INTRODUCTION:

Anemia can be defined as a condition in which the number of red blood cells or the concentration of hemoglobin in the blood is unable to meet the physiological needs of the body¹. Anemia due to iron deficiency is a common phenomenon in the world's population, especially women. The prevalence of anemia in the world is reported to be between 40-80% in young women. India and Indonesia, 2 countries with large populations have a prevalence of anemia reaching 45% and 32%, respectively. Ironically, the incidence of anemia in young women in Indonesia has increased from 37.1% to 48.9% in just 5 years (2013-2018)².

Losing a lot of blood due to menstruation is often considered a cause of anemia in young women, as well as pregnancy. Pregnancy causes an increase in energy metabolism thereby causing an increase in energy and nutrient requirements during pregnancy. Increased energy and nutrients are needed for the growth and development of the fetus, the increase in the size of the uterine organs, and changes in the composition and metabolism of the mother's body. So a lack of certain nutrients needed during pregnancy can cause the fetus to grow imperfectly. For pregnant women, all nutrients require addition, but they often become deficient in energy, protein, and several minerals such as iron and calcium³.

Anemia due to blood loss (menstruation) and pregnancy can be prevented if the quality and quantity of nutritional intake meet these conditions' needs^4,5. In terms of quantity, this is due to the low intake of iron consumed daily. Meanwhile, in terms of quality, iron absorption is greatly influenced by interference with other types of food factors such as phytate, polyphenols, calcium/phosphate, and protein consumed in the same food⁶. This condition occurs in non-heme iron which comes from vegetable sources as a contributor of 90% of body iron. In contrast, iron from animal sources such as meat, fish, or poultry in the form of iron-heme complexes is not affected by absorption inhibiting factors but only contributes 10% of body iron^7,8.

Studies on increasing the absorption of active compounds into the body have been reported^9,10,11. Therefore, efforts to increase the absorption of non-heme iron need to be made considering that vegetable sources of nutrition are easier to obtain than animal sources. Ascorbic acid, one of the important substances found in various types of fruits and vegetables, it’s a model of a substance that has been shown to be able to increase the absorption of non-heme iron by four times by forming iron-ascorbate complexes that dissolve easily in intestinal fluids¹². Even ascorbic acid given together with iron supplements also has a better effect on increasing hemoglobin levels when compared to iron supplementation alone¹³. Increased blood hemoglobin levels with increased intake of ascorbic acid and non-heme iron by 2-20% have also been reported in groups of women of childbearing age¹⁴. Ascorbic acid facilitates iron absorption by forming chelates with iron at an acidic pH that remains soluble at an alkaline pH in the duodenum.

The mechanism of ascorbic acid in increasing iron absorption is by converting ferric iron (Fe³⁺) to ferrous (Fe²⁺) which is catalyzed by the enzyme "human duodenal cytochrome b" (Dcytb) and or can form soluble ascorbic acid-iron complexes so that Iron is easily absorbed by the intestine. The mechanism of ascorbic acid's electron transfer from non-haem Fe⁺³ ions to Fe⁺² ions also occurs in many other molecules that have carboxyl or hydroxyl groups bonded to 2 neighboring C atoms. Phenol and flavonoid compounds usually have this kind of structure so it is suspected that they also can donate electrons to non-hem Fe⁺³ ions to become Fe⁺² ions which are easily absorbed through the DMT1 channel.

Traditionally, the leaves of the Moringa oleifera plant have been commonly used by some people in Indonesia as vegetables because they are believed to be rich in nutrients. The pharmacological activities of Moringa oleifera are very broad, including as a strong antioxidant, anticancer, anti-arthritic, antiasthmatic, antibacterial, anti-diabetic, antifertility, antifungal, anti-inflammatory, coagulant activity, antiulcer, hepatoprotective activity, etc^15,16,17,18. Mahmood et al.,¹⁹ reported that the leaves of this plant contain nutrients such as vitamins A and ascorbic acid with high concentration; minerals such as Na⁺, Fe⁺³, and Ca⁺²; as well as all kinds of essential amino acids. The ascorbic acid of this plant even reaches 7x more than orange fruit ²⁰. Ascorbic acid in high concentrations allows increased absorption of non-haem Fe⁺³ ions into the circulatory system. This plant is also very good to be given as a daily nutritional supplement as well as a booster in boosting the immune system as reported by Popoola and Obembe²¹. Limited clinical studies such as a review article written by Susilowati et al.,²² showed an increase in Hb levels of 1.4 gr/dL in female adolescents after consuming Moringa oleifera leaf powder 25mg/day for 3 months. When administered at a dose of 210mg/day for 1 month it increased the Hb level to 1.75gr/dL.

In addition to nutritional content, various bioactive compounds have also been reported from this plant ²³ such as alkaloids (marumoside A and B)²⁴, steroids (β-Sitosterol)²⁵, phenolic acids (gallic acid, ellagic acid, ferulic acid, caffeic acid, etc)²⁶, glucosinolates such as the compound 4-[(3′-O-acetyl-α-L-rhamnosyloxy) benzyl] glucosinolate and some of its derivatives²⁷, terpenes (luteolin, lupeol, etc)^28,29 and flavonoids (rutin, quercetin, rhamnetin, kaempferol, apigenin, myricetin or in the form of glycosides)²⁷. Most of these plant flavonoids are in the form of flavanol and glycoside compounds³⁰.

Phenolics and flavonoids are bioactive metabolites that are often associated with the ability to reduce Fe⁺³ ions to Fe⁺² ions as well as ascorbic acid through their electron donors. Quercetin is reported to facilitate the effective loading of metal ions onto iron transport proteins^31,32. The role of quercetin is suspected as a substrate for the enzyme "ferric reductase" just like ascorbic acid through the reduction of non-heme Fe⁺³ ions to Fe⁺² so that they are easily absorbed through the DMT1. Quercetin is also able to form a quercetin-Fe⁺³ complex which allows entry into the intracellular fluid through the GLU1. However, absorption is limited at concentrations of quercetin ≤ 100 nM. This limitation was reported due to a blockade mechanism by cytochalasin B or phloretin as reported by Vlachodimitropoulou et al.,³³. In vivo in rat models, the administration of quercetin was also reported to affect iron storage by increasing the expression of hepcidin and several cytokines such as IL-1, IL-6, and IL-10³¹. Thus, in addition to the high content of iron and ascorbic acid, the secondary metabolite components contained in the Moringa oleifera plant are thought to be able to increase iron absorption so that the use of Moringa oleifera extract as an iron supplement to treat iron deficiency anemia seems very promising.

This research is in the form of a digital simulation in modeling the potential of each active compound contained in Moringa oleifera plant in reducing Fe⁺³ ions to Fe⁺² ions through the enzyme "human duodenal cytochrome b" (Dcytb) with ascorbic acid as the control. The method used includes "molecular docking" to describe the affinity strength and visualization of the interaction of the ligand compound to the receptor protein. The docking results show rigidity during receptor-ligand interaction, therefore it needs to be evaluated under conditions where the receptor-ligand moves dynamically with each other when interacting through the "molecular dynamics" method. Some of the ligand compounds with the strongest affinity were analyzed using this method to observe the stability strength of the interaction during dynamics simulations run at 2000pc time intervals.

MATERIALS AND METHODS:

Materials:

This study used the 3D structure of the enzyme “human duodenal cytochrome b” (Dcytb) as the receptor target obtained from the online protein database http://rscb.org (pdb id 5ZLG), has a resolution of 2.80 Å through the “X-RAY DIFFRACTION” method. The ligands were obtained from the results of GC-MS analysis of the ethanol extract of Moringa oleifera leaves by the authors themselves and reports on the content of bioactive substances in Moringa oleifera leaves by Abd Rani et al., 30. The “SMILES” structure of all test ligands obtained from the online compound database https://pubchem.ncbi.nlm.nih.gov/ was converted into a 3D structure using the “MOE Builder tool”. Meanwhile, the 3D structure of the control ligand is a native ligand that forms a complex with the “human duodenal cytochrome b” (Dcytb) (5ZLG) enzyme.

The software uses the Windows 10 Operating System. The in silico test uses MOE 10, 2008 (Montreal Canada) software and is supported by an online web server such as pkCSM https://biosig.lab.uq.edu.au/pkcsm/. While the hardware used is a computer equipped with High-Performance Computing (HPC) facilities with Intel (R) Core i5-8500 processor specifications CPU @ 4.30GHz (6 CPUs), 4096 MB RAM, 2TB hard drive, 120GB solid-state drive, and VGA Intel HD Graphics NVIDIA GeForce GTX 1080 Ti.

Methods:

Molecular Docking:

Molecular docking treatment is intended to observe the bond-free energy (∆G_binding) resulting from the enzyme-ligand complex formed. First, a molecular docking validation was carried out on the native ligand ascorbic acid attached to the binding site of the “human duodenal cytochrome b” (Dcytb) (5ZLG) enzyme. If the position of the copy ligand is more similar to the native ligand (<2Å), then the docking protocol is more valid so that it is feasible to continue with other ligands ³⁴. The molecular docking process begins with file docking preparation through the optimization of enzymes and ligands (adding hydrogen atoms, setting partial energy with the MMFF94x force field, and minimizing energy under the most optimum conditions to obtain the most stable interaction of the enzyme-ligand complex). The next stage is the selection of the "binding site" referring to the position as the native ligand. Furthermore, the docking process was carried out on all selected test ligands using the MOE-Simualtion dock program with scoring function parameters using London dG, refinement forcefield with 1000x population repetition configuration. The first repetition is 100x and the second setting only shows one best result out of 100 repetitions. The result of docking is free energy (∆G_binding). Bond-free energy is seen in the mdb viewer format, while 2D and 3D structure visualization is depicted through Lig Plot.

Molecular Dynamics Simulation:

The molecular dynamics simulation files were prepared by optimizing the geometry and minimizing the energy of the 3-dimensional structures of the selected docking enzyme-ligand complexes using MOE 2008 software. Optimization of the partial charge geometry of the enzyme-ligand complexes was carried out using the current forcefield parameter method. Furthermore, energy minimization was carried out with the MMFF94x forcefield, the solvation used was born, and the “Root Means Square Deviation” (RMSD) gradient was 0.05 kcal/mol Ǻ. The other parameters use the default and the output file is in moe format.

The molecular dynamics simulation process for inhibitor ligands was carried out using the MOE-dynamics program. The parameters used are by the default MOE dynamics, namely the NVT ensemble (N: number of atoms; V: volume; T: temperature) with the NPA algorithm. The other parameters conform to the defaults in MOE dynamics. Furthermore, an analysis of the results of the dynamics of the inhibitor enzyme complex was carried out based on the results of molecular dynamics simulation calculations. Determination of the conformational stability of the enzyme complex to the solvent was carried out for 100 pico seconds at the initialization stage. The simulation was carried out for 2000 picoseconds, at a temperature of 310 ºK (normal temperature conditions for humans). And ends in the cooling stage for 20 picoseconds. Position, velocity, and acceleration results are stored every 0.5 picoseconds. The rest of the parameters used follow the MOE-dynamics default protocol.

Enzyme-ligand complex interactions during the molecular dynamics simulation process can be seen in the MOE database viewer output in .mdb format. At the end of the simulation, the parameters analyzed were conformational deviations of the enzyme-ligand complex through RMSD values and hydrogen bonding conditions.

Predictive Analysis of Ligand Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Profiles:

Predictive analysis of absorption, distribution, metabolism, elimination, and toxicity (ADMET) profiles using the pkCSM web server (https://biosig.lab.uq.edu.au/pkcsm/). The absorption profile used parameters (Caco2 permeability, HIA, and skin permeability), the distribution profile used parameters (VDss, BBB permeability, and CNS permeability), the metabolism profile used parameters (CYP2D6 substrates and CYP2D6 inhibitors), and excretion profile used parameters (total clearance and Renal (OCT2) substrate) and toxicity profile using parameters (AMES toxicity, LD₅₀, LOAEL, hepatotoxicity, and skin sensitization).

RESULT:

The results of molecular docking of 50 bioactive compounds of Moringa oleifera leaves against the enzyme “human duodenal cytochrome b” obtained different affinity values, the lowest was produced by p-cymene with a ∆G_binding value of -6.26 kcal/mol, and the highest was produced by Quercetin-3-O-glucoside with a ∆G_binding value of -23.10 kcal/mol. Molecular docking results obtained ∆G_binding and hydrogen bonding values of 50 bioactive compounds of Moringa oleifera leaves as shown in Table 1.

Table 1. ∆G_binding and hydrogen bonding of 50 bioactive compounds of Moringa oleifera leaves

Compounds	PubChem CID	∆G_binding (kcal/mol)	Hydrogen Bond
Flavanoids And Flavanol Glycosides
Rutin	5280805	-18.07	Lys 79, Lys 83(2x), Phe 142, Lys 225, Arg 226, Glu 229(3x)
Quercetin	5280343	-14.82	Lys 83(2x), Lys 225(2x)
Isoquersetin	5280804	-16.57	Tyr 68(2x), Lys 79(3x), Lys 83, Pro 227
Isorhamnetin	5281654	-14.71	Lys 79, Lys 83, Lys 225, Glu 229
Kaempferol	5280863	-12.46	Lys 83(2x), Lys 225
Apigenin	5280443	-11.98	Lys 83 (2x), Lys 225
Luteolin	5280445	-14.49	Lys 83(2x), Lys 225(2x)
Genistein	5280961	-11.85	Lys 228
Daidzein	5281708	-12.16	Lys 83, Lys 225, Arg 226
Myricetin	5281672	-13.05	Lys 83, Lys 225
Epicatechin	72276	-15.26	Lys 79(2x), Arg 226
Vicenin-2	442664	-17.31	Tyr 69(2x), Lys 79, Lys 228, Glu 229
Quercetin-3-O-glucoside	25203368	-23.10	Ty2 69(2x), Lys 79(3x), Lys 83(2x), Glu 229
Kaempferol-3-O-glucoside	25203515	-18.99	Tyr 69(2), Lys 79, Lys 225, Arg 226
Kaempferol-3-O rutinoside	5318767	-16.11	Tyr 69(2x), Lys 79(2x), Lys 83, Lys 228, Glu 229, Pro 230
Glucosinolate
Sinalbin	656568	-14.76	Lys 79, Phe 142, Lys 228, Lys 229(2x)
Glucoconringin	656537	-15.64	Tyr 69(4x), Lys 79, Lys 83, Lys 225, Glu 229
Phenolic Acid
Gallic acid	370	-10.74	Lys 79, Lys 83. Phe142(2x)
Salicylic acid	338	-10.26	Lys 83
Gentisic acid	3469	-12.06	Lys 78, Lys 228, Glu 229(2x)
Syringic acid	10742	-10.63	Lys 79, Lys 83(2x), Phe 142, Lys 228
Ellagic acid	5281855	-13.59	The 142(2), Lys 225(2x)
Ferulic acid	445858	-13.58	Lys 79, Lys 83, Glu 229
Caffeic acid	689043	-12.06	Lys 79, Lys 83, Glu 229
o-Coumaric acid	5280841	-8.23	Lys 83, Glu 229
p-Coumaric acid	637542	-11.40	Lys 79, Lys 83
Sinapic acid	10743	-11.22	Lys 79, Lys 83, Glu 229
Chlorogenic acid	1794427	-15.51	Tyr 69(2x), Lys 79(2x), Lys 83(2x), Phe 142, Lys 225
Cryptochlorogenic acid	92135678	-14.69	Lys 79(2x), Lys 83, Lys 225, Pro 227
Alkaloid and Sterol
Marumoside A	101794623	-13,35	Lys 79(4x), Lys 83, Val 141, Pro 227
Marumoside B	101794624	-13.92	Lys 79, Glu 229
Pyrrolemarumine-4′′-O-α-L- rhamnopyranoside	101794622	-14.21	Lys 79(2x), Lys 83, Lys 225
Niazimicin A	10247749	-12.05	Lys 79, Lys 225(2x)
β-sitosterol	222284	-12.52	Lys 83 Phe 142
Lupeol	259846	-12.16	Lys 225
N, α-L-Rhamnopyranosyl vincosamide	71717770	-12.94	Lys 79, Lys 225
Others
Stearic acid	5281	-10.33	Lys 83
Arachidic acid	444899	-9.69	Lys 83
Linolenic acid	5280934	-10.22	Lys 79, Pro 227
Behenic acid	8215	-14.32	Lys 83
Paullinic acid	5312518	-10.30	Ile 66, Arg 70
Niazidin	11792427	-11.87	Lys 79, Lys 83, Phe 142(2x)
Niazirinin	10426197	-12.07	Lys 79, Lys 83. Lys 225
Moringyne	131751186	-12.34	Lys 83(2x), Phe 142, Lys 228
α-Phellandrene	7460	-6.46	-
Eugenol	3314	-9.71	Lys 79, Glu 229
Vanillin	1183	-9.08	Lys 79, Lys 228, Glu 229
Benzylamine	7504	-7.12	Lys 79, Glu 229
D-allose	439507	-9.80	Lys 79(2x), Lys 228(2x), Glu 229
p-Cymene	7463	-6.26	-
Ascorbic acid (Control)		-13.29	Lys 79, Lys 83, Arg 152

Following is a 2D and 3D visualization of the interaction of 7 ligands with the best affinity for 5ZLG (Glucoconringin, Epicatechin, Kaempferol-3-O-rutinoside, Kaempferol-3-O-glucoside, Quercetin-3-O-glucoside, Rutin, Vicenin-2) and control Ascorbic acid

2D Structure	3D Structure	2D Structure	3D Structure
Glucoconringin		Epicatechin

Kaempferol-3-O-rutinoside		Kaempferol-3-O-glucoside

Quersetin-3-O-glucoside		Rutin

Vicenin-2		Ascorbic acid (Control)

Figure 1. Visualization of 2D and 3D structures of 7 active compounds of Moringa oleifera with the strongest affinity and control of Ascorbic acid for the enzyme “human duodenal cytochrome b” (Dcytb) (5ZLG)

Table 3. Pharmacokinetic and toxicity profiles of 8 selected compounds and Ascorbic acid as control

Fase	Parameter	Clr	Epic	Ka3Or	Ka3Og	Qu3Og	Rtn	Vcn2	Qst	Asc
Absorption	Water solubility	-2.449	-3.117	-2.9	-2.627	-2.848	-2.892	-2.844	-2.925	-1.556
	Caco2 permeability	-0.84	-0.283	0.189	0.378	0.336	-0.949	-1.127	-0.229	-0.255
	Intestinal absorption (human)	36.377	68.829	30.743	50.269	42.972	23.446	14.664	72.07	39.154
	Skin Permeability	-2.735	-2.735	-2.735	-2.735	-2.735	-2.735	-2.735	-2.735	-2.955
	P-glycoprotein substrate	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	No
	P-glycoprotein I inhibitor	No	No	No	No	No	No	No	No	No
	P-glycoprotein II inhibitor	No	No	No	No	No	No	No	No	No
Distribution	VDss (human)	0.581	1.027	1.71	0.858	1.423	1.663	0.567	1.559	0.218
	Fraction unbound (human)	0.658	0.235	0.157	0.212	0.215	0.187	0.276	0.206	0.825
	BBB permeability	-1.407	-1.054	-1.669	-1.318	-1.491	-1.899	-1.927	-1.098	-0.985
	CNS permeability	-3.856	-3.298	-5.015	-3.906	-4.068	-5.178	-4.772	-3.065	-3.217
Metabolism	CYP2D6 substrate	No	No	No	No	No	No	No	No	No
	CYP3A4 substrate	No	No	No	No	No	No	No	No	No
	CYP1A2 inhibitor	No	No	No	No	No	No	No	Yes	No
	CYP2C19 inhibitor	No	No	No	No	No	No	No	No	No
	CYP2C9 inhibitor	No	No	No	No	No	No	No	No	No
	CYP2D6 inhibitor	No	No	No	No	No	No	No	No	No
	CYP3A4 inhibitor	No	No	No	No	No	No	No	No	No
Excretion	Total Clearance	0.307	0.183	-0.16	0.196	0.215	-0.369	-0.108	0.407	0.631
Excretion	Renal OCT2 substrate	No	No	No	No	No	No	No	No	No
Toxicity	AMES toxicity	No	No	No	No	No	No	No	No	No
	Max. tolerated dose (human)	-0.134	0.438	0.481	0.544	0.588	0.452	0.46	0.499	1.598
	hERG I inhibitor	No	No	No	No	No	No	No	No	No
	hERG II inhibitor	No	No	Yes	No	Yes	Yes	Yes	No	No
	Oral Rat Acute Toxicity (LD50)	1.973	2.428	2.513	2.548	2.543	2.491	2.484	2.471	1.063
	Oral Rat Chronic Toxicity (LOAEL)	2.982	2.5	3.569	4.493	4.54	3.673	5.859	2.612	3.186
	Hepatotoxicity	No	No	No	No	No	No	No	No	No
	Skin Sensitisation	No	No	No	No	No	No	No	No	No
	T.Pyriformis toxicity	0.285	0.347	0.285	0.285	0.285	0.285	0.285	0.288	0.285
	Minnow toxicity	5.741	3.585	6.252	5.772	7	7.677	11.609	3.721	4.386

*Information

Clr : Clorogenic acid Ka3Or : Kaempferol-3-O-rutinoside Qu3Og : Quersetin-3-O-glucoside

Epic : Epicathecin Ka3Og : Kaempferol-3-O-glucoside Rtn : Rutine

Vcn2 : Vicenin-2 Qst : Quersetin Asc : Ascorbat acid

DISCUSSION:

The docking of the ligand of the Moringa oleifera test for the enzyme “human duodenal cytochrome b” (Dcytb) refers to the position of ascorbic acid (native ligand) contained in the 5ZLG complex which has been reported by Ganesan et al.,³⁶. Orientation, position, and prediction of the affinity of a ligand for the enzyme as a target are important outputs obtained from this molecular docking process. The estimated affinity value represents the binding free energy (∆G_binding) in kcal/mol. ∆G_binding represents the energy released when forming an enzyme-ligand bond to achieve complex stability. The value of ∆G_binding can also be associated with the strength of the bond that occurs between the enzyme-ligand, the greater the energy released, the stronger the strength of the enzyme-ligand interaction³⁵. The value of ∆G_binding is described in the following thermodynamic equation:

[EI]

∆G⁰ = -RT ln K_A K_A = ki^-1 = --------

[E] [I]

Before molecular docking is processed, docking validation is first carried out to determine whether the docking protocol used meets the requirements. The deviation value of the copy native ligand position against the native ligand is used as a docking protocol standard and is declared to have met the validity requirements. The deviation parameter is known as “Root Means Square Deviation” (RMSD) with a deviation value limit of ≤ 2Å. Ascorbic acid as a native ligand experienced a deviation of 0.723 Å, so the docking protocol that was prepared met the validity requirements³⁴.

Figure 2. Pose of native ligand ascorbic acid (Green) against native ligand ascorbic acid (Yellow)

Based on Table 1, it was observed that 19 Moringa oleifera ligand compounds had a stronger affinity than the ascorbic acid control. These compounds are generally in the form of phenolic-flavonoid groups and their glycoside derivatives. The ligand compounds with the strongest affinity were Quercetin-3-O-glucoside with a ∆G_binding value of -23.10kcal/mol, Kaempferol-3-O-glucoside with a ∆G_binding value of -18.99 kcal/mol, Rutin with a ∆G_binding value of -18.07kcal/mol, Vicenin-2 with a ∆G_binding value of -17.31kcal/mol. The 4 compounds are in the form of flavonoid glucoside compounds which have 1 or more bonds with sugar. The presence of this bond with sugar increases its affinity compared to when it is not bound with sugar such as Quercetin and Kaempferol which release free energy (∆G_binding) of -14.82kcal/mol and 12.46kcal/mol, respectively. The next strongest affinities were Isoquersetin with a ∆G_binding value of -16.57kcal/mol, Kaempferol-3-O rutinoside with a ∆G_binding value of -16.11kcal/mol, Epicathecin with a ∆G_binding value of -15.26kcal/mol, Glucoconringin with a ∆G_bindingvalue of -15.64kcal/mol, Chlorogenic acid with a ∆G_binding value of -15.51 kcal/mol, etc. While the Ascorbic acid control has an affinity with a ∆G_binding value of -13.29kcal/mol.

The interaction of 7 ligand compounds with adjacent amino acids (pockets) is similar with Ascorbic acid as control (figure 1), but the hydrogen bonds formed between the hydroxyl groups of the ligand and 3 amino acids (Lys 79, Lys 83, Arg 152) of the Dcytb enzyme are being a "site binding" Ascorbic acid is not the same. Only Lys 79 and Lys 83 amino acids were observed to form hydrogen bonds with several compound ligands such as Quercetin-3-O-glucoside and Rutin. Some ligands only form one hydrogen bond with the Lys 79 amino acid such as Sinalbin, and some do not even form hydrogen bonds such as Ellagic acid (table 1).

Molecularly, the process of electron transfer ascorbic acid in reducing Fe⁺³ ions to Fe⁺² ions was observed in the "apical side" of enterocyte cells. The electron transfer mechanism model involves 2 ascorbic acid hydroxyl groups which form hydrogen bonds with the amino acids Lys 79, Lys 83, and Arg 152 of the Dcytb enzyme as reported by Ganasen et al.,³⁶.

The flavonoid, phenolic, and glucosinolate groups which have a stronger affinity than ascorbic acid have the opportunity to have the ability to act as substrates for the Dcytb enzyme as well as ascorbic acid. These three groups of compounds have hydroxyl groups that can release electrons in reducing Fe⁺³ ions to Fe⁺² ions. Both of them also interact on the same amino acid "pocket" as ascorbic acid, forming hydrogen bonds with at least one of the amino acids which becomes the "site binding" of ascorbic acid in the "apical side" region. While the other groups of compounds (alkaloids, steroids, and other groups) do not meet all the criteria as the flavonoids and phenolic groups.

Based on Table 2, the interaction of hydrogen bonds between ligands and amino acids in the "site binding" of the Dcytb enzyme during the dynamics simulation was not significantly different, except for quercetin. Changes in epicatechin hydrogen bonds are described as observed in Table 2 where at the count of 500pc there are 5 hydrogen bonds (Lys 79(2x), Arg 152, Glu 229(2x)). At the 1000pc, 2 hydrogen bonds are added to the amino acid Lys (2x) so that the total hydrogen bonds become 7, conversely at the 1500pc, there is a decrease of 3 hydrogen bonds so that the total hydrogen bonds become 4, and at the 2000pc it is formed again the addition of 2 bonds to 6. In addition to changes in the number of hydrogen bonds, there was also an inconsistency in the types of amino acids that form hydrogen bonds. In the epicatechin compound, 1 hydrogen bond (Lys 83) was also found which was observed at the 2000pc. In contrast, the kaempferol-3-O-glucoside and control ascorbic acid did not change the number and types of amino acids that form hydrogen bonds.

Changes in hydrogen bonding at the site binding site of the Dcytb enzyme do not cause a conformational deviation of the ligand-enzyme complex which can be observed through the RMSD value. RMSD is a measure often used in 3D molecular geometry analysis to compare changes or shifts in molecular conformation. To ensure the stability of the dynamics of the ligand-enzyme complex, RMSD values of the enzyme backbone atoms were calculated from the start of the simulation and plotted against time. An increase in the RMSD value indicates that the structure of the enzyme is starting to open and the ligands are starting to look for the appropriate bonding sites or coordinates on the protein. Meanwhile, the RMSD value which is starting to stabilize indicates that the maximum conformation of the protein after being bound with the ligand has begun to be reached so that the protein can maintain its position. In addition, the interaction between residues in enzymes makes proteins tend to maintain their structure ³⁷.

Overall, it can be seen in Figure 3 that the time required for the three compounds (quercetin, epicatechin, and kaempferol-3-O-glucoside) to form a stable complex with the Dcytb enzyme is relatively the same (± 500pc). In the next molecular dynamics simulation, it can be seen that the conformation of the enzyme-ligand complex changes with the quercetin compound showing the largest deviation with RMSD > 2Å, ascorbic acid also deviates with RMSD values > 2Å. Meanwhile, the kaempferol-3-O-glucoside and epicatechin compounds tend to have smaller deviations between 1-2 Å. This event was observed in a molecular dynamics simulation count between 600-1800 pc. When the molecular dynamics simulation approaches the count of 2000 pc, it can be seen that quercetin and ascorbic acid show a sharp increase in deviation. In contrast, the kaempferol-3-O-glucoside and epicatechin compounds still show stable dynamic conditions.

Figure 3. RMSD of the Quercetin, Epicatechin, Kaempferol-3-O-glucoside, and Ascorbic acid compounds against the Dcytb enzyme

The epicatechin and kaempferol-3-O-glucoside compounds have a better affinity and stability profile than ascorbic acid. While quercetin has a better affinity profile, its stability is no different from that of ascorbic acid during molecular dynamics simulations. Thus in silico, the three compounds are thought to have a better ability than ascorbic acid in reducing Fe⁺³ ions to Fe⁺² through the activation of the Dcytb enzyme. In silico analysis using molecular docking methods and molecular dynamics is often associated with digital pharmacodynamic analysis. This pharmacodynamics analysis always coincides with a pharmacokinetics analysis which includes absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies which are also easily accessible digitally.

Increased absorption of non-heme iron by ascorbic acid has been observed at daily doses of 30-1000 mg when ascorbic acid is given as a single supplement. However, if given with foods rich in phytate and polyphenols such as fruit and vegetable foods, the supporting effect of ascorbic acid on iron absorption will be reduced^38,39,40,41. Although ascorbic acid supports increased iron absorption, improvements in iron status in humans with chronic supplementation with ascorbic acid have not been observed⁴². The low absorption of ascorbic acid (39.15%) and the relatively high rate of elimination (0.631) in Table 3 may be the reason why iron status in chronic supplementation is not so significant.

Absorption and metabolism of flavonoids are in principle not much different from quercetin which is the reference. Generally quercetin is found in the glycosylated form with the main forms often found in plants being quercetin-3-O-rutinoside (rutin), quercetin-3-O-galactoside (hyperoside), quercetin-3-O-glucoside (isoquercitrin), quercetin- 3-O-rhamnoside (quercitrin) and quercetin-4-O-glucoside (spiraeoside)⁴³. Absorption of glycosylated quercetin is through the sodium-dependent glucose transporter channel (SGLT1), whereas quercetin without sugar bonds is absorbed by passive diffusion^44,45,46. In Table 3, it can be seen that the absorption of quercetin is predicted to be quite high, reaching 72.07% higher than the glycosylated quercetin type, the same thing can be seen in the absorption of epicatechin reaching 68.83%. However, in general, the absorption of the glycosylated form of quercetin is not different from that of ascorbic acid, the same thing can be seen in their metabolic profile.

In the distribution profile, it can be seen that quercetin has a VD of 1.559, while ascorbic acid has a VD of 0.218. Distribution is low if Log VD < -0.15, otherwise high if > 0.45⁴⁷. Thus the distribution of quercetin, glycosylated quercetin, and other types of flavonoids has a wider volume of distribution than ascorbic acid. In contrast, the total clearance of ascorbic acid shows the highest value (table 3). Thus the speed of elimination of ascorbic acid is faster than quercetin, a type of other polyphenol-flavonoids. Toxicity prediction shows similar data between ascorbic acid and quercetin and their glycosylated forms, especially in the absence of carcinogenic, hepatotoxic, and skin sensitization potential. The results of molecular docking analysis of several phenolic compounds such as glucoconringin and almost all polyphenol-flavonoid compounds such as quercetin, kaempferol, epicatechin, isorhamnetin as well as in their glycosylated forms showed better affinity than ascorbic acid. The same results were also observed for the stability value of the interaction between the Dcytb enzyme and 3 representative ligands (glucoconringin, epicatechin, and kaempferol-3-O-glucoside) using the molecular dynamics method. Likewise, the results of the analysis of the absorption profile of some of these compounds showed a better ability than ascorbic acid (table 3).

The role of this polyphenol-flavonoid compound as an iron enhancer is still not fully agreed upon. Vlachodimitropoulou et al.,³³ reported the role of quercetin as a substrate for the Dcytb enzyme in increasing the availability of more Fe²⁺ ions for cellular absorption through the DMT1 channel. In contrast, quercetin and several other types of polyphenols have also been reported as inhibitors of iron absorption in the duodenum, presumably due to their ability to chelate iron, but this mechanism has not been fully proven⁴⁸. Apart from quercetin, phenolic compounds of the myricetin type, myricetin 3-glucoside, and quercetin derivatives (quercetin 3-glucosides) are 3 compounds that have also been reported to suppress iron absorption. Conversely, some polyphenols such as catechins and kaempferol increase iron absorption^48,49,50.

The difference between the statuses of polyphenols as enhancers or as inhibitors of iron absorption is still being debated. Petry et al.,⁵¹ investigated the effect of giving peanut polyphenols on the ability to absorb iron. At a dose of 20 mg polyphenols did not show any changes, on the contrary, at a dose of 50mg and 200mg polyphenols showed a decrease in iron bioavailability by 18% and 45%, respectively. This indicates a decrease in the bioavailability of iron is highly dependent on the dose of polyphenols. Another study said that ascorbic acid was able to suppress the lowering effect of small doses of polyphenols⁵².

This research reveals the potential of bioactive metabolites of the Moringa oleifera plant as substrates for the Dcytb enzyme in duodenal enterocyte cells. Almost all of the polyphenol-flavonoid compounds in this plant are expected to be able or even better to reduce Fe⁺³ ions to Fe⁺² ions like ascorbic acid. However, the results of the in silico analysis that the authors report seem to be only supported by the report of Vlachodimitropoulou et al.,³³. It happened because this study was based on digital tests, on the other hand quite different conclusions were reported by several authors ^{48, 49,50} through in vitro experimental studies. Another argument is that the scope that the authors describe focuses on the role of the iron-reducing enzyme of human duodenal enterocyte cells. After entering the enterocyte cells, most of the iron is stored in the form of ferritin, and a small part is released into the blood due to the role of several regulatory proteins such as hepcidin. Quercetin compounds and other types of flavonoids play an important role in systemic iron metabolism by controlling the expression of this hepcidin to prevent various pathological conditions due to excess iron ⁵². High levels of iron, ascorbic acid, and other nutritional content from the Moringa oleifera plant, as well as the hemostatic role of iron by other metabolites strongly support its role in therapy or supports the treatment of anemia in pregnant women²². The role of increasing systemic iron levels and hemostasis by various polyphenol-flavonoid metabolites Gynura bicolor plant extract (Roxb. and Willd.) has also been reported by Chih-Chung et al.,⁵³ through regulation of MTD1 and FPN expression in vivo in rat duodenal enterocytes. Thus the supplementation of Moringa oleifera leaves is quite promising supplementation of nutraceuticals as previously reported by several researchers^54,55,56.

CONCLUSION:

The Moringa oleifera plant is rich in nutrients such as high concentrations of non-hem iron which is considered to be able to treat anemia due to iron deficiency. This plant also contains ascorbic acid which supports the process of reducing Fe⁺³ ions to Fe⁺² through the Dcytb enzyme so that it is easily absorbed by duodenal enterocyte cells. Many phenol-flavonoid compounds are also reported to be contained in this plant such as quercetin, epicatechin, and kaempferol in the singular or in the glycosylated form which are thought to have the ability to reduce ions like ascorbic acid. This has been proven by several phenol-flavonoid compounds such as glucoconringin, epicatechin, and kaempferol-3-glucoside through the molecular docking method which are known to have a better affinity than ascorbic acid, through the molecular dynamics method it is known that these compounds also have better interaction stability than ascorbic acid on the Dcytb enzyme binding site. The same thing can be seen in its absorption profile which is similar to or even better than ascorbic acid. Thus, apart from having a high content of iron and ascorbic acid, this plant also contains bioactive metabolite compounds that can maintain iron hemostasis through protein regulation which plays an important role in systemic iron regulation as an effort to support anemia therapy in pregnant women.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 15.08.2023 Modified on 02.03.2024

Research J. Pharm. and Tech 2024; 17(10):4785-4795.

DOI: 10.52711/0974-360X.2024.00737

Time in picosecond (pc)	Dcytb (5ZLG)
Time in picosecond (pc)	Epicathecin	Kaempferol-3-O-glucoside	Quersetin	Ascorbic acid
500 pc	Lys 79(2x), Arg 226	Arg 70, Lys 79, Lys 83, Gly 138, His 159, Lys 225	Lys 79(2x), Arg 152, Glu 229(2x)	Lys 79(2x), Lys 83, Lys 228(2x)
1000 pc	Lys 79(2x), Arg 226	Arg 70, Lys 79, Lys 83, Gly 138, His 159, Lys 225	Lys 79(2x), Arg 152, Lys 225(2x), Glu 229(2x)	Lys 79(2x), Lys 83, Lys 228(2x)
1500 pc	Lys 79(2x), Arg 226	Arg 70, Lys 79, Lys 83, Gly 138, His 159, Lys 225	Lys 79(2x), Lys 225, Glu 229	Lys 79(2x), Lys 83, Lys 228(2x)
2000 pc	Lys 79(2x), Lys 83, Arg 226	Arg 70, Lys 79, Lys 83, Gly 138, His 159, Lys 225	Lys 79(2x), Arg 152, Lys 83, Lys 225, Glu 229	Lys 79(2x), Lys 83, Lys 228(2x)