Part 1 Using Dates (Phoenix Dactylifera Linn And Cistanche) To Improve Energy Metabolism in Fatigue-induced Sprague Dawley Rats

Hana Kaduna,b, Azizah Hamida,∗, Faridah Abas a,c, Nurul Shazini Ramli a, Ahmad Haniffff Jaafar a,∗, Mohd Sabri Pak Deka, Belal J. Muhialdina,d, Hani Hafeeza Halima, Abdul Karim Sabo Mohammed,e, Salam A. Ibrahim

a Department of Food Science, Faculty of Food Science and Technology, UPM Serdang, Universiti Putra Malaysia, Selangor 43400, Malaysia

b Faculty of Science, Al-Muthanna University, Iraq

c Institute of Bioscience, UPM Serdang, Universiti Putra Malaysia, Selangor 43400, Malaysia

d Halal Products Research Institute, UPM Serdang, Universiti Putra Malaysia, Selangor 43400, Malaysia

e Faculty of Science, The Federal University of Dutse, Dutse, Jigawa, Nigeria

f Food and Nutritional Sciences Program, North Carolina A&T State University, Greensboro, NC 27411, USA

Abstract

fatigue increases oxidative stress which damages body cells and increases the risk for the development of various chronic diseases. In this study, the anti-fatigue activity of Piyarom date extract was evaluated in fatigue-induced rats using a forced swimming test (FST). Results showed that rats treated with 500 mg/kg BW date extract exhibited enhanced energy production with the highest endurance capacity (632.9±28.7 min). The date-treated rats also had lower blood lactate, indicating efficient energy utilization with lower fatigue as compared to normal and fatigued rats. Moreover, the serum lactate dehydrogenase levels in 250 and 500 mg/kg BW date extracts and caffeine groups were significantly lower compared to the fatigued rats. Creatine kinase activity was the lowest in the group fed with 500 mg/kg BW date extract. The treated rats showed amelioration of lipid profiles, while the histological study revealed improvement in various organs. Rats treated with 500 mg/kg BW date extract demonstrated an enhancement in energy production and improved energy metabolism which could be due to the presence of bioactive compounds in the dates. Piyarom date extract demonstrated anti-fatigue properties and could be used as a functional ingredient in the development of beverages or snacks that address fatigue-associated maladies.

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1. Introduction

Fatigue is a common, non-specific symptom experienced by many people and demonstrated in different health conditions. Physical fatigue is associated with a reduction in performance as a result of inefficient energy utilization (Kim et al., 2002; Wan et al., 2017) and is further defined as an overwhelming sense of tiredness, lack of energy, and a feeling of exhaustion that is related to difficulty in initiating or sustaining voluntary activities (Kadum et al., 2018; Li et al., 2020). Fatigue increases oxidative stress that can damage cells and increase the risk for the development of various chronic diseases (Lee et al., 2018; Osman and Mohamed, 2018).

Ergogenic property is the ability of substances to enhance energy production and utilization (Halim et al., 2018). For example, athletes and active people benefit from ergogenic aid that helps to boost their competitive performance and to improve energy utilization (Kreideret al., 2010; Silver, 2001). Ergogenic aid helps to improve the production of energy and thus prevent fatigue by lowering oxidative stress and improving health in general (Halim et al., 2017; Osman and Mohamed, 2018). There is an increasing trend toward the use of natural products as an ergogenic aid to replace and/or reduce the use of synthetic compounds in preventing oxidative stress-related fatigue.

Dates (Phoenix dactylifera Linn and cistanche) are rich in various bioactive compounds and have been reported to be an effective part of a diet that helps to protect against chronic diseases (Al-Alawi et al., 2017). Moreover, dates are known to be rich in antioxidant compounds such as phenolics including flavonoids, phenolic acids, ascorbic acid, tocopherols, and carotenoids (Al-Farsi and Lee, 2008; Shabani et al., 2016). In previous studies, we demonstrated that Piyarom and Rabbi dates exhibited the highest antioxidant, antimicrobial and anti-elastase activities among five tested varieties of dates (Kadum et al., 2019). The beneficial activities noted in the studies could be attributed to the identified metabolites in the dates which include ascorbic acid, epicatechin, citric acid, and gallic acid. This outcome was done based on a correlation study from Partial Least Squares biplot obtained from a metabolomics study of the different date extracts (Kadum et al., 2019). The above-mentioned antioxidants are known to exhibit radical scavenging activity which is crucial in alleviating oxidative stress and associated fatigue (Zhu et al., 2021). In addition, we also showed that Piyarom dates consist of adequate levels of electrolytes and sugars that are known to have ergogenic attributes (Kadum et al., 2018). In the present study, we aimed to evaluate the anti-fatigue and ergogenic properties of Piyarom date extract and determine its effects on the energy metabolism of fatigue-induced Sprague Dawley rats.

2. Materials and methods

2.1. Plant materials and extraction

Piyarom dates were purchased from a local store in Kuala Lumpur, Malaysia, and were free from defects and contaminants. The seeds were removed, and the edible parts were cut into pieces and dried at 40°C. The dried pieces were then ground into powder, aseptically dispensed into sterile plastic bags, and stored at −80°C. Extraction was carried out using 80% ethanol at 25°C for 24h as optimized in the previous study (Kadum et al., 2019). The solvent was evaporated using a rotary vacuum evaporator (EYELA A-1000S, USA) at 40°C, and the resultant extract was freeze-dried and stored in dark bottles at 4°C.

2.2. Animal experimentation

Male Sprague Dawley rats (aged 5-6 weeks, average body weight of204.5 g) were purchased from the Animals Resource Unit (ARU), Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM), and approval for the study was obtained from the Animal Ethics Committee ofUPM (UPM\IACUC\AUP-R037\2017). The rats were allowed to acclimatize to the standard laboratory conditions for 1 w and were also given free access to tap water and chow diet throughout the one-week period. They were randomly divided into groups including Normal Group (NG), Normal Fatigue Group (NFG), Group subjected to 250 mg/kg BW DateExtract (LDFG) (250 Date), Group subjected to 500 mg/kg BW of date extract (500 Date) and Group subjected to 5 mg/kg BW of caffeine (5C).

2.3. Experiment design

Male Sprague Dawley rats (n = 60) were randomly divided into two clusters as follows: cluster 1 (NG) (n = 12) which was named the nonfatigue control group; and cluster 2 (n = 48) which was subjected to the Forced Swimming Test (FST). Once becoming fatigued, the rats in cluster 2 were divided into four different groups. Two groups were fed with date extract at 250 mg/kg BW (low dose), and 500 mg/kg BW (high dose) as described in a previous study (Sheikh et al., 2016). One group served as a positive control and was fed 5 mg/kg BW caffeine, and a control group was subjected to the FST but did not receive any treatment. The animals were fed their diets, and dosages of the date extract were administered through gastric intubation. Date extract and caffeine were given to the respective groups continuously every day for 8 w per the study design.

2.4. Forced swimming test

The rats were subjected to the FST in order to induce fatigue and determine their endurance capacities following the method described by Prasad and Khanum (2012). Briefly, a water tank containing water 50 cm in depth and maintained at 25 ± 2°C was used. Steel washers weighing approximately 7% of each rat’s body weight were tied to the tails. The animals were then carefully placed into the tank and the time is taken for each one to swim before exhaustion was recorded. Exhaustion/fatigue was determined when the rat’s head failed to surface within 7 s. The experiment was repeated every 2 w over a period of 8 w of treatment.

2.5. Blood and organ collection

Fasting blood samples were collected from the rats at weeks 0 and 4by retro-orbital vein collection, and, at the end of the treatment (week8), by cardiac puncture under general anesthesia using ketamine and xylazine. All of the blood samples were transferred into EDTA-containing tubes, centrifuged at 3500 rpm at room temperature for 15 min, and plasma was collected and stored at -80 °C until further analysis. After8 w of treatment, the organs (liver, kidney, heart, lung, muscle) were collected for analysis.

2.6. Biochemical analyses

Various biochemical parameters related to energy metabolism were measured in this study. Serum was used to analyze glucose, serum urea nitrogen (SUN), lactate (Lac), lactate dehydrogenase (LDH), creatine kinase (CK), and creatine (Cr). All biochemical parameters were assayed based on standard procedures provided by commercially available assay kits (Span diagnostic Ltd., India). The units for SUN andCr were mg/dL, while LDH and CK were expressed in IU/L. Serum lipid profiles were also assayed (triglycerides, TG, total cholesterol, TC, low-density lipoprotein cholesterol, LDL-C, and high-density lipoprotein cholesterol HDL-C). Liver and kidney function tests were performed, and their assayed markers were alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT). The Roche/Hitachi Cobas C system procedure was followed.

2.7. Histological study of selected organs

The collection of organs of interest (liver, kidney, heart, and muscle), was followed by weighing and then storage in 10% formalin. The heart tissue was cut transversely in order to obtain ventricular sections, the four-chamber cross-sections and the liver and muscle tissues (soleus) were minced and dipped in paraffin and then hematoxylin and eosin (H&E) for staining. The tissue examination by a veterinary pathologist (Huang et al., 2015) was conducted under a light microscope equipped with a CCD camera (BX-51, Olympus, Tokyo, Japan).

2.8. Statistical analysis

One-way ANOVA followed by Tukey’s HSD test was used to obtain statistical differences between the samples and the controls. Results were expressed as mean ± standard deviation (SD). A difference in the mean values with p < 0.05 was considered to be statistically significant, and obtained data was analyzed using MINITAB version 16 (Minitab, Inc., State College, Pennsylvania, USA).

3. Results and discussion

3.1. Food intake and water consumption

Results of the study showed no significant difference in the food and water intake among the rats (Fig. 1 A and B). However, rats fed with date extract showed a slight body weight gain throughout the experiment (Fig. 1 C).

3.2. Forced swimming test

The Forced Swimming Test (FST) represents a valid animal model for testing the anti-fatigue property of bioactive compounds (Prasad and Khanum, 2012; Hao et al., 2014; Halim et al., 2017). The FST results clearly showed an anti-fatigue effect of the date extract on the rats during swimming (Fig. 2). The rats receiving the higher dose of date extract (500 mg/kg BW) exhibited the longest endurance capacity of approximately 632.9 ± 28.7 s, which was significantly (p < 0.05) higher compared to that of the groups treated with 250 mg/kg BW extract and 5 mg/kg BW caffeine. Interestingly, the enhanced swimming capacity of the rats fed with date extract was observed as early as the fourth week of the experiment. The prolonged swimming time of the rats indicated higher resistance towards fatigue during the FST (Tanaka et al., 2003; Ma et al., 2007). Similar results have been reported by several researchers who tested the anti-fatigue potential of plant extracts (Prasad and Khanum, 2012; Xu et al., 2013). For instance, in one previous study, rats fed with holy basil (Ocimum sanctum) exhibited better performance against fatigue and prolonged their swimming times (Singh et al., 2012b). In a recent study, Peruvian ginseng (Lepidium meyenii) fed to mice at three different doses for 28 d resulted in anti-fatigue effects, and the researchers suggested that L. meyenii was an effective ergogenic aid (Zheng et al., 2018). In another report, four different doses of moringa (Moringa oleifera) extracts were given to rats in order to test their endurance capacity. In that study, the extract was found to have delayed the fatigue time for the rats and enhanced their swimming capacities (Lamou et al., 2016). In the present study, dates have been shown to exhibit potential for application in energy drinks and are comparable to other natural plant extracts.

3.3. Blood glucose, lactate, and lactate dehydrogenase levels

The extent of fatigue in muscles after exercise can be measured by various biochemical markers including glucose, lactate, and lactate dehydrogenase. The blood glucose levels of the rats after 8 w of treatment were thus measured in order to determine the effects of the date extract. The results showed no significant differences in glucose levels among the rat groups except for the Normal Fatigue Group (NFG) which signified- cantly (p < 0.05) lower (Table 1). The results indicated protective effects of the date extract that may be attributed to the ergogenic property of dates that is ostensibly as good as that of caffeine. In skeletal muscles, glucose is the fuel for energy generation during exercise, and glycogen supplies the bulk of it through glycogenolysis in order to maintain the fellow of glycolysis for energy generation. It has been shown that nutritional interventions via the consumption of dates may help in boosting or maintaining muscle glycogen concentrations both before and during exercise (Li et al., 2018, 2017). However, it is important to keep blood glucose levels lower than 6 mol/L in order to avoid complications from diabetic conditions that may occur when hyperglycemia is allowed to persist over a period of time. In a previous study, mice fed with Cistanche deserticola extract for 3 w showed increased blood glucose levels which resulted in improved energy levels (Cai et al., 2010). In another study, the extract of Radix Rehmannia was reported to be responsible for anti-fatigue activity due to the increased glycogen stored in the liver and decreased build-up of glucose in the blood as a result of glucose consumption by muscles (Liu and Liu, 2016; Tan et al., 2012).

Blood lactate, a crucial marker for fatigue during heavy exercise, is used to measure an existing pathology in a routine stress test (Halim et al., 2017). Lactic acid is a product of glycolysis due to anaerobic conditions which is the primary form of metabolism in the muscles during exercise (Fu et al., 2010; Sun et al., 2014). Lactate accumulation is considered to be a major inducer of fatigue, and thus the inhibition of lactate accumulation or the reduction of lactate levels represents an anti-fatigue effect (Blasiak et al., 2014). The results obtained in the current study demonstrated a significant (p < 0.05) reduction in lactate levels between rat groups treated with date extract and caffeine as compared to that of normal and normal fatigue groups (Table 1). The lactate levels were 10.75 ± 0.90, 11.08 ± 0.26, and 12.09 ± 0.761 mmol/L for the rat groups treated with 500 mg/kg BW, caffeine 5 mg/kg BW, and 250 mg/kg BW date extract, respectively. The lactate levels for the normal group and fatigue groups were 14.41 ± 0.45 and 16.35 ± 0.18 mmol/L, respectively. The results indicated positive effects from the date extract that caused the reduction in lactate levels in the rat groups. The observed effects could be attributed to the improved performance of the rats during the FST. This result, in turn, could be due to the reduction in muscle damage caused by the lower lactate accumulation. In a previous

study, rats fed with Amarkand extract had decreased blood lactate and increased endurance associated with fatigue (Narkhede et al., 2016). The previous report is in agreement with the findings of the present study, indicating the anti-fatigue activity of different plant extracts that we're able to decrease blood lactate levels in the rats, and thereby improve the animals’ performance.

The presence of high levels of LDH in the blood is an indication of excessive muscle use and is thus a marker for fatigue (Halim et al., 2017). Excessive LDH is also an indication of necrosis in cells and damage to tissues (Li et al., 2017). Our results showed that FST significantly (p < 0.05) increased the blood LDH levels of the normal fatigue rats (588 ± 13.30 U/L) which was significantly (p < 0.05) higher than that of rats fed with 500 mg/kg BW of date extract (297 ± 29.21 U/L), the lower dose (316.16 ± 27.58 U/L) of date extract and caffeine (305 ± 25.91 U/L) after 8 w treatment. It can thus be suggested that the date extract was able to enhance energy production in rats and consequently reduced damage to the rat muscle. Our results agreed with that reported previously by Narkhede et al., (2016). In that study, Amarkand extracts were given to Wistar rats, and after 2 w, there was a significant reduction in the animals’ blood LDH levels. Similar results have also been reported in another study in which fatigued rats treated with water extract from fermented rice bran showed a decrease in serum LDH levels (Kim et al., 2002).