Hypoglycemic, anti‐inflammatory, and neuroprotective potentials of crude methanolic extract from Acacia nilotica L. – results of an in vitro study

Abstract Acacia nilotica L., also known as babul, belonging to the Fabaceae family and the Acacia genus, is typically used for ornamental purposes and also as a medicinal plant found in tropical and subtropical areas. This plant is a rich source of bioactive compounds. The current study aimed to elucidate the hypoglycemic, anti‐inflammatory, and neuroprotective potential of A. nilotica's crude methanolic extract. The results of the in vitro antidiabetic assay revealed that methanolic extract of A. nilotica inhibited the enzyme α‐glucosidase (IC50: 33 μg mL−1) and α‐amylase (IC50: 17 μg mL−1) in a dose‐dependent manner. While in the anticholinesterase enzyme inhibitory assay, maximum inhibition was shown by the extract against acetylcholinesterase (AChE) (637.01 μg mL−1) and butyrylcholinesterase (BChE) (491.98 μg mL−1), with the highest percent inhibition of 67.54% and 71.50% at 1000 μg mL−1, respectively. This inhibitory potential was lower as compared to the standard drug Galantamine that exhibited 82.43 and 89.50% inhibition at the same concentration, respectively. Moreover, the methanolic extract of A. nilotica also significantly inhibited the activities of cyclooxygenase 2 (COX‐2) and 5‐lipoxygenase (5‐LOX) in a concentration‐dependent manner. The percent inhibitory activity of 5‐LOX and COX‐2 ranged from 42.47% to 71.53% and 43.48% to 75.22%, respectively. Furthermore, in silico, in vivo, and clinical investigations must be planned to validate the above‐stated bioactivities of A. nilotica.


| INTRODUC TI ON
Inflammation is a painful and distressing immune system response to injury, manifested by symptoms of redness, swelling, and pain (Chen et al., 2018).The inflammatory response, which includes local inflammation of blood vessels, is developed and amplified through a series of chemical reactions, including the release of inflammatory agents from various immune cells and cells that have been injured (Ahmed, 2011).The pain accompanying inflammation results from endogenous substances, specifically prostaglandins, produced from arachidonic acid (AA) through the action of cyclooxygenases, i.e., cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) (Duarte et al., 1988;Khan et al., 2010).The overexpression of lipoxygenase (LOX) and COX converts AA to leukotriene and prostaglandins, respectively (Shah et al., 2014).These released LOX and prostaglandin products increase the sensitivity of nociceptors, causing pain and promoting vasodilation that leads to increased capillary permeability and, eventually, edema (Muoghalu et al., 2016).Therefore, COX and LOX inhibitors are favored for peripheral pain relief and inflammation reduction (Laveti et al., 2013).Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to alleviate inflammation (Moore et al., 2019).Even prolonged use of NSAIDs can result in gastric and renal toxicity (Magni et al., 2021), mainly due to the nonselective inhibition of COX-1 and COX-2 (Roedder et al., 2017).It is believed that currently used anti-inflammatory drugs, including opiates and non-NSAIDs, have associated toxic effects like gastric and renal ulcers and low potentiality (Coban & Degim, 2020;Phueanpinit et al., 2018).Due to these side effects, searching for new and effective pain management and anti-inflammatory treatments is as necessary today as ever.
Alzheimer's disease (AD) is one of the most prevailing neurological disorders affecting older adults and is the primary cause of dementia (Coban & Degim, 2020).Clinically, AD is characterized by a range of symptoms, including declining memory and language abilities, cognitive impairments, and behavioral disorders such as psychosis, anxiety, and depression, all of which deteriorate over time (Syad et al., 2012).Acetylcholine (ACh) is essential in transmitting impulses through synapses (Ayaz et al., 2017;Patri, 2019).The concentration of this neurotransmitter is reduced due to the increased activity of two key enzymes, i.e., acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) (Ayaz et al., 2019).Hence, inhibitors of AChE and BChE are considered one of the therapeutic choices available to restore cholinergic activity.Most currently available approved commercial drugs for AD treatment work principally by inhibiting cholinesterase activity (Ayaz et al., 2015).On the other hand, these commercial synthetic drugs available for the treatments for AD help in managing the symptoms associated with the condition temporarily and are harmful when used for a more extended period (Yiannopoulou & Papageorgiou, 2020).Hence, developing effective, safe, and easily available drugs for treating AD is needed.
Diabetes mellitus (DM) is a group of metabolic disorders with various causes characterized by persistent high blood glucose serum concentration (American Diabetes Association, 2010;Hussain et al., 2019).Uncontrolled DM leads to many complications, such as diabetic retinopathy, nephropathy, and neuropathy, arising from damage to critical tissues due to microvascular changes (Sadiq et al., 2020;Tomic et al., 2022).In addition, the risk of macrovascular changes, such as peripheral vascular disease, ischemic heart disease, stroke, and reduced quality of life, increases mortality and shortens life expectancy (Aslam et al., 2018;Moreno-Díaz et al., 2008).
To reduce hyperglycemic conditions, targets for therapy include αamylase and α-glucosidase (Olaokun et al., 2013).Slowing down carbohydrate digestion and reducing glucose absorption by inhibiting both α-amylase and α-glucosidase help curb elevated blood glucose levels (Kajaria et al., 2013).However, commercially available drugs for inhibiting α-amylase and α-glucosidase have associated adverse side effects, such as flatulence, diarrhea, and stomachache, reducing their appeal as treatments (Poongunran et al., 2015).The scientific community is currently focused on finding alternatives to overcome commercial drugs' low potency and harmful effects in treating hyperglycemia and diabetes.For this purpose, natural products are being investigated extensively and have been reported as safe, effective, and less harmful options to already available drugs.
Traditional medicine utilizes medicinal plants with a long history in developing countries (Pan et al., 2014).They are often used as folk remedies to treat various ailments, such as neurological, inflammatory, and diabetic conditions (Oguntibeju, 2018).For centuries, herbal remedies have been used effectively in medication against various syndromes.Due to their relatively harmless effects, natural products have garnered modern scientists' attention for treating and managing various challenging diseases (Yeung et al., 2018).Plantderived extracts and isolates have been investigated as a safe and effective alternative to currently available synthetic compounds.
Besides being a source of antioxidants, these natural constituents possess various pharmacological properties like antiviral, anticancer, anti-inflammatory, neuroprotective, antidiabetic, and many more (Uwineza & Waśkiewicz, 2020).Acacia nilotica (L.) Wild, a member of the Fabaceae family, is commonly referred to as the Indian gum arabic tree, kikar, or babul, which has gained recognition as a versatile and valuable tree species.Although it is considered a nuisance weed due to its invasive nature, it has significant environmental value and holds the potential to boost the economy.A. nilotica has medicinal properties as per Unani, traditional Chinese medicine (TCM), and Ayurvedic texts.On the other hand, several preclinical and clinical studies support the potential functional and nutraceutical use of A. nilotica, owing to its diversified phytochemistry and bioactivities RAUF et al. (Abduljawad, 2020;Saeedi et al., 2020).This plant's polyphenolic extract reportedly helps reduce induced neurotoxicity and oxidative stress conditions in an animal model (Foyzun et al., 2022).Moreover, A. nilotica extract restored glucose homeostasis by inhibiting JNK (c-Jun N-terminal kinase) and upregulation of insulin (Majeed et al., 2021).In diabetic rats, A. nilotica's extracts lowered cholesterol levels and imparted renoprotective effects (Niyodusenga et al., 2019;Sirajo et al., 2022).Quercetin, kaempferol, and ellagic acid present in the extracts of A. nilotica are considered responsible for this plant's diverse biological activities (Al-Nour et al., 2019).
Given the importance of A. nilotica, the current study was designed to investigate the hypoglycemic, anti-inflammatory, and neuroprotective potential of Acacia nilotica's crude methanolic extract.

| Plant collection
The stems of A. nilotica were collected from Swabi, Khyber Pakhtunkhwa province (KPK), Pakistan, and were brought to the Department of Botany, University of Swabi (UoS), Khyber Pakhtunkhwa (KP), Pakistan, for authentication.Specimens were placed in the herbarium of UoS with voucher no.(UOS/Bot-121).

| Extraction
The A. nilotica stems were collected thoroughly, washed with tap water, and shade-dried for 10 days.The dried stem was ground into a fine powder (40 mesh) using a laboratory mill (Haier, HJE-1024).
Stem powder (100 g) was placed in a thimble, and methanol (70%) was used for extraction.The extract was placed in a clean flatbottomed flask, left to soak for 72 h with periodic stirring, and was later stored at room temperature in sterile bottles.Whatman No. 1 filter paper was used to filter the extract, which was subsequently concentrated using a rotary concentrator (Rotavapor® R-300, BUCHI Corporation, USA) (Revathi et al., 2017).

| In vitro α-amylase inhibition assay
Already established protocols for the evaluation of the α-amylase inhibitory potential of A. nilotica's crude methanolic extract were followed in this study (Huneif et al., 2022).Phosphate buffer was used for the preparation of the α-amylase solution.Varied concentrations (62.5-1000 μg mL −1 ) of crude methanolic extract (30 μL) were mixed with already prepared α-amylase solution (10 μL) and phosphate buffer (pH: 6.9; 2 mM).This experiment used acarbose (64 mg mL −1 ) as a positive control.The prepared mixtures were initially incubated at 25°C for 10 min.Afterwards, the reaction was initiated by adding starch solution (1%; 40 mL) in test samples and control-containing tubes and was incubated again (10 min; 25°C).Later, iodine solution (75 μL) and hydrochloric acid (HCl) (20 μL; 1 M) were added to end the reaction, and absorbance (Abs) was measured at 540 nm.The below-mentioned formula was used for calculating the percent inhibition of α-amylase activity:

| In vitro α-glucosidase inhibitory assay
In this assay, A. nilotica's crude methanolic extract was examined for its α-glucosidase inhibitory potential by following the protocols adopted by Revathi et al. (2017).For this purpose, varied concentrations (62.5-1000 μg mL −1 ) of 50 μL of crude extract and acarbose were allowed to incubate (37°C; 20 min) with α-glucosidase solution (10 μL; 1 U mL −1 ) and phosphate buffer (0.1 M; 125 μL; pH: 6.9).Afterwards, the reaction was initiated by adding substrate (1 M 4-nitrophenyl β-D-glucopyranoside (pNPG); 20 μL).This mixture was allowed to incubate at 37°C for further 30 min.Later, 50 μL of sodium carbonate (Na 2 CO 3 ) (0.1 N) was added to this mixture to stop the reaction.Finally, the absorbance (Abs) of the control and the test samples (containing crude methanolic extract) was calculated at 405 nm.Herein, acarbose was used as a positive control.α-Glucosidase inhibition was deduced from the following formula:

| In vitro anticholinesterase inhibitory activity
To assess the AChE and BChE inhibitory potentials of A. nilotica's crude methanolic extract, the in vitro anticholinesterase assay followed the already reported protocols (Huneif et al., 2022).Varied concentrations (62.5-1000 μg mL −1 ) of crude extract and standard drug Galantamine were prepared and used in this assay.The IC 50 values were calculated by creating a graph in Excel that compared the inhibition and concentration of the sample solution being tested.

| Cyclooxygenase (COX-2) assay
The COX-2 assay was performed to assess the anti-inflammatory potential of A. nilotica's crude methanolic extract through the protocols reported by Jan et al. (2020).This assay prepared a solution containing 300 U mL −1 of the COX-2 enzyme.For the activation of the enzyme, its solution (10 mL) was kept on ice for 5-10 min.
Additionally, the enzyme solution was combined with a cofactor solution [(50 mL); hematin: 1 mM; glutathione: 0.9 mM; TMPD The COX-2 enzyme's percent inhibition was calculated using the absorbance value per unit of time.The IC 50 values were calculated by creating a graph comparing the inhibition and concentration of the tested sample solution.In this assay, Celecoxib was used as a reference drug.

| 5-LOX assay
The 5-LOX assay was also performed to evaluate the antiinflammatory potential of A. nilotica's crude methanolic extract by following the methods of Jan et al. (2019).Purposely, different concentrations of the extract (62.5-1000 mg mL −1 ) were made in this assay.Subsequently, a 5-LOX enzyme solution comprising 10,000 U mL −1 of 5-LOX enzyme was prepared.Linoleic acid (80 mM) was used as a substrate to start the reaction.Moreover, a phosphate buffer (250 mL; 50 mM; pH 6.3) was mixed with crude extracts, followed by adding enzyme solution (250 mL) and incubating for 5 min.
After that, a substrate solution (500 mL) was mixed with an enzyme solution with continuous agitation.The absorbance (Abs) of control and test samples was examined at 234 nm via a ultraviolet-visible (UV-vis) spectrophotometer.Montelukast was used as a positive control in this assay.In the end, the percent inhibition of 5-LOX was calculated using the following formula:

| Statistical analysis
Results in this study were presented as mean ± SEM.Student's t test was performed to compare the inhibitory effects of standard drugs and A. nilotica's methanolic extracts.p-Value <.05 values was considered significant.

| Cyclooxygenase 2 (COX-2) inhibitory effect
The dose-dependent activity of the A. nilotica in

| DISCUSS ION
Natural compounds in plant extracts have shown hypoglycemic properties, besides their antioxidant capabilities by binding to glucose transporters and inhibiting digestive enzymatic activity (Baldea et al., 2010;Lacroix & Li-Chan, 2014).α-Amylase and α-glucosidase are the most critical hydrolyzing enzymes that break down starch from food and convert oligosaccharides into glucose, leading to a spike in blood sugar levels after a meal.Therefore, inhibiting their functionality is crucial for managing hyperglycemia in type 2 DM patients.The current study revealed a significant inhibitory effect of crude methanolic extract of A. nilotica against α-amylase and αglucosidase (Table 1).The results of our study are in accordance with the previous research conducted by Elbashir et al. (2018).They reported that an extract of A. nilotica found in Sudan demonstrated significant in vitro antidiabetic activity by inhibiting α-glucosidase activity.Earlier, Vadivel and Biesalski (2012) also showed a similar inhibitory (72.91%) potential of A. nilotica's seed extract against α-amylase.
The widespread prevalence and associated high mortality of neurological disorders have made them a primary global health concern.Currently, in clinical practice, few drugs help effectively treat neurological problems without causing harmful side effects.
Natural components are a promising solution due to their significant bioactivities and accessibility to humans.Thus, they are considered an essential alternative for developing novel drugs to treat neurological disorders (Anand et al., 2017) (Osama et al., 2015).Another group of researchers revealed the AChE inhibitory potential (0.35-5.40 μg mL −1 ) of extracts (ethyl acetate, butanol, aqueous, and chloroform) of A. nilotica pods (Crowch & Okello, 2009).
The COX and LOX pathways are vital in causing inflammation and related pain (American Diabetes Association, 2010).
However, further experimental investigations are needed to validate these potentialities.

| CON CLUS IONS
It can be concluded from the results of our current research that A. nilotica possesses varied bioactivities.Herein, we evaluated the in vitro antidiabetic property of A. nilotica's crude methanolic extract.
We confirmed that A. nilotica inhibited the activity of α-amylase and α-glucosidase with IC 50 values of 17 and 33 μg mL −1 , respectively.
Moreover, the experimented extract significantly inhibited the activity of cholinesterase (AChE and BChE).In a nutshell, these results will provide the scientific community with baseline data in planning and executing further in vivo and clinical studies to investigate the use of A. nilotica to treat or support treatment diabetes, inflammation, and AD.

Table 2
Antidiabetic activity of methanolic extract of Acacia nilotica.
Note:The effect of tested crude extract on antidiabetic activity.The values are expressed as mean ± SEM. ns (non-significant) compared to standard drugs (Acarbose).Data were analyzed via Student's t test.TA B L E 1 The effect of tested crude extract on anticholinesterase activity.The values are expressed as mean ± SEM. ***p < .001compared to standard drugs (Galantamine).Data were analyzed via student t test.Anticholinesterase activity of methanolic extract of Acacia nilotica.The effect of tested crude extract on anti-inflammatory activity.The values are expressed as mean ± SEM. ***p < .001compared to standard drugs (Celecoxib for COX-2 and Montelukast for 5-LOX).Data were analyzed via Student's t test.