IMR Press / FBL / Volume 29 / Issue 5 / DOI: 10.31083/j.fbl2905183
Open Access Original Research
Anti-Diabetic, Anti-Cholinesterase, and Anti-Inflammatory Potential of Plant Derived Extracts and Column Semi-Purified Fractions of Ficus benghalensis
Show Less
1 Department of Chemistry, University of Swabi, 23561 Anbar, Khyber Pakhtunkhwa, Pakistan
2 Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, 11671 Riyadh, Saudi Arabia
3 University Institute of Diet and Nutritional Sciences, Faculty of Allied Health Sciences, The University of Lahore, 54000 Lahore, Pakistan
4 Department of Nutrition and Dietetics, National University of Medical Sciences, 00666 Rawalpindi, Pakistan
5 Institute of Molecular Biology and Biotechnology, The University of Lahore, 54000 Lahore, Pakistan
6 Department of Pharmacy, Bacha Khan University, 24420 Charsadda, Khyber Pakhtunkhwa, Pakistan
7 Department of Botany, School of Basic and Applied Sciences, Shri Guru Ram Rai University, 248001 Dehradun, Uttarakhand, India
8 Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, 20133 Milan, Italy
9 National Interuniversity Consortium of Materials Science and Technology [INSTM], 50121 Firenze, Italy
10 Department of Rasa Shastra and Bhaishajya Kalpana, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, 221005 Varanasi, Uttar Pradesh, India
*Correspondence: mashaljcs@yahoo.com (Abdur Rauf); marcello.iriti@unimi.it (Marcello Iriti)
Front. Biosci. (Landmark Ed) 2024, 29(5), 183; https://doi.org/10.31083/j.fbl2905183
Submitted: 14 August 2023 | Revised: 15 September 2023 | Accepted: 26 October 2023 | Published: 11 May 2024
Copyright: © 2024 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
Abstract

Background: The present study aimed to investigate the in-vitro anti-diabetic, anti-cholinesterase, and anti-inflammatory potential of extracts from different parts of Ficus benghalensis, including leaves, stem, and roots, as well as isolated column fractions (F-B-1 C, F-B-2 C, F-B-3 C, and F-B-4 C). Methods: The extracts and subsequent fractions were evaluated for their inhibitory activity against key enzymes involved in diabetes [α-glucosidase and α-amylase], neurodegenerative diseases [acetylcholinesterase and butyrylcholinesterase], and inflammation (cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX)). Results: The results showed that F. benghalensis leaf extract exhibited the highest α-glucosidase inhibitory activity (73.84%) and α-amylase inhibitory activity (76.29%) at 1000 µg/mL. The stem extract (65.50%) and F-B-2 C fraction (69.67%) also demonstrated significant α-glucosidase inhibitory activity. In terms of anti-cholinesterase activity, the extracts of roots, leaves, and stem showed promising inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with half maximal inhibitory concentration (IC50) values ranging from 50.50 to 474.83 µg/mL. The derived fractions (F-B-1 C, F-B-2 C, F-B-3 C, and F-B-4 C) also exhibited notable inhibition of AChE and BChE, with IC50 values from 91.85 to 337.94 µg/mL. Moreover, the F-B-3 C fraction demonstrated the highest COX-2 inhibitory potential (85.72%), followed by F-B-1 C (83.13%), the stem extract (80.85%), and the leaves extract (79.00%). The F-B-1 C fraction showed the highest 5-LOX inhibitory activity (87.63%), while the root extract exhibited the lowest inhibition (73.39%). Conclusions: The results demonstrated promising bioactivity, suggesting the potential of F. benghalensis as a source of natural compounds with therapeutic applications. Further studies are required to identify and isolate the active components responsible for these effects and to evaluate their in-vivo efficacy and safety.

Keywords
Ficus benghalensis
α-amylase
α-glucosidase
AChE
BChE
COX-2
5-LOX
1. Introduction

Nowadays, health professionals are seriously concerned regarding the escalating prevalence of diabetes mellitus in both developing and developed countries. World Health Organization (WHO) stats reveal that 30 million people were diagnosed with diabetes in the year 1985, which increased to 135 million by 1995. It has been further projected that nearly 300 million population will be suffering from diabetes by the year 2025 [1]. This rise in the prevalence of diabetes is causing substantial financial problems in healthcare settings of underdeveloped countries [2, 3]. There are two main categories of diabetes: type-I accounts for nearly 5 to 10% of cases, while type-II diabetes accounts for approximately 90% of cases [4]. Impaired secretion of insulin results in type-I diabetes, whereas insulin resistance causes type-II diabetes [5, 6]. Hyperglycemia is a state associated with uncontrolled diabetes that results in elevation of blood sugar levels. Prolonged hyperglycemic conditions may result in significant deterioration of different body organs, specifically blood vessels and the central nervous system. Presently, diversified synthetic drugs have been developed for the management of diabetes, such as meglitinides, biguanides, incretin mimetics, thiazolidinediones, sodium/glucose co-transporter 2 (SGLT2) inhibitors, and dipeptidyl peptidase-IV inhibitors [7]. Even though various developed anti-diabetic drugs have demonstrated significant results, they are associated with different side effects [8]. These adverse effects include visual complications, headache, diarrhea, impotency, and hypoglycemia [9]. Therefore, research and development of innovative anti-diabetic medications that have significant effects against diabetes along with the least adverse effects remains a vital opportunity for scientists [10, 11]. For this purpose, investigations on alternative natural medications have attained significant importance owing to their safe nature. Various folk medications, such as administration of different herbal plants and their resultant extracts, have been efficient alternatives to synthetic drugs in managing diabetes. Herbal plants have been a potential source of novel drug development, as nearly 90% of pharmaceuticals are directly or indirectly derived from them [12, 13].

Alzheimer’s disease (AD) is characterized as a neurodegenerative ailment known for its deteriorating effect on the central nervous system [14]. AD is related to different factors such as neuroinflammation, oxidative stress, aggregation of amyloids, and cholinergic deficiency [15, 16, 17, 18]. To date, the underlying origin and optimum treatment of AD is still unclear and unidentified, while managing AD might include targeting its associated conditions. Effective management of the progression of AD can be possible by significantly dealing with oxidative stress, cholinergic deficiency, aggregation of amyloids, and neuroinflammation [19]. Mostly, the treatments of AD focus on easing the symptoms associated with its progression rather than providing an ultimate cure. These therapies help in improving cognitive functionality, managing behavioral symptoms, and improving overall quality of life [19]. Even though these treatments are of significant benefits, continual research to hunt for a definitive cure is needed [20]. As a result, recent investigations focus on exploring improved and alternative therapies for effective treatment of neurodegenerative ailments. Acetylcholine-a, neurotransmitter that is consumed by cholinergic neurons via the central nervous system, plays a vital role in normal functionality [21]. However, acetylcholinesterase (AChE), an enzyme, is considered to be responsible for the reduction of acetylcholine (ACh), hence causing problems in neurotransmission. Recently, AChE inhibitors have been used as an effective alternative approach for the management of AD as they elevate the levels of acetylcholine in the brain [22]. Various investigations have shown the anti-cholinesterase potential of different plant extracts. In spite of recent advancements in the field of modern medication, plant constituents play a significant part in the health and well-being of the community. Pharmaceutical companies are increasingly interested in exploring higher plants as potential sources for discovering new lead compounds and developing effective drugs. This renewed interest highlights the potential therapeutic value of plant-based medicines in addressing various health conditions [23].

Inflammation is a complex biological process that plays a significant role in the development and progression of various disorders, including arthritis and cardiovascular disease [24]. The current approach for symptomatic treatment of inflammation primarily relies on nonsteroidal anti-inflammatory drugs (NSAIDs), which exert their anti-inflammatory effects by inhibiting cyclooxygenase (COX). COX exists in two isoforms: cyclooxygenase-1 (COX-1), which is constitutively expressed in most cells under normal conditions, and cyclooxygenase-2 (COX-2), which is induced by pro-inflammatory agents like tumor necrosis factor-α (TNF-α), Lipopolysaccharide (LPS), and tumor-promoting factors [25]. As a consequence, novel dual COX-2/5-LOX inhibitors with anti-inflammatory properties have been identified through investigations involving both natural and synthetic sources. In an effort to contribute to advancements in this area, studies have been conducted to provide a comprehensive summary of the structural characteristics and pharmacological activities of heterocyclic scaffolds and natural products that have been investigated as dual COX-2/5-LOX inhibitors [26]. Another limitation of COX inhibition is that it can lead to an increased production of leukotrienes (LT) by 5-lipoxygenase (5-LOX). While 5-lipoxygenase (5-LOX) inhibitors have demonstrated a protective effect, their usage is hindered by several limitations. These limitations include poor bioavailability, lack of inhibitor specificity, potential hepatic and renal abnormalities, myelosuppression, gastrointestinal disturbances, and an inability to provide protection in chronic inflammation models [27]. The complex and potentially adverse side effects associated with COX and 5-LOX inhibitors have indeed restricted their long-term use as treatments for inflammatory disorders [28]. The limitations and risks associated with these inhibitors highlight the need for alternative therapeutic approaches that can effectively manage inflammation with a more favorable side effect profile.

Ficus benghalensis, a member of the Moraceae family, is widely recognized as the Banyan tree in English. It is native to a vast region of Asia. This plant is grown in several botanical gardens throughout different tropical areas across the world [29]. Studies have been performed to assess the phytochemistry and health-promoting benefits of F. benghalensis. Nevertheless, very few investigations have been conducted on leaves of F. benghalensis, which is of great importance owing to its medicinal applications [30]. F. benghalensis was reported to own different health-promoting benefits such as antibacterial [31], anti-diabetic, anticancer, and anti-inflammatory [32]. Moreover, it has demonstrated beneficial effects against skin ailments, gastric ulcers, and other gastrointestinal problems [33]. F. benghalensis methanolic extract was found to be effective against inhibiting acetylcholinesterase activity [21]. The composition of the leaves of F. benghalensis reveals the presence of crude protein (9.63%), Crude fiber (2.53%), phosphorous (0.4%), and calcium oxalate (2.53%). Phytochemical screening of leaves shows the presence of tannins, sterols, phenolic acids, saponins, and flavonoids. On the other hand, compounds like triterpenoids, volatile oils, and aromatic acids were not present in this plant [30]. In Ayurveda medications, this plant has been consumed for treating piles, dysentery, and diarrhea. Additionally, it is also employed as a hypoglycemic agent, indicating its potential in managing blood sugar levels [34]. Different extracts of Ficus bengalensis were assessed for their potential anti-allergic and anti-stress effects in asthma using milk-induced leucocytosis and milk-induced eosinophilia as indicators [35]. Literature reveals the potential of different Ficus species as anti-diabetic, antimicrobial, anticancer, and anti-inflammatory activities [36, 37]. The current study is unique as we have compared the in-vitro anti-diabetic, anti-cholinesterase, and anti-inflammatory potential of extracts and sub-fractions of different parts of F. bengalensis. In light of this, current study was designed to evaluate the in-vitro anti-diabetic [α-amylase and β-glucosidase inhibition], anti-inflammatory (5-LOX and COX-2 inhibition), and anti-cholinesterase (AChE and butyrylcholinesterase [BChE] inhibition) activity of different extracts and subfractions from stem, leaves, and roots of F. bengalensis.

2. Materials and Methods
2.1 Plants Collections

In June 2022, plant samples of F. benghalensis were gathered from Anbar Swabi. The collected specimen was taken to the Department of Botany, where it was examined and identified by Dr. Muhammad Ilyas, a member of the Botany Department at the University of Swabi. The voucher specimen number UOS-BOT/103 was then placed in the herbarium of the aforementioned department.

2.2 Extractions and Preparation of Subfractions

All information about software/equipment/drugs/reagents can be found in Supplementary Material and article. After collecting the plant material, consisting of stems, leaves, and roots, each weighing 2.2 kg, they were dried in the shade. Subsequently, the dried plant material was subjected to extraction using methanol. The obtained extracts were concentrated using a rotary evaporator (Rotavapor® R-300, BUCHI Corporation, New Castle, DE, USA), resulting in crude extracts of 18.00 g for the stems, 14.76 g for the leaves, and 16.1 g for the roots. Thin Layer Chromatography (TLC) profiling revealed that stem extract possessed the highest number of constituents, which were subjected to chromatographic analysis yielding different sub-fractions like F-B-1 C: CC chloroform: methanol (7:3); F-B-2 C: (chloroform: methanol (7.5:3.5); F-B-3 C: chloroform: methanol (6:4) and F-B-4 C: chloroform: methanol (6.5:3.5). The extracts and sub-fractions obtained were carefully stored in a freezer to maintain their integrity and preserve their biological activity. Later, these stored samples were utilized for various biological studies and investigations.

2.3 In-Vitro Anti-Diabetic Activity
2.3.1 α-amylase Inhibitory Activity Assay

An investigation was conducted to examine the impact of extracts and column semi-purified fractions of F. benghalensis on α-amylase activity. This study employed an enzyme-starch system to assess the effects of extracts and column semi-purified fractions of F. benghalensis on α-amylase activity [38]. Purposely, to prepare the experimental mixture, each extract, and column of semi-purified fractions of F. benghalensis powder (1%) was thoroughly stirred with 25 mL of a 4% potato starch solution in a beaker. Following that, 100 mg of α-amylase was added to the starch solution, and the mixture was vigorously stirred. Subsequently, the prepared mixture was incubated at a temperature of 37 °C for a duration of 60 minutes. At the end of the incubation period, 0.1 M NaOH was introduced to halt the enzymatic activity. The mixture was subjected to centrifugation at 3000 ×g for a duration of 15 minutes. Following centrifugation, the supernatant was collected, and the glucose content within it was measured.

2.3.2 α-glucosidase Inhibitory Activity Assay

The α-glucosidase inhibitory activity of extracts and column semi-purified fractions obtained from F. benghalensis was evaluated utilizing the methodology outlined by Pistia-Brueggeman and Hollingsworth in 2001 [39]. In order to assess the α-glucosidase inhibitory activity of extracts and fractions derived from F. benghalensis, varying concentrations of the extracts/fractions (62.5–1000 µg/mL) were combined with a solution containing 10 µL of α-glucosidase (1 U/mL) and 125 µL of phosphate buffer (pH: 6.8; 0.1 M). The mixture was subjected to an incubation period of 20 minutes at a temperature of 37 °C. To initiate the reaction, a 20 µL solution of 1 M pNPG (4-Nitrophenyl-β-d-glucopyranoside) substrate was added, and the mixture was further incubated for 30 minutes. To halt the reaction, 50 µL of Na2CO3 (0.1 N) was introduced into the reaction mixture. A UV-Vis spectrophotometer was utilized to measure the optical density of both the sample and the control at 405 nm wavelength. The following formula was employed to calculate the inhibitory activity:

Inhibitory Activity ( % ) : ( OD ( control ) - OD ( sample ) OD ( control ) ) × 100

Inhibitory Activity ( % ) : ( A b s C - A b s S A b s C ) × 100

Here: OD (control) = absorbance of control, while OD (sample) = absorbance of extracts/fractions (AbsC: Absorption of control; AbsS: Absorption of sample).

2.4 In-Vitro Anti-Cholinesterase Activity

The anti-cholinesterase activity of both the extracts and column semi-purified fractions of F. benghalensis was assessed using the Ellman method [40]. In summary, the cholinesterase enzyme breaks down the acetyl thiocholine substrate, resulting in the formation of thiocholine. Thiocholine then reacts with Ellman’s reagent (DTNB), generating 2-nitrobenzoic-5-mercaptothiocholine (thiocholine-thionitrobenzoate disulfide) and 5-thio-2-nitrobenzoic acid (thionitrobenzoate). These reaction products can be detected at a wavelength of 405 nm. For this assay, the reaction mixture had a total volume of 1 mL. Within a 1 mL cuvette, 50 µL of 3.5 mmol L-1 acetyl thiocholine iodide (ATCI) in a buffer was combined with 920 µL of a 0.125 mmol L-1 DTNB/buffer mixture. Subsequently, 20 µL of cholinesterase enzyme was added, and the absorbance was measured at 405 nm every 30 seconds for a total of 3 measurements. This process allowed the determination of the enzyme’s overall activity prior to inhibition. Afterward, 10 µL of the sample [extract/fractions] was introduced into the mixture, and the absorbance was once again recorded at the same wavelength, with three measurements taken at 30-second intervals. Following the addition of the sample, the enzyme activity inhibition percentage was calculated. To evaluate the inhibition of BChE (Butyrylcholinesterase), a similar method as described earlier was employed with slight adjustments. In this case, 25 µL of 5 mM butyrylthiocholine iodide was utilized as the substrate, while the enzyme concentration was set at 0.05 U/mL of BChE. Galantamine served as the positive control for both enzymes. The experiments were conducted in triplicate to ensure the accuracy and reliability of the results. The IC50 values, which represent the concentration at which there is 50% enzyme inhibition, were determined for the selected samples [extracts/fractions] using GraphPad Prism-7 software. Galantamine, a standard reference, was utilized for comparison and analysis.

2.5 In-Vitro Anti-Inflammatory Activity
2.5.1 COX-2 Inhibitory Assay

In this study, an in-vitro assay was performed to assess the anti-inflammatory effectiveness of extracts and column semi-purified fractions derived from F. benghalensis. In this study, the evaluation of COX-2 inhibitory potential was conducted using the methods outlined by Jan et al. [41] in 2020. Initially, 300 U/mL of COX-2 enzyme was prepared, from which 10 mL of COX-2 solution was ice-cooled for activation of enzymatic activity. To activate the enzymatic activity, the whole procedure was carried out for 5 to 10 minutes. Moreover, a co-factor solution (hematin: 1 mM, N,N,N,N-tetramethyl-p-phenylenediamine (TMPD): 0.24 mM, and glutathione: 0.9 mM) containing 50 mL that was prepared in Tris-HCl buffer (pH: 8, 0.1M) was mixed with this activated enzymatic solution. Afterwards the plant extracts and fractions were mixed with enzyme solution at varied concentrations (62.5–1000 µg/mL). Later, this mixture was incubated at 25 °C for 5 minutes. To initiate the reaction, 20 mL of a 30 mM arachidonic acid solution was introduced to the mixture. The resulting solution was then incubated for a duration of 4–5 minutes. In this assay, celecoxib was employed as a reference drug to enable comparison with the tested samples. Finally, absorbance was noticed at 540 nm wavelength using a UV-vis spectrophotometer.

Inhibitory Activity ( % ) : ( A b s C - A b s S A b s C ) × 100

Here: AbsC = absorbance of control, while AbsS = absorbance of sample [extract/fractions].

2.5.2 5-LOX Inhibitory Assay

Additionally, this study included a 5-LOX (5-lipoxygenase) assay to assess the in-vitro anti-inflammatory activity of extracts and column semi-purified fractions obtained from F. benghalensis [42]. For the 5-LOX assay, different doses (ranging from 62.5 to 1000 µg/mL) of extracts and fractions derived from F. benghalensis were prepared. Subsequently, a solution of the 5-LOX enzyme was prepared with a concentration of 10,000 units per milliliter. To initiate the enzymatic reaction, an 80 mM linoleic acid (L1376-5G, Sigma-Aldrich, St. Louis, MO, USA) substrate was added to the solution. To create the desired reaction mixture, 250 µL of crude extracts were combined with a phosphate buffer (50 mM, pH 6.3). Next, an enzyme solution of 250 mL was introduced to the mixture, and the resulting mixture was incubated for a duration of 5 minutes. Following the incubation, the substrate solution was added to the enzyme mixture and thoroughly mixed together. The absorbance (Abs) of both the control and test samples was measured at a wavelength of 234 nm using a UV-Vis spectrophotometer. A graph was constructed to examine the correlation between different extract concentrations and the extent of enzyme inhibition. This analysis allowed for the calculation of the IC50 values, which represent the concentration at which the enzyme inhibition reaches 50%. Finally, the percentage inhibition was determined using the following formula:

Percent inhibtion (%): ( A b s C - A b s S A b s C ) × 100

Here: AbsC = absorbance of control, while AbsS = absorbance of sample (extract/fractions).

2.6 Statistical Analysis

The results in this study were expressed as mean ± S.D. Level of significance (p < 0.05) was assessed using two-way ANOVA. Tukey’s HSD (honest significant difference) was performed to evaluate the mean pairwise comparison among groups.

3. Results
3.1 In-Vitro Anti-Diabetic Potential

Supplementary Table 1 shows the results of the anti-diabetic activity of extracts from different parts and isolated column fractions of F. benghalensis. At 1000 µg/mL, the highest α-glucosidase inhibitory was demonstrated by F. benghalensis leaves extract (73.84%) followed by F-B-2 C (69.67%), F. benghalensis stem extract (65.50%), F-B-3 C (65.50%), F-B-1 C (61.22%), and F. benghalensis roots extract (55.44%). In the case of α-amylase inhibitory activity, F. benghalensis leaves extract showed maximum (76.29%) potential, while F. benghalensis root extract had minimum inhibitory activity (57.33%). The standard (acarbose) used in this study showed a percent reduction of 81.85 and 83.53% in the activity of α-glucosidase and α-amylase, respectively.

3.2 In-Vitro Anti-Cholinesterase Potential

In this study, extracts of F. benghalensis roots, leaves, and stem, along with derived fractions (F-B-1 C, F-B-2 C, F-B-3 C, and F-B-4 C) were analyzed for their potential anti-cholinesterase activity. Glutamine was used in this study as a reference standard drug. Results revealed a significant reduction in activity of AChE and BChE in a concentration-dependent manner (Supplementary Table 2). It is evident from Supplementary Table 2 that extracts of roots, leaves, and stems demonstrated potential inhibition of AChE and BChE with IC50 values ranging from 50.50 to 474.83 µg/mL. In comparison, four different fractions (F-B-1 C, F-B-2 C, F-B-3 C and F-B-4 C) inhibited the activity of AChE and BChE with IC50 values in the range of 91.85 to 337.94 µg/mL.

3.3 In-Vitro Anti-Inflammatory Potential

The results of the anti-inflammatory activity of extracts from different parts and isolated column fractions F. benghalensis against COX-2. At 1000 µg/mL, the highest COX-2 inhibitory potential was demonstrated by F-B-3 C (85.72%), followed by F-B-1 C (83.13%), F. benghalensis stem extract (80.85%), F. benghalensis leaves extract [79.00%], F-B-2 C (73.08%), and F. benghalensis roots extract (69.47%). In the case of 5-LOX inhibitory activity, F-B-1 C showed maximum (87.63%) potential, while F. benghalensis root extract had minimum inhibitory activity (73.39%). The standard (Montelukast and Celecoxib) used in this study showed a percent reduction of 95.20 and 93.55% in the activity of COX-2 and 5-LOX, respectively as shown in Figs. 1,2. The IC50 values of the crude extracts/fractions and isolated column fractions of F. benghalensis were shown in Figs. 3,4, respectively.

Fig. 1.

Percent COX-2 inhibition activity of crude extracts/fractions and isolated column fractions of Ficus benghalensis. Data is represented as mean ± Standard Error of the Mean (S.E.M); n = 3; Two Way ANOVAs followed by the Bonferroni test were followed for test sample comparison to the standard drug. Values significantly different as compared to positive control; *** = p < 0.001. COX-2, cyclooxygenase-2.

Fig. 2.

Percent 5-LOX inhibition of crude extracts/fractions and isolated column fractions of Ficus benghalensis. Data is represented as mean ± S.E.M; n = 3; Two Way ANOVAs followed by the Bonferroni test were followed for test sample comparison to the standard drug. Values significantly different as compared to positive control; *** = p < 0.001. 5-LOX, 5-lipoxygenase.

Fig. 3.

The IC50 (half-maximal inhibitory concentration) values of various tested samples against COX-2. Data is represented as mean ± S.E.M; n = 3; Values significantly different as compared to positive control.

Fig. 4.

The IC50 values of various tested samples against 5-LOX. Data is represented as mean ± S.E.M; n = 3.

4. Discussion

The global population’s reliance on plant-based remedies is on the rise, primarily attributed to their easy accessibility and affordability [43, 44]. Consequently, researchers are persistently engaged in the pursuit of significant natural-based remedies that can be employed in the treatment, diagnosis, mitigation, or prevention of various disorders [45]. Validating the therapeutic potential of these readily accessible natural products holds the promise of offering more cost-effective treatment alternatives, particularly in light of the prevailing high inflation rates globally. It is worth noting that the pharmacodynamics of many drugs are associated with the inhibition of enzymes present in various biological compartments. Local physicians have recognized the therapeutic properties of the Ficus plant. The local physicians have long recognized the therapeutic properties of various parts of the F. benghalensis plant. The milky fluid obtained from the plant is known for its external application in alleviating pain, rheumatism, bruises, backaches, and swollen soles of the feet. In India, the roots of F. benghalensis are traditionally used to treat conditions such as dysentery, biliousness, gonorrhea, and liver swelling. Additionally, the aerial roots and tips of the plant are employed for their medicinal benefits in the treatment of dysentery and vomiting [46]. The infusion of small branches is consumed to alleviate hemoptysis, while the bark is believed to possess potent tonic properties and is used as a cure for diabetes [47]. Different components of F. benghalensis are employed in the treatment of diarrhea, leucorrhea, wound healing, and skin diseases [48]. According to folkloric practices, the aerial parts of F. benghalensis are employed to alleviate persistent vomiting and as an anti-asthmatic remedy [49]. Additionally, F. benghalensis leaves and stems have been documented for their therapeutic applications in various ailments [50]. However, there is a limited exploration of the phytochemical composition of this plant. Some compounds identified in its leaves include β-sitosterol, psoralen, β-amyrin, quercetin-3-galactoside, leucopelargonon, rutin, and leucodelphinidin derivatives [51].

The present study was performed to assess the anti-cholinesterase, anti-inflammatory, and anti-diabetic properties of extracts/fractions of F. benghalensis. In-vitro anti-diabetic assay revealed a significant [p < 0.05] concentration-dependent reduction in the activity of α-amylase and α-glucosidase due to the application of different extracts and fractions of F. benghalensis. In α-glucosidase and α-amylase assays, as compared to standard (Acarbose, IC50: 26.58 and 21.30 µg mL-1), the IC50 values of different extracts and fractions were in the range of 50.50–474.83 and 91.85–337.4 µg mL-1, respectively. The results of our study are in accordance with the findings of Blickle in 2008 [51]. They reported that the bark of F. benghalensis significantly inhibited the activity of both α-glucosidase and α-amylase [52]. Glucosidases play a vital role in various biological processes, including the breakdown of dietary carbohydrates [53]. α-glucosidase is one of several glucosidases found on the brush-border surface membrane of intestinal cells, and it plays a crucial role in the digestion of carbohydrates [54]. Daniel et al. [55] in 1998 reported that the presence of phenolics and flavonoids in F. benghalensis bark contributes to its inhibitory effect on α-glucosidase. These compounds possess potential antioxidant activity, and various investigations have shown that phenolic-enriched extracts of F. benghalensis demonstrate moderate free radical scavenging ability and strong inhibitory activity against α-glucosidase. In a study conducted by Madiwalar et al. [56], it was reported that 17 phytoconstituents derived from F. benghalensis demonstrated blood glucose-lowering effects. Notably, the highest drug-likeness score has been shown by 4-methoxybenzoic acid, while maximum (–8.02 kcal/mol) binding affinity was demonstrated by lupeol acetate. Lupeol acetate formed nine pi-interactions with Ala451, Ile319, Phe323, Phe24, Ile200, Tyr324, and Ile28. Additionally, the extract displayed the most significant glucose uptake efficacy in yeast cells at a concentration of 500 µg/mL [56]. Aqueous extract of F. bengalensis bark had inhibitory (IC50: 4.4 µg/mL) effects of α-amylase as experimented by Ponnusamy et al. [57].

In this study, the extracts and fractions of F. benghalensis were also examined for their potent anti-inflammatory properties. These two assays, namely COX-2 and 5-LOX inhibitory assays, were performed in this study. Results of both assays demonstrated a significant [p < 0.05] reduction in the activity of COX-2 and 5-LOX activity. According to a study conducted by Kothapalli et al. [58], extracts of F. benghalensis leaves showed potential anti-inflammatory activity [58]. Moreover, an in-vivo study further investigated the anti-inflammatory potential of F. bengalensis bark extract in a rat model. They administrated varied content of extract [50–200 mg/kg] of F. bengalensis bark in carrageenan-induced paw edema. Results showed that administration of this extract reduced the inflammation in a concentration-dependent manner. They were of the view that tannins and flavonoids present in the bark extract of F. bengalensis were responsible for this anti-inflammatory potential. Oxidative stress induced by induction of carrageenan injection was also reduced on the application of experimented extracts [59]. Likewise, bark and leaves extract of F. curtipes—another species of genus Ficus-have been reported to downregulate the activity of 5-LOX in a dose-dependent manner. The bark extract of F. curtipes significantly inhibited 5-LOX activity with an IC50 value of 10.75 µg/mL. The inhibitory potential of bark was reported to be more owing to the presence of more phenolic compounds in stem (5374.1 mg/kg dry extract) as compared to leaves (611.5 mg/kg dry extract) [60]. In general, phenolic constituents are recognized as predominant inhibitors of 5-LOX activity [61]. The results of these studies are in accordance with the findings of the current study, which revealed the inhibitory potential of F. benghalensis extracts and fractions against the activity of 5-LOX and COX-2. Bengalensinone and benganoic acid have been isolated from roots of F. benghalensis and possessed inhibitory (164.5 and 154.5 µM) action against acetylcholinesterase, respectively. Both these compounds inhibited the activity of butyrylcholinesterase with an IC50 value of 224.9 and 120 µM, respectively [62]. Moreover, Hassan et al. [21] have shown the anti-acetylcholinesterase (IC50: 194.6 µg mL-1) activity of F. benghalensis.

Phytochemical profiling of F. benghalensis extracts have indicated the presence of diverse phytomolecules like steroids, tannins, flavonoids, alkaloids, anthraquinones, and glycosides [63]. Roots extract of F. benghalensis inhibited the activity of acetylcholinesterase in an in-vitro model. The ethyl acetate extract of roots showed inhibitory action with an IC50 value of 67 µg/mL as compared to the control (Donepezil), i.e., 33 µg/mL. The results of this study were comparable to the outcomes reported by Ramasamy et al. [63]. Moreover, Hassan et al. [21] 2020 also assessed the in-vitro AChE inhibitory potential of extracts (methanolic), fractions (ethyl acetate), and isolated compounds of F. benghalensis.

5. Conclusions

The findings of this study revealed the in-vitro potential of Ficus benghalensis extracts and fractions in various pharmacological activities. The leaf extract exhibited the highest alpha-glucosidase and alpha-amylase inhibitory activities among all the tested samples, indicating its potential as an anti-diabetic agent. Furthermore, the roots, leaves, and stem extracts, as well as the derived fractions, exhibited significant anti-cholinesterase activity, highlighting their potential for the management of neurodegenerative diseases. Additionally, the F-B-3 C fraction showed the highest COX-2 inhibitory activity, suggesting its potential as an anti-inflammatory agent. Overall, this study provides valuable insights into the medicinal potential of F. benghalensis and supports its traditional use in the treatment of diabetes, neurodegenerative diseases, and inflammatory conditions. Further studies are warranted to identify and characterize the active compounds responsible for these observed bioactivities and to evaluate their in-vivo efficacy and safety profiles.

Availability of Data and Materials

The data generated in the present study are included in the figures and/or tables of this article.

Author Contributions

AR was responsible for conceptualization, visualization, supervision, project administration and writing-original draft. MIbr and MIri contributed by conducting formal analysis, interpreting data, investigation and writing-original draft. AAK, NA, TSA, TK and MUK contributed by writing-original draft, analyzing, data curation, reviewing and editing of manuscript. KB, MSJ, and RS made significant revisions, visualization, and editorial changes. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Ethics Approval and Consent to Participate

Plant samples of F. benghalensis were gathered from Anbar Swabi. The collected specimen was taken to the Department of Botany, where it was examined and identified by Dr. Muhammad Ilyas, a member of the Botany Department at the University of Swabi. The voucher specimen number UOS-BOT/103 was then placed in the herbarium of the aforementioned department.

Acknowledgment

Authors would like to acknowledge Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia for supporting this study.

Funding

Princess Nourah bint Abdulrahman University Researchers Supporting Project number [PNURSP2024R18], Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Conflict of Interest

The authors declare no conflict of interest. Given his role as Guest Editor and Editorial Board Member of Frontiers in Bioscience-Landmark, Marcello Iriti had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Jen-Tsung Chen.

References
[1]
Global report on diabetes 2016. 2016. Available at: https://www.who.int/publications-detail-redirect/9789241565257 (Accessed: 12 June 2023).
[2]
Adams GG, Imran S, Wang S, Mohammad A, Kok S, Gray DA, et al. The hypoglycaemic effect of pumpkins as anti-diabetic and functional medicines. Food Research International. 2011; 44: 862–867.
[3]
Chew NWS, Ng CH, Tan DJH, Kong G, Lin C, Chin YH, et al. The global burden of metabolic disease: Data from 2000 to 2019. Cell Metabolism. 2023; 35: 414–428.e3.
[4]
American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2010; 33: S62–S69.
[5]
Beidokhti MN, Jäger AK. Review of antidiabetic fruits, vegetables, beverages, oils and spices commonly consumed in the diet. Journal of Ethnopharmacology. 2017; 201: 26–41.
[6]
Wang J, Jiang J, Zhao C, Shan H, Shao Z, Wang C, et al. The Protective Effect of Theaflavins on the Kidney of Mice with Type II Diabetes Mellitus. Nutrients. 2022; 15: 201.
[7]
Rath P, Ranjan A, Chauhan A, Verma NK, Bhargava A, Prasad R, et al. A Critical Review on Role of Available Synthetic Drugs and Phytochemicals in Insulin Resistance Treatment by Targeting PTP1B. Applied Biochemistry and Biotechnology. 2022; 194: 4683–4701.
[8]
Feingold KR. Oral and Injectable (Non-Insulin) Pharmacological Agents for the Treatment of Type 2 Diabetes. [Updated 2022 Aug 26]. In Feingold KR, Anawalt B, Blackman MR, Boyce A, Chrousos G, Corpas E, et al. (eds.) Endotext [Internet]. MDText.com, Inc.: South Dartmouth (MA). 2000.
[9]
Safavi M, Foroumadi A, Abdollahi M. The importance of synthetic drugs for type 2 diabetes drug discovery. Expert Opinion on Drug Discovery. 2013; 8: 1339–1363.
[10]
DeFronzo RA, Ferrannini E, Groop L, Henry RR, Herman WH, Holst JJ, et al. Type 2 diabetes mellitus. Nature reviews Disease Primers. 2015; 1; 15019
[11]
Davies MJ, D’Alessio DA, Fradkin J, Kernan WN, Mathieu C, Mingrone G, et al. Management of hyperglycaemia in type 2 diabetes, 2018. a consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) Diabetologia. 2018; 61: 2461–2498.
[12]
Sharma R, Singla RK, Banerjee S, Sinha B, Shen B, Sharma R. Role of Shankhpushpi (Convolvulus pluricaulis) in neurological disorders: an umbrella review covering evidence from ethnopharmacology to clinical studies. Neuroscience & Biobehavioral Reviews. 2022; 140: 104795.
[13]
Tran N, Pham B, Le L. Bioactive compounds in anti-diabetic plants: From herbal medicine to modern drug discovery. Biology. 2020; 9: 252.
[14]
Sharma R, Kuca K, Nepovimova E, Kabra A, Rao MM, Prajapati PK. Traditional Ayurvedic and herbal remedies for Alzheimer’s disease: from bench to bedside. Expert Review of Neurotherapeutics. 2019; 19: 359–374.
[15]
Kumar K, Kumar A, Keegan RM, Deshmukh R. Recent advances in the neurobiology and neuropharmacology of Alzheimer’s disease. Biomedicine & Pharmacotherapy. 2018; 98: 297–307.
[16]
Chen ZR, Huang JB, Yang SL, Hong FF. Role of cholinergic signaling in Alzheimer’s disease. Molecules. 2022; 27: 1816.
[17]
Sharma R, Kabra A, Rao MM, Prajapati PK. Herbal and Holistic Solutions for Neurodegenerative and Depressive Disorders: Leads from Ayurveda. Current Pharmaceutical Design. 2018; 24: 2597–2608.
[18]
Qureshi T, Chinnathambi S. Histone deacetylase-6 modulates Tau function in Alzheimer’s disease. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2022; 1869: 119275.
[19]
Sharifi-Rad J, Rapposelli S, Sestito S, Herrera-Bravo J, Arancibia-Diaz A, Salazar LA, et al. Multi-target mechanisms of phytochemicals in Alzheimer’s disease: Effects on oxidative stress, neuroinflammation and protein aggregation. Journal of Personalized Medicine. 2022; 12: 1515.
[20]
Kumar A, Singh A, Ekavali. A review on Alzheimer’s disease pathophysiology and its management: an update. Pharmacological Reports. 2015; 67: 195–203.
[21]
Hassan HA, Allam AE, Abu-Baih DH, Mohamed MF, Abdelmohsen UR, Shimizu K, et al. Isolation and characterization of novel acetylcholinesterase inhibitors from Ficus benghalensis L. leaves. RSC Advances. 2020; 10:36920–36929.
[22]
Taqui R, Debnath M, Ahmed S, Ghosh A. Advances on plant extracts and phytocompounds with acetylcholinesterase inhibition activity for possible treatment of Alzheimer’s disease. Phytomedicine Plus. 2022; 2: 100184.
[23]
Valarezo E, Ludeña J, Echeverria-Coronel E, Cartuche L, Meneses MA, Calva J, et al. Enantiomeric composition, antioxidant capacity and anticholinesterase activity of essential oil from leaves of Chirimoya [Annona cherimola Mill.]. Plants. 2022; 11: 367.
[24]
Cacoub P, Choukroun G, Cohen-Solal A, Luporsi E, Peyrin-Biroulet L, Peoc’h K, et al. Towards a common definition for the diagnosis of iron deficiency in chronic inflammatory diseases. Nutrients. 2022; 14: 1039.
[25]
Doiphode S, Lokhande KB, Ghosh P, Swamy KV, Nagar S. Dual inhibition of cyclooxygenase-2 [COX-2] and 5-lipoxygenase [5-LOX] by resveratrol derivatives in cancer therapy: in silico approach. Journal of Biomolecular Structure and Dynamics. 2023; 41: 8571–8586.
[26]
Meshram MA, Bhise UO, Makhal PN, Kaki VR. Synthetically-tailored and nature-derived dual COX-2/5-LOX inhibitors: Structural aspects and SAR. European Journal of Medicinal Chemistry. 2021; 225: 113804.
[27]
El-Miligy MMM, Al-Kubeisi AK, Bekhit MG, El-Zemity SR, Nassra RA, Hazzaa AA. Towards safer anti-inflammatory therapy: synthesis of new thymol–pyrazole hybrids as dual COX-2/5-LOX inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry. 2023; 38: 294–308.
[28]
Jaismy Jacob P, Manju SL. Novel approach of multi-targeted thiazoles and thiazolidenes toward anti-inflammatory and anticancer therapy—dual inhibition of COX-2 and 5-LOX enzymes. Medicinal Chemistry Research. 2021; 30: 236–257.
[29]
Gopukumar ST, Praseetha PK. Ficus benghalensis Linn–the sacred Indian medicinal tree with potent pharmacological remedies. International Journal of Pharmaceutical Sciences Reviews Research. 2015; 32: 223–227.
[30]
Shi Y, Mon AM, Fu Y, Zhang Y, Wang C, Yang X, et al. The genus Ficus (Moraceae) used in diet: its plant diversity, distribution, traditional uses and ethnopharmacological importance. Journal of Ethnopharmacology. 2018; 226: 185–196.
[31]
Hassan HA, Abdelwahab SF, Desoukey SY, Mohamed KM, Kamel MS. Comparative Study of Antimicrobial Activity of Seven Ficus Species Cultivated in Egypt. Indian Journal of Public Health Research Development. 2019; 10: 1938–1943.
[32]
Gayathri M, Kannabiran K. Antidiabetic and ameliorative potential of Ficus bengalensis bark extract in streptozotocin induced diabetic rats. Indian Journal of Clinical Biochemistry. 2008; 23: 394–400.
[33]
Etratkhah Z, Ebrahimi SE, Dehaghi NK, Seifalizadeh Y. Antioxidant activity and phytochemical screening of Ficus benghalensis aerial roots fractions. Journal of Reports in Pharmaceutical Sciences. 2019; 8: 24–27.
[34]
Murugesu S, Selamat J, Perumal V. Phytochemistry, pharmacological properties, and recent applications of Ficus benghalensis and Ficus religiosa. Plants. 2021; 10: 2749.
[35]
Taur DJ, Nirmal SA, Patil RY, Kharya MD. Antistress and antiallergic effects of Ficus bengalensis bark in asthma. Natural Product Research. 2007; 21: 1266–1270.
[36]
Logesh R, Vivekanandarajah Sathasivampillai S, Varatharasan S, Rajan S, Das N, Pandey J, et al. Ficus benghalensis L. (Moraceae): a review on ethnomedicinal uses, phytochemistry and pharmacological activities. Current Research in Biotechnology. 2023; 6: 100134.
[37]
Singh B, Sharma RA. Updated review on Indian Ficus species. Arabian Journal of Chemistry. 2023; 16: 104976.
[38]
Ou S, Kwok K, Li Y, Fu L. In Vitro Study of Possible Role of Dietary Fiber in Lowering Postprandial Serum Glucose. Journal of Agricultural and Food Chemistry. 2001; 49: 1026–1029.
[39]
Pistia-Brueggeman G, Hollingsworth RI. A preparation and screening strategy for glycosidase inhibitors. Tetrahedron. 2001; 57: 8773–8778.
[40]
Ellman GL, Courtney KD, Andres V, Jr, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology. 1961; 7: 88–95.
[41]
Jan MS, Ahmad S, Hussain F, Ahmad A, Mahmood F, Rashid U, et al. Design, synthesis, in-vitro, in-vivo and in-silico studies of pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory agents. European Journal of Medicinal Chemistry. 2020; 186: 111863.
[42]
Jan MS, Shahid M, Ahmad S, Hussain F, Ahmad A, Mahmood F, et al. Synthesis of pyrrolidine-2, 5-dione based anti-inflammatory drug: in vitro COX-2, 5-LOX inhibition and in vivo anti-inflammatory studies. Latin American Journal of Pharmacy. 2019; 38: 2287–2294.
[43]
Dome P, Tombor L, Lazary J, Gonda X, Rihmer Z. Natural health products, dietary minerals and over-the-counter medications as add-on therapies to antidepressants in the treatment of major depressive disorder: a review. Brain Research Bulletin. 2019; 146: 51–78.
[44]
Layek B, Mandal S. Natural polysaccharides for controlled delivery of oral therapeutics: a recent update. Carbohydrate Polymers. 2020; 230: 115617.
[45]
Fadilah NI, Isa IL, Zaman WS, Tabata Y, Fauzi MB. The effect of nanoparticle-incorporated natural-based biomaterials towards cells on activated pathways: a systematic review. Polymers. 2022; 14: 476.
[46]
Rauf A, Ibrahim M, Muhammad N, Naz S, Wadood A, Khan B, et al. Enzyme Inhibitory Activities of Extracts and Carpachromene from the Stem of Ficus benghalensis. BioMed Research International. 2022; 2022: 1–6.
[47]
Manocha N, Chandra SK, Sharma V, Sangameswaran B, Saluja M. Anti-rheumatic and antioxidant activity of extract of stem bark of Ficus bengalensis. Research Journal of Chemical Sciences. 2011; 1: 2–8.
[48]
Suryanarayanan TS, Vijaykrishna D. Fungal endophytes of aerial roots of Ficus benghalensis. Fungal Diversity. 2001; 8: 155–160.
[49]
Taur DJ, Nirmal SA, Patil RY. Effect of various extracts of Ficus bengalensis bark on clonidine and haloperidol-induced catalepsy in mice. Pharmacologyonline. 2007; 3: 470–477.
[50]
Joseph B, Raj SJ. Phytopharmacological and phytochemical properties of three Ficus species-an overview. International Journal of Pharmacy and Biological Sciences. 2010; 1: 246–253.
[51]
Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P, Vidal N. Antioxidant activity of some algerian medicinal plants extracts containing phenolic compounds. Food Chemistry. 2006; 97: 654–660.
[52]
Blickle JF, Andres E, Brogard JM. Current status of the treatment of type 2 diabetes mellitus. Alpha-glucosidase inhibitors. La Revue de Medecine Interne. 1999; 20: 379s–383s.
[53]
Abadan Ş, Saglam MF, Koca MS, Bingul M, Sahin H, Zorlu Y, et al. Synthesis and molecular modeling studies of naphthazarin derivatives as novel selective inhibitors of α-glucosidase and α-amylase. Journal of Molecular Structure. 2023; 1278: 134954.
[54]
Dirir AM, Daou M, Yousef AF, Yousef LF. A review of alpha-glucosidase inhibitors from plants as potential candidates for the treatment of type-2 diabetes. Phytochemistry Reviews. 2022; 21: 1049–1079.
[55]
Daniel RS, Mathew BC, Devi KS, Augusti KT. Antioxidant effect of two flavonoids from the bark of Ficus bengalensis Linn in hyperlipidemic rats. Indian Journal of Experimental Biology. 1998; 36: 902–906.
[56]
Madiwalar VS, Dwivedi PSR, Patil A, Gaonkar SMN, Kumbhar VJ, Khanal P, et al. Ficus benghalensis promotes the glucose uptake- Evidence with in silico and in vitro. Journal of Diabetes & Metabolic Disorders. 2022; 21: 429–438.
[57]
Ponnusamy S, Ravindran R, Zinjarde S, Bhargava S, Ravi Kumar A. Evaluation of traditional Indian antidiabetic medicinal plants for human pancreatic amylase inhibitory effect in vitro. Evidence-Based Complementary and Alternative Medicine. 2010; 2011: 515647.
[58]
Kothapalli PK, Sanganal SJ, Shridhar NB, Narayanaswamy HD, Narayanaswamy M. In-vivo anti-inflammatory and analgesic screening of Ficus bengalensis Leaf extract in rats. Asian Journal of Research and Pharmaceutical Sciences. 2014; 4: 174–178.
[59]
Wanjari M, Kumar P, Umathe SN. Anti-inflammatory Effect of Ethanolic Extract of Ficus bengalensis Linn. in Carrageenan Induced Paw Edema in Rats. Pharmacognosy Journal. 2011; 3: 96–99.
[60]
Andrade C, Ferreres F, Gomes NG, Duangsrisai S, Srisombat N, Vajrodaya S, et al. Phenolic profiling and biological potential of Ficus curtipes corner leaves and stem bark: 5-lipoxygenase inhibition and interference with NO levels in LPS-stimulated RAW 264.7 macrophages. Biomolecules. 2019; 9: 400.
[61]
Yahfoufi N, Alsadi N, Jambi M, Matar C. The immunomodulatory and anti-inflammatory role of polyphenols. Nutrients. 2018; 10: 1618.
[62]
Riaz N, Naveed MA, Saleem M, Jabeen B, Ashraf M, Ejaz SA, et al. Cholinesterase inhibitory constituents from Ficus bengalensis. Journal of Asian natural Products Research. 2012; 14: 1149–1155.
[63]
Ramasamy A, Anandakumar K, Kathiresan K. In-vitro antioxidant potential and acetylcholinesterase inhibitory effect of Ficus benghalensis aerial root extract. African Health Sciences. 2022; 22: 291–299.

Publisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share
Back to top