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Biological Activities of Ashwagandha (Withania somnifera L.) Roots and their Effect on the Neurological Complications of Obesity in Rats

Yousif A. Elhassaneen*, Reem A. Boraey, Amal Z. Nasef

Department of Nutrition and Food Science, Faculty of Home Economics, Menoufia University Shebin El-Kom, Egypt

Abstract: The current study aims to see if eating Ashwagandha roots can help with neurological issues in an obese rat model. This study will also look at the biochemical roles of Ashwagandha roots. Alkaloids were shown to be the most prevalent bioactive chemicals in Ashwagandha roots, followed by triterpenoids, carotene, flavonoids, tannins, total phenolics, and saponins. Furthermore, the hydro-ethanolic extract of Ashwagandha roots demonstrated a wide range of biological properties, including antioxidant and free radical scavenging activity, suppression of low-density lipoprotein, and antibacterial activity. Normal rats fed diet-induced obesity (DIO) (model control) had higher body weight (BW), feed intake (FI), and feed efficiency ratio (FER) than the control group. At the end of the trial (8 weeks), rats in the normal group had BW, FI, and PER values of 0.91%, 13.10 g/day/rat, and 0.076, respectively, whereas these values had grown by rates of 54.95, 23.82, and 21.05% in the model control group. Intervention with Ashwagandha roots (2, 4, 6, and 8 g/100g diet) in feeding rats for 8 weeks resulted in a significantly (p0.05) lower BWG, FI, and FER, improved serum lipid profile parameters, decreased liver functions, improved neurological disorders (dopamine and serotonin content and acetylcholine esterase activity), and positively manipulated obesity-related histopathological changes in brain and adipose tissues. These data support the use of Ashwagandha roots for obesity treatment and prevention. Furthermore, the findings suggest the benefits of dietary changes, such as Ashwagandha root supplementation, in reducing the complications linked with obesity, such as neurological problems.

Keywords: body weight, organs weight, liver functions, serum lipid profile, acetylcholine esterase, dopamine, serotonin

Received August 18, 2023; Revised September 20, 2023; Accepted September 27, 2023

Citation: Yousif A. Elhassaneen, Reem A. Boraey, Amal Z. Nasef, Biological Activities of Ashwagandha (Withania somnifera L.) Roots and their Effect on the Neurological Complications of Obesity in Rats. American Journal of Food and Nutrition, 11 (3). 71-88. January 2023. doi:10.12691/ajfn-11-3-3

1. Introduction

Obesity is a complicated condition caused by improper management of the body's energy balance, which results in excess adipose tissue accumulation. In Organization for Economic Cooperation and Development member nations, more than half of adults are overweight, and 19.5% of adult populations are obese. [1]. Obesity is responsible for around 4.7 million premature deaths per year. It was placed fifth among the main avoidable causes of mortality, accounting for 12.3% of all global fatalities and 8.4% of total disability-adjusted life years (DALYs) lost to non-communicable illnesses. [2,3]. Obesity is well established to be a risk factor for a variety of cancers, including colon, thyroid, pancreatic, hepatic, and uterine cancer, as well as diabetes mellitus and cardiovascular disease. [4,5,6]. Also, epidemiological studies revealed that obesity is identified as a risk factor for neurological disorders such as Alzheimer's disease [7,8]. Thus, the attention of scientists around the world turned to innovating many pharmaceutical methods to prevent or treat obesity, but what was developed represented a small number of effective and safe products from a therapeutic standpoint [9]. Also, most pharmaceutical therapy is expensive and accompanied by many adverse effects, leading to patient non-compliance. Hence, there was an urgent need to look for alternative remedies mainly, from natural sources, since they are cost-effective and have low side effects. In this direction, many studies indicate that applying the use of medicinal plants to prevent or treat obesity is an effective and safe method with great economic advantages [10,11].

Ashwagandha (Withania somnifera L., Family: Solanaceae) is a xerophytic plant found in the dried parts areas of the world including and is distributed in the Mediterranean regions in including Egypt [12,13,14]. In Unani system of medicine, roots of Ashwagandha are employed for the therapeutic characteristics. However, leaves of the plant are also said to be used for medicinal purposes [15]. Fresh roots are harvested from January to March, placed in the shade, and dried for several days. The medicine maintains its therapeutic effectiveness for a maximum period of less than two years [15-17]. This plant roots includes several hundred typical phytochemicals like phenolics, steroidal, saponins, alkaloids, glycosides and volatile oils [18]. Such compounds possess several biological roles including antioxidant and anti-inflammatory properties as well as leading role in Ashwagandha roots therapeutic effects [18-20]. It has been used to treat infertility, the immune system, cardiovascular disease and nervous issues [19,21]. Ashwagandha roots also have the ability to treat arthritis, nervous system disorders, hyperlipidemia, diabetes, attention deficit disorder, hiccups, hyperactivity, bronchitis, sleep deprivation, back pain, tumors, Parkinson's disease, tuberculosis, and irregular menstruation. And chronic liver disease. [20,22]. Furthermore, various studies have explored the preventive impact of medicinal plants on several obesity consequences, such as serum lipid profile, hyperglycemia, cardiovascular disease, atherosclerosis, and liver and kidney functions in rat and human models. [10,11,23]. But till now, there is a dearth of information related to the effect of medicinal plants including Ashwagandha consumption on neurological complications of obesity. To fill such information gap, we designed this study to investigate whether consumption of Ashwagandha roots would alleviate neurological disorders in obesity rat model. Also, the biological roles of Ashwagandha roots will be in the scope of this investigation.

2. Materials and Methods

2.1. Materials

2.1.1. Ashwagandha Samples

Dried Ashwagandha (Withania somnifera L.) root samples were acquired by Agriculture Seeds, Spices, and Medical Plant Company (Harraz), El-Darb El-Ahmar, Cairo Governorate, Egypt. Samples were validated in the Department of Agricultural Plant, Faculty of Agriculture, Menoufia University, Shebin El-Kom, Egypt.

2.1.2. Chemicals and Machines

Scale of biologically active compounds [gallic acid (GA), vanellic acid (VA), catechine (CA), linalool, ursolic acid and butylated hydroxytoluene (BHT), DDPH (2,2-diphenyl-1-picrylhydrazyl), AAPH [2,2'-Azobis(2-methylpropionamidine) dihydrochloride] and dimethyle sulfoxide (DMSO)were purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals (Except as otherwise mentioned), solvents and reagents were of analytical quality were purchased from El-Ghomhorya Company for Trading Drug, Chemicals and Medical Instruments, Cairo, Egypt. Throughout this investigation absorbance for different assays were determined using UV-160A; Shimadzu Corporation, Kyoto, Japan.

2.2. Methods

2.2.1. Preparation of Ashwagandha Roots Powder

According to Gharib et al. [24], Ashwagandha root samples were cleaned manually and sorted to remove foreign bodies before being dried in a hot air oven (Horizontal Forced Air Drier, Proctor and Schwartz Inc., Philadelphia, PA) at 90°C until the moisture content in the final product reached 10%. The dried materials were ground to a fine powder using a high-speed mixer (Moulinex Egypt, ElAraby Co., Benha, Egypt). The material that went through a sieve with a mesh of 70 was kept in tight glass bottle and stored in refrigerator at 4°C for using in different chemical and biological experiments later.

2.2.2. Preparation of Ashwagandha Roots Hydro-Ethanolic Extract

Ashwagandha roots hydro-ethanolic extract was prepared according to the described manner by Abd Elalal et al., [25]. In brief, 20 g of Ashwagandha roots powder were extracted with 180 ml ethanol/water (80:20) on an orbital shaker (Unimax 1010, Heidolph Instruments GmbH & Co. KG, Germany) for 120 min at 70°C. The resulting mixture was then filtered (Whatman No. 5) via a Buchner funnel, and the remaining solvents were evaporated using a rotary evaporator (Laborata 4000; Heidolph Instruments GmbH & Co. KG, Germany) at 40°C. The extracted solution was kept in a sealed glass container at 4°C until use.

2.2.3. Bioactive Compounds Determination in Ashwagandha Roots Powder

The total phenolics in Ashwagandha root powder were measured using the Folin-Ciocalteu reagent, as described by Singleton and Rossi [26], and Wolfe et al. [27]. The data are expressed as gallic acid and equivalents (GAE). The total carotenoids were calculated using Litchenthaler's technique [28]. The total flavonoid content was calculated using Zhisen et al.'s colorimetric technique. [29]. Gallic acid and equivalents are used to express the results (GAE). Tannins were determined using the Van-Burden and Robinson [30] technique. Gallic acid (GA) was utilized as a standard to create the standard curve, which was then used to estimate the tannin concentration. The Harbome technique [31] was used to detect alkaloids. The saponin content was calculated using the Fenwick and Oakenfull technique [32]. Gallic acid was utilized as a standard to create the standard curve, which was then used to calculate the saponin concentration of the sample. Triterpenoids were isolated and quantified using the technique described by Schneider et al. [33]. Ursolic acid was employed as a control, and the findings were represented in milligrams of ursolic acid.100 g⁻¹.

2.2.4. Antioxidant Activity Determination

2.2.4.1. Antioxidant Activity

Antioxidant activity (AA) of Ashwagandha roots hydro-ethanolic extract (ARE) and standards (α-tocopherol and butalated hydroxytoluene, BHT) were measured using the β-carotene bleaching test, as reported by Marco, [34].

2.2.4.2. Assay for Radical Scavenging DPPH

Desmarchelier et al. [35] method used the DPPH radical scavenge experiment to investigate the free radical scavenging activity of Ashwagandha roots hydro-ethanolic extract (ARE).

2.2.4.3. Inhibition of low Density Lipoprotein (LDL) Oxidation

The inhibition of LDL oxidation was discovered in Ashwagandha roots hydro-ethanolic extract (ARE) according to the method of Princen et al., [36].

2.2.4.4. Assays for Antibacterial and Antifungal

As test microorganisms, Escherichia coli, Staphylococcus aureus, and Candida albicans (from the Microbiology Department, Faculty of Agriculture, Damietta University, Damietta, Egypt) were employed. Spooner and Sykes [37] devised agar cup techniques for determining antibacterial and antifungal properties in Ashwagandha root hydro-ethanolic extract (ARE).

2.2.5. Biological Experiments

2.2.5.1. Ethical Approval

The current study's biological experiments were ethically approved by the Scientific Research Ethics Committee, SREC (Animal Care and Use), Faculty of Home Economics, Menoufia University, Shebin El-Kom, Egypt (Approval # 26- SREC- 11-2020).

2.2.5.2. Animals

Adult male albino rats, Sprague Dawley strain (1507.6 g each), were procured from the Research Institute of Ophthalmology, Medical Analysis Department, Giza, Egypt, for use in this study.

2.2.5.3. Diets

The baseline diet (BD) was made using the following AIN, [38] recipes: casein/protein (10%), corn oil (10%), vitamin mixture (1%), mineral combination (4%), choline chloride (0.2%), methionine (0.3%), cellulose (5%), and corn starch (69.5%). DIO was made according to Research Diets, Inc. NJ, USA, as follows: casein, 80 mesh (23.3%), L-cystine (0.35%), maltodextrin (11.65%), corn starch (8.48%), sucrose (20.14%), soybean oil (2.91%), animal/lamb fat (20.69%), mineral mixture (1.17%), dicalcium phosphate (1,52%), calcium carbonate (0.64%), and potassium citrate.1 H₂O (1.92%), vitamin blend (1.17%), and choline bitartrate (0.23%). Salt and vitamins mixtures recipes were constituted such as mentioned by Reeves et al., [39].

2.2.5.4. Experimental Design

Biological experiments were carried out in accordance with the rules of the Institute of Laboratory Animal Resources, the Commission on Life Sciences, and the National Research Council. [40]. Rats (n=36) were housed separately in wire cages in a room kept at 25 °C and under typical healthy circumstances. For accommodation, all rats were fed BD for two weeks before the trial began. After two weeks, the rats were divided into two main groups, the first being a normal control group (Group 1, 6 rats) that was still fed on BD, and the other being an obesity induction group (30 rats) that was fed with DIO for eight weeks and was classified into five sub groups as follows: group (2), model control, fed on BD only as a positive control (rats with obesity), and groups (3-6) fed on BD containing 2 to 8% Ashwagandha roots powder (ARP). Many of the prior research' data [10,41] were used to select ARP doses for tests. For 8 weeks, each of the aforementioned groups was housed in a separate cage.

2.2.5.5. Biological Evaluation

During the 56-days trial, the diet was recorded every day and the body weight was recorded every week. According to Chapman et al., [42], the body weight gain (BWG, %), food intake (FI), and food efficiency ratio (FER) were calculated using the following equations: FER = Grams growth in body weight (g/56 day)/ Grams feed intake (g/56 day), BWG (%) = (Final weight - Initial weight)/ Initial weight×100.

2.2.5.6. Blood Sampling

At the end of the experiment, after twelve hours of fasting, rats were scarified under ethyl ether anesthesia, and blood samples were obtained from rats via the abdominal aorta. According to Drury and Wallington [43], blood samples were placed in clean, dry centrifuge tubes and allowed to coagulate at 4°C before being spun for ten minutes at 3000 rpm to separate the serum. Serum was aspirated carefully, transferred to clean glass tubes, and kept frozen at -20°C until analysis. Internal organs (liver, heart, lungs, kidneys, spleen, and brain) were also removed immediately after the rats were sacrificed, weighted, and submerged in 10% neutral buffered formalin for histological investigation.

2.2.6. Hematological Analysis

2.2.6.1. Liver Functions

Liver functions were determined using the following methods: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities according to Yound, [44], Tietz, [45], and Yound, [44], respectively.

2.2.6.2. Blood Lipid Profile

Triglycerides (TGs), Total cholesterol (TC) and HDL-Cholesterol were determined in serum according to the methods of Fossati and Prenape [46], Richmod [47], and Lopes-Virella et al., [48], respectively. Low density lipoprotein cholesterol (LDL-c) was assayed according to the equation of Fniedewald et al., [49] as follow: LDL-c = TC – (HDL-c + TGs/5).

2.2.6.3. Level of Serotonin and Dopamine

Levels of serotonin and dopamine in serum were assayed according to the method of Mahood and Hamzah, [50] and Mitra and Guha, [51], respectively.

2.2.6.4. Acetylcholinestrase (AChE) Determination

Gowenlock's [52] colorimetric approach was used to measure serum AChE activity (Rappaport units/mL).

2.2.7. Histopathological Examination

Internal organ specimens (adipose tissue and brain) were collected immediately after slaughtering animals and submerged in 10% neutral buffered formalin. Fixed specimens were then trimmed and dehydrated with increasing concentrations of alcohol, cleared in xylene, embedded in paraffin, sectioned (4-6 µm thickness), stained with hematoxylin and eosin and examined microscopically [53].

2.3. Statistical Analysis

All analyses were performed in triplicate, and results were represented as mean± standard deviations (SD). The student t-test and the MINITAB 12 computer software (Minitab Inc., State College, PA) were used for statistical analysis. A P 0.05 result was deemed to be statistically significant.

3. Results and Discussion

3.1. Bioactive Compounds in Ashwagandha Root Powder (ARP)

Bioactive compounds (total phenolics, flavonoids, carotene, tannins, saponins, triterpenoids and alkaloids) were determined in Ashwagandha root powder (ARP) as shown in Table (1). The most abundant were alkaloids, followed by triterpenoids, carotene, flavonoids, tannins, total phenolics, and saponins. Such findings are consistent with Munir et al.'s [54] observations on the effects of several bioactive constituents in Ashwagandha extract are flavonoids, tannins, alkaloids, saponins, steroids, glycosides and triterpenoids. Also, The concentrations of total phenolics and flavonoids in Ashwagandha extract were significant (P.05) at 148.79 ± 6.39 μg CE/g and 372.81 ± 7.71 mg GAE/g, respectively. Gulati et al. [55] have observed the presence of alkaloids and tannins in Ashwagandha root extract. Furthermore, Saxena et al. [56] discovered that the roots of Ashwagandha contain a variety of essential elements such as alkaloids, steroids, starch, reducing sugars, amino acids, volatile oils, glycosides, and alkaloids. Several previous studies have demonstrated that bioactive compounds such as phenolics, flavonoids, carotenoids, and triterpenoids found in Ashwagandha play important roles in the prevention and/or treatment of many diseases such as diabetes, cancer, atherosclerosis, obesity, bone, and aging [10,11,57-61]. All of the these effects compounds are due mainly to their biological activities including antioxidant. Also, several studies have reported that phenolic compounds and flavonoids act as antioxidative, anticancer, antibacterial, cardio-protective, anti-inflammatory, and immune-stimulating agents [24,25,62]. Furthermore, the most significant groups, such as flavonoids as natural antioxidants, are crucial natural phenolic agents. The findings demonstrated that these natural flavonoids have the ability to detoxify reactive oxygen species (ROS) as well as limit the oxidation of low-density lipoproteins [63]. Carotene/carotenoids, on the other hand, are significant because of their nutritional and technical features, particularly their anti- and pro-oxidant capabilities [64]. Okwu and Okwu [65] discovered that alkaloids present in or produced from medicinal plants such as Ashwagandha have therapeutic uses such as analgesic, antimicrobial, and antispasmodic. Other bioactive chemicals, such as triterpenoids, have been shown in preclinical animal models to have cytotoxicity against a range of tumor cells as well as anticancer effectiveness. [66] Bishayee et al. Saponins, triterpenoid glycosides found in Ashwagandha, have been shown to stimulate the mammalian immune system, sparking interest in their potential as vaccine adjuvants [67].

Table 1. Total content of bioactive compounds in Ashwagandha root powder (ARP)

3.2. Biological Activities of Ashwagandha Root Hydro-Ethanolic Extract (ARE)

Ashwagandha root hydro-ethanolic extract showed considerable antioxidant activity (AA, 74.45%) and even compared well with the most common standards even i.e. α -tocopherol and butalyted hydroxytoluene (Table 2). In general, antioxidant activity was measured using β-carotene bleaching (BCB), which is based on the coupled oxidation of β-carotene and linoleic acid and estimates the relative ability of antioxidants to scavenge the radical of linoleic acid peroxide (LOO•) that oxidizes (loses the double bonds) in the emulsion phase. The BCB approach has been used to assess antioxidant activity in a wide range of plant components, including medicinal plants [59,41,61,68–73]. All of this research indicated that polyphenols, flavonoids, carotenoids, and triterpenoids, which are abundant in Ashwagandha, were substantially linked with antioxidant activity. Zheng et al., [74] in the same situation, It has been found that the presence of phenolic compounds in Ashwagandha contributes to its considerable antioxidant capacity. Also, the most significant categories of natural antioxidants, such as flavonoids, are key natural phenolic agents. According to research, these natural flavonoids have the ability to detoxify ROS and also limit the oxidation of low-density lipoproteins [63]. Furthermore, Jin et al. [75] discovered a link between antioxidant activity in plant components and phenols, or alkaloids.

Table 2. Antioxidant activity of Ashwagandha root hydro-ethanolic extract (ARE)

3.3. DPPH Radical Scavenging Activity

DPPH radical scavenging activity (%) and half maximal inhibitory concentration (IC₅₀) of Ashwagandha root hydro-ethanolic extract (ARE) and standard (Butylated hydroxytoluene, BHT). are illustrated in Figure 1. Such data indicated that ARE possessed the high activity compared to the BHT. The radical scavenging activity of ARE at a concentration of 100 μg/mL was 77.2%, whereas BHT standard was 89.43%. For the IC50, ARE was recorded 15.07 μg/mL while BHT standard was 10.73 μg/mL. In general, The radical-scavenging activity of the DPPH test measures diene conjugation absorption in the presence of the reagent (DPPH, 2,2-diphenyl-1-picrylhydrazyl) [76]. According to survey research, the DPPH test has been effectively utilized to evaluate the scavenging activity of various plant sections, including medicinal plants [60,70,71,77-80]. In Ashwagandha, Kumar and Dinesh, [81] found that ethanolic extract had significant (p<0.01) DPPH- free radicals scavenging (82%) which is due to the high polyphenolic content. Also, Kateina et al., [82] discovered that alkaloids present in Ashwagandha were examined using the DPPH assay and demonstrated that some of them were either equivalent to or much more active than typical antioxidants, with the quantity of aromatic hydroxyl groups appearing to be the key predictor of activity. The current study's findings demonstrated that the free radical scavenging ability of ARE might be employed to avoid the negative effects of free radicals in a variety of disorders, including obesity, diabetes, cancer, neurological, pulmonary, renal, and cardiovascular disease.

Figure 1. DPPH radical scavenging activity (%) and half maximal inhibitory concentration (IC₅₀) of Ashwagandha root hydro-ethanolic extract (ARE) and standard (Butylated hydroxytoluene, BHT). Each value is the average of three replicates

3.4. Inhibition of Low Density Lipoprotein (LDL) Oxidation

Dose-dependent inhibition of CuSO4-induced LDL oxidation in vitro by Ashwagandha root hydro-ethanolic extract (ARE) and standard (caffeic acid, CA) was shown in Figure (2). These findings suggest that ARE has an inhibitory effect on CuSO4-induced LDL oxidation, as seen by a dose-dependent reduction in conjugated dienes formation. Data from this study and others demonstrated that the inhibitory action of ARE was due to the high amount of bioactive chemicals as antioxidants (polyphenols, flavonoids, carotenoids, alkaloids, triterpenoids, and so on) [60,67] [70,71] [79,80,81] [83,84]. Also, Jin et al., [75] The researchers evaluated the relationship between the antioxidant activity of plant parts and bioactive chemicals such as lipid peroxidation inhibition ability (LPIA), total phenolic contents, total alkaloid contents, and total sterol contents. In general, ROS-induced oxidative stress causes cellular abnormalities/damage to membranes (cell wall, mitochondria, lysosomes, etc.) by peroxiding lipid moieties, particularly polyunsaturated fatty acids, in a chain process known as lipid peroxidation [85]. As a result, the prevention of lipid peroxidation is regarded as the most significant indicator of antioxidant activity. The current study's findings suggest that ARE can protect cells from free radical damage by slowing the chain events that produce lipid peroxidation. In this manner, data of experimental animals indicated that bioactive compounds such as found in ARE including polyphenols, flavonoids, carotenoids, alkaloids and triterpenoids demonstrated LDL oxidation protection by raising the amounts of reduced glutathione (GSH) and glutathione reductase (GSH-Rd) in the liver and lungs, as well as an increase in prevention of NADPH-dependent lipid peroxidation [11,60] [86,87,88]. Also, fractions extracted from the roots of Ashwagandha demonstrated dose-dependent anti-stress efficacy in mice [89]. Furthermore, it was shown that combining Ashwagandha with other medicines at rats provided protection against a number of biological, physical, and chemical stresses [90,91]. On the other side, a number of scientists discovered that the concept of oxidative alteration of lipoproteins posits that LDL oxidation plays a crucial role in early atherosclerosis [92,93]. The current study's findings demonstrated that ARE might be employed successfully as a potential drug in the prevention of atherosclerosis by slowing the LDL oxidation process.

Figure 2. Dose-dependent inhibition of CuSO4-induced LDL oxidation in vitro by Ashwagandha root hydro-ethanolic extract (ARE) and standard (caffeic acid, CA) Results are expressed as the area under the curve (AUC)

Table 3. The correlation coefficients (r2) between bioactive compounds content measured and antioxidant activities of Ashwagandha root hydro-ethanolic extract (ARE)

Table 4. Antibacterial and antifungal activities of Ashwagandha root hydro-ethanolic extract (ARE)

Table 5. Effect of a dietary intervention with Ashwagandha root powder (ARP) on body weight (BW), feed intake (FI) and feed efficiency ratio (FER) of obese rats

3.5. Relationship Between Bioactive Compounds Content and Antioxidant Activities of Ashwagandha Root Hydro-Ethanolic Extract (ARE)

Table 3 shows the correlation coefficients (r²) between the bioactive components concentration and antioxidant activities of Ashwagandha root [β-carotene bleaching rate, BCB, free radical scavenging efficiency, DPPH-RSA, and suppression of low-density lipoprotein (LDL) oxidation]. The concentration of phenols, flavonoids, carotenoids, saponin, triterpenoids, alkaloids, and tannins in Ashwagandha root exhibited a significant (p≤0.05) and substantial positive connection with all antioxidant activity testing. Tannins also demonstrated a significant (p≤0.05) and somewhat favorable connection for the same relationship. The current statistics are consistent with the findings of Woldegiorgis et al. [94] who discovered a clear association between total phenolic and total flavonoid components in quantitative estimations as well as antioxidation efficacy. Also, Paul, [95] It was shown that Ashwagandha root has substantial abilities to scavenge free radicals such as DPPH. The differences observed in the studied relationships may be due relatively to several factors including the composition of each of the active compounds (the number of hydroxyl groups and their distribution in the compound) and the scientific basis on which each test is built on. With the same context, According to Woldegiorgis et al., [94], the radical scavenging test responses are mostly dependent on hydroxylation and structural stability owing to hydrogen donation by antioxidant chemicals found in medicinal plants. Thus, our findings indicate that phenolic compounds, in addition to flavonoids, triterpenoids, and alkaloids, play a more major role in antioxidant activation; nevertheless, other compounds such as carotene, saponin, and tannins were also involved in those biological activities, albeit to a lower extent.

3.6. Antibacterial and Antifungal Activities of Ashwagandha Root Hydro-Ethanolic Extract (ARE)

Antibacterial and antifungal activities of Ashwagandha root hydro-ethanolic extract (ARE) were Table (4). Such data indicated that the activity (inhibition zones) against the gram-positive bacteria Staphylococcus aureus (17.3 mm) and fungus (Candida albicans) were recorded. Also, the ARE showed no efficacy against the Gram-negative bacteria Escherichia coli. These findings are consistent with those obtained by Abd Elalal et al., [70] and Gharib et al., [24] using algae extracts. Finally, the results of this investigation supported the potential use of Ashwagandha as an antibacterial and antifungal agent against gram-positive bacteria.

Figure 3. Effect of a dietary intervention with Ashwagandha root powder (ARP) on body weight (BW), feed intake (FI), and feed efficiency ratio (FER) (as a % of change) of obese rats

Figure 4. Effect of a dietary intervention with Ashwagandha root powder (ARP) on weight organs (as a % of change ) of obese rats

3.7. Biological Part

3.7.1. Effect of a Dietary Intervention with Ashwagandha Root Powder (ARP) on Body Weight (BW), Intake of Feed (FI) and Feed Effectiveness Ratio (FER) of Obese Rats

Data in Table (5) and Figure (3) illustrate the impact of an Ashwagandha root powder (ARP) dietary intervention on body weight (BW), intake of feed (FI), and feed effectiveness ratio (FER) of obese rats. Such data indicated that feeding of rats on DIO (model control) leads to increase the BWG, FI and FER than the normal group. At the end of the experiment (8 weeks), rats of the normal group recorded 0.91%, 13.10 g/day/rat and 0.076 for the BW, FI and FER while these values were increased by the rates of 54.95, 23.82 and 21.05% in model control group. Intervention with ARP (2, 4, 6 and 8 g/100g diet) in feeding rats for 8 weeks led to significantly (p≤0.05) decrease on the BWG, FI and FER of the obese rats (model control) by the rates of 48.35, 40.66, 23.08 and 17.58; 20.61, 15.34, 7.86 and 5.80; and 18.42, 10.53, 5.26 and 3.95% of the normal group, respectively. The rate of decreasing in BWG, FI and FER of the obese rats were exhibited a dose- dependent manner. The present data are corresponding well with several studies reviewed by Raakhee et al., [96] and Ali, [97]. They observed that Ashwagandha boosts the immune system, diverting energy back to the weight reduction process. It also contains antioxidants, which are necessary for weight loss and overall health. Furthermore, some previous studies revealed that the good effects of ARP on obesity control might be due to its high level content of several types of bioactive components such as alkaloids, triterpenoids, carotene, flavonoids, tannins, phenolics, and saponins [10,41,61]. All previous studies with others indicated that the antiobesity effects of ARP could be attributed to a variety of mechanisms, including antioxidant, anti-inflammatory, reduced fat accumulation, decreased leptin and resistin levels, increased lipolysis, increased adiponectin, inhibited adipocyte differentiation, and reduced adipogenesis [71] [96-99].

Figure 5. Effect of a dietary intervention with Ashwagandha root powder (ARP) on liver functions (as a % of change) of obese rats

3.7.2. Effect of a Dietary Intervention with Ashwagandha Root Powder (ARP) on Weight Organs of Obese Rats

Data in Table (6) and Figure (4) were shown the effect of a dietary intervention with Ashwagandha root powder (ARP) on organs body of obese rats. Such data demonstrated that feeding rats DIO (model control) causes an increase in organ weight compared to the normal group. At the conclusion of the trial (8 weeks), the rats from the normal group reported 5.76, 1.34, 0.67, 0.61, 1.31 and 1.61 g for the liver, kidneys, spleen, heart, lungs and brain while these values were increased by the rates of 5.76, 1.59, 2.99, 14.75, 6.11 and 8.07% in model control group. Intervention with ARP (2, 4, 6 and 8 g/100g diet) in rats feeding for 8 weeks led to considerably (p≤0.05) decrease on the organs body of the obese rats (model control) by different rates. The rate of decreasing in BWG, FI and FER of the obese rats were exhibited a dose- dependent manner. In population studies, Mandal et al. [100] revealed that there were statistically significant increases in organ weights with body mass index (BMI) in both men and women. Furthermore, Waxler and Enger [101] discovered that the organs of obese animals are heavier than those of control mice, and this weight gain is seen in the wet, dry, and defatted states. This rise in organ weight in obese animals cannot be explained only by adipose tissue.

3.7.3. Effect of a Dietary Intervention with Ashwagandha Root Powder (Arp) on Liver Functions of Obese Rats

The Effect of a dietary intervention with Ashwagandha root powder (ARP) on liver functions of obese rats was shown in Table (7) and Figure (5). From such data it could be noticed that DIO caused a considerably (p≤0.05) increased in AST, ALT and ALP activities with 77.45, 40.86 and 28.05% compared to normal control group, respectively. Intervention with ARP (2, 4, 6 and 8 g/100 diet) in feeding rats for 8 weeks led to considerably (p≤0.05) reduce the levels of activity of these enzymes which recorded 71.43, 63.73, 57.43 and 55.85% (AST), 38.32, 27.68, 11.04 and 9.13% (ALT), and 23.34, 20.28, 11.37 and 8.30 (ALP), respectively. The rate of reduction in serum liver enzyme activity was exhibited in a dose-dependent manner. In similar study, Ashwagandha at a 10 mg/kg dosage substantially protected rats from CCl₄-induced hepatotoxicity [89,102]. Also, Elhassaneen et al., [72] found that the consumption of bioactive compounds such as found in Ashwagandha on CCl₄ hepatotoxic rats lead to significantly improve in liver functions and histology through its antioxidant mechanism. In several previous studies, different parts rich in phenolic constituents rather than Ashwagandha could lower liver serum enzyme activity through many suggested effects, including blocking the hepatocellular uptake of bile acids, improving the antioxidant capacity of the liver, diminishing the bilirubin concentration, reducing the damage to hepatocytes, and acting as scavengers of reactive oxygen species [71,97,103–105]. Furthermore, Administration of active principles of Ashwagandha consisting of alkaloids was found to increase antioxidant enzymes in liver cells. The antioxidant activity of Ashwagandha alkaloids may explain, at least in part, the recorded anti-inflammatory, immunomodulatory, anti-stress, and anti-aging benefits in experimental animals and clinical settings [106].

3.7.4. Effect of Intervention with Ashwagandha Root Powder (ARP) on Serum Lipid Profile of Obese Rats

Table (8) and Figure (6) indicate the effect of an Ashwagandha root powder (ARP) intervention on the blood lipid profile of obese rats. According to the findings, DOI therapy resulted in a significant (p0.01) rise in serum TGs, TC, LDL-c, and VLDL-c by the ratios of 47.66, 62.58, 135.04, and 47.66% as compared to the control group. Intervention with ARP (2, 4, 6, and 8 g/100 diet) in nutrition rats for 8 weeks resulted in a substantial (p≤0.05) decrease in the levels of serum lipid profile, which documented 42.88, 18.98, 10.87, and 11.32% (TGs); 56.49, 22.96, 9.45, and -2.47% (TC); 121.37, 58.80, 25.89, and -7.35% (LLP); and 42.88, 18.98, 10.87, and 11.32% (VLDL), accordingly. The pace at which serum lipid profile values decreased was dose-dependent. The reverse tendency was also found for HDL-c levels. Obesity was related to hyperlipidemic and hypercholesterolemic states, which raised the serum bad lipid particles (TGs, TC, LDL-c, and VLDL-c) and decreased the good (HDL-c), which was significantly (p0.01) improved by ARP intervention. Several other publications [10,41,61,107-111] observed a similar response with different plant components other than ARP. In general, high blood lipid and lipoprotein levels, particularly LDL and VLDL, increase the risk of cardiovascular disease, fatty liver disease, and diabetes. carcinogenesis, peripheral vascular disease, and atherosclerosis in human [61,112]. Several novel synthetic oral antihyperlipidemic and antihypercholesterolemic medicines have been developed in the same area, but they have negative side effects such as myopathy, a rise in hepatic aminotransferases, and rhabdomyolysis [113]. Data of the present study reported the effectiveness of ARP in improving the blood lipid profile inducing by DIO without any side effects. ARP's beneficial effects on blood lipid profiles may be attributed to a variety of mechanisms, including antioxidant, anti-inflammatory, reduced fat accumulation, decreased leptin and resistin levels, increased lipolysis, increased adiponectin, inhibited adipocyte differentiation, and reduced adipogenesis [71,96-99]. The current study's data, with others, revealed that he antihyperlipidemic and antihypercholesterolemic effects of ARP could be attributed to several processes, such as stimulating the creation of hepatic LDL-c the receptors, which increased clearance of plasma LDL-c and VLDL-c, increasing endogenous cholesterol conversion to bile acid, inhibiting acyl-CoA such as cholesterol acyltransferase, which is a key pathway of the metabolism of lipids and may be effective in reducing intestinal cholesterol absorption. and the polymeric structure of ARP reactive compounds content which may bind to cholesterol and bile acids.

Table 6. Effect of a dietary intervention with Ashwagandha root powder (ARP) on weight organs of obese rats

Table 7. Effect of a dietary intervention with Ashwagandha root powder (ARP) on liver functions of obese rats

Table 8. Effect of a dietary intervention with Ashwagandha root powder (ARP) on blood lipids profile of obese rats

Figure 6. Effect of a dietary intervention with Ashwagandha root powder (ARP) on the blood lipid profile (as a % of change) of obese rats

3.7.5. Effect of a Dietary Intervention with Ashwagandha Root Powder (ARP) on Neurological Disorder of Obese Rats

3.7.5.1. Serum Acetylcholine Esterase (AChE) Activity

The effect of dietary intervention with Ashwagandha root powder (ARP) on the acetylcholine esterase of the obese rats has been shown in Table (9) and Figure (7). Obesity caused a substantial (p0.05) increase in serum AChE by 21.22% when compared to the control group, according to the results. ARP (2, 4, 6, and 8 g/100 food) intervention in feeding rats for 8 weeks resulted in significantly (p0.05) lower levels of AChE (18.77, 15.35, 7.41, and 4.74%, respectively) compared to the regular group. The rate of decreasing AChE was shown to be dose-dependent. AChE is a cholinergic enzyme that is found largely at postsynaptic neuromuscular junctions, particularly in muscles and nerves. It rapidly hydrolyzes or breaks down acetylcholine (ACh), a neurotransmitter that occurs naturally, into acetic acid and choline. Choline, which is present in almost all tissues in animals, is a fundamental component of the neurotransmitter acetylcholine and works as an essential component of lecithin with inositol. Choline also helps to avoid fat accumulation in the liver and promotes fat transport into cells. Therefore, AChE is known to be for the termination of cholinergic response. Both nicotinic and muscarinic nerve receptors are excessively stimulated during a cholinergic crisis, resulting in an excessive parasympathetic response (muscle cramping, excessive secretions, weakness/paralysis, increased gastric motility [diarrhea], memory deficits [Alzheimer's disease], and bradycardia) [114,115]. Increased AChE activity in the brain also causes memory impairments and oxidative stress [116]. With this context, Rastogi et al., [102] discovered that the entire alkaloidal fraction of Ashwagandha root extract had long-lasting hypotensive, bradycardic, and respiratory stimulating effects in experimental mice. The hypotensive impact was primarily caused by autonomic ganglion-blocking activity, which was enhanced by the depressive action on higher cerebral centers. In numerous experimental animals, the total alkaloids tamed and had a slight depressive impact (tranquillizer-sedative type) on the central nervous system. Furthermore, systemic injection of Ashwagandha root extract had varying effects on AChE activity in basal forebrain nuclei. The lateral septum and globus pallidus have somewhat increased AChE activity [117]. Our findings, together with those of others, most likely explain the mechanism of Ashwagandha's AChE activity, which may be attributed to the antioxidant activities of their bioactive constituents such as phenolics, alkaloids, flavonoids, carotenoids, and so on. These chemicals shield neuronal cells from oxidative stress insults through the biological roles that it possesses, such as antioxidant and scavenging activities, and inhibition the lipid oxidation.

Table 9. Effect of dietary intervention with Ashwagandha root powder (ARP) on acetylcholine esterase (ACE, Rappapor U/mL) of obese rats

3.7.5.2. Serum Neurotransmitters (Dopamine and Serotonin)

The effect of dietary intervention with Ashwagandha root powder (ARP) on Serum neurotransmitters (dopamine and serotonin) of obese rats was shown in Table (10) and Figure (8). From such data, obesity was shown to cause a substantial (p0.05) decrease in blood dopamine and serotonin by rates of -17.54 and -14.86%, respectively, when compared to the regular group. Intervention with ARP (2, 4, 6 and 8 g/100 diet) in feeding rats for 8 weeks led to significantly (p≤0.05) increase the levels of dopamine and serotonin which recorded -15.98, -13.75, -8.10 and -6.06%, and -13.66, -11.98, -7.23 and -5.93%, respectively, compared to the regular group. The rate of decreasing in dopamine and serotonin was exhibited a dose- dependent manner. Dopamine and serotonin are neurotransmitters (chemical messengers) that help control numerous body activities. Dopamine is involved in movement, coordination, and pleasure and reward sensations. Serotonin is similarly implicated in emotions, but it also has an impact on digestion and metabolism [118]. Dopamine and serotonin system dysfunction have been linked to several nervous system illnesses, such as obesity [119]. In this context, multiple studies have found that dopamine and serotonin, two neurotransmitters, have a role in the control of food intake and body weight [120]. Nam et al. [119] also looked at the connection between obesity and the availability of striatal dopamine and extra-striatal serotonin transporters. on the other hand Morton et al. [121] reported that the brain is important in modulating and suppressing prepotent reactions to meals. Previous research demonstrated that a wide range of bioactive chemicals, such as those present in the Ashwagandha root that exhibited neurological disorders potential amelioration effects. Treatment of neurological problems is defined as the capacity of bioactive chemicals, such as antioxidants, to support healthy brain processes. Certain bioactive substances have been demonstrated in experimental research to exhibit neurostimulant qualities; that is, they appear to boost neurotransmitance processes by activating catecholamine transmitters such as dopamine and serotonin [8,122]. To interpret such mechanism, Lopez-Perez et al., [123] It is explained that dopamine is broken down enzymatically to its inactive metabolites by various enzymes, the most important of which is monoamine oxidase (MAO). In neurodegenerative illnesses, inhibiting MAO, which may be produced by obesity and its consequences, has been investigated as an additional treatment. Another process, the formation of hydrogen peroxide (H2O2) as a result of oxidative stress caused by obesity and its comorbidities, might be a co-factor in neurodegenerative illnesses [8,104]. To deal with H2O2 generation, neurons contain many antioxidant mechanisms, including catalase (CAT) and glutathione (GSH). In this respect, Mahran et al. [104] and [87] demonstrated that various plant sections contain the same bioactive chemicals, such as those found in Ashwagandha, that induce diverse antioxidant systems in living cells, such as serum glutathione (GSH) and antioxidant enzymes such as CAT, which cope with H2O2 and neutralization/prevents MAO inhibitors.

Table 10. Effect of a dietary intervention with Ashwagandha root powder (ARP) on serum dopamine and serotonin of obese rats

Figure 7. Effect of a dietary intervention with Ashwagandha root powder (ARP) on acetylcholine esterase (as a % of change) of obese rats

3.7.6. Histopathological Studies

3.7.6.1. Adipose Tissue

Effect of feeding intervention with Ashwagandha root powder (ARP) on adipose tissue histopathological examination of obese rats was shown in Figure 9. Microscopic examination of the adipose tissue of rats from Group 1 revealed typical unilocular adipocytes that were polygonal in form and resembled signet rings (Photo 1). In adverse, Adipose tissue from rats in Group 2 revealed histopathological changes characterized by congestion of blood vessel, large size unilocular adipocytes (Photo 2) associated with inflammatory cells infiltration (Photos 2 and 3). Meanwhile, adipose tissue of rats from group 3 demonstrated few large size unilocular adipocytes and some small size adipocytes as well as few inflammatory cells infiltration (Photo 4). Furthermore, examined sections from group 4 revealed few large size unilocular adipocytes and some small size adipocytes (Photo 5). On the other hand, some examined sections from groups 5 and 6 exhibited apparent histologically normal unilocular adipocytes (Photos 6 and 7). Such observation are in accordance partially with that reported by Elhassaneen et al., [41] who showed fat accumulation in major fat white adipose tissues and the expansion of adipocytes were both suppressed by Silybum marianum extract treatment containing the same bioactive compounds found in Ashwagandha root.

Figure 8. Effect of a dietary intervention with Ashwagandha root powder (ARP) on serum dopamine and serotonin of obese rats

3.7.6.2. Brain Tissue

Effect of feeding intervention with Ashwagandha root powder (ARP) on brain tissue histopathological examination of obese rats was shown in Figure (10). Brain (Cerebral cortex) of rats from group 1 showed no histopathological alterations (Photo 1). Meanwhile, sections from group 2 revealed necrosis, shrunken and pyknosis of neurons neuronophagia, cellular edema, vacuolation of neuropil, congestion of blood vessel and perivascular lymphocytic cuffing (photos 2 and 3). Meanwhile, cerebral cortex of rats from group 3 exhibited necrosis of some neurons and vacuolation of neuropil (Photo 4). Furthermore, cerebral cortex of rats from group 4 described necrosis of some neurons, cellular edema and vacuolation of neuropil (Photo 5). On the other hand, cerebral cortex of rats from groups 5 and 6 revealed marked improvement, examined sections showed necrosis of sporadic neurons (Photo 6) and slight vacuolation of neuropil (Photo 7). Such as according to Pannacciulli et al. [124] and Pistell et al. [125]. Obesity has been linked to decreased brain volume, altered brain structure, and function deficits in learning, memory, and executive function, as well as alterations and reductions in focal gray matter volume. As shown in Figure (11), Ashwagandha roots supplementation can overcome the high fat diet- induced obesity complications through many intracellular pathways.

Figure 9. Effect of feeding intervention with Ashwagandha root powder (ARP) on adipose tissue histopathological examination of obese rats (H & E, scale bar 100 µm, X 100)

Figure 10. Effect of feeding intervention with Ashwagandha root powder (ARP) on brain tissue histopathological examination of obese rats (H & E, scale bar 100 µm, X 100)

Figure 11. Graphical summary showing the effect of Ashwagandha roots powder on high fat diet- induced obesity complications

4. Conclusion

The study's findings validated our hypothesis that Ashwagandha (Withania somnifera L.) root samples include a variety of bioactive components such as phenolics, alkaloids, triterpenoids, carotenoids, flavonoids, tannins, and saponins. These chemicals, like the others, have a variety of biological actions, including antioxidant and free radical scavenging, low-density lipoprotein inhibition, and antibacterial which leading role(s) in Ashwagandha roots therapeutic effects. Such therapeutic effects include those related to the prevention or treatment of obesity and its related complications, which include decrease on body weight, enhance the serum lipid profile parameters, minimize the liver functions, improve neurological illnesses (dopamine and serotonin levels, as well as acetylcholine esterase activity), and positively manipulate the obesity-related histopathological changes in brain an adipose tissues of the obese rats. These findings support the benefits of dietary modification, Ashwagandha roots supplementation, in alleviating the complication associated obesity including neurological disorders.

ACKNOWLEDGMENT

The current work was partially funded by the Research Support Unit (RSU) of Menoufia University in Egypt. The authors would like to express their heartfelt gratitude to the technical staff (Animal House Unit) at the Department of Nutrition and Food Sciences, Faculty of Home Economics, Minoufiya University, Egypt, for their tireless efforts during the chemical analysis. It's not a simple chore. The professors of Plant Taxonomy, Faculty of Agriculture, Menoufia University, Egypt, were also thanked for their constructive support in evaluating and validating Ashwagandha root samples. Finally, the cooperation of the microbiology technician's team at Menoufia University's Faculty of Science was much appreciated.

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