Date seeds powder alleviate the aflatoxin B1 provoked heart toxicity in male offspring rat

Abstract

Date (Phoenix dactylifera L.) seeds (PDL) have recently evoked significant attention for their therapeutic potential against numerous diseases. Aflatoxin B1 (AFB1) is an inevitable environmental hazard that pollutes foods and may harm the heart. This study investigated the beneficial effect of PDL against cardiac toxicity induced by AFB1 in male offspring. Female albino rats received PDL (200 mg/kg) orally for 14 days before mating till weaning and AFB1 (50 μg/kg) intramuscularly throughout gestation and lactation. At postnatal day 60, male offspring hearts were collected. Compared to AFB1 intoxicated group, PDL-treated offspring displayed improved cardiac biomarkers, an increase in their antioxidant defense, and a decrease in the cardiac proinflammatory cytokines. Additionally, a reduction in the expression levels of Bcl2 and Nrf2 was observed, with genes linked to increased cardiac caspase-3, Bax, ACE1, P53, and cytochrome C levels. In conclusion, PDL acts as a potential adjuvant agent for ameliorating cardiac toxicity and apoptosis resulting from exposure to AFB1. This is attributed to its antioxidative and anti-inflammatory effects, as well as its capacity to sequester free radicals within cardiac tissue.

Introduction

Aflatoxins are a class of compounds that are derived from di-furanocoumarin and are synthesized by fungi Aspergillus flavus and Aspergillus parasiticus¹. The ubiquitous nature of environmental pollutants in herbs, nuts, grains, fruits, veggies, and pet products has been found to have deleterious effects on human and animal health². The highest authorized levels of aflatoxins in food have been established at 20 ppm. Aflatoxin B1 (AFB1) is considered to be the most lethal form of aflatoxin¹. AFB1 has been categorized as a category I carcinogen by the International Agency for Research on Cancer³. Also, its metabolite, aflatoxin-exo-8,9-epoxide (AFO), is a highly reactive cytotoxic metabolite that cause tissue damage. AFO is produced from aflatoxins through the hepatic microsomal system, facilitated by cytochrome-P450-mediated epoxidation⁴. It binds irreversibly with nucleophilic hetero-atoms of biological macromolecules, such as nitrogen, oxygen, and sulphur, causing tissue damage⁵. The process of conjugating AFO with reduced glutathione (GSH), a crucial intrinsic antioxidant necessary for reversing the deleterious effects of AFO, is facilitated by glutathione-S-transferases⁶.

Several studies have reported the incidence of oxidative stress subsequent to exposure to AFB1, as indicated by the reduction of intracellular antioxidant enzymes and the excessive generation of reactive oxygen species (ROS)⁷. Ma and his colleagues have reported that ROS overproduction activates inflammatory pathways⁵. Thus, antioxidants may prevent oxidative damage and promote tissue regeneration, reducing AFB1-induced tissue damage. AFB1 has been linked to immunosuppression, oncogenesis, teratogenesis, mutagenesis, and genotoxicity^8,9. Recently, AFB1 induced cardiotoxicity has been reported in several studies^10,11.

Natural antioxidants have recently sparked global interest because of their significant therapeutic value and are increasingly used as a substitute medication¹². Date (Phoenix dactylifera L.) seeds (PDL), a member of the Palmaceae family, have been utilized in medicinal applications due to their significant concentration of polyphenol constituents¹³. Polyphenols exhibit antioxidative properties and anti-inflammatory capabilities^14,15.

Several flavonoids, such as quercetin, rutin glycoside, and luteolin, have been identified in different parts of the date palm, including the seeds, fruit, and pollen^16,17. Also, several terpenes, including pinene and its isomer camphene exhibit anti-inflammatory, antioxidant, and anticancer properties¹⁸. Sabinene also has anti-inflammatory and antioxidant effects¹⁹ while δ-Tocotrienol exhibits anticancer effects by suppressing lipid peroxidation processes and ROS²⁰. Through GC–MS analysis, numerous studies have shown that terpenes can be found in date seeds, palm fruit, and leaves²¹.

With these findings, we hypothesized that PDL supplementation could reduce AFB1-induced cardiac oxidative stress and inflammation through the regulation of apoptosis, antioxidant enzymes, and inflammatory processes.

Materials and methods

Material and preparation of date seeds powder

PDL was purchased at a nearby market in El Kharga, Egypt, in the New Valley region. Date seeds were prepared according to the protocol of Horwitz and Latimer²².

Chemical analyses

The chemical composition of date seed samples regarding moisture, ash, protein, crude fiber, and lipids was determined using a previous method²². Each sample was subjected to three replicate measurements to determine its sugar content. The Lane and Eynon volumetric method were employed to determine the levels of total sugars and reducing sugars, as described by Oberoi and Sogi²³. The quantification of non-reducing sugars was achieved through the mathematical operation of subtracting the amount of reducing sugars from the overall sugar concentration.

Analysis of minerals

Total mineral quantification was performed using a spectrophotometer with atomic absorption (model 4200 MPAES, Agilent Technologies, Santa Clara, CA, USA), and the outcomes were expressed in mg/100 g (dry weight basis)²².

Bioactive compounds determination

The phenolic compounds and flavonoids present in date seeds were determined by High-Performance Liquid Chromatography (HPLC), as the methodology outlined by previous studies^24,25.

Fatty acids determination

The content of fatty acids was ascertained through the preparation of methyl esters of fatty acids following²⁶ and subsequently subjected to gas chromatography (GC) analysis as per the methodology described previously²⁷.

Experimental design

Ethical statement

The animal study protocol and the methods were approved by the ethical committee of New Valley University (Approval number: NVREC 033620231). Also, they were carried out in accordance with ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.

Methods

A cohort of Forty-eight female albino rats and 16 male albino rats (purchased from VACERA, Egypt) weighed 200–260 g, all of mature age and in good health, were kept in a laboratory setting with standard hygienic conditions for two weeks to allow for acclimation. At day fourteen, estrus phase was detected in the female rats by taking vaginal smear and detect the cornified epithelial cells microscopically. The smears are often examined for several consecutive days to ensure the accuracy and to track the progression of the cycle. Three female albino rats were observed to be in the estrus stage and were individually housed with one adult male rat in a distinct cage.

Female rats were checked daily for the presence of a vaginal plug and/or sperm in a vaginal smear to confirm successful mating. According to Scorza Barcellona and his colleagues²⁸, the detection of spermatozoa indicates a gestational age of zero days (GD 0). After being weighed, forty pregnant female rodents were separated into two major groups four equal groups:

Control group

Female rats were administered distilled water daily via stomach tube at a dose of 2 ml/kg/ day from 14 days before mating until 21 days postnatal and administrated DMSO intramuscularly at a dose of 50 μg/kg of DMSO from zero-day of pregnancy until 21 days postnatal.

PDL group throughout pregnancy and lactation

Rats received PDL daily via stomach gavage at a dose of 200 mg/kg/day in distilled water from 14 days prior to mating until 21 days postnatal²⁹.

AFB1-treated group throughout pregnancy and lactation

Rats received AFB1 intramuscularly (Sigma Chemicals, Inc. St. Louis, Mo, USA) every day at a dose of 50 μg/kg from the zero-day of pregnancy until 21 days after birth^30,31.

PDL-AFB1 treated group throughout pregnancy and lactation

Rats received PDL daily at a dose of 200 mg/kg/day in distilled water from 14 days before mating until 21 days postnatal and administrated AFB1 intramuscularly at 50 μg/kg/ day from zero-day of pregnancy until 21 days after birth. The first generation (F1) male offspring were weaned from their mother at 21 days postnatal and maintained till 60 days postnatal.

Blood and tissue sampling

The survival index of the pups was evaluated at 60 days postpartum. Ten male rat offspring were selected randomly from each group, weighed and euthanized by cervical dislocation to collect blood and heart samples. The cardiac mass was quantified, and part of the heart was cryopreserved in liquid nitrogen for genetic analysis. The other part of cardiac tissue was removed, washed, and perfused with a chilled solution of 50 mmol/L sodium phosphate-buffered saline (100 mmol/L Na₂HPO₄/NaH₂PO₄, pH 7.4) that contained 0.1 mmol/L EDTA to eliminate any red blood cells and clots. One gram of cardiac tissue was homogenized with 5 ml phosphate buffer at a pH of 7.4. The tissue homogenates were centrifuged at 14,000×g for 15 min at 4 °C. The supernatant was used to quantify the oxidative metabolites and proinflammatory cytokines.

Biochemical assay

The study involved the analysis of heparinized plasma samples for various biomarkers, including lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), creatine kinase (CK), total lipids, cholesterol, triacylglycerol (TAG), and high-density lipoprotein cholesterol (HDL-c) according to the manufacture instructions (Laboratory Biodiagnostic Co., Giza, Egypt). The calculation of very low-density lipoprotein cholesterol (VLDL c) involved the division of TAG values by a factor of 5. The calculation determined the value of low-density lipoprotein-cholesterol (LDL-c) by utilizing the formula: LDL-c = cholesterol -(HDL c + vLDL c). ThermoFisher Scientific Invitrogen, Waltham, MA, supplied the Apolipoprotein B (Apo B) ELISA kit. Assays followed manufacturer instructions. Also, in accordance with previous methods^32–35.

Oxidative biomarkers assay

The present study assessed the antioxidant enzymatic activities of superoxide dismutase (SOD), total (enzymatic plus non-enzymatic) superoxide radical scavenger activity (TSSA), non-enzymatic superoxide radical scavenger activity (NSSA), glutathione peroxidase (GPX), glutathione -s- transferase (GST), glutathione reductase (GRD), and catalase (CAT). Additionally, the levels of reduced glutathione (GSH), the lipid peroxidation marker, malondialdehyde (MDA), and nitric oxide levels (NO) were assessed according to the manufacture instructions (Laboratory Biodiagnostic Co., Giza, Egypt). Also, in accordance with previous methods^36–40.

Proinflammatory cytokine assay

The level of interleukin-6 (IL-6), interleukin-1-beta (IL-1β), interleukin‑1 (IL‑1), interleukin‑2 (IL‑2), tumour necrosis factor- α (TNF-α), nuclear factor erythroid 2-related factor 2(Nrf2), ICAM1, p53, beclin 1, caspase-3, Paraoxonase-1 (PON1), and 8-hydroxy-20-deoxyguanosine (8-OHdG), were quantified in the supernatant of the heart using affordable ELISA kits from Thermo Fisher scientific Invitrogen (Waltham, MA, USA) and Abcam (Cambridge, UK), corresponding to the instructions provided by the manufacturer. Also, in accordance with previous methods^41–43.

Quantitative of Nrf-2, Bcl-2, Bax, P53, ACE-1, Caspase-3,9, and cytochrome C gene expression by qRT-PCR

Using the RNA purification kit (Thermo Scientific, Waltham, MA, USA, GeneJET #K0746), RNA was isolated from 100 mg of rat heart tissue in accordance with the manufacturer’s guidelines. RNA concentration was calculated by spectrophotometric analysis (Thermo Scientific, USA). Using specified primers, reverse transcription of the extracted RNA produced cDNA, which was then used in PCR. http://www.ncbi.nlm.nih.gov/tools/primer-blast/ produced. The sequence of these primers were presented in Table 1. The studied genes were evaluated utilizing the S.Y.B.R. Green PCR Master Mix. The cDNA was incorporated into an SYBR Green qPCR Master Mix (Thermo Scientific, USA) with 30 pg/mL of each primer. The amplification of the product was achieved through a series of 40 cycles involving denaturation at a temperature of 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 45 s. The 95 °C steps were prolonged to 1 min during the first cycle. In the same reaction, the β-Actin gene was amplified to serve as the reference gene. Each specimen was examined twice for analysis. The relative cycle threshold approach was used to quantify gene expression according to a previous study⁴⁴.

Table 1.

Forward and reverse primer sequence of target gene.

Target gene	Forward sequence (5′–3′)	Reverse sequence (5′–3′)	Tm [°C]
mRNA RT-PCR primers sequences
Cytochrome C	5′-TCCTCTACCCTCTATGCCAGGA-3′	5′-AGCTGGGAACCATCATGTGC-3′	58
Bax	5′-ACAGGGTTTCATCCAGGATCGAG-3′	5′-AGCTCCATGTTGTTGTCCAGTTC-3′	58
Bcl2	5′-GGATTGTGGCCTTCTTTGAGTTC-3′	5′-AGAGCGATGTTGTCCACCAG-3′	58
P 53	5′-TTTTCACCCCACCCTTCCCC-3′	5′-CCTCAGACACACAGGTGGCA-3	58
ACE 1	5′-GGAGACGACTTACAGTGTAGCC-3′	5′-CACACCCAAAGCAATTCTTC-3	58
Nrf 2	5′-GCCAGCTGAACTCCTTAGAC-3′	5′-GATTCGTGCACAGCAGCA-3′	58
RNF157	5′-AACAGCCAAGGGCTCAAACT 3′	5′-TCTGACTCACTGCAAGAGCG-3′	58
Caspase-3	5′-GGTATTGAGACAGACAGTGG-3′	5′-CATGGGATCTGTTTCTTTGC-3′	58
Caspase-9	5′-GGAGACGACTTACAGTGTAGCC-3′	5′-CACACCCAAAGCAATTCTTC-3′	58
mRNA RT-PCR primers of housekeeping gene
β-Actin	5′-CACCCGCGAGTACAACCTTC-3′	5′-CCCATACCCACCATCACACC-3′	58

Statistical analysis

A one-way analysis of variance (ANOVA) was employed to assess the differences between the categories followed by Tukey’s honestly significant difference post hoc test. The results are displayed as the mean ± standard error (S.E.). A P value of < 0.05 was deemed significant. The data analysis was conducted utilizing GraphPad Prism version 9 for Windows (GraphPad Software, California USA). All assays were conducted in duplicate.

Result and discussion

Chemical analyses

Table 2 summarizes date seed chemical components. The moisture level was low (3.16 ± 0.03%), with crude fat at 12.18%, protein at 8.85%, and ash at 3.81% of dried matter. This is in line with the findings a previous study⁴⁵, who also observed that seeds had high crude protein and fat content but low moisture content. Due to their minimal moisture content, date seeds can be stored at room temperature for subsequent analysis⁴⁶. Palm seed chemical compositions vary due to cultivar variability and climate. However, its mineral content was 56.44, 216.34, 168.02, 16.80, 75.38, 0.19, 6.17, 0.68, and 0.29 (Ca, P, K, Na, Mg, Cu, Fe, Mn, and Zn mg/100g). According to various research, date variety, soil type, and fertilizer application determine palm flesh and seed mineral content⁴⁷.

Table 2.

Chemical composition and minerals content of date seeds.

Moisture%	Crude protein%	Crude fat%	Ash%	Crude fiber%	Total sugar%
3.16 ± 0.03	8.85 ± 0.03	12.18 ± 0.01	3.81 ± 0.01	13.03 ± 0.02	20.93 ± 0.03

Ca	P	K	Na	Mg	Cu	Fe	Mn	Zn
Minerals content of date seeds (mg/100g)
56.44	216.34	168.02	16.80	75.38	0.19	6.17	0.68	0.29

Bioactive compounds determination

The results in Table 3 indicate that pyrogallol exhibited the highest concentration among the phenolic substance, followed by catechin. Catechin has been found to potentially prevent cardiac disease and the manifestation of anticancer properties⁴⁸. The composition of the aforementioned substances includes various phenolic acids, including gallic, isovanillic, chlorogenic, p-coumaric, ferulic, caffeic, Alpha-coumaric, and P-OH-benzoic. The primary phenolic acid identified throughout the samples analyzed was p-coumaric, with concentrations ranging from 116.96 to 143.60 mg/100 g. The results indicate that caffeic was the second most prevalent phenolic, exhibiting a concentration range of 59.40 to 88.64 mg/100 g. Gallic and ferulic followed, with concentrations ranging from 10.35 to 7.62 mg/100 g and 5.51 to 12.16 mg/100 g, respectively. Date seeds contain various amounts of vanillic, gallic, p-coumaric, caffeic, and syringic phenolic acids⁴⁹.

Table 3.

The concentration of bioactive phenolic compounds in date seeds, measured in parts per million (ppm).

Phenolic compounds	(ppm)	Phenolic compounds	(ppm)
Gallic	154.17	Vanillic	40.12
Pyrogallol	2850.25	P-coumaric	6.08
Epicatechein	325.12	Ferulic	29.87
e-vanillic	340.87	Iso-ferulic	21.54
Chlorogenic	29.02	Ellagic	10.09
Caffeic	50.11	3,4,5-methoxy- cinnamic	10.1
Catechein	596.77	Alpha-coumaric	12.41
4-Amino-benzoic	29.1	Benzoic	91.7
Caffeine	42.22	Protocatchuic	130.31
P-OH-benzoic	232.12	Coumarin	3.81
Catechol	49.2	Salycillic	78.38

The flavonoid composition of the date seeds was displayed in Table 4. Hesperidin was found to be the predominant flavonoid with a concentration of 16.22 mg/100g, followed by naringenin (3.17 mg/100g), luteolin (3.03 mg/100g), and quercetin (2.49 mg/100g).

Table 4.

Flavonoids compounds in date seeds (mg/100 g).

Flavonoids	mg/100 g	Flavonoids	mg/100 g
Luteolin	3.03	Quercetrien	2.49
Narengin	3.17	Quercetin	1.30
Rutin	1.88	Hispertin	1.33
Hisperidin	16.22	Kampferol	0.50
Rosmarinic	1.23	Apegnin	0.49

previous research reported that the Barhi cultivar date fruit contained hesperidin (3.53 mg/100 g), isorhamnetin (5.76 mg/100 g), and kaempferol (2.13 mg/100 g) as the primary flavonoids⁵⁰. Furthermore, an examination of HPLC data utilizing a diode-array detector (DAD) showed that the predominant flavonoid in palm seeds is rutin, ranging from 72.74 to 86.62 mg per 100 g. Subsequently, luteolin (9.17–13.24 mg/100 g) and quercetin (23.71–34.06 mg/100 g) were identified as the subsequent most abundant flavonoids²⁹.

Fatty acid determination

The predominant fatty acids detected in date seeds comprise linoleic (C18:2), oleic (C18:1), myristic (C14:0), lauric (C12:0), and palmitic (C16:0) acids, which collectively constitute over 90% of the overall fatty acid composition. Palm seed fatty acid profiles vary widely depending on variety, climate, and cultivation⁵¹. As indicated by Table 5, date seeds contain the most saturated fatty acids: lauric (19.82%), myristic (11.6%), palmitic (11.07%), and stearic (3.59%). The most abundant monounsaturated fatty acids are palmitoleic acid (0.19%), gadoleic (0.46%), and oleic (43.62%). In contrast, linoleic acids (8.02%) and linolenic acids (0.18%) are the most abundant polyunsaturated fatty acids, albeit in relatively smaller quantities⁵². Although linoleic acid and linolenic acid are key for developing the epidermis, they have less oxidative balance than oleic acid⁵³.

Table 5.

Fatty acids compositions of date seeds (%).

Fatty acids (F.A.)	(%)
A. Saturated fatty acid	47.51%
Caprylic (C 8:0)	0.20
Capric (C10:0)	0.43
Lauric (C 12:0)	19.82
Myristic (C14:0)	11.60
Palmitic (C 16:0)	11.07
Arachidic (C 20:0)	0.42
Heptadecounic (C17:0)	0.17
Stearic (C 18:0)	3.59
Behenic (C 22:0)	0.21
B. Unsaturated fatty acid	52.47%
Linoleic (C 18:2)	8.02
Linolenic (C 18:3)	0.18
Palmitoleic (C16:1)	0.19
Gadoleic (C 20:1)	0.46
Oleic (C 18:1)	43.62
Saturated fatty acid	47.51
Monounsaturated fatty acid	44.27
Polyunsaturated fatty acid	8.20

AFB1 is one of the most dangerous toxins found in grains, herbs, nourishment, and feed products and maintains its stability during food manufacturing stages, posing significant health concerns³. It is believed that ROS, excessive oxidant production, cellular antioxidant deficiency, and mitochondrial dysfunction are critical processes in their development³.

The current research indicates PDL date seed extract is protected against cardiac toxicity. As shown in Table 6, AFB1 triggers atrophy in body and heart weight, with a decline in pups’ survival index. Cardiac biomarkers, including LDH, CK-MB, and CK are significantly increased as shown in Fig. 1. Figures 1 and 2 show that AFB1 has substantially increased cholesterol, triglycerides, total lipid, LDL-C, VLDL-C, and Apo B. At the same time, HDL-C levels lowered when compared with the prophylactic and control groups. A hyperlipidemic AFB1 during pregnancy and lactation significantly increased rats’ morbidity and death and caused body and heart weight atrophy. PDL extracts were efficacious in mitigating the reduction in body and heart weight, morbidity, and mortality without a dose-dependent response^54,55.

Table 6.

The effect of PDL extract and AFB1 adminstration on body weight, heart weight, and survival index of pups in different groups.

Groups	Body weight (g)	Heart weight (mg)	Survival index (%)
Control	53.67 ± 1.19	156.80 ± 1.03	89.66
PDL	51.8 ± 1.08*	155.97 ± 0.67*	96.97*
AFB1	40.83 ± 1.38*	140.47 ± 1.08*	58.82*@
AFB1 + PDL	48.07 ± 0.79*$	152.2 ± 0.78*$	79.17*@$

Data are reported as mean ± S.E.M., P < 0.05 (n = 10).

*Significant variance from the control, ^@significant difference from PDL group, and ^$considerable variation from the AFB1 group.

Fig. 1.

Effect of PDL date seed extract and AFB1 administration on cardiac toxicity biomarkers in different groups of male offspring rats. (a) LDH. (b) CK-MB. (c) CK. (d) Apo B *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05 (n = 10).

Fig. 2.

Effect of PDL date seed extract and AFB1 administration on (a) cholesterol, (b) triglycerides, (c) HDL-c, (d) total lipid, (e) LDL-c, (f) VLDL-c in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05 (n = 10).

PDL extracts are a noteworthy reservoir of phenolic compounds with robust antioxidant activity. Our investigation revealed the presence of seven phenolic acids (namely p-coumaric, caffeic, syringic, vanillic, ferulic, gallic, caffeic, and chlorogenic) and three flavonoids (quercetin, rutin, and luteolin), as evidenced by the chromatographic profile. This result was in line with a previous study that found similar phenolic profiles in Saudi date-seed variants⁵⁶. The higher levels of declared compounds in PDL extract may account for its heightened efficacy in reserving pups rats’ body weight, heart weight, and survival index⁵⁴.

Biochemical assay

The present investigation found that co-administration of an AFB1 with a PDL extract can mitigate cardiotoxicity. AFB1 causes severe myolysis, myocardial degeneration, and cardiac apoptosis in cardiac tissue⁵⁷. The administration of PDL has been observed to decrease the levels of cardiovascular markers, specifically L.D.H., CK-MB, and C.K, indicating its potential cardiovascular protective effects⁵⁸. This result is in agreement with a previous research showing date extract has cardioprotective and nephroprotective properties⁵⁹. Also, the administration of PDL date extracts decreased oxidative injury, inflammatory processes, and apoptosis in heart tissue as discussed previously⁵⁸.

The current investigation suggests that the offspring subjected to AFB1 exhibited a noteworthy elevation in the plasma levels of cholesterol, triglycerides, Apo B, VLDL-C, and LDL-C, while concurrently inducing a significant reduction in HDL-C levels. Consuming AFB1 can induce hyperlipidemia in rats^60,61. Fasting AFB1-induced hyperlipidemia suggests endogenous lipid synthesis. AFB1 can potentially inhibit the hydrolysis rate of triglycerides by lipoprotein lipase, thereby causing a decline in the absorption of triglycerides by tissues. Furthermore, it resulted in an upsurge in cholesterol synthesis in the liver and a rise in the activity of 3-hydroxy, 3-methyl-glutaryl CoA (HMG CoA). Also, it has been observed to induce the splitting of apolipoprotein A-I (apo A-I) and apo C-II from HDL-C. The study observed the protective efficacy of PDL extracts against hyperlipidemia impact of AFB1 during pregnancy and lactation, which shows that plasma cholesterol, triglycerides, total lipid, Apo B, VLDL-C, and LDL-C were decreased. At the same time, HDL-C was significantly increased. It can be inferred that the PDL extract affects lipid metabolism, reducing lipid levels. The findings indicate that the bioactive constituents present in the extracts of PDL extracts may potentially enhance the functions of lipoprotein lipase and hepatic lipase, which are recognized to have a major effect on lipids metabolism⁶². PDL extracts primarily contain caffeic, p-coumaric, and quercetin, all of which have been shown to suppress pancreatic lipase activity⁶³. Cholesterol-metabolizing enzymes such as HMG-CoA reductase, acyl CoA cholesterol acyl transferase, and lecithin-cholesterol acyl transferase may be modulated by PDL extract, leading to a reduction in cholesterol levels⁶⁴. Also, PDL extract lowers cholesterol by increasing bile acid excretion⁵⁴. Lowering hepatocyte cholesterol and circulating LDL-C and its precursors raises hepatic LDL-C receptors⁶⁵. Previous study revealed that PDL extract are rich in epicatechin and catechin⁵⁴. These substances may form complexes with lipids, hindering lipid hydrolysis, emulsification, micellar solubilization, and absorption in the luminal compartment^66–68.

Oxidative biomarkers assay

The results of the present study indicate that AFB1 caused a notable elevation in MDA, NO, and 8-OHdG levels among male rat pups, as illustrated in Fig. 3. This was accompanied by a significant reduction in the activities of GST, GRD, GPX, CAT, NSSA, TSSA, SOD, and GSH concentration, as depicted in Fig. 4 when compared to both the prophylactic and control pups. AFB1 bio-transforms into a highly toxic secondary intermediate (AFO)⁴. The AFO can rapidly bind to cellular biomolecules like RNA, DNA, and protein constituents, producing intracellular ROS as hydroxyl radicals (OH•), superoxide anions (O2•–), nitric oxide (NO), and hydrogen peroxide (H₂O₂)⁶⁹. This leads to the creation of MDA, NO, 8-OHdG and hastens the development and progression of atherogenesis. This was in line with a previous research who concluded that cardiac muscle antioxidant enzyme activity decreases; this leads to rapid ROS production and increases oxidative status, peroxidation, and mitochondrial membrane damage⁷⁰. Furthermore, it has been observed that the cardiolipin phospholipid found in heart tissues exhibits rapid reactivity, leading to an escalation in cardiotoxicity⁷¹. In response to heightened levels of ROS, endogenous antioxidant enzymes endeavor to counteract the ROS to safeguard the heart³.

Fig. 3.

Effect of PDL date seed extract and AFB1 administration on the levels of (a) MDA, (b) No, and (c) 8-OHdG in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05(n = 10).

Fig. 4.

Effect of PDL date seed extract and AFB1 administration on the activity of (a) GST, (b) GRD, (c) GPX., (d) CAT, (e) NSSA, and (f) TSSA in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05(n = 10).

The lipid peroxidation inhibitory effect of PDL extracts was potent upon evaluation through the decline levels of MDA, NO, 8-OHdG. Previous research demonstrated that they exhibited exceptional chelating activity against ferrous ions, demonstrated a robust capacity for PDL to scavenge radicals⁵⁴. GSH, an antioxidant abundant in all biological systems, mitigates AFO’s harmful effects. AFO-GSH conjugates and ROS scavenging. As a result, the depletion of the GSH store disrupts cellular redox equilibrium, accelerating ROS⁷. GPX is an endogenous enzyme with antioxidant properties that plays a vital role in catalyzing the conversion of H₂O₂ into O₂ and H₂O and reducing ROS. It is the primary enzymatic antioxidant defense mechanism³. The present findings indicate that the PDL extract exhibited proficient ROS scavenging activity and stimulated the production of endogenous antioxidant enzymes. The progeny of rats treated with AFB1 and PDL had significantly higher cardiac GST, GRD, GPX, CAT, NSSA, TSSA, SOD, and GSH. These findings suggest that the PDL extract vigorously protects against oxidative damage in the heart. The results of this study are in line with those of previous studies showing the fact of upregulation of GST, GRD, GPX, CAT, NSSA, TSSA, SOD, and GSH in cardiac muscles can effectively mitigate heart injury and cardiac toxicity by safeguarding contrary to oxidative stress-induced impairment. Consequently, inhibiting apoptosis⁷², this agrees with a previous study showing that PDL date extract boosted myocardial antioxidant activity^54,73–75.

PDL date extract’s terpenes and polyphenols were extensively investigated in this study, showing several antioxidant components, including δ-tocotrienol, Linalool, camphene, caryophyllene, Sabinene, and α-pinene that may contribute to its antioxidant capabilities. These chemicals have been shown to have antioxidant, anticancer, and anti-inflammatory properties¹⁸. This result is in line with a previous study that reported PDL extract possesses antioxidant terpenes⁷⁶. Polyphenolic components in the PDL extract are responsible for ROS scavenging and antioxidant capabilities^54,66,76,77.

Proinflammatory cytokine assay

The levels of cardiac apoptotic proteins, including the expressions of IL-6, IL-1β, IL‑1, IL‑2, beclin-1, TNF-α, PON1, p53, caspase-3, ICAM1, cytochrome C, bax, ACE, and caspase-9 were assessed (Figs. 5, 6). Nrf2 and Bcl-2 are recognized as anti-apoptotic regulators, whereas IL-6, IL-1β, IL-1, IL-2, beclin-1, TNF-α, PON1, p53, caspase-3, ICAM1, cytochrome C, bax, A.C.E., and caspase-9 are identified as proteins that promote apoptosis, as disclosed in a previous study⁷⁸. The results indicated an upregulation of these proteins while Nrf2 and Bcl2 proteins were found to be significantly downregulated compared to the control and prophylactic off springs. Both Nrf2 and Bcl2 signaling pathways are widely recognized as crucial regulators of the cellular detoxification process and redox state. Apoptosis, a genetically planned cell death mechanism, causes tissue harm through caspase-3 and caspase-9 enzymes. The results of our investigation demonstrate a noteworthy significant increase in programmed cell death in the heart tissues of AFB1-exposed rats, together with an increase in caspase-3 and caspase-9 gene expression³. The occurrence of apoptosis in cardiomyocytes can be attributed to elevated levels of ROS, NO, and 8-OHdG, as discussed previously⁷⁸. After elevated ROS levels, ATP production was hindered, leading to the depletion of energy reserves in cardiomyocytes and necrosis. In agreement with prior investigations, our results suggest a significant involvement of apoptotic pathways in the damage caused by AFB1³. Death ligands and mitochondrial pathways activate apoptosis-related caspase-3 and -9⁷⁸.

Fig. 5.

Effect of PDL date seed extract and AFB1 administration on the level of (a) IL-6, (b) IL-1, (c) IL-1β, (d) IL-2, (e) beclin-1 (f) Nrf2, in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05 (n = 10).

Fig. 6.

Effect of PDL date seed extract and AFB1 administration on the level of (a) TNF-α, (b) PON1, (c) P53, (d) caspase-3, (e) I.C.A.M.-1 in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05 (n = 10).

Quantitative of Nrf-2, Bcl-2, Bax, P53, ACE-1, Caspase- 3,9, and cytochrome C Gene Expression by qRT-PCR

AFB1 exposure increases inflammation-related gene mRNA expression⁷. TNF-α is the primary and initial inflammatory mediator implicated in the pathogenesis of inflammation⁷⁹. The up-regulation of proinflammatory cytokines is initiated by the TNF-α and p53 pathways, which also induce adhesion molecules that facilitate the recruitment of leukocytes to the inflammation site⁸⁰.

Recent findings demonstrate that AFB1 induces oxidative stress and mitochondrial inflammation to signal apoptosis^3,81,82. AFB1 permanently damages the mitochondrial membrane, releasing cytochrome C, bax, Bcl2, and ACE into the cytosol and activating caspase-3, caspase-9, and other apoptotic proteins downstream (Figs. 6, 7, 8)^82–84. According to previous findings, there was a notable reduction in the percentage of apoptosis observed in the PDL groups⁷³. The biological activities of terpenes have been observed, with α-pinene and its isomer camphene exhibiting anti-inflammatory, antioxidant, and anticancer activity¹⁸. In accordance with previous research Linalool demonstrates properties that reduce inflammation⁸⁵. Also, sabinene demonstrates antioxidative and anti-inflammatory characteristics¹⁹ while δ-Tocotrienol demonstrates anti-inflammatory and anti-cancer characteristics by counteracting ROS and impeding lipid peroxidation reactions^8,20,86,87. Furthermore polyphenols and δ-Tocotrienol exhibit anti-inflammatory properties through the upregulation of Nrf2 and Bcl2 protein expressions⁸⁸. Our findings made it a paradoxical observation that the downregulation of Nrf2 and Bcl-2 expression occurred despite its known role in provoked apoptosis. It is postulated that augmented expression of Nrf2 and Bcl-2 in the progeny subjected to PDL treatment may serve as a cellular mechanism of defense against apoptosis provoked by AFB1^89–91. It is worth noting that Bcl-2 can be cleaved by caspases, resulting in the production of a pro-apoptotic fragment that bears a resemblance to Bax⁷⁸. In contrast, the Bcl-2 antibody employed in our study could not detect the aforementioned cleaved fragment.

Fig. 7.

Effect of PDL date seed extract and AFB1 administration on the mRNA expression of (a) cytochrome C, (b) bax (c) Bcl2, (d) P 53, (e) ACE 1, (f) Nrf 2 in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as Mean ± S.E.M., P < 0.05 (n = 10).

Fig. 8.

Effect of PDL date seed extract and AFB1 administration on the mRNA expression of A. caspase-3 and B. caspase-9 in different groups of male offspring rats. *: significant variance from the control, @: significant difference from PDL group, and $: considerable variation from the AFB1 group. Data are reported as mean ± S.E.M., P < 0.05 (n = 10).

Conclusions

AFB1 can potentially induce significant cardiac injury through mechanisms involving oxidative stress, lipid peroxidation, and inflammatory responses. The efficacy of PDL seed powder extract’s supplementation to protect heart cells from AFB1-induced damage. It is hypothesized that PDL extract’s antioxidant, anti-inflammatory, and anti-apoptotic properties are responsible for these improvements. Our data support that PDL extracts could act as a preventative and curative candidate against AFB1-induced heart injury.

Author contributions

Conceptualization, HMAK, WAME, and HMAE; methodology, HMAK, WAME, and HMAE; software, WAME, ME, JSA, and EME; validation, HMAK, WAME, JSA and HMAE; formal analysis, HMAK, WAME, ME, and EME, and HMAE; investigation, HMAK, WAME, and HMAE; resources, WAME, ME, JSA, and EME; data curation, HMAK, WAME, JSA and HMAE; writing—original draft preparation, HMAK, WAME, ME, JSA and HMAE; writing—review and editing, HMAK, WAME, EME, and HMAE; project administration, ME and EME; funding acquisition, ME, and EME. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by Deanship of Scientific Research at King Khalid University through large group Research Project under grant number RGP2/262/45.

Data availability

Data will be available upon request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.