Keywords: Ajwa Dates Price in Pakistan date fruits, sickness behaviour, antioxidants, quercetin, cytokines

Dates (Phoenix dactylifera L., family—Arecaceae) are widely grown as a food and commercial crop in countries including India and Pakistan . Ajwa dates are a unique variety of date fruit grown in Al-Madina Al-Munawwarah, Saudi Arabia and they have medicinal properties [1,2]. The carb-rich Ajwa date is a fantastic source of proteins, vitamins, high dietary fibre, minerals, and lipids . They contain typical minerals such as iron, copper, zinc, calcium, potassium, cobalt, fluorine, sulphur, magnesium, manganese, phosphorus, selenium, and boron . A variety of phytochemicals of Ajwa date include glycosides, polyphenols, flavonoids, and sterols. Ajwa dates have hepatoprotective , cardioprotective , nephroprotective , antioxidant [7,8], antihyperlipidemic , anti-inflammatory , antibacterial and anti-cancer properties.

In the Middle East, dates have been used as a staple meal for thousands of years . Various religions give high importance to date fruits . In Islam, to break the daylong fast during the holy month of Ramadan, dates are used . Date palms are the best assets, according to the Prophet Muhammad, who also advised consuming dates and developing a preference for date palms . Jews celebrate Palm Sunday and consider dates to be one of the seven holy fruits . Many people in the Middle East believe that eating date fruits on an empty stomach can counteract the poisonous effects of any substances to which the individual may have been exposed . In traditional medicine, the date plant is used to treat fever, inflammation, paralysis, nerve disorders, and memory loss [2,6]. All these overlap with the signs and symptoms of sickness behaviour . The impact of dates on the CNS is extensively documented , on the other hand, there are no reports of its advantageous benefits on sickness behaviour.

A sophisticated and coordinated adaptive change brought on by tissue damage or acute infections is known as sickness behaviour [13,14,15]. The sickness behavioural pattern includes malaise, hyperalgesia, fever, lethargy, social retreat, inhibition, decreased locomotor activity, exploration, grooming, loss of libido, anhedonia, sleepiness, anorexia weight loss, disrupted concentration, and anxiety [14,16]. Even while sick behaviour is a normal immune response for faster recovery from an infection or damage, it nonetheless causes discomfort in the victims if it lasts for a longer period . So, it is important to address sickness behaviour to reverse the patients’ altered social, cognitive, and mental functioning.

The effect of medications on sickness behaviour is preclinically evaluated using a variety of animal models. Preclinical research uses lipopolysaccharide (LPS)-induced sickness behaviour in rodents the most frequently among others [12,17].

Based on the aforementioned information, the current investigation was carried out to determine the effectiveness of date fruit extracts against LPS-induced illness behaviour in rats by evaluating behavioural and biochemical markers.

2.1. Standardisation of PDF

Total phenolic content in PDF was found to be 5.57 ± 0.31 mg/g of gallic acid equivalent weight (mean ± SEM, n = 3) and total flavonoid content was found to be 171.2 ± 1.15 mg/g of quercetin equivalent weight (mean ± SEM, n = 3).

The HPLC fingerprint profile of PDF was standardised with biomarker and it served as a standard for comparison in the subsequent preparation of PDF. HPLC chromatogram of the extract was found to contain constituents eluting between 2.0 min and 7.0 min. The presence of Quercetin in PDF at RT 6.171 min was confirmed by comparing its retention time and UV spectra with that of the standard Quercetin. The amount of Quercetin present in PDF was found to be 56.9 µg/mg. HPLC Chromatogram of PDF and quercetin is shown in Figure 1.

HPLC Chromatogram of PDF showing the presence of Quercetin (Inset), separated on a Waters xbridge ® RP C18 column (5 µm, 4.6 × 250 mm) using isocratic elution was performed using 70% methanol, 30% milli-q water and 0.1% trifluoro acetic acid at a total flow rate of 1.0 mL/min with a run time of 10 min. The chromatogram at 254 nm was analysed.

Treatment with LPS leads to reduced water and food intake in the rats by 63.77% and 55.66%, respectively, when compared to the vehicle control group. Administration of PDF 250 and PDF 500 mg/kg to LPS-challenged animals restored the water and food intake by 84.04% and 134.66%, respectively, when compared to the LPS control animals. Animals administered with PDF 500 mg/kg (15.3 ± 0.56 and 4.66 ± 0.33, respectively) consumed almost the same amount of water and food as animals treated with 1 mg/kg of dexamethasone (15.6 ± 0.59 and 4.83 ± 0.40, respectively). The effect of PDF on water and food intake in LPS-treated rats is shown in Table 1.

All the values are expressed in mean ± SEM. (n = 6). Where, LPS—lipopolysaccharide, PDF—Phoenix dactylifera fruit extracts and Dex—dexamethasone.

2.3.1. Locomotor Activity Scores

Compared to the vehicle control group, the locomotor score in LPS control rats was drastically decreased (p < 0.001). Treatment with both doses of PDF improved (p < 0.05 and p < 0.01, respectively) the locomotor score in LPS-treated rats when compared to LPS control rats. The improvement in locomotor score in a higher dose of PDF was comparable with the scores of the standard drug dexamethasone (p < 0.001). The effect of PDF on locomotor score in LPS-treated rats is shown in Figure 2.

Effect of Phoenix dactylifera fruit extracts on locomotor activity score in actophotometer test of lipopolysaccharide treated rats. Numerical values are represented as mean ± SEM (n = 6), * p < 0.05, ** p < 0.01 and *** p < 0.001.

2.3.2. Tail Suspension Test

In the tail suspension test, compared to the vehicle control rats LPS control rats had significantly (p < 0.001) higher periods of immobility. Treatment of both the doses of PDF and the standard drug dexamethasone significantly reduced the immobility in LPS-treated rats when compared to the LPS-control rats. The effect of PDF on tail suspension test-induced immobility in LPS-treated rats is shown in Figure 3.

Effect of Phoenix dactylifera fruit extracts on tail suspension test induced-immobility in lipopolysaccharide treated rats. Numerical values are represented as mean ± SEM (n = 6), *** p < 0.001.

2.3.3. Despair Behaviour Test

The despair behaviour of rats was measured by the forced swim-induced immobility in the rats. Compared to the vehicle control rats the immobility time and the number of immobility states are significantly (p < 0.001) greater in LPS-control rats. Treatment with PDF at the dose of 250 mg/kg and 500 mg/kg drastically (p < 0.01 and p < 0.001, respectively) reduced the immobility time and number of immobility states in LPS-treated rats. The higher dose of PDF administration gives almost equivalent results to that of the standard control drug dexamethasone (p < 0.001). The results of the effect of PDF on forced swim test-induced immobility in LPS-treated rats are shown in Figure 4.

Effect of Phoenix dactylifera fruit extracts on forced swim test-induced immobility in lipopolysaccharide treated rats, (A) Immobility time and (B) Number of immobility state. Numerical values are represented as mean ± SEM (n = 6), ** p < 0.01 and *** p < 0.001.

The administration of LPS elevated the levels of liver marker enzyme ALT and AST in the serum when compared to the vehicle control rats and the values are found to be statistically significant with a p-value less than 0.001. The administration of PDF to LPS-treated rats signi