Seven types of phenolic acid and inhibitory activity for protein tyrosine phosphatase 1B (PTP1B) from the stem of Sandoricum koetjape

Sandoricum koetjape has been traditionally used in Indonesian medicine for generations. The stem was used as a remedy for helminthiasis, sneezing, stomachache, indigestion, abdominal pain, leucorrhea, colic, and fever in Indonesia. The purpose of this study was to identify the phenolic acids found in the stem of S. koetjape and to test their ability to inhibit the protein tyrosine phosphatase 1B (PTP1B). P-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid, syringic acid, 4-hydroxybenzoic acid, and gallic acid were identified as phenolic acids found in the S. koetjape stem. The interaction of phenolic acids with the PTP1B was predicted using molecular docking. PTP1B has been linked to diabetes, obesity, tumors, and Alzheimer’s. The autodock 4.2 program, which is integrated with the pyrx v.09.8 virtual screening tool, was used to conduct the docking studies. Interacting between p-coumaric acid with PTP1-B on amino acids Glu115, Lys116, Lys120, Cys215, Ala217, Gly218, Ile219, Gly220, and Arg221. P-coumaric acid fulfills Lipinski’s rule and therefore can be taken orally. P-coumaric acid is expected to have a high oral bioavailability in humans, great absorption in the intestine, and an equivalent distribution in blood plasma and intestine. P-coumaric acid’s acute toxicity is also expected to be low. P-coumaric acid is also non-toxic to the liver, immune system, mutagenic, and cytotoxic. Sandoricum koetjape phenolic acids, particularly p-coumaric acid, appeared to be an effective PTP1-B inhibitor based on docking results.


Introduction
For centuries, traditional medicines have been traditionally used for centuries to sustain great wellness and to treat a variety of illnesses [1].Indonesia, as a mega biodiversity country, has an abundance of plants for medicinal purposes [2].Sandoricum koetjape has been traditionally used in Indonesian medicine for generations.Sandoricum koetjape is classified as a member of the Meliaceae family.The stem was used as a remedy for helminthiasis, sneezing, stomachache, indigestion, abdominal pain, leucorrhea, colic, and fever [3].Numerous research investigations on plants for medicinal purposes have been carried out to verify and figure out their therapeutic properties and mechanism of action through evidence from science [1].According to phytochemical screening, Sandoricum koetjape stems contained alkaloids, flavonoids, quinone, triterpenoids, and tannin [4].The major volatile compounds in Sandoricum koetjape stem extract were fonenol, 1H-cycloprop[e]azule-7-ol, and 1-H-Cyclopenta [1,2]benzene [5].
Phenolic acids are common plant metabolites with bioactive properties that are used in the formulation of functional foods [13].Phenolic acid is made up of phenol (an aromatic compound containing no less than a single hydroxyl substituent) and at least one organic carboxylic acid [14].Ferulic acid and coumaric acid are low molecular phenolic acids with antioxidant and antimicrobial properties [13].MCF-7 cells from humans with breast cancer are cytotoxic to gallic acid and caffeic acid, inducing apoptosis [15].Koetjapic acid, a phenolic acid found in the bark of Sandoricum koetjape, has anticarcinogenic properties [12].Katonic acid, another phenolic acid derived from Sandoricum koetjape, was found to be cytotoxic to ten cancer cell lines [16].Gentisic acid, a phenolic acid, inhibits the activity of α-glucosidase and α-amylase in silico and in vitro [17].Based on docking studies, chlorogenic acid in Sandoricum koetjape appears to be an effective MMP-9 inhibitor [18].
The Republic of Indonesia's Ministry of Health has mandated six health transformation programs by 2022, one of which is the transformation of the health resilience system [19].Noncommunicable diseases, stunting, tuberculosis, and maternal-child health account for up to 60% of health-system transformation [20].Heart disease, stroke, and cancer are the three noncommunicable diseases that kill the most people in Indonesia [19].Overexpression of PTP1B has implications for signaling the formation of cancer, tumors, and inflammatory processes [21].Furthermore, PTP1B is a key inhibitor of the leptin and insulin pathways of signaling [22].The enzyme protein tyrosine phosphatase 1B (PTP1B) interacts with the insulin receptor (dephosphorylation) and downstream signaling components.PTP1B-deficient mice showed resistance to weight gain and significant insulin sensitivity when fed a high-fat diet [17].So, PTP1B may be an option for therapy for type 2 diabetes, cancer, overweight, and various metabolic disorders [21].
Chlorogenic acid and cichoric acid have strong interactions with the -3, -6, and -7 helices of PTP1B.The study found that chlorogenic acid interacts with key residues Gln 288, Glu 200, and Lys 197 of PTP1B, whereas cichoric acid does not because of its binding orientation.Chlorogenic acid was discovered to interact better with the -7 helix, shedding light on protein inhibition [22].Furthermore, Caffeic acid isolated from Artemisia minor aerial parts was discovered to be a PTP1B inhibitor with an IC50 value of 3.06 mol/L [23].Thus, the purpose of this study was to determine the type of phenolic acids found in the Sandoricum koetjape stem and to predict PTP1B inhibition using in silico study.

Plant Materials
Sandoricum koetjape stems were purchased in February 2020 from a common natural market in Bogor, Indonesia.Herbarium Bogoriense, National Research and Innovation Agency, identified taxonomic identification and verification, using standard botanical techniques for species identification.

Phenolic acids analysis using UPLC
One gram of Sandoricum koetjape stem extract was placed in an Erlenmeyer flask.It was then dissolved in 50 ml of Methanol: HCl 2M 1:1 and sonicated for 60 minutes at 60°C.Then filtered with Whatman paper.At 60°C, the filtrate is evaporated.The next step is to optimize the solvents using three different solvents.The first solvent used is ethyl acetate, the second is n-hexane, and the third is water.The obtained residue was mixed with 50 ml of distilled water before being separated multiple times with 25 ml of each solvent, taking the solvent phase each time.The organic solvent was vaporized for 30 minutes at 40°C.Two milliliters of methanol were used to dissolve the residue and then filtered through a millipore.The filtrate is then introduced into the Ultrahigh Performance Liquid Chromatography system.The mobile phase was an 82:18 v/v mixture of 2% acetic acid and methanol.Acquity UPLC BEH C18 1.7 m (2.1 x 50 mm) column with a wavelength of 280 nm was used.The injection volume is one milliliter, and the flow rate is one hundred milliliters per minute.The standards used are p-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid, syringic acid, 4-hydroxybenzoic acid, and gallic acid.

Preparation of phenolics acid as a ligand
Each phenolic acid compound obtained from the database was examined for its isomeric simplified molecular input line entry system (SMILE) and canonical structure in the PubChem database.The previously obtained canonical SMILE structure was entered as a keyword for searching ADMETSAR data using the LMMD AdmetSAR database version 2.0 and Protox version II.

Molecular docking and visualization
The RSCB PDB database PTP1B (PDB ID: 1nny) was used to obtain the 3D structures of the chosen target proteins.Meanwhile, the 3D structure of each active compound of Sandoricum koetjape was obtained using the PubChem database.In addition, the protein was created by eliminating molecules of water in the Discovery Studio 2019 software, and the ligands were minimized in the Pyrx v software.0.9.8.Autodock 4.2, which has been combined with Pyrx v.09.8, is used for docking.Docking is accomplished using a targeted docking technique and the Lamarckian GA algorithm.Based on the literature, the size of the grid box is adjusted based on the position of the amino acid residues.As a result of compound-protein interactions, docking outcomes appear in a format of affinity energy or binding affinity.BioVia Discovery Studio 2019 was also used to visualize the chemical bond between the substance and the docked protein.

Results and Discussion
The chromatogram obtained from the identification of phenolic acid in Sandoricum koetjape stem extracts using UPLC is shown in Figure 1.P-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid, syringic acid, 4-hydroxybenzoic acid, and gallic acid were all found in the Sandoricum koetjape stem.

Figure 1. UPLC chromatogram of phenolic acids from Sandoricum koetjape stem extract
The affinity bonds of phenolic acids from Sandoricum koetjape stem on PTP1B protein were predicted and analyzed using molecular docking studies.Molecular docking is a virtual environment that allows for the support of complex details of ligand-protein interactions (14).Figure 2   The phenolic acid from the Sandoricum koetjape stem extract with the lowest binding affinity is pcoumaric acid, while gallic acid has the highest energy affinity.Table 1 shows the interaction residue of phenolic acids from Sandoricum koetjape stems extract on PTP1B.The results of molecular docking of the PTP1B protein with phenolic acids from Sandoricum koetjape stem revealed that p-coumaric acid had the highest binding affinity value of the phenolic acids tested in this study, compared to the other phenolic acids tested.P-coumaric acid has hydrogen bond interaction with PTP1B on amino residues GLU115 and CYS215.It is known that CYS215 was the active site of PTP1B [17], [21], [22], [24].Gallic acid, caffeic acid, chlorogenic acid, and 4hydroxybenzoic acid also have interaction with CYS215 residue in PTP1B.
The signature motif of the PTPases is the PTP loop, which contains the active site CYS215 [22].The phosphorus atom is attacked by CYS215, which causes the P-O bond to be cleaved and the formation of a phosphocysteine intermediate, which is then hydrolyzed to yield the final products [22].The WPD loop is normally in an open conformation in the Apo (inactive) form, but on linking of the pTyr substrate, it twists over the active site CYS215, causing the catalytic loop to be in a "closed" conformation (WPD closed), resulting in protein activation [22].
P-coumaric acid also has pi-alkyl interaction with ALA217 and ARG221 residue in PTP1B.Chlorogenic, gallic, and syringic acids all interact with the ARG221 amino acid in PTP1B.PTP1B activation is found in residue 214-221, where CYS215 and ARG221 are critical factors in catalytic activity [21].PTP1B's catalytic loop is defined by the amino acids ILE219, GLY220, ALA217, and ARG221 as well as the active site on CYS215 [17].PTP1B targeted molecular dynamics (MD) computational research studies have revealed rotation of the catalytic WPD loop closure and hypothesized that decreased mobility of the WPD loop is followed by decreased movement of the Sloop in conjunction with the TRP179-ARG221 H-bond interaction [22].In the molecular docking study of several representative compounds, information was obtained about the presence of hydrogen bonds on several important residues from the PTP1B active site such as GLY220 and ARG221 [21].
P-coumaric acid also interacts with LYS116, LYS120, ILE219, and GLY220 via Van der Waals forces.All of the phenolic acids in Sandoricum koetjape stem extracts except 4-hydroxybenzoic acid have interacted with LYS120 in PTP1B.Lys120 in PTP1B contributes significantly to peptide structure detection via the interaction of hydrogen-bond, hydrophobic, and electrostatic interactions [22], [23].
The physicochemical properties of phenolic acids in the Sandoricum koetjape stem are shown in Table 2. Phenolic acids had molecular weights (MW) ranging from 138 to 355 Da (500).The values of Log P of the phenolic acids ranged from -0.65 to 1.5 (5), indicating that all of the phenolic acids were lipophilic.Except for chlorogenic acid, the hydrogen bond characteristics (as acceptors or donors) and total polar surface area (TPSA) of most phenolic acids were within acceptable limits.Overall, the findings indicate that all phenolic acids satisfy Lipinski's rule and can thus be consumed orally.According to HIA analysis, all phenolic acids are easily absorbed compounds that move from the intestine to the bloodstream.The blood-brain barrier (BBB) keeps exogenous substances out of the brain.
When decreasing adverse reactions and toxic effects or enhancing the effectiveness of medicines with pharmacological activity in the brain, the potential for the active ingredient of a drug to cross into brain tissue is a crucial aspect to consider.When the BBB is greater than 0.3, molecules can rapidly traverse the blood-brain barrier [25].According to Table 3, the BBB values of phenolic acids range from 0.3005 to 0.8938, implying that all of the phenolic acids in the Sandoricum koetjape stem are present.During the early stages of identifying and developing drugs, human oral bioavailability is used to select potential drug candidates and reject those with a lower likelihood of success [26].Table 3 shows that the HOB values for phenolic acids range from 51.43 to 71.43, implying that all phenolic acids have a high human oral bioavailability (HOB > 50).
Protox II is an internet-based server that estimates hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity in small molecules.The most significant safety issue in developing drugs is drug-induced damage to the liver, which additionally contributes to drug attrition.Hepatotoxic compounds were defined as those that caused no less than one physiological or pathological liver event IOP Publishing doi:10.1088/1755-1315/1255/1/0120707 and were strongly associated with liver disruption [25].According to Table 3, none of the phenolic acids are hepatotoxic.
The toxicological endpoint that raised the most concerns about human health was carcinogenicity [27].Phenolic acids with an inactive prediction were found to be non-carcinogenic.P-coumaric acid, chlorogenic acid, and gallic acid are three phenolic acids that have the potential to cause cancer.Mutagenesis is also linked to carcinogenesis, according to evidence from mutation spectra and tumor genomic sequencing.Mutagenicity refers to the induce long-lasting modifications in the sequence of DNA [28].Table 3 shows that none of the phenolic acids are mutagenic.
Another important method for determining product safety is immunotoxicity testing.Immunotoxicity testing is required not only for product safety evaluation but also for product market approval [29].Two phenolic acids that may be immunotoxic are chlorogenic acid and ferulic acid.Furthermore, cytotoxicity is the test used to determine physiological effects at the cellular level [30].According to Table 3, none of the phenolic acids are cytotoxic.
A prospective phenolic acid's potential toxicity must be evaluated.The LD50 (median lethal dose) of a test substance is used to calculate the dose that kills 50% of animals in a given species.It is used to assess the potential toxicity of chemicals to humans [31].The relative toxicity and acute toxicity of multiple substances can be calculated the lethal dosage value [25].Gallic acid, syringic acid, and ferulic acid are three phenolic acids that fall into category 4 (LD50 = 300 to 2000 mg/kg), indicating that they are slightly toxic.Four phenolic acids in Sandoricum koetjape stem extracts, such as pcoumaric acid, caffeic acid, 4-hydroxybenzoic acid and chlorogenic acid are classified as having low acute toxicity (LD50 = 2000 to 5000 mg/kg).

Conclusions
P-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid, syringic acid, 4-hydroxybenzoic acid, and gallic acid are the seven identified phenolic acids in the Sandoricum koetjape stem extract.P-coumaric acid binds more strongly than the other phenolic acid found in the stem extract of Sandoricum koetjape.Interacting between p-coumaric acid with PTP1-B on amino acids Glu115, Lys116, Lys120, Cys215, Ala217, Gly218, Ile219, Gly220, and Arg221.P-coumaric acid fulfills Lipinski's rule and therefore can be taken orally.P-coumaric acid is expected to have a high oral bioavailability in humans, great absorption in the intestine, and an equivalent distribution in the blood plasma and intestine.P-coumaric acid's acute toxicity is also expected to be low.P-coumaric acid is also non-toxic to the liver, immune system, mutagenic, and cytotoxic.Sandoricum koetjape phenolic acids, particularly p-coumaric acid, appeared to be an effective PTP1-B inhibitor based on docking results.Further, an in vivo study to be conducted to assess the inhibitory activity of Sandoricum koetjape phenolic acid is to down-regulate PTP1B activity

Figure 2 .
Figure 2. Sandoricum koetjape stem extract phenolic acid binding affinity on PTP1B depicts the binding affinity of phenolic acids from Sandoricum koetjape stem extract on PTP1B.

Table 2 .
PTP1B active site physiochemical properties of phenolic acids compound in S. koetjape stem

Table 3
shows the in silico ADMET (adsorption, distribution, metabolism, excretion, and toxicity) properties of phenolic acids in Sandoricum koetjape stem extracts.

Table 3 .
ADMET of phenolic acids compound in Sandoricum koetjape stem into the active site of PTP1B in Silico