNA damage, and liver death [1], [24], [72]. Cisplatin's mode of action is related to its capacity to create crosslinks with purine bases on DNA, therefore upsetting DNA repair systems, generating DNA damage, and finally inducing death in cancer cells [19]. Reduced antioxidant enzyme activity and subsequent higher levels of malondialdehyde (MDA) suggest oxidative stress and enhanced lipid peroxidation that is linked with the amplification of liver enzymes. The damage caused by lipid peroxidation raised the cellular permeability of hepatocytes, which caused the leakage of proteins including amino transferases into the serum, indicating hepatic damage and necrosis [70], [9]. Crucially, a protein thiol, glutathione (GSH) scavenges free radicals and reactive oxygen species [23] therefore bolstering the defence system of the body against oxidative stress. The present work showed that injection of cisplatin drastically raised MDA levels, and lowered GSH concentration, thereby diminishing the activity of the phase II metabolising enzyme, glutathione peroxidase (GPx). On the other hand, pre- and post-treatment with DFP in rats exposed to cisplatin showed a significant drop in MDA levels and an increase in GSH level, GPx, catalase and superoxide dismutase (SOD) activity. Increased reactive oxygen species (ROS) explains the noted decline in catalase activity in rats treated exposed to cisplatin [55]. The drop in GPx activity under cisplatin suggests a drop in GSH levels and an increase in peroxides [32]. The findings implies that the decrease of lipid peroxidation and protein oxidation is connected to the observed rise in GSH level in groups pre- and post-treated with DFP extract. In the livers of rats sub-chronically exposed to trichloroacetic acid, a previous study showed that date fruit extract reduced lipid peroxidation and increased antioxidant status [22]. Given their abundance of natural antioxidants, date fruits are a possible functional diet for controlling oxidative stress-related diseases [10]. The results of the present work show generally, the strong effect of DFP in reducing cisplatin-induced hepatic oxidative stress and lipid peroxidation.

Commonly used to evaluate liver activities are alkaline phosphatase (ALP) and acid phosphatase (ACP) [40]. Although mostly found in the liver, bones, intestines, and kidneys, ALP is an enzyme linked to liver function [40]. Administration of cisplatin increased ALP and ACP, suggesting substantial hepatic cell damage most likely caused by tissue disintegration and allowing intracellular enzymes to escape from the cytosol into the bloodstream [41]. When cisplatin was administered along with DFP, the liver enzymes ALP and ACP were significantly lower than in the group only administered cisplatin. These hepatoprotective effects of DFP have been confirmed in previous reports involving hepatotoxicity induced by various agents such as paracetamol [15], doxorubicin [29], diclofenac, carbon tetrachloride [6], and mercuric chloride [62]. The present work exposed several histological anomalies in the liver: blocked sinusoids, fragmented and vacuolized hepatocytes, hepatocytes with perinuclear spacing, and morphologically distorted central portal veins. Second only to the kidneys, the liver is well documented to accumulate notable levels of cisplatin [25]. Pre- and post-treatment with DFP, however, was observed to lower the histopathological effects brought about by cisplatin. Possibly due to its significant antioxidant content, free radical scavenging activities, and hepatoprotective actions, Phoenix dactylifera L. shows better protection for the hepatocytes [55]. Apart from liver protection, our results indicate that DFP may also play a possible supportive (adjuvant) role during chemotherapy. By limiting cisplatin-induced toxicity, these polyphenolics could make it possible to adjust the dose or extend treatment without compromising its anticancer action. However, this potential needs to be verified in future studies using tumour-bearing models.

5. Conclusion

The efficiency of Date Fruit Phenolics in mitigating the harmful effects of cisplatin administration by both approaches was demonstrated in this work. This was clear in the serum biomarkers (ALP, ACP, and bilirubin) reduction and the increase in both non-enzymatic (GSH) antioxidant levels and enzymatic (SOD, CAT, and GPx) activities. Strong antioxidant activity, membrane-stabilizing effect, and their capability to increase enzymatic antioxidants in the liver help to explain the preventive efficacy of the extracts. These results support the conventional use of date seeds as a liver detoxifier and a possible adjuvant in combination chemotherapy based on cisplatin.

CRediT authorship contribution statement

Omowumi O. Adewale: Conceptualization, Methodology, Investigation, Data curation, Project administration, Writing – original draft, Writing – review & editing, Funding acquisition. Roseline F. Oyelola: Funding acquisition, Methodology, Investigation, Writing – original draft. Adeola B. Adenmosun: Methodology, Investigation, Writing – original draft, Proofreading. Oluwaseun A. Adebisi: Methodology, Investigation, Resources, Proofreading. Isaac O. Babatunde: Methodology, Resources. Adedayo D. Adekomi: Methodology, Resources, Proofreading. Johnson O. Oladele: Methodology, Resources, Proofreading.

Funding

This research received no external funding.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Prof. L.H. Lash

Data Availability

Data will be made available on request.