Journal of Herbal Medicine

Phytochemical Screening and In-Silico Antidiabetic Activity of Ethyl Acetate Extract of

Sanrego (Lunasia amara Blanco)
--Manuscript Draft--

Manuscript Number:

Article Type: Research paper

Keywords: Sanrego
α-glucosidase
molecular docking
hesperidin
scopoletin
acarbose.

Corresponding Author: Adriani Adriani

Universitas Brawijaya
INDONESIA

First Author: Adriani Adriani

Order of Authors: Adriani Adriani

Noorhamdani Noorhamdani, Profesor

Tri Ardyati, Dr

Sri Winarsih, Profesor

Abstract: This study aimed to know the active compound from the ethyl acetate extract (EEA) of
Sanrego ( Lunasia amara Blanco) stems and leaves and predict  its ability as an anti-
diabetic by in silico. The dried leaves and stems of Sanrego are ground-up powder and
extracted using ethyl acetate. The detection of active compounds using TLC and LC-
HRMS. Anti-diabetic prediction conducted by molecular docking approach compared to
acarbose and vildagliptin. The TLC results show that Sanrego EEA contains alkaloid
and flavonoid compounds include scopoletin. The LC-HRMS results showed 11 active
compounds in EEA and all of them have anti-diabetic activity. The main compounds
detected were hesperidin, scopoletin, tangeritin, and trigonelline. Based on the results
of molecular docking, the four compounds have anti-diabetic activity through the
mechanism of α-glucosidase inhibition and DPP4 inhibition. Hesperidin has the highest
energy affinity value as an α-glucosidase inhibitor (-7.4) and DPP4 inhibitor (-9.8),
followed by tangeritin, scopoletin, and trigonelline. The EEA of Sanrego contains
hesperidin, tangeritin, scopoletin, and trigonelline which have anti-diabetic activity
through α-glucosidase inhibition mechanism and DPP4 inhibition.

Suggested Reviewers: Noor Mat Mahanem

mahanem@ukm.edu.my
She is an expert in the field of natural products

Haroon Khan
haroonkhan@awkum.edu.pk
He is expert in molecular docking and secondary metabolite of plant

Aryy Yanuar
arry.yanuar@ui.ac.id
He is evpert in pharmacy and molecular docking

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Cover Letter

Cover letter

March 19, 2021

Editorial Department Journal of Herbal Medicine

The National Institute of Medical Herbalists Ltd. Exeter, United Kingdom

Dear Editor J. Herb. Med.

I am submitting a manuscript for consideration of publication in Journal of Herbal Medicine. The

manuscript is entitled “Phytochemical Screening and In-Silico Antidiabetic Activity of Ethyl

Acetate Extract of Sanrego (Lunasia amara Blanco)”.

It has not been published elsewhere and that it has not been submitted simultaneously for

publication elsewhere.

Sanrego (Lunasia amara Blanco) is empirically used by Indonesians as an antidiabetic. The

results of TLC and LCHRMS tests showed that the sanrego extract consisted of hesperidin,

scopoletin, tangeritin, and trigonelline. The active compound acts as an antidiabetic through the

mechanism of alpha-glucosidase inhibitor and DPP4 inhibitor based on the in silico approach.

Thank you very much for your consideration.

Yours Sincerely,

Prof. Sri Winarsih

Departement of Pharmacy, Faculty of Medicine,

Universitas Brawijaya, Malang, Indonesia.

Email : wien23.fk@ub.ac.id
Title page with Author details

Phytochemical Screening and In-Silico Antidiabetic Activity of Ethyl Acetate Extract of

Sanrego (Lunasia amara Blanco)

Adriani Adriani1,2, Noorhamdani3, Tri Ardyati4, Sri Winarsih5*

1
Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang,

Indonesia. Email: adrimarsya@gmail.com

2
Departemen of Biology Education in STKIP Pembangunan Indonesia, Makassar, Indonesia
3
Departement of Microbiology Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

Email: dr.noorhamdani@gmail.com
4
Departemen of Biology, Faculty of Mathematics and Science Universitas Brawijaya, Malang,

Indonesia. Email: triardy@ub.ac.id

5
Departement of Pharmacy, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

Email : wien23.fk@ub.ac.id

*Corresponding author:

Sri Winarsih

Departement of Pharmacy, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

Email : wien23.fk@ub.ac.id
Graphical Abstract
Manuscript without Author details Click here to view linked References

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5 Phytochemical Screening and In-Silico Antidiabetic Activity of Ethyl Acetate
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7 Extract of Sanrego (Lunasia amara Blanco)
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1. Introduction
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15 Diabetes is a chronic metabolic syndrome characterized by hyperglycemia. Currently, the
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17 number of diabetes patients worldwide reaches 171 million. This number is estimated to increase
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20 to reach 366 million people in 2030 and 693 million people in 2040 (Adil et al. 2017) .
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22 Postprandial hyperglycemia is one of the triggers for type 2 diabetes mellitus. Therefore, blood
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25 sugar control is vital to prevent diabetes. One way to control blood glucose levels is by inhibiting
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27 the activity of α-glucosidase and DPP-4 enzymes (Mohapatra et al. 2015; Purnomo et al. 2015;
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Yin et al. 2014) .
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32 The enzymes of α-glucosidase and DPP-4 are both produced in the small intestine.
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34 Alpha-glucosidase functions to hydrolyze α-1,4 glycosidic bonds in polysaccharides to form
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37 glucose (R. Subramanian et al. 2008; A. J. Zhang et al. 2017). Inhibition of α-glucosidase
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39 activity will reduce the amount of glucose produced in the small intestine. The role of DPP-4 is
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42 to degrade the incretin hormone in a short time so that blood glucose remains stable. Incretin
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44 hormone is a stimulator of insulin secretion. In diabetics, the production of incretin is low, due to
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47 the activity of the DPP-4 enzyme. Inhibition of DPP-4 enzyme activity causes incretin levels to
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49 increase so that postprandial hyperglycemia does not occur (Tabopda et al. 2008) . Currently,
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inhibition of the activity of α-glucosidase and DPP-4 enzymes is one of the targets of diabetes
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54 treatment because it is considered capable to control blood sugar levels well (Xie et al. 2017;
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56 Zafar et al. 2016) . Recent research states that α-glucosidase inhibitor drugs circulating in the
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59 community cause various side effects, such as nausea, vomiting, flatulence, and various other
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4 digestive disorders (Gong et al. 2017; Zubair et al. 2016). Based on these, the solution offered is
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7 to use herbal plants as an alternative medicine for diabetes management.
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9 Sanrego (Lunasia amara Blanco) is one of the antidiabetic plants from the Rutaceae
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12 family in the Sulawesi people. Sanrego has many empirical uses, such as anticancer, aphrodisiac,
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14 anti- inactivators of CYP2D6, and also antidiabetic (Hasnaeni et al. 2017; Luthfi et al. 2017;
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17 Ratnadewi et al. 2020; Takahashi et al. 2012). An in vitro study found that Sanrego extract
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19 lowered blood glucose levels; however, it is still unknown whether Sanrego's active compounds
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21 act as an antidiabetic agent. The purpose of this study was to detect the active compounds from
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24 the Sanrego ethyl acetate extract (EEA) using LC-HRMS and predict their ability as an
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26 antidiabetic through a molecular docking approach.
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29 2. Materials and methods
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31 2.1 Sample preparation
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34 The Sanrego simplisia in the form of leaves and stems was taken from Bantimurung-
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36 Bulusaraung National (Babul) National Park, South Sulawesi. The simplisia was identified
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taxonomically by a botanist at Purwodadi Botanical Gardens-LIPI Indonesia
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41 (No.882/IPH.06/HM/VIII/2019).
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43 2.2 Sanrego plant extraction
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46 The dried leaves and stems of Sanrego are made into powder using a grinding machine. Sanrego
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48 stem powder and leaf powder respectively added with ethyl acetate (1: 4 v/v) then macerated for
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51 48 hours at room temperature. The extract was then filtered using Whattman paper no.40 and
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53 evaporated in an evaporator.
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56 2.3 Phytochemical screening active compounds of EEA
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4 Phytochemical screening active compounds conducted by thin-layer chromatography
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7 (TLC) and LC-HRMS. The separation of active compounds using G60 F254 silica plates as the
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9 stationary phase. The mobile phase used a mixture of dichloromethane (DCM): ethyl acetate
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12 (91:9 to 99:1 v/v). The TLC results were observed under UV light at a wavelength 254 nm and
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14 366 nm.
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17 2.4 LC-HRMS analysis of Sanrego EEA
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19 EEA was diluted with 1300 μL of methanol and vortex for 1 minute and spindown for 2
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21 minutes. The supernatant was filtered and ready to be injected into the LC-HRMS apparatus.
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24 Chromatographic separation using a Hypersil Gold aQ column (50x1 mm x 1.9 u). The mobile
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26 phase used 0.1% formic acid dissolved in distilled water (eluent A) and 0.1% formic acid
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29 dissolved in acetonitrile (eluent B). The flow rate analysis of 40 µL/min during for 30 minutes at
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31 300C. Data analysis using Compound Discoverer with MzCloud MS/MS Library. LC-HRMS
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34 analysis of Sanrego EEA was carried out at the Central Laboratory of Biological Sciences UB
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36 (LSIH UB)
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2.5 Receptor and ligand preparation for in silico studies
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41 The 3D structure of the ligands obtained from Pubchem. The design of the 3D receptors
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43 (α-glucosidase PDB ID: 2QMJ and DPP4 PDB ID: 5Y7K) is downloaded from the Protein Data
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46 Base (RSCB) and has X-Ray values < 2.5 Ȃ (Karumanchi et al. 2019; Mohapatra et al. 2015; Y.
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48 Zhang et al. 2016).
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51 2.6 Pharmacokinetic analysis of the Sanrego EEA
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53 Pharmacokinetic analysis was done using Pubchem analysis. The ability of active
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compounds to cross the cell membrane and interact with the target protein using the Lipinski
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58 Rule of Five Test and molinspiration property prediction tool (Hari 2019; Kaloni et al. 2020;
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4 Peasari et al. 2018). Canonical smile ligands were downloaded from Pubchem and inputted into
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7 the online Molinspiration software to get the data.
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9 2.7 Ligand-receptor preparation and optimization
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12 Ligand preparation in a minimizing manner the structure using PyRx 2.0 software.
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14 Receptor preparation by removing water molecules and drug ligands that are still attached to the
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17 protein using the PyMol software.
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19 2.8 Molecular docking
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21 Inserts the receptors and ligands designed into Pyrx software version 2.2.3 which has the
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24 Lamarckian genetic algorithm (Mohapatra et al. 2015; Nguyen Vo et al. 2016). The ligands were
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26 arranged by placing them in the gird box on the active site of the receptors and running the
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29 receptor-ligands three times. The visualization of molecular docking results using Pymol
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31 software, while the 2D ligand-receptor interactions using Discovery Studio software. Confirmed
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34 docking results were then aligned with the original ligands from crystallographic measurements
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36 and expressed as the root mean square deviation (RMSD) value. The docking method is declared
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as valid if the RMSD value is < 2 (Adriani et al. 2019; Ernawati et al. 2021)
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3. Results and Discussion
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44 Sanrego is one of Indonesia's endemic herbal plants with various active compounds. Its
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47 active compounds include alkaloids, coumarin, steroids, phenolics, essential oils, and terpenoids
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49 (Hasnaeni et al. 2017; Patrick, Macabeo, and Aguinaldo 2008) . Several studies have succeeded
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52 in revealing Sanrego's potential as an antibacterial, anticancer, aphrodisiac, increase sperm
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54 quality, and anti-inflammatory (Hasnaeni et al. 2017; Patrick, Macabeo, and Aguinaldo 2008;
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57 Zubair et al. 2016). Sanrego extract can reduce levels of blood glucose in vitro (Ratnadewi et al.
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4 2020). However, until now, it is not known that the active compound of Sanrego has an
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7 antidiabetic role.
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9 Based on the optimization results, the suitable solvent ratio for screening Sanrego EEA
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12 active compounds is DCM: ethyl acetate (94:6). The results of TLC showed that the EEA
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14 contained a scopoletin compound characterized by blue fluorescence under UV light (366 nm).
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17 This is consistent with previous research that found scopoletin in sanrego stems (Hasnaeni et al.
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19 2017). EEA of Sanrego stem showed contains almost scopoletin. Other scopoletin, EEA of
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21 Sanrego leaf showed red and blue stains color, which is estimated to be alkaloids and flavonoids
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24 (Figure 1).
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26 Detection of active compounds in EEA put on LC-HRMS analysis was carried out and
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29 antidiabetic prediction of active compounds through molecular docking. Analysis with LC-
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31 HRMS is used because it is sensitive, does not require standard solutions, and can detect
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34 minority compounds in a sample. The results of phytochemical screening and LC-HRMS
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36 analysis of Sanrego EEA (leaves and stem) showed 11 active compounds detected. EEA leaves
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and stem of sanrego contain almost the same secondary metabolites except for hesperidin and
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41 oleamide. These two compounds were only found in stem EEA and not found in leaves EEA.
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43 The structure of the active compounds shown in Table 1.
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46 Based on the results of the LC-HRMS analysis, we know that Sanrego EEA contains
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48 flavonoids, alkaloids, coumarin, fatty acids, and esters. Flavonoid compounds include hesperidin,
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51 tangeritin, nobiletin. Other components are scopoletin, trigonelline, α-linoleic acid, oleamide, α-
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53 eleostearic, chlorogenic acid, dibutyl phthalate. The results of this analysis indicate conformity
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with the results of screening using TLC, which states that Sanrego EEA contains scopoletin. The
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58 compounds that are dominant in the EEA are trigonelline with [Retention Time (RT) = 1.203
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4 minutes], scopoletin (RT = 7.105 minutes), tangeritin (5.097 minutes), and hesperidin (8.192
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7 minutes). The chromatogram of the component is shown in Figure 2.
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10 The results of the pharmacokinetic analysis showed that not all of the Sanrego active
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12 compounds met the Lipinski and Veber-Egan rules. These two rules are related to the ability and
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15 polarity of active material to penetrate the cell membrane. The requirements of the Lipinski rule
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17 are compounds with molecular weight ≤ 500 Da, H-donor ≤ 5, H-acceptor ≤ 10, and logP ≤ 5
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(Lipinski et al. 1997). Hesperidin and chlorogenic acid do not meet the Lipinski rule because
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22 they have H-donor> 5. Hesperidin has a molecular weight of> 500 Da, and H-acceptor>10.
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24 Besides, the value of the topological polar surface area (TPSA) hesperidin is 234 Ȃ and does not
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27 meet the rule Veber -Egan (≤ 140 Ȃ). (Table 2). This explanation indicates that hesperidin is not
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29 able to penetrate cell membranes and works extracellularly. This property is similar to acarbose.
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32 In contrast to hesperidin, scopoletin, tangeritin, and trigonelline can pass through the cell
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34 membrane because they meet the Lipinski and Veber-Egan rules. Thus it can be assumed that the
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37 three compounds have intracellular work targets like vildagliptin.
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The docking molecular method is used because the analysis time is short, cheap, and
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42 gives results that are not much different from the effects of in vitro test (Lin et al. 2020; Safitri et
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44 al. 2020). Based on the molecular results of docking, it explains that all active compounds of
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47 Sanrego have activity as α-glucosidase inhibitors. Hesperidin had the highest affinity value, -7.4,
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49 and it was higher than the acarbose control. Hesperidin has hydrogen bonds in the amino acids
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52 SerA 394 (3.40 Ȃ) and SerA 459 (3.25 Ȃ) and binds to the O-group of the receptors (Figure 3).
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54 Tangeritin, scopoletin, and trigonelline have energy affinity values of -5.8 and -4.3, respectively.
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57 They do not have hydrogen bonds but have carbon-hydrogen bonds, van der Waals, and
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59 electrostatic bonds in the form of Pi anions, Pi alkyl, Pi sigma (Figure 3).
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4 Hesperidin has the highest energy affinity value as a DPP-4 inhibitor compared to
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7 vildagliptin control. The affinity value for hesperidin was -9.8 while for vildagliptin was only -
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9 7.8. The cause is hesperidin has the amino acid TyrB 238 (2.77Ȃ), which is attached to the O
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12 group of the protein (enzyme) receptor (Figure 4). Tangeritin has an energy affinity value that is
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14 also higher than vildagliptin, but its amino acids are not attached to the active site of the receptor
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17 protein’s active site. Because of this the energy affinity value of tangeritin to be lower than
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19 hesperidin. Scopoletin and trigonelline both have low energy affinity values, respectively (Table
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21 3).
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24 Based on the results of docking's analysis, it shows that hesperidin has the highest energy
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26 affinity value as an α-glucosidase and DPP-4 inhibitor. The 2D structure shows that hesperidin
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29 occupies the active site and forms the same hydrogen bonds as the control drug. This reality
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31 proves that the energy affinity value will increase if the ligands include hydrogen or hydrophobic
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34 bonds on the active site of the protein. Hydrogen bonds show the stability of the ligand and
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36 receptor interactions (Arthur 2019; Srinivasan and Sadasivam 2018). Other factors that affect the
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energy affinity value are the number of hydrogen bonds and the distance between the ligands and
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41 proteins target. The more hydrogen bonds and the smaller the resulting space, the more stable the
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43 ligand-receptor interaction. The presence of alkyl bonds and Pi and N-element bonds also helps
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46 to increase hydrophobic interactions between ligands and receptors. Hesperidin is similar to the
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48 control of drug acarbose. Both of them cannot penetrate the cell membrane because they have
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51 large molecular weights and are non-polar. The massive molecular weight causes slow molecular
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53 diffusion. Acarbose itself works extracellularly in inhibiting the hydrolysis of α-glycosidic (1-4)
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bonds in polysaccharides Hesperidin as candidate α-glucosidase inhibitor expects to do as well as
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4 Several previous studies stated that hesperidin has antidiabetic activity. Hesperidin is
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7 known to be involved in glucose regulation in the liver, inhibits glucose 6-phosphatase (G6Pase)
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9 activity, and protects the heart (Akiyama et al. 2010; Jung et al. 2004; Xie et al. 2017). Besides
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12 that, hesperidin also increases GLUT2 translocation for glucose uptake in tissues, increases
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14 PPAR activity and insulin sensitivity (Agrawal et al. 2014; Elshazly et al. 2018; Fitrawan et al.
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17 2018). Recent studies suggest that hesperidin has effects as a hypolipidemic agent, antioxidant,
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19 and reduce complication risk because of diabetes (Dokumacioglu et al. 2019). Apart from
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21 hesperidin, another flavonoid compound in the EEA is tangeritin.
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24 Tangeritin is a flavone compound and is acceptable in the citrus family. Previous studies
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26 stated that tangeritin has bioactivity as an antioxidant, antitumor, anti-inflammatory,
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29 hepatoprotective and neuroprotective (Ashrafizadeh et al. 2020). As an antidiabetic, tangeritin
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31 increases glucose uptake in adipocyte cells increases glycogen synthase activity, and regenerates
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34 pancreatic β-cells so that insulin production is better (Onda et al. 2013; Sundaram et al. 2015).
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36 Hesperidin and tangeritin are both flavonoid compounds, which flavonoids themselves have long
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been known as antidiabetic agents. Besides flavonoids, coumarin compounds in the form of
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41 scopoletin also have antidiabetic activity.
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43 Scopoletin mechanism as an antidiabetic includes inhibition of α-glucosidase,
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46 inhibition of gluconeogenesis activity, and production of methylglyoxal and AGE-s (Chang et al.
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48 2015; Cho et al. 2013; Jang et al. 2018). Furthermore, scopoletin inhibits the activation of the
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51 JKN pathway so that β-pancreatic cell apoptosis is reduced (Kalpana et al. 2018) . Recent
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53 studies have found that scopoletin activates the PI3K and AMPK signaling pathways, thereby
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triggering translocation of GLUT4 to the cell membrane for glucose uptake (Jang et al. 2020) .
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58 Uptake of glucose in muscle tissue and adiposity is important to reduce hyperglycemia and
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4 maintain glucose homeostasis. Similar to scopoletin, trigonelline also acts as an anti-
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7 hyperglycemic and antidyslipidemic (S. P. Subramanian and Prasath 2014) . Recent data have
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9 found that trigonelline reduces inflammation-causing retinopathy and insulin resistance through
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12 inhibition of PPAR-γ (Li et al. 2019)
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14 There have been many studies on hesperidin as an antidiabetic, but an α-glucosidase
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17 and DPP-4 inhibitor has never been reported. This study indicates the Sanrego EEA contains
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19 hesperidin, tangeritin, scopoletin, and trigonelline that have antidiabetic activity through α-
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21 glucosidase and DPP-4 inhibition mechanisms. This study only predicts by in-silico about the
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24 ability of Sanrego active compounds as α-glucosidase and DPP4 inhibitors. Therefore, in vitro
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26 and in vivo tests are still needed to prove the effect.
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29 4. Conclusion
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32 All the active compounds of the Sanrego EEA have antidiabetic activity. Hesperidin has the
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34 highest energy affinity value as α-glucosidase and DPP-4 inhibitors compared to scopoletin,
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37 tangeritin, trigonelline, and control drugs. Based on this, the hesperidin is considered a potential
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39 candidate for antidiabetic compounds.
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42 Declaration of Competing Interest
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45 All authors declare no potential conflict of interest.
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48 CRediT authorship contribution statement
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51 Adriani Adriani: Conceptualization, Methodology, Resources, Investigation, Wtiting-original
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53 draft. Noorhamdani: Review and Supervision. Tri Ardyati: Review and Supervision. Sri
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56 Winarsih: Review and Supervision.
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59 Acknowledgments
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4 This work was supported by Badan Penelitian dan Pengabdian Kepada Masyarakat (BPPM)
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7 Faculty of Medicine Universitas Brawijaya (grant number: 214.45/SK/UN10.F08.06/PN/2020).
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9 We acknowledge the Central Laboratory of Biological Sciences UB (LSIH-UB) which helped
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12 during the research process.
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15 5. CONFLICTS OF INTEREST
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18 The authors declared no conflict of interest.
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51 Table 1. Result of LC-HRMS EEA analysis from leaves and stem Sanrego
52 No Active compounds Leaves Stem Formula Rt mz
53 (minutes) Cloud
54 1 Trigonelline √ √ C7H7NO2 1.203 97.8
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56 2 Scopoletin √ √ C10 H8 O4 7.105 96.7
57 3 Bis(2-ethylhexyl) √ √ C24 H38 O4 23.3 95.3
58 phthalate
59 4 Α--eleustearic acid √ √ C18 H30 O2 17.322 94
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4 5 Nobiletin √ √ C21 H22 O8 13.598 93.5
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6 6 Dibuthyl phthalate √ √ C16 H22 O4 18.36 93.4
7 7 Α-linoleic acid) √ √ C18 H30 O2 20.39 93.3
8 8 Oleamide - √ C18 H35 N O 22.194 92.5
9 9 Tangeritin √ √ C20 H20 O7 14.749 91.6
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10 Chlorogenic acid √ √ C16 H18 O9 5.097 91.4
12 11 Hesperidin - √ C28 H34 O15 8.192 91
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Table 2. Result of analysis pharmacokinetic active compounds from Sanrego EEA and control
22 (acarbose and vildagliptin)
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24 Ligan Moleculer H- H- Log P TPSA (Ȃ)
25 weight donor acceptor
26 (g/mol)
27 Trigonelline 137 0 2 1,2 44
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29
Scopoletin 192 1 4 1,5 55,8
30 Bis(2-ethylhexyl) phthalate 390 0 4 7,4 52,6
31 α-eleostearic acid 278 1 2 6,4 37,3
32 Nobiletin 402 0 8 3 81,7
33 Dibutyl phthalate 278 0 4 4,7 52,8
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35 α-linoleic acid 280 1 2 6,8 37,3
36 Oleamide 281 1 1 6,6 43,1
37 Tangeritin 372 0 7 3 72,4
38 Hesperidin 610 8 15 -1,1 234
39 Chlorogenic acid 354 6 9 -0,4 72,4
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41 Acarbose 645 14 19 -8,5 321
42 Vildagliptin 303 2 4 0,9 76,4
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54 Table 3. Energy affinity of Sanrego EEA active compounds
55 against α- glucosidase and DPP-4
56 Active compounds Energy affinity Energy affinity
57 α- glucosidase DPP-4
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59 Hesperidin -7,4 -9,8
60 Tangeritin -5,8 -9,6
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4 Scopoletin -5,8 -6,5
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6 Trigonelline -4,3 -5,3
7 Acarbose -7,1 -
8 Vildagliptin - -7,8
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Figure Click here to access/download;Figure;Figure_1_300 DPI.jpg
Figure Click here to access/download;Figure;Figure2_300 DPI.JPG
Figure Click here to access/download;Figure;Figure_3_300 DPI.jpg
Figure Click here to access/download;Figure;FIGURE_4-300 DPI.jpg
Conflict of Interest Statement

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Author’s name Affiliation

Adriani, Adriani 1. Doctoral Program of Medical Science, Faculty of
Medicine, Universitas Brawijaya, Malang,
Indonesia
2. Departemen of Biology Education in STKIP
Pembangunan Indonesia, Makassar, Indonesia

Noorhamdani Departement of Microbiology Faculty of Medicine,

Universitas Brawijaya, Malang, Indonesia.
Tri Ardyati Departemen of Biology, Faculty of Mathematics
and Science, Universitas Brawijaya, Malang,
Indonesia
Sri Winarsih Departement of Pharmacy, Faculty of Medicine,
Universitas Brawijaya, Malang, Indonesia.