Objective: Mitragyna inermis is important in traditional pharmacopeia and is used in treating diabetes and neurological diseases among others. This study involves inhibition of cholinesterases, α-amylase, α-glucosidase, tyrosinase and urease by compounds from M. inermis. Methods: Compounds were isolated from roots of M. inermis, identified using 1D and 2D NMR data and evaluated for their enzymes inhibitory effect. Results: The compounds were identified as emodin (MGA-1), citreorosein (MGA-2), a mixture (MGA-3) of 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) and (+)-bornesitol (MGA-4). These compounds are isolated from this plant for the first time and complete 1H NMR and 13C NMR data of MGA-3a and MGA-3b properly assigned. Compounds exhibited good acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition, with MGA-3 (IC50 = 25.65 ± 0.63 µg/mL) more active than galantamine (IC50 = 42.10 ± 0.44 µg/mL). All compounds, MGA-1 (IC50 = 76.89 ± 0.50 µg/mL), MGA-2 (IC50 = 43.87 ± 0.46 µg/mL), MGA-3 (IC50 = 74.73 ± 0.59 µg/mL) and MGA-4 (IC50 = 39.18 ± 0.33 µg/mL) were more active than acarbose (IC50 = 87.70 ± 0.68 µg/mL) in the α-glucosidase assay while in α-amylase inhibitory assay, only MGA-4 (IC50 = 28.69 ± 0.52 µg/mL) was more active than acarbose (IC50 = 32.25 ± 0.36 µg/mL). Compounds inhibited metalloenzymes (urease and tyrosinase) with MGA-1 (IC50 = 22.94 ± 0.61 µg/mL) and MGA-2 (IC50 = 20.48 ± 0.53 µg/mL) having greater antityrosinase activity than kojic acid (IC50 = 23.75 ± 0.30 µg/mL). Molecular docking suggested good structure-activity relationship revealed through binding affinities and negative binding energies. ADME predictions suggested that all compounds had good lipophilicities, only MGA-1, MGA-2 and MGA-4 had acceptable molecular weights, topological surface area, hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA). No compound could cross the blood-brain-barrier (BBB) and none showed alerts suggesting that the compounds do not contain substructures that are medicinally problematic. Conclusion: The compounds showed medicinal potential by inhibiting enzymes related to Alzheimer's disease, diabetes, hyperpigmentation and ureolytic bacterial infections.
Hundreds of metabolic stepwise reactions are catalyzed by enzymes which are capable of tapping chemical energy from breakdown of compounds and producing biological macromolecules.1 Though metabolic enzymes are important in the regulation of biochemical processes in living systems, their deficiencies, changes in expression levels or disruption can lead to metabolic impairments and illnesses.2,3 Some of the enzymes which are involved in some illnesses include acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, tyrosinase and urease.4,5 A balance in the amounts of various neurotransmitters including acetylcholine (ACh), dopamine, noradrenaline, serotonin, gamma-aminobutyric acid and glutamate is necessary for proper functioning of cognitive processes of the brain.6,7 This implies that a fall in the amounts of any of them such as acetylcholine can lead to deficient cholinergic neurotransmission causing pathophysiological conditions such as memory and learning impairments and this is common cause of adult dementia and Alzheimer's disease (AD).8 AD is a brain deteriorating condition which leads to cognitive impairment and dementia accompanied by cognitive dysfunctions such as loss of memory, thinking capacity, language problems, reasoning and behavioral patterns. Serine hydrolases such as cholinesterases, which are of two types, butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) can breakdown acetylcholine neurotransmitter leading to decrease in its amounts and termination of its action.9,10 The major cause of AD is associated with cholinergic deficiency, thereby making cholinesterase inhibitors from synthetic, natural and hybrid analogue sources to be considered as a suitable therapeutic strategy to remedy the disease AD.11,12 Available anticholinesterase drugs which are approved and commercially available include rivastigmine, metrifonate, donepezil, tacrine, galantamine and physostigmine that can inhibit acetylcholinesterase at the synapse and palliate AD.13 These conventional drugs are considered as non-cost-effective treatments and cases of undesirable side effects attributed to their use are reported, thereby, making natural therapeutics as suitable remedies for AD.14,15
Diabetes mellitus (DM) is a serious metabolic disease caused by high amounts of glucose in the blood. Type 1 diabetes involves extremely low, or inexistence of insulin caused by destruction of dysfunction of beta cells in the pancreas. Type 2 diabetes, also known as insulin resistance is a hyperglycemic condition in which insufficient amounts of insulin is produced or the insulin produced is unable to function properly in the conversion of glucose leading to the accumulation of the latter in the blood.16 Though the amounts of glucose in the blood can be regulated through physical exercise and feeding habits, there are carbohydrate metabolic enzymes inhibitors such as miglitol, acarbose and voglibose which inhibit α-glucosidase and α-amylase and delay glucose generation and absorption.17,18 These carbohydrate-hydrolyzing enzymes, α-amylase and α-glucosidase convert carbohydrate to monosaccharides such as glucose which can be assimilated and causing rise in blood glucose levels.19–21 This implies that the inhibition of these enzymes can slow hydrolysis and absorption of glucose and reduce diabetes risk or manage to keep sugar at safe levels in type 2 diabetes management.22,23
Tyrosinase is a copper-containing metalloenzyme that plays a key role in enzymatic browning and melanogenesis and therefore, its inhibitors are employed in medicinal and cosmetic industries to overcome hyperpigmentation and in food industries as antibrowning agents.24 Several synthetic tyrosinase inhibitors exist but some of which have side effects and are carcinogenic, thereby making natural antityrosinase compounds as suitable options for skin whitening agents and anti-melanogenetic agents.25,26 Urease is a nickel-containing metalloenzyme which hydrolyzes urea to gaseous ammonia and carbon dioxide and many ureolytic bacteria are responsible for infections and emission of ammonia from fertilizers.27 Urease plays a key role in the growth milieu and immune system of Helicobacter pylori which causes gastrointestinal ulcers, gastric cancer, duodenal and peptic ulcerations.28 Urease inhibitors can therefore find applications in agriculture to reduce ammonia losses through volatilization, and in human and animal health since ureolytic bacteria can cause ulcerations, kidney stone formation, hepatic encephalopathy, pyelonephritis and hepatic coma.29,30
Mitragyna inermis (Willd.) Kuntze (Rubiaceae), a small bushy tree or shrub is distributed in West and sub-saharan Africa where it is used in traditional medicine to treat hypertension, fever, anorexia, constipation, leprosy, epilepsy, diabetes, jaundice, syphilis, arthritis, stomach pains, tuberculosis, dysentery, schistosomiasis, malaria and mental illnesses.31–33 Phytochemical investigation of this plant has revealed the presence of tannins, alkaloids, anthraquinones, flavonoids, triterpenes, steroids, carotenoids, coumarins, leucoanthocyanins, anthocyanins, saponosides and cardiac glycosides with various biological activities.31,32,34 However, the roots of this plant are understudied.
It is imperative to surmount constraints associated with traditional drug identification methodologies by employing affordable, efficient, and all-encompassing computational techniques. High-throughput virtual screening is a screening method that medicinal chemists typically use. It is currently widely used in pharmaceutical industry research. Virtual screening is more feasible when a high-resolution crystal structure of the protein target is available to use as a template for computer screening. This work comprised of the isolation of metabolites from the roots of M. inermis and evaluation of their inhibitory potential against acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, tyrosinase and urease. Molecular docking was used to investigate the binding affinities of the compounds and structure-activity relationship while the druglikeness, and pharmacokinetics properties were performed through ADME (absorption, distribution, metabolism and excretion) predictions.
Materials and Methods
Plant Collection and Extraction
The plant was collected from Moundou town, Lac Wey Division, Logone Occidental Region in Chad during the month of May, 2022. It was identified by the botanist Mr B. Abouna of the Farcha Laboratory and deposited at the national herbarium of Chad where an authentic specimen number 0043 exists. The plant material was further authenticated by Mr Fulbert, a botanist of the National Herbarium of Cameroon under the voucher specimen numbered 8886/SRFcam. The work was done as part of ongoing research in the University of Ngaoundere in Cameroon and plant collection and processing was performed in accordance to the rules and regulations on the collection of biological materials for biological testing and drug discovery in Cameroon.35 Permissions for the study was granted by the University of Ngaoundere.
The roots of the plant were dried in the shade and ground into powder. Five hundred grams (500 g) of the powder was mixed with 5 L of water:dichloromethane (1:1). Water was chosen because it is the aqueous extract that is used in traditional medicine and dichloromethane is used so that it breaks cell walls of plant material for the release of active compounds into the solvent during extraction. The mixture was then immersed into an ultrasonic device (Caliskan ultrasonic cleaner bath LAB.ULT.4030) containing distilled water and irradiated at 40 kHz and 100 W for a preset extraction time of 1 h.36 The supernatant was filtered and evaporated to dryness under vacuum at 50 °C on a rotary evaporator. This extraction process was repeated 3 times and yielded a crude extract. The crude extract was re-extracted with hexane to get rid of fatty substances and the residue was dried to afford 20 g.
Isolation and Identification of Compounds
Isolation of Compounds
A portion of the defatted extract (15 g) was used in preparing slurry with silica gel before being subjected to column chromatography. 200 g of silica gel were filled up in a column by wet filling and the slurry was introduced and eluted on a gradient polarity scale as follows: DCM:EtOAc (0-100%) then EtOAc:MeOH (0-100%) while collecting equal volumes of 100 mL which were subsequently pooled into 5 major fractions based on their TLC profiles. The fractions were denoted A to E. Fraction A (1.3 g) was rechromatographed on silica gel column using an isocratic eluent DCM:EtOAc (30%) to give 24 sub-fractions which were regrouped into two which precipitated and were separately filtered and washed with diethyl ether to afford MGA-1 (68.5 mg) and MGA-2 (51.0 mg). Fraction C was subjected on a silica gel column using an isocratic eluent EtOAc:MeOH (5%) and afforded fractions which precipitated on standing and were filtered to give a mixture of compounds MGA-3a and MGA-3b (1:0.45) (35.0 mg). Fraction D was eluted using gradient scale of EtOAc:MeOH (10-20%) and yielded compound MGA-4 (22.0 mg) as a precipitate on standing.
Identification of Isolated Compounds
The compounds were identified using a combination of one-dimensional (1D) and two-dimensional (2D) NMR experiments, optical rotation and comparing the data with those described in the literature. Optical rotation [a]D20 + 30.02° (c 0.91, H2O) for MGA-4 was measured with a Jasco P-2000 polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCEII + 400 NMR spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. 1D and 2D NMR spectra of which enabled the identification of the compounds are presented in Figures S1-S4. 1H and 13C NMR data of the mixture MGA-3 (MGA-3a and MGA-3b) are presented in Table S1.
Enzymes Inhibition Assays
Anticholinesterase Activity
The inhibitory activities of acetylcholinesterase and butyrylcholinesterase by the compounds were determined using spectrophotometer following the protocol described elsewhere, with slight modifications.37,38 The Galantamine was used as a reference compound. The IC50 values were calculated using a program derived from the anticholinesterase graph. Percentages of inhibitory activity (% inhibition) were developed relative to sample concentrations (μg/mL).
In Vitro α-Amylase and α-Glucosidase Inhibitory Assay
The α-amylase inhibitory activity was evaluated by using starch-iodine method with some modifications.39 50 µL of α-amylase from porcine pancreas in pH 6.9 phosphate buffer prepared with 6 mM NaCl and 25 µL of sample solutions were mixed in a 96-well microplate. The mixture was pre-incubated for 10 min at 37 °C and 50 µL of starch solution (0.05%) was added and incubated additionally for 10 min at 37 °C. Following incubation, the reaction was completed by adding HCl (0.1 M, 25 µL) and Lugol (100 µL) solutions, and the absorbance was recorded at 630 nm.
The α-glucosidase inhibitory activity was evaluated according to the method described previously.40 50 µL of glutathione, 50 µL of sample solution, 50 µL of α-glucosidase from Saccharomyces cerevisiae in phosphate buffer (0.01 M pH 6.8) and 50 µL of PNPG (4-N-nitrophenyl-α-D-glucopyranoside) in phosphate buffer (0.01 M pH 6.8) were mixed in a 96-well microplate. Then the solution was incubated for 15 min at 37 °C. The reaction was then stopped with the addition of 50 μL sodium carbonate (0.2 M) and the absorbances were read at 400 nm. Acarbose was used as a standard compound for both analyses. Results were given as percentage inhibition (%) at 100 µg/mL and 50% inhibition concentration (IC50).
Anti-Urease Activity
The inhibitory activity of the urease enzyme by each compound were evaluated by determining ammonia production with the indophenol method using a microplate reader.41,42 Briefly, 25 μL of enzymatic urease solution (jack bean source), 50 μL Urea (100 mM) and (100 mM) of sodium phosphate buffer (pH 8.2) were mixed and incubated at 30 °C for 15 min after adding the sample (10 μL). Then 70 μL of alkali reagent and 45 μL Phenol reagent were added to each well. After 50 min of incubation, the absorbance was recorded at 630 nm using a microplate reader. The reference compound used was thiourea, and the results were expressed as a 50% inhibitory concentration (IC50).
Anti-Tyrosinase Activity
Tyrosinase enzyme inhibitory activity was measured by the spectrophotometric method as described previously.43 L-DOPA was utilized as substrate of the reaction. 150 μL of sodium phosphate buffer (pH 6.8, 100 mM), 10 μL of sample and 20 μL of tyrosinase enzyme solution in buffer were mixed and incubated for 10 min at 37 °C. Following incubation, 20 μL of L-DOPA was added. The absorbances in a 96-well microplate were monitored at 475 nm after 10 min of incubation at 37 °C. Kojic acid was used as a reference compound. Results were stated as percentage inhibition (%) at 100 µg/mL and 50% inhibition concentration (IC50).
Molecular Docking Details
The binding affinities of the isolated compounds emodin (MGA-1), citreorosein (MGA-2), 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a), 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), and (+)-bornesitol (MGA-4) into the binding sites of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease were determined using the Autodock4 package.44 X-ray coordinates of the targeted α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease along with their corresponding original docked ligands were downloaded from the RCSB data bank website of PDB codes 3W37, 1B2Y, 4EY7, 1P0I, 2Y9X, and 1FWE respectively.45–50 Structural geometries of isolated constituents emodin (MGA-1), citreorosein (MGA-2), 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a), 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), and (+)-bornesitol (MGA-4) were minimized using Merck molecular force field 94 (MMFF94) level44 and they were saved as PDB files. Molecular docking stepwise procedure is found in our previous reported studies.15,21 The binding interactions of the docked emodin (MGA-1), citreorosein (MGA-2), 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a), 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), and (+)-bornesitol (MGA-4) into the binding sites of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease were visualized using the Discovery Studio Client (Discovery Studio Client is A Product of Accelrys Inc., San Diego, CA, USA).
ADME and Druglikeness Properties
The predictions of ADME (absorption, distribution, metabolism and excretion), druglikeness, pharmacokinetics, and physico-chemical properties of emodin (MGA-1), citreorosein (MGA-2), 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a), 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), and (+)-bornesitol (MGA-4) were carried out using the Swiss ADME tool available at the https://www.swissadme.ch/ website. Further, the Swiss target prediction tool (https://www.swisstargetprediction.ch/) was employed to predict the probable targets of emodin (MGA-1), citreorosein (MGA-2), 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a), 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) and bornesitol (MGA-4).
Statistical Analysis
Activity assays were performed in triplicate analyses. The data were recorded as means ± Standard Error of the Means (SEM). Minitab 16 statistical software were used to determine the significant differences between means using one-way ANOVA (analysis of variance), in which p < 0.05 were regarded as significant.
Results
Isolated Compounds
The structures of the isolated compounds were elucidated by a combination of 1D and 2D data, optical rotation and comparison with literature data. The compounds were identified as the anthraquinones emodin (MGA-1)51 and citreorosein (MGA-2).52MGA-3 was identified as a mixture of two xanthone diglycosides: 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (1-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl]-8-hydroxy-3,7-dimethoxyxanthone) (MGA-3a)53 and 1-O-primeverosyl-37,8-trimethoxyxanthone (1-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl]-37,8-trimethoxyxanthone) (MGA-3b)54 in ratio 1:0.45, respectively. Compound MGA-4 was a cyclitol identified as (+)-bornesitol.55,56 The structures of the isolated compounds are provided on Figure 1.
Structures of the Compounds Isolated from M. Inermis Roots.
Anticholinesterase Activity
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes hinders neurotransmission activity of acetylcholine (Ach) since it reduces its levels. By inhibiting cholinesterases (AChE and BChE) the levels of ACh in the central nervous system can be boosted. The inhibition of cholinesterase enzymes by the compounds are presented on Table 1. The compounds MGA-1, MGA-2 and MGA-3 exhibited IC50 values within tested concentrations (maximum test concentration of 200 µg/mL). The mixture MGA-3 (IC50 = 29.43 ± 0.58 µg/mL) was the most active in the AChE assay. In the BChE assay, MGA-1 (IC50 = 40.28 ± 0.31 µg/mL), MGA-2 (IC50 = 29.10 ± 0.25 µg/mL) and MGA-3 (IC50 = 25.65 ± 0.63 µg/mL) were more active than the standard galantamine (IC50 = 42.10 ± 0.44 µg/mL).
Cholinesterase, Antidiabetic, Antiurease and Antityrosinase Activities of the Compoundsa.
Cholinesterase Inhibitory Activity
Anti-Diabetic Activity
Samples /
Standards
AChE
BChE
α-Glucosidase
α-Amylase
Urease Inhibitory
Tyrosinase Inhibitory
IC50 (µg/mL)
MGA-1
53.21 ± 0.14
40.28 ± 0.31
76.89 ± 0.50
32.60 ± 0.26
62.47 ± 0.38
22.94 ± 0.61
MGA-2
38.52 ± 0.27
29.10 ± 0.25
43.87 ± 0.46
37.33 ± 0.63
49.14 ± 0.75
20.48 ± 0.53
MGA-3
29.43 ± 0.58
25.65 ± 0.63
74.73 ± 0.59
58.38 ± 0.45
31.53 ± 0.22
59.83 ± 0.81
MGA-4
>200
> 200
39.18 ± 0.33
28.69 ± 0.52
> 200
> 200
Galantamine
5.50 ± 0.25
42.10 ± 0.44
NT
NT
NT
NT
Acarbose
NT
NT
87.70 ± 0.68
32.25 ± 0.36
NT
NT
Thiourea
NT
NT
NT
NT
8.20 ± 0.35
NT
Kojic acid
NT
NT
NT
NT
NT
23.75 ± 0.30
Values represent the means ± SEM of three parallel sample measurements (p < 0.05).
NT: not tested.
Inhibition of α-Amylase and α-Glucosidase
Postprandial hyperglycemia can be reduced by inhibiting α-amylase and α-glucosidase which are enzymes that hydrolyze carbohydrates to produce glucose. The inhibitory potential of the isolated compounds on both enzymes was evaluated and the results are presented in Table 1. All substances inhibited α-amylase and α-glucosidase as in the α-glucosidase assay and the tested compounds were more active than acarbose (IC50 = 87.70 ± 0.68 µg/mL). While against α-amylase, only MGA-4 (IC50 = 28.69 ± 0.52 µg/mL) was more active that acarbose (IC50 = 32.25 ± 0.36 µg/mL) though MGA-1 and MGA-2 exhibited almost the same inhibition as acarbose.
Tyrosinase and Urease Inhibitory Activities
The inhibition of metalloenzymes, urease and tyrosinase by the compounds is presented in Table 1. Though no compound was more active than the standard thiourea (IC50 = 8.20 ± 0.35 µg/mL) in the urease inhibitory assay, MGA-3 (IC50 = 31.53 ± 0.22 µg/mL) was the most active while MGA-1 (IC50 = 62.47 ± 0.38 µg/mL) and MGA-2 (IC50 = 49.14 ± 0.75 µg/mL) were appreciably active. The compounds displayed good antityrosinase activity with the exception of (+)-bornesitol. The two anthraquinones MGA-1 (IC50 = 22.94 ± 0.61 µg/mL) and MGA-2 (IC50 = 20.48 ± 0.53 µg/mL) were more active than the standard kojic acid (IC50 = 23.75 ± 0.30 µg/mL).
Molecular Docking Results
The observed inhibitory of the isolated compounds emodin (MGA-1), citreorosein (MGA-2), 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a), 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), and (+)-bornesitol (MGA-4) towards the targets α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease are given in Table 1. From IC50 values, it is evident that the inhibitory of the isolated compounds may strongly depend on the basic skeleton features of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4. These compounds may divide into three subsets MGA-1 and MGA-2forms the first set with anthracene-9,10-dione as the basic skeleton. MGA-3a and MGA-3b form the second set with 9H-xanthen-9-one as the basic skeleton. MGA-4 forms its subset with cyclohexane as its corresponding basic skeletons. In an attempt to explain the observed inhibitions of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 against α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease the targets, molecular docking is employed to determine the binding modes between the tilted compounds from one side and the active residues of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease. Table 2 summarizes the free binding energies, the number of hydrogen bonds, and the number of interactions in the complexes formed between MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 isolated compounds and the active residues of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease.
Free Binding Energies, Hydrogen Bonding, and Number of Closest Residues to the Docked Compounds MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 into the Binding Sites of α-Amylase, Acetylcholinesterase, Butyrylcholinesterase, Tyrosinase, and Urease Along with Their Corresponding IC50 Values.
No. of
Compound
Free Binding
Energy
(kcal/mol)
H-Bonds
(HBs)
Number of Closest Residues
to the Docked Ligand in the
Active Site/Number of Interactions
IC50 ± SEM
α-glucosidase
MGA-1
–6.00
4
7/12
76.89 ± 0.50
MGA-2
–6.54
5
7/11
43.87 ± 0.46
MGA-3a
–6.59
5
9/14
MGA-3b
–7.26
5
8/11
MGA-4
–6.34
7
4/7
39.18 ± 0.33
α-amylase
MGA-1
–6.52
2
4/5
32.60 ± 0.26
MGA-2
–6.63
5
5/6
37.33 ± 0.63
MGA-3a
–7.72
7
11/15
MGA-3b
–7.88
9
9/11
MGA-4
–4.79
6
6/6
28.69 ± 0.52
acetylcholinesterase
MGA-1
–7.74
4
7/10
53.21 ± 0.14
MGA-2
–7.97
4
6/8
38.52 ± 0.27
MGA-3a
–10.55
7
10/13
MGA-3b
–10.78
8
12/15
MGA-4
–4.70
5
3/5
> 200
Butyrylcholinesterase
MGA-1
–7.12
6
8/8
40.28 ± 0.31
MGA-2
–7.26
2
7/10
29.10 ± 0.25
MGA-3a
–8.35
7
9/11
MGA-3b
–8.22
6
15/16
MGA-4
–3.68
7
4/7
> 200
Tyrosinase
MGA-1
–6.61
4
9/13
22.94 ± 0.61
MGA-2
–6.38
3
8/11
20.48 ± 0.53
MGA-3a
–5.91
6
7/9
MGA-3b
–6.34
5
9/12
MGA-4
–4.09
6
5/7
> 200
Urease
MGA-1
–5.46
3
9/13
62.47 ± 0.38
MGA-2
–5.76
5
7/10
49.14 ± 0.75
MGA-3a
–6.85
7
11/15
MGA-3b
–6.55
6
13/16
MGA-4
–5.03
6
5/6
> 200
From molecular docking results, MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 compounds are relatively well fit into the binding site of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease. They form stable complexes into the binding sites of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease with negative binding energies. The binding energies were in the ranges −6 to −7.26 kcalmol−1 (α-glucosidase), −4.79 to −7.88 kcalmol−1 (α-amylase), −4.7 to −10.78 kcalmol−1 (AChE), −3.68 to −8.35 kcalmol−1 (BChE), −4.09 to −6.61 kcalmol−1 (tyrosinase) and −5.03 to −6.85 kcalmol−1 (urease) respectively (Table 1). Those have negative binding energies more than −5.00 kcalmol−1may considered as weak/inactive inhibitors, which is the case of the inhibitory potency of MGA-4 towards acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease. However, those display negative binding energies below −5.00 kcalmol−1 may have the potency to inhibit α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, urease, and that the inhibition process may be considered thermodynamically favorable (Table 1). Figure 2 displays the most stable complexes established between the docked MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 compounds into the binding site of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease. MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 have different basis skeletons, which may explain the difference in the observed inhibitory in regards to each target. 2D closest interactions of the most stable complexes formed by the isolated compounds into the binding site of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase, and urease are given on Figure 2. Complete 3D and 2D binding interactions of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 in the binding sites of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase and urease are given on Figures S5-S10.
2D Closest Interactions of the Most Stable Complexes Formed Between the Isolated Compounds into the Binding Site of α-Glucosidase, α-Amylase, Acetylcholinesterase, Butyrylcholinesterase, Tyrosinase, and Urease.
Predicted ADME and Druglikeness of the Isolated Compounds
The predicted ADME and druglikeness properties of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 are summarized in Tables S2-S7. Except MGA-3a and MGA-3b, all isolated compounds, that is, emodin (MGA-1), citreorosein (MGA-2) and (+)-bornesitol (MGA-4) adhere to Lipinski's rule of five. All compounds have lipophilicity value (MlogP) lower than the threshold value 4.15 (Tables S3 and S6), indicating their good druglikeness properties. Emodin (MGA-1), citreorosein (MGA-2) and (+)-bornesitol (MGA-4) have topological surface area (TPSA) within the range 78–125 Ų, suggesting that they are likely to be orally absorbed, with bioavailability scores of 0.55 (Table S6). The bioavailability radars of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 are presented in Figure 3. The bioavailability radars analysis reveals that MGA-4 falls within the pink area of the polygon. Emodin (MGA-1), and citreorosein (MGA-2) fall within the pink area of the polygon except for the unsaturation parameter. This suggests that these compounds may have good oral bioavailability. The pharmacokinetic properties in Table S5 indicate that emodin (MGA-1), citreorosein (MGA-2) have high gastrointestinal (GI) absorption, while MGA-3a, MGA-3b, and MGA-4 have low GI. MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4 didn’t exhibit penetration through the Blood-Brain Barrier (BBB). The Boiled-egg model as shown in Figure 4 indicates poor BBB penetration and good GI absorption. The generated pie chart in Figure 5 shows the probable and predicted biological targets of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4.
Bioavailability Radars of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4.
Boiled-Egg Model of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4.
Predicted Biological Targets of MGA-1, MGA-2, MGA-3a, MGA-3b, and MGA-4.
Discussion
The compounds isolated from the roots of M. inermis could be classified as two anthraquinones emodin (MGA-1), citreorosein (MGA-2), a mixture of two xanthone diglycosides (MGA-3) 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), and one cyclic polyol (+)-bornesitol (MGA-4). These compounds are isolated from M. inermis for the first time to the best of our knowledge. Although some quinone derivatives have been described in this plant, anthraquinones have not been isolated from it. In the same light, certain phenolic glycosides have been isolated from this plant previously, but xanthone glycosides have not been described. In terms of the classes of compounds isolated, the work described here corroborates in part with works reported.33 It can be understood that the roots of M. inermis are rich in cyclic ketone derivatives since in previous study 3-acetyl-2,6-dimethyl-4H-pyran 4-one and 3-acetylpentane-2,4-dione were identified by GC-MS in root extracts of the plant.57
The isolated compounds demonstrated in vitro enzymes inhibitory effects against cholinesterases, α-amylase, α-glucosidase, urease and tyrosinase. They inhibited AChE and BChE which are enzymes that cause fall in Ach amounts and neurodeterioration associated with AD. This means that, the compounds could be used to develop remedies for AD. The plant M. inermis has been shown to have various effects on the central nervous system including anticonvulsant, muscle relaxant, stimulant, antispasmodic, antiamnesique and neuroprotective effects.32,35,58,59 It could be explained by the fact that the components isolated and tested in this study contribute to the beneficial effects of this plant on the central nervous system. Though no direct cure exists for AD, medicinal plants and their constituents can provide symptomatic treatments and relieve neurodegeneration and dementia and they intervene through various mechanisms of action with lower risks of side effects.12,60–64 The compounds in this study thus provide alternative AD medications. Phytochemicals are able to decelerate onset of AD and its progression, eliminating amyloid-beta (Aβ) plaques alongside other multitargeting approaches involving antioxidant, anti-inflammatory and enzyme-mediated mechanisms to provide neuroprotection and neurodegeneration.65 The diverse chemical scaffolds isolated from M. inermis in this study are possible anticholinesterase pharmacophores. Anthraquinones including emodin have been described previously as natural dual inhibitors of both AChE and BChE.66 The effects of anthraquinones including emodin (6-methyl-13,8-trihydroxyanthraquinone) in treating AD involves anti-inflammatory effects as well as prevention of abnormal aggregation of tau proteins in the brain.67–69 Citreorosein (13,8-trihydroxy-6-(hydroxymethyl) anthraquinone) also possess antiinflammtory effects which could contribute to neuroprotection.70
Lowering blood glucose is an inevitable strategy towards the management of hyperglycemia and diabetes. The isolated compounds inhibited α-glucosidase and α-amylase indicating that they could be used in lowering postprandial blood glucose. Carbohydrate metabolism by α-amylase and α-glucosidase contributes to increasing blood glucose levels since both enzymes are involved in glucose breakdown into absorbable monosaccharides and their intake into the blood system.71 Several natural inhibitors of α-glucosidase and α-amylase from natural sources and medicinal plants are alternative antidiabetic drugs with great efficacy and little side effects.72–75 The plant M. inermis is used in managing diabetic conditions and it has been shown experimentally to be able to lower blood glucose levels in rat models and also inhibiting advanced glycation end-products.76–78 The compounds contained in this plant could be responsible for its hypoglycemic effects. Emodin and citreorosein exhibited appreciable inhibition of α-amylase and α-glucosidase activities and they were more active than the standard drug acarbose in the α-glucosidase assay. Anthraquinones have been shown to be good inhibitors of carbohydrate digestive enzymes and also to possess antidiabetic effects.79,80 Anthraquinones exercise antidiabetic effects through phosphoinositide-3-kinase and Akt-ser473 expression, upregulation of insulin receptor substrates-1, elevation of glucagon-like peptide-1 levels, activation of peroxisome proliferator-activated receptors gamma and inhibition of α-glucosidase and the number of hydroxyl groups in the chemical scaffold greatly contributes to the overall antidiabetic activity.79 This could explain why citreorosein was more active than emodin. The compound with the best antidiabetic effects was (+)-bornesitol, exhibiting greater activity than the standard acarbose. Cyclitols such as D-pinitol and (+)-bornesitol is a natural polyols with known antidiabetic activity together with antioxidant, anticancer and anti-inflammatory properties.81 In one study D-pinitol which resembles (+)-bornesitol structurally was in high concentrations in plant extracts which were shown to correlate with antidiabetic effects.82
H. pylori exhibits tolerance to acidity because of urease which triggers hydrolysis of urea, releasing ammonia which in turn creates an alkaline milieu that is favorable for the survival of the bacteria and also causes inflammation in immune cells, fibroblasts and uroepithelium causing various ailments.83,84 The anthraquinones in this work exhibited moderate inhibitory effects of urease and this conforms to previous studies in which anthraquinones including emodin and its derivatives showed antiurease effects.84,85 Accumulation of ammonia in the body causes undesirable rise in pH leading various diseases like pyelonephritis, urolithiasis, hepatic coma, catheter scab, hepatic and ammonia encephalopathy.86 The compounds emodin (MGA-1) and citreorosein (MGA-2), therefore may be used as potential urease inhibitors which can find application in health for the treatment of diseases caused by ureolytic bacteria and also in reducing degradation of urea based fertilizers in agriculture.
Emodin (MGA-1), citreorosein (MGA-2) as well as MGA-3 which is a mixture of 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b), exhibited good tyrosinase inhibition. The two anthraquinones (emodin and citreaosein) showed greater tyrosinase inhibition than kojic acid which is used as the standard antityrosinase compound. Tyrosinase plays an important part in the production of melanin by converting tyrosine to L-DOPA and finally to melanin proper which gives colour to skin, hair and eyes. Though melanin protects the skin from harmful radiations and environmental stresses, an over-expression of tyrosinase leads to excessive melanin pigmentation, causing senile plaques and melanogenesis.87 Besides causing skin problems in humans, tyrosinase over-expression also causes browning of fruits, fungi and vegetables and there is need to develop new tyrosinase inhibitors from natural and synthetic sources which find applications in medicine, food and cosmetics.24,25,88 Anthraquinones are one of the most potent tyrosinase inhibitory compounds from natural sources.87,89 This corroborates with the profound tyrosinase inhibitory effect of emodin and citreorosein in this study. In one study, anthraquinones including emodin and citreorosein showed strong inhibition of tyrosinase compared to kojic acid and they were able permeate the skin, indicating strong potency as skin whitening agents and therapy for melanogenesis and hyperpigmentation.90 The results indicate that the isolated compounds are potent natural tyrosinase inhibitors which could be used to reduce enzyme browning reactions, skin melanization, unwanted pigmentation and skin spots.
The results of the in vitro assays were further substantiated with the in silico evaluations. The compounds (+)-bornesitol (MGA-4), emodin (MGA-1), citreorosein (MGA-2) as well as MGA-3 which is a mixture of 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) exhibited interactions binding sites of α-glucosidase, α-amylase, acetylcholinesterase, butyrylcholinesterase, tyrosinase and urease. 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) had the lowest binding energy in the α-glucosidase assay while all other compounds had binding energies lower than 5 kcalmol−1, which indicates good potency. 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) showed the lowest binding energy and the highest number of H-bonds when docked in the α-amylase receptor while (+)-bornesitol had the highest binding energy though with the lowest IC50 value. 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) had the lowest binding energy and the highest number of H-bonds when docked in the acetylcholinesterase receptor while in the butyrylcholinesterase docking, it had lowest binding energy and the highest number of H-bonds was exhibited by 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) and (+)-bornesitol. In the tyrosinase receptor, emodin (MGA-1) had the lowest binding energy while 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and (+)-bornesitol had the highest number of H-bonds. Within the urease receptor site, 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) showed the lowest binding energy and the highest number of H-bonds. In molecular docking, binding energy describes how strongly a protein interacts with a ligand and predicting a ligand's location in the receptor's active site that has the lowest binding energy and the optimizations. It is useful to compare the binding energies in kcal/mol and bonding interactions of various proteins and ligands including hydrophobic interactions and hydrogen bonding patterns to predict bioactivity.91,92 Predicting the ligand-protein complex's binding orientation and stability is the aim of molecular docking, and the binding energy offers useful information in this respect and gives insights in structure-activity relationship as well as prediction of bioactivities.93,94 The molecular docking results indicate that all the compounds have good potent activities in all assays and bind effectively to the active sites of the receptor proteins except for (+)-bornesitol in the α-amylase, AChE, BChE and tyrosinase. The compounds have different basic skeletons, which may explain the observed inhibitory in regards to each target. For antidiabetic tests towards α-glucosidase and α-amylase, (+)-bornesitol shows the strongest efficiency, which may be attributable to the number of hydrogen bonds formed between it and the amino acids of α-glucosidase, and α-amylase (Table 1). The good anticholinesterase and antiurease effects of the mixture of 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) is attributable to their low binding energies and high number of hydrogen bonds formed with the corresponding receptor protein. In the same way, the good antityrosinase activity of emodin (MGA-1) and citreorosein (MGA-2) is reflected by their low binding energies.
In silico predictions of some molecular descriptors of the compounds including physicochemical properties, lipophilicity, solubility, pharmacokinetic, druglikeness and medicinal properties are reported on Tables S2-S7. Compounds MGA-3a and MGA-3b have molecular weights greater than 500 g/mol, indicating that they do not meet the mass criterion for suitable drug. However, some authors predict that molecular weights below 1000 g/mol makes compounds easy to be absorbed, diffused and transported within the body.95 The number of hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) are within acceptable values for MGA-1, MGA-2 and MGA-4 as indicated on Table S3. Good values for rotatable bonds number should be less than 12, number of HBA should be less than or equal to 10 while HBD should be less than or equal to 5.96 Topological polar surface area (TPSA) scores were suitable for MGA-1, MGA-2 and MGA-4 as it is suggested that PSA scores below 140 Å is an indication that the molecule is appropriate for human absorption and does not have passive cellular permeability.97 Total polar surface area (TPSA) less than 150 Å, revealing strong polarity with good oral absorption and membrane permeation. The lipophilicity of molecules affects their toxicological and pharmacodynamics properties and influences membrane permeability and solubility. The lipophilic predictions of the compounds are presented on Table S3. lipophilicity is usually expressed as the logarithmic of n-octanol-water partition coefficient (logP) which is characteristic for each molecule. It is believed that lipophilicity values of logP > 5 is responsible for undesirable effects such as strong plasma protein binding, fast metabolic turnover, tissue accumulation and poor water solubility.98 All the compounds in this study were characterized by lipophilicity values below the maximum value, indicating that they all meet the criterion of suitable lipophilicity. Appropriate lipophilicities of these compounds are an indication that they have good permeability, with high chances of reaching targets and bind specifically to plasma proteins. One of the biggest challenges in the drug development and discovery process is poor drug solubility. The appropriate solubility in water and intestinal fluids is relevant in absorption and getting sufficient blood concentrations of drug molecules to achieve desired therapeutic effects.99 The aqueous solubilities of the compounds were predicted and presented on Table S4. It is observed that all compounds were soluble in water according to the in silico predictions and this was observed experimentally as well. Aqueous solubility and intestinal permeability are preliminary of oral bioavailability. The analysis of bioavailability radars indicates that MGA-4 falls within the pink area of the polygon while MGA-3a and MGA-3b fall within the polygon except for size and polarity. In addition, emodin (MGA-1), and citreorosein (MGA-2) fall within the pink area of the polygon except for their unsaturation parameter. The overall bioavailability information as presented on the bioavailability radars (Figure 3) suggest that these compounds may have good oral bioavailability. The boiled egg model suggests that emodin (MGA-1), citreorosein (MGA-2) exhibit good gastrointestinal (GI) absorption, while MGA-3a, MGA-3b, and MGA-4 have low GI. None of the compounds exhibited Blood-Brain Barrier (BBB) penetration. Predicted pharmacokinetic properties presented on Table S5 shows high gastrointestinal absorption of compounds MGA-1 and MGA-2 which were also non-substrates to P-glycoprotein and CYP3A4 inhibitors. Only MGA-1 was CYP1A2 inhibitor while none of the compounds could cross the blood-brain-barrier (BBB). Cytochrome P are relevant for drug metabolism and detoxification, but reduced drug clearance, toxicity or ineffectiveness of drugs may arise from their inhibition or activation.100 The skin permeability (log Kp) for the compounds were good and meets requirements. A substance's skin permeability is measured in Log Kp values; if the log Kp value is larger than 2.5 (Log Kp > 2.5), the compound is considered to have low skin permeability.101 Generally, the Lipinski rule of 5 is important in the predictions. The rule states that a molecule or an inhibitor can be orally absorbed/active if two (2) or more of these thresholds; molecular weight (Mw) of molecule < 500, octanol/water partition coefficient (iLOGP) ≤ 5, number of hydrogen bond acceptors (nHBA) ≤ 10, number of hydrogen bond donors (nHBD) ≤ 5, and topological polar surface area (TPSA) < 140 Å2) are not violated.102,103 Compounds MGA-3a and MGA-3b violated Lipinski rules and others while compound MGA-4 violated one Ghose rule and two Muegge rules as shown on Table S6. As concerns the medicinal properties of the compounds, potentially problematic fragments were predicted and presented on Table S7. Also, the failure to satisfy the criteria for drug “leadlikeness” was simulated as well as the synthetic accessibilities of the compounds. Synthetic accessibility is crucial in computer-aided drug design as it predicts that the most promising virtual molecules can possibly be synthesized and put through biological assays or other experiments.104 The results suggest that the compounds do not contain substructures that are medicinally problematic and that compounds could be synthesized with relative ease. The overall in silico studies indicate the pharmacokinetic and pharmacodynamics parameters of the studied compounds and support the results from the in vitro studies. Pharmacokinetic and pharmacodynamic behavior of natural compounds can be understood through comprehensive ADME (absorption, distribution, metabolism, and excretion) evaluations.105 The anthraquinones and xanthone glycosides possess aromatic rings and hydroxide groups attached to the rings and this could help to improve the inhibitory effects against cholinesterase enzymes either through direct inhibition or antioxidant process. The compounds were more active than the standard galantamine in the BChE assay. This was substantiated by the molecular dicking as they shiwed good binding scores ranging from −7.74 to −10.78 kcal/mol in the AChE assay and from −7.12 to −8.35 kcal/mol in the BChE assay. The presence of aromatic rings and hydroxyl groups in anthraquinones and xanthone glycosides compounds is believed to play a role in modulating anticholinesterase activity and this has been demonstrated that aromatic the rings undergoes a π–π stacking against aminophenol residues of AChE, and the structure–activity relationship proven through molecular docking.106 These compounds also possess carbonyl functional groups which can interact with cholinesterases and influence the inhibitory activity and binding affinity. The phenolic nature of the anthraquinones and xanthone glycosides compounds, plays a significant role in determining the inhibitory activity and mechanism of action against α-amylase and α-glucosidase, contributing to alleviation of hyperglycemia. Conjugated π-system in benzene ring and presence of hydroxyl groups enhance binding affinity to α-amylase and α-glucosidase while reducing their catalytic efficiency through hydrogen bonds, hydrophobic force, or π-π interaction as proven by experimental and docking.107 The structure of anthraquinones and xanthone glycosides can significantly influence their ability to inhibit tyrosinase, acting as substrates or competitive inhibitors. Among natural phenolic compounds acting as tyrosinase inhibitors, anthraquinones are among the most active and their substructure are similar to that of standard tyrosinase inhibitors such as kojic acid. The anthraquinones and xanthone glycosides can act as copper chelators or structural mimics of tyrosinase substrates, thereby causing competitive inhibition.108 The anthraquinones and xanthone glycosides can have a big impact on urease inhibition, especially those with particular configurations of aromatic rings and hydroxyl groups. The capacity of the compounds to bind with and inhibit the urease enzyme is largely dependent on its size, shape, and availability of hydroxyl groups, particularly in ortho or para orientations. Compounds which are phenolic in nature can inhibit urease either by targeting the Ni2+ in the active site directly or by interaction with the flexible peptide covering the active or by nonspecific hydrophobic/hydrophilic linkages.109 Considering the above aspects, some insights can be made to the enzymes inhibitory effects of the anthraquinones and xanthone glycoside compounds.
This studies is an in vitro and in silico evaluation and may involve some limitations as follows. The results will require in vivo studies to ascertain the antidiabetic and anticholinesterase properties of the compounds and their potential application in treating AD and diabetes. Additionally, the toxicity of the compounds need to be elucidated.
Conclusion
Medicinal plants such as Mitragyna inermis have proven their virtues within the traditional pharmacopoeia as a remedy for many ailments including diabetes and neurological disorders. Some biological activity tests carried on this plant's extracts have been able to prove scientifically its important bioactivities. The aerial parts of the plant are well studied while little interest is paid to its roots. In the present study, M. inermis roots were collected and some of its components were isolated and identified. These were emodin (MGA-1), citreorosein (MGA-2), a mixture of 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) (MGA-3) and (+)-bornesitol (MGA-4). It should be noted that this is one of the prime studies on the metabolites of M. inermis roots. The isolated compounds showed important enzyme inhibitory properties. They inhibited cholinesterases (AChE and BChE) with MGA-3, a mixture of 1-O-primeverosyl-8-hydroxy-3,7-dimethoxyxanthone (MGA-3a) and 1-O-primeverosyl-37,8-trimethoxyxanthone (MGA-3b) being the most active against cholinesterase which suggests that the constituents could be responsible for the potential of the plant in relieving symptoms of Alzheimer's disease. The tested components inhibited carbohydrate hydrolyzing enzymes α-amylase and α-glucosidase, with (+)-bornesitol (MGA-4) being the most active, showing greater inhibition than the standard drug acarbose. These suggest that the studied compounds could be used to slow glucose production from carbohydrate breakdown. Urease was also inhibited by the tested substances which indicates that they could remedy ailments caused by ureolytic bacteria. The isolated compounds have potential in preventing melanogenesis, hyperpigmentation and fast ripening and browning of fruits and vegetables due to their good antityrosinase effect especially that of the anthraquinones emodin (MGA-1) and citreorosein (MGA-2). The activities were substantiated with molecular docking studies, where compounds showed good binding affinities to respective proteins with negative binding energies. The isolated components showed medicinal potential since most of the enzymes they inhibit intervene in pathological conditions, and altering their activities is a strategy to treat the respective illnesses. To ascertain the use of the compounds as in the treatment of AD and diabetes, in vivo and toxicity studies will be necessary.
Supplemental Material
sj-docx-1-npx-10.1177_1934578X251367153 - Supplemental material for Enzymes Inhibitory Properties of Compounds from Roots of Mitragyna inermis Willd (Rubiaceae): In Vitro, Molecular Docking and ADME Evaluations
Supplemental material, sj-docx-1-npx-10.1177_1934578X251367153 for Enzymes Inhibitory Properties of Compounds from Roots of Mitragyna inermis Willd (Rubiaceae): In Vitro, Molecular Docking and ADME Evaluations by Alfred Ngenge Tamfu, Monde Gaye, Ndoubalem Roland, Sameh Boudiba, Selcuk Kucukaydin, Milena Popova, Boryana Trusheva, El Hassane Anouar, Rodica Mihaela Dinica and Vassya Bankova in Natural Product Communications
Footnotes
Acknowledgements
We sincerely appreciate the Institute of Organic Chemistry with Center for Phytochemistry of the Bulgarian Academy of Science for spectral analysis. The authors are also grateful to the participating Universities.
ORCID iDs
Alfred Ngenge Tamfu
Sameh Boudiba
Selcuk Kucukaydin
Boryana Trusheva
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Statement of Human and Animal Rights
This article does not contain any studies with human or animal subjects.
Statement of Informed Consent
There are no human subjects in this article and informed consent is not applicable.
Supplemental Material
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