INTRODUCTION:

Diabetes is a chronic metabolic syndrome characterized by hyperglycemia. Currently, the number of diabetes patients worldwide reaches 171 million. This number is predicted to increase to 366 million people in 2030 and 693 million people in 2040¹. Postprandial hyperglycemia is one of sign for type 2 diabetes mellitus. Therefore, blood sugar control is vital to diabetes management². One way to control blood glucose levels is by inhibiting the activity of α-glucosidase and DPP-4 enzymes^3-5.

α-glucosidase and DPP-4 enzymes are produced in the small intestine. α-glucosidase work as hidrolize 1,4 glycosidic bonds in polysaccharides to form glucose^6,7. Inhibition of α-glucosidase activity will reduce glucose production in the small intestine. Incretin is a digestive hormone produced by the small intestine to stimulate insulin secretion. The role of DPP-4 is rapidly degrading incretin hormone to keep blood glucose stable. In DM patients, low incretin but high DPP-4, this causes decreased insulin secretion. Inhibition of DPP-4 activity can increases insulin secretion and reduces postmeal hyperglycemia⁸. Currently, inhibition of the activity of α-glucosidase and DPP-4 enzymes are one of potential the targets of diabetes treatment^8-10. Unfortunately, recent research showed that α-glucosidase inhibitor drugs can cause various side effects, such as nausea, vomiting, flatulence, and various other digestive disorder¹¹. Therefore, herbs containing α-glucosidase inhibitory campounds can be offered as alternative medicine for diabetes management^12-15.

Sanrego (Lunasia amara Blanco) is one of the antidiabetic plants from the Rutaceae family in the Sulawesi Sanrego has many empirical uses, such as anticancer, aphrodisiac, activator of CYP2D6, and antidiabetic^16-21. An in vitro study showed that Sanrego extract lowered blood glucose levels²²; however, it is still unknown which Sanrego's active compounds act as an antidiabetic agent. The non-targeted screening method by LC-HRMS is a technique that allows quick detection and identification of unknown or unexpected compounds in a natural substance. Generally, this method does not need standard compounds for analysis because it has used the reference library^16-19. The purpose of this study was to detect the active compounds from the Sanrego ethyl acetate extract (EEA) using LC-HRMS and predict their ability as an antidiabetic through a molecular docking approach.

MATERIAL AND METHODS:

Materials:

Scopoletin were purchased from Sigma Aldrich. All other chemicals purchased from local suppliers.

Sample preparation:

The Sanrego simplisia in the form leaves and stems was taken from Bantimurung-Bulusaraung National (Babul) National Park, South Sulawesi. The simplisia was identified at Purwodadi Botanical Gardens-Institution Indonesian Science (LIPI) Indonesia (No.882/IPH.06/HM/VIII/2019).

Sanrego plant extraction:

The dried leaves and stems of Sanrego was each made into powder separately using a grinding machine. The solvent (ethyl acetate) was added to each powder (1:4 v/v) and then macerated for 48 hours at room temperature. The extract was then filtered using Whatman paper no.40 and evaporated in an evaporator.

Phytochemical screening active compounds of Sanrego EEA:

Phytochemical screening of compounds conducted by thin-layer chromatography (TLC) and LC-HRMS. The separation of active compounds was carried out using G60 F254 silica plates as the stationary phase. A mixture of dichloromethane (DCM): ethyl acetate (91:9 to 99:1 v/v was used as the mobile phase. The TLC results were observed under UV light at wavelengths of 254 nm and 366 nm.

LC-HRMS analysis of Sanrego EEA:

EEA was diluted with 1300μL of methanol and vortex for 1 minute and spindown for 2 minutes. The supernatant was filtered and ready to be injected into the LC-HRMS apparatus. Chromatographic separation was achieved using Hypersil Gold aQ column (50x1mm x 1.9u). The mobile phase used was a mixture of 0.1% formic acid dissolved in distilled water (eluent A) and 0.1% formic acid dissolved in acetonitrile (eluent B). The flow rate analysis of 40µL/min during for 30 minutes at 30⁰C. LC-HRMS of Sanrego EEA was carried out at the Central Laboratory of Biological Sciences Universitas Brawijaya (LSIH UB) and the data analyzed by Compound Discoverer with MzCloud MS/MS Library

Receptor and ligand preparation for in silico studies:

The 3D structure of the ligands was obtained from Pubchem. The design of the 3D receptors (α-glucosidase PDB ID: 2QMJ and DPP4 PDB ID: 5Y7K) was downloaded from the Protein Data Base (RSCB) and had an X-Ray values <2.5 _Ȃ4,27-28_.

Pharmacokinetic analysis of the Sanrego EEA:

Pharmacokinetic analysis was carried out using Pubchem analysis. The ability of active compounds to cross the cell membrane and interact with the target protein was analyzed by the Lipinski Rule of Five Test and molinspiration property prediction tool^29-30. Canonical smile ligands was obtained from Pubchem and entered into the online Molinspiration software to get the topological polar surface area (TPSA) data.

Ligand-receptor preparation and optimization:

Ligand preparation conducted by minimizing the size of the structure  was used PyRx 2.0  software. Receptor was preparated by remove water molecules and drug ligands that were still attached to the protein using the PyMol software.

Molecular docking:

The designed receptors and ligands were entered into Pyrx software version 2.2.3 which had have the Lamarckian genetic algorithm^4,29. The ligands were arranged by placing them in the gird box on the active site of the receptors and run the docking process. This process was repeated three times. The molecular docking results was visualized  by Pymol software, and  Discovery Studio software for the 2D ligand-receptor interactions. Confirmed docking results were then aligned with the original ligands from crystallographic measurements and expressed as the root mean square deviation (RMSD) value. The docking method was accepted as valid if the RMSD value is < 2^31-33

RESULTS:

Phytochemical screening of Sanrefo EEA active compounds:

Based on the optimization results, the suitable solvent ratio for screening Sanrego EEA active compounds were DCM: ethyl acetate (94:6). The results of TLC showed that the EEA contained a scopoletin compound characterized by blue fluorescence under UV light (366 nm). EEA of Sanrego leaf also showed red and blue spots which were predicted to be alkaloids and flavonoids (Figure 1).

Figure 1. Visualization of TLC results of Sanrego EEA stem and leaf extract with DCM: ethyl acetate eluent (94:6). Visualization with a wavelength of 366 nm EEA of Sanrego stem (a), EEA of Sanrego leaf (b), and standard scopoletin (c).

Phytochemical screening and analysis by LC-HRMS:

The results of phytochemical screening and LC-HRMS analysis of Sanrego EEA (leaves and stem) identified 11 active compounds. EEA leaves (EEA-L) and stem (EEA-S) of Sanrego contain almost the same secondary metabolites except for hesperidin and oleamide. These two compounds were only found in EEA-S and not found in EEA-L. The characteristic of the active compounds shown in Table 1. The dominant compounds were scopoletin trigonelin, tangeritin and hesperidin. All of these compounds were presented in EEA-S but trigonelline and tangeritin were not detected in EEA-L. The chromatograms The chromatograms of each component were shown in Figure 2.

Pharmacokinetic analysis of Sanrego EEA active compounds

The results of the pharmacokinetic analysis showed that not all of the Sanrego active compounds met the Lipinski and Veber-Egan rules. These two rules are related to the ability and polarity of active material to penetrate the cell membrane. The requirements of Lipinski rule are compounds with molecular weight ≤ 500 Da, H-donor ≤ 5, H- acceptor ≤ 10, and logP ≤ 5. Hesperidin and chlorogenic acid did not meet the Lipinski rule because they had H- donor > 5, H-acceptor >10 and sesperidin has molecular weight of > 500 Da, and. Besides, the value of topological polar surface area (TPSA) of hesperidin was 234 Ȃ, therefore it did not meet the Veber –Egan rule (≤ 140 Ȃ). (Table 2).

Table 1. Result of LC-HRMS analysis from EEA-L and EEA-S Sanrego

S.No	Active compounds	Leaves	Stem	Formula	Molecular Weight	Rt (minutes)	mz Cloud
1	Trigonelline	√	√	C7H7NO2	137,05	1.203	97.8
2	Scopoletin	-	√	C10 H8 O4	192,04	7.105	96.7
3	Bis(2-ethylhexyl) phthalate	√	√	C24 H38 O4	390,28	23.3	95.3
4	Α-eleustearic acid	√	√	C18 H30 O2	278,22	17.322	94
5	Nobiletin	√	√	C21 H22 O8	402,13	13.598	93.5
6	Dibuthyl phthalate	√	√	C16 H22 O4	278,15	18.36	93.4
7	Α-linoleic acid	√	√	C18 H30 O2	278,22	20.39	93.3
8	Oleamide	-	√	C18 H35 N O	281,27	22.194	92.5
9	Tangeritin	√	√	C20 H20 O7	372,12	14.749	91.6
10	Chlorogenic acid	√	√	C16 H18 O9	354,09	5.097	91.4
11	Hesperidin	-	√	C28 H34 O15	610,19	8.192	91

Figure 2. LC-HRMS chromatograms and mass spectra of the Sanrego plant include (A) hesperidin, (B) scopoletin, (C) trigonelline, and (D) tangeritin.

Table 2. Pharmacokinetic analysis of Sanrego EEA avtive compounds and control (Acarbose and Vildagliptin)

Ligan	Moleculer weight (g/mol)	H-Donor	H-acceptor	Log P	TPSA (Ȃ)
Trigonelline	137	0	2	1,2	44
Scopoletin	192	1	4	1,5	55,8
Bis(2-ethylhexyl) phthalate	390	0	4	7,4	52,6
α-eleostearic acid	278	1	2	6,4	37,3
Nobiletin	402	0	8	3	81,7
Dibutyl phthalate	278	0	4	4,7	52,8
α-linoleic acid	280	1	2	6,8	37,3
Oleamide	281	1	1	6,6	43,1
Tangeritin	372	0	7	3	72,4
Hesperidin	610	8	15	-1,1	234
Chlorogenic acid	354	6	9	-0,4	72,4
Acarbose	645	14	19	-8,5	321
Vildagliptin	303	2	4	0,9	76,4

Molecular docking of Sanrego EEA active compounds against α-glucosidase

Based on the results of docking, all active compounds of Sanrego EEA had activity α- glucosidase inhibitor. Hesperidin had the highest affinity value (-7.4), that was higher than the acarbose (Table 3). Hesperidin has hydrogen bonds with receptor in the amino acids SerA 394 (3.40 Ȃ) and SerA 459 (3.25 Ȃ) and bound the O-group of the receptors (Figure 3). Tangeritin, scopoletin, and trigonelline had energy affinity values of -5.8 and -4.3, respectively. They did not have hydrogen bonds which will interact with receptor but they have carbon-hydrogen bonds, van der Waals, and electrostatic bonds in the form of Pi anions, Pi alkyl, and Pi sigma (Figure 3).

Table 3. Energy affinity of Sanrego EEA-L and EEA-S active compounds against α- glucosidase and DPP-4

Active compounds	Energy affinity with α- glucosidase	Energy affinity withDD DPP-4
Hesperidin	-7,4	-9,8
Tangeritin	-5,8	-9,6
Scopoletin	-5,8	-6,5
Trigonelline	-4,3	-5,3
Acarbose	-7,1	-
Vildagliptin	-	-7,8

Figure 3. Interactions between hesperidin (I) and acarbose (II) with the protein (enzyme) receptor. (A) 3D structure of the documented results of hesperidin and acarbose. (B) 3D visualization of the interaction of hesperidin and acarbose and (C) 2D visualization of the interaction hesperidin and acarbose

Molecular docking of Sanrego EEA active compounds against DPP4:

Hesperidin had the highest energy affinity value as a DPP-4 inhibitor compared to vildagliptin. The affinity value for hesperidin was -9.8 while for vildagliptin was -7.8 (Table 3). This result could be caused by the interaction of hesperidin’s amino acid TyrB 238 (2.77Ȃ) with DPP-4’s O group of the protein (enzyme) receptor (Figure 4). Tangeritin had an energy affinity value that was also higher than vildagliptin, but its amino acids were not attached to the active site of the receptor protein’s active site. The difference in the involvement of amino acids between the two compounds made the energy affinity value of tangeritin lower than hesperidin. Both scopoletin and trigonelline both had low energy affinity values (Table 3).

Figure 4. Interactions between hesperidin (I) and vildagliptin (II) with the target protein. (A) 3D structure of the documented results of vildagliptin and hesperidin. (B) 3D visualization of the interaction of vildagliptin and hesperidin, and (C) 2D visualization of the interaction hesperidin and vildagliptin.

DISCUSSION:

Sanrego is one of Indonesia's endemic herbal plants with various active compounds. Its active compounds include alkaloids, coumarin, steroids, phenolics, essential oils, and terpenoids^22-24,
34. Several studies had succeeded in revealing the potency of Sanrego's as antibacterial, anticancer, aphrodisiac, increases sperm quality, and anti- inflammatory^22-24. The results of LC-HRMS analysis found that Sanrego EEA contains flavonoids compounds including hesperidin, tangeritin, and nobiletin. This compound has never been reported before. Other components in EEA was included coumarin like scopoletin, alkaloid like trigonelline, α- linoleic acid, oleamide, α-eleostearic, chlorogenic acid, dibutyl phthalate. This analysis indicates onformity with the results of screening using TLC and consistent with previous research²⁴.

The results of the pharmacokinetic analysis predicted that hesperidin was not able to penetrate cell membranes and would work extracellularly. This property was similar to acarbose. In contrast to hesperidin, scopoletin, tangeritin, and trigonelline could pass through the cell membrane because they meet the Lipinski and Veber-Egan rules. Thus it can be assumed that the three compounds have intracellular work targets.

The docking molecular method is preferred because its short analysis time, inexpensive and comparable results with in vitro test^36-39. Based on the molecular docking results, all of Sanrego active compounds exhibited α-glucosidase inhibitors activity. Hesperidin had the highest energy affinity value as α-glucosidase and DPP-4 inhibitor. The 2D structure showed that hesperidin occupies the active site and formed the same hydrogen bonds as the control drugs. The energy affinity value will increase if the ligands form were hydrogen or hydrophobic bonds on the active site of the protein⁴. Hydrogen bonds cause the stability of ligand and receptor interactions^38-39. Other factors that affect the energy affinity value are the number of hydrogen bonds and the distance between the ligands and proteins target. The more hydrogen bonds and the closer of the distance, the more stable of ligand-receptor interaction. The presence of alkyl bonds and Pi and N- element bonds also contribute to increase hydrophobic interactions between ligands and receptors. Hesperidin is similar to the control of acarbose. Both of them cannot penetrate the cell membrane because they have large molecular weights. The massive molecular weight causes slow molecular diffusion. Acarbose itself works extracellularly in inhibiting the hydrolysis of α-glycosidic (1-4) bonds in polysaccharides after meal period. That so, hesperidin as the candidate of α- glucosidase inhibitor is expected to act like acarbose.

Previous studies stated that hesperidin had antidiabetic activity. Hesperidin has been shown to regulate glucose metabolism in the liver, inhibits glucose 6-phosphatase (G6Pase) activity, and protects the heart^12,40,41. Hesperidin also increases GLUT2 translocation for glucose uptake, increases PPAR activity and insulin sensitivity^43-44. Hesperidin has effects as a hypolipidemic agent, antioxidant, and reduces complication risk because of diabetes⁴⁵. Recent in silico and in vitro studies reported that hesperidin reduces hyperglycemia through α-glucosidase and DPP-4 inhibition^46-47.

Apart from hesperidin, another flavonoid compound in the EEA is tangeritin. Tangeritin is a flavone compound and is mostly found in the citrus family. Previous studies stated that tangeritin has bioactivity as an antioxidant, antitumor, anti-inflammatory, hepatoprotective and neuroprotective⁴⁸. As an antidiabetic, tangeritin increases glucose uptake in adipocyte cells increases glycogen synthase activity, and regenerates pancreatic β-cells thus improving insulin production^49-50. Tangeritin acts as an alpha glucosidase inhibitor⁴⁷, but its role as a DPP4 inhibitor is unknown. angeritin and coumarin are both known antidiabetic agents. One of the derivatives of coumarin is scopoletin, also has antidiabetic activity.

Scopoletin act as antidiabetics through inhibition of α-glucosidase, inhibition of gluconeogenesis activity, and production of methylglyoxal and AGE-s^51-54. The activity of scopoletin as a DPP-4 inhibitor has not been previously reported. Furthermore, scopoletin inhibits the activation of JKN pathway that will reduce β-pancreatic cell apoptosis⁵¹. Recent studies have found that scopoletin activates the PI3K and AMPK signaling pathways, thereby triggering translocation of GLUT4 to the cell membrane for glucose uptake⁵⁵. Uptake of glucose in muscle tissue and adiposit are important to reduce hyperglycemia and maintain glucose homeostasis. Similar to scopoletin, trigonelline also acts as an anti-hyperglycemic and antidyslipidemic⁵⁶. Recent data have found that trigonelline reduces inflammation induced retinopathy and insulin resistance through inhibition of PPAR-γ^57-58.Another mechanism of tangeritin is an α- glucosidase inhibitor and DPP-4 inhibitor based on in vitro test results^59-60. Based on this research results, the Sanrego EEA had antidiabetic potential based on in silico. In vitro study is ongoing research

CONCLUSION:

All the active compounds of the Sanrego EEA had antidiabetic activity. Hesperidin had the highest energy affinity value as α-glucosidase and DPP-4 inhibitors compared to scopoletin, tangeritin, trigonelline, and control drugs. Therefore hesperidin is considered as a potential candidate for antidiabetic compound.

ACKNOWLEDGMENTS:

The authors would like to thank the Faculty of Medicine Universitas Brawijaya for funding this research and the Central Laboratory of Biological Sciences Universitas Brawijaya (LSIH-UB), for facilitating the research process.

CONFLICTS OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 04.06.2021 Modified on 02.07.2021

Research J. Pharm.and Tech 2022; 15(3):1077-1084.

DOI: 10.52711/0974-360X.2022.00180