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Article On the Inhibitability of Natural Products Isolated from ruticarpum towards Tyrosine Phosphatase 1B (PTP1B) and α-Glucosidase (3W37): An In Vitro and In Silico Study

Dao-Cuong To 1,2,†, Thanh Q. Bui 3,† , Nguyen Thi Ai Nhung 3 , Quoc-Toan Tran 4 , Thi-Thuy Do 4, Manh-Hung Tran 5 , Phan-Phuoc Hien 6 , Truong-Nhan Ngu 7, Phan-Tu Quy 7, The-Hung Nguyen 8, Huu-Tho Nguyen 8, Tien-Dung Nguyen 8,9,* and Phi-Hung Nguyen 4,*

1 Nano Institute (PHENA), Phenikaa University, Yen Nghia, Ha Dong District, Hanoi 12116, Vietnam; [email protected] 2 A&A Green Phoenix Group JSC, Phenikaa Research and Technology Institute (PRATI), 167 Hoang Ngan, Cau Giay District, Hanoi 11313, Vietnam 3 Department of Chemistry, University of Sciences, Hue University, Hue City 530000, Vietnam; [email protected] (T.Q.B.); [email protected] (N.T.A.N.) 4 Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay District, Hanoi 122100, Vietnam; [email protected] (Q.-T.T.);   [email protected] (T.-T.D.) 5 Faculty of Hi-Tech Agricultural and Food Sciences, Dong A University, Da Nang City 550000, Vietnam; Citation: To, D.-C.; Bui, T.Q.; Nhung, [email protected] 6 N.T.A.; Tran, Q.-T.; Do, T.-T.; Tran, Institute of Applied Science and Technology, Van Lang University, Ho Chi Minh City 700000, Vietnam; [email protected] M.-H.; Hien, P.-P.; Ngu, T.-N.; Quy, 7 Department of Natural Sciences & Technology, Tay Nguyen University, Buon Ma Thuot, P.-T.; Nguyen, T.-H.; et al. On the Dak Lak 630000, Vietnam; [email protected] (T.-N.N.); [email protected] (P.-T.Q.) Inhibitability of Natural Products 8 College of Agriculture and Forestry, Thai Nguyen University (TUAF), Quyet Thang 24119, Vietnam; Isolated from [email protected] (T.-H.N.); [email protected] (H.-T.N.) towards Tyrosine Phosphatase 1B 9 Institute of Forestry Researh and Development, TUAF, Quyet Thang 24119, Vietnam (PTP1B) and α-Glucosidase (3W37): * Correspondence: [email protected] (T.-D.N.); [email protected] (P.-H.N.) An In Vitro and In Silico Study. † These authors equally contributed to this work. Molecules 2021, 26, 3691. https:// doi.org/10.3390/molecules26123691 Abstract: Folk experiences suggest natural products in Tetradium ruticarpum can be effective inhibitors towards diabetes-related . The compounds were experimentally isolated, structurally Academic Editors: Dezs˝oCsupor, elucidated, and tested in vitro for their inhibition effects on tyrosine phosphatase 1B (PTP1B) and Fang-Rong Chang and Mohamed α-glucosidase (3W37). Density functional theory and molecular docking techniques were utilized El-Shazly as computational methods to predict the stability of the ligands and simulate interaction between the studied inhibitory agents and the targeted . Structural elucidation identifies two natural Received: 3 June 2021 1 Accepted: 14 June 2021 products: 2-heptyl-1-methylquinolin-4-one ( ) and 3-[4-(4-methylhydroxy-2-butenyloxy)-phenyl]-2- Published: 17 June 2021 propenol (2). In vitro study shows that the compounds (1 and 2) possess high potentiality for the inhibition of PTP1B (IC50 values of 24.3 ± 0.8, and 47.7 ± 1.1 µM) and α-glucosidase (IC50 values of Publisher’s Note: MDPI stays neutral 92.1 ± 0.8, and 167.4 ± 0.4 µM). DS values and the number of interactions obtained from docking with regard to jurisdictional claims in simulation highly correlate with the experimental results yielded. Furthermore, in-depth analyses published maps and institutional affil- of the structure–activity relationship suggest significant contributions of amino acids Arg254 and iations. Arg676 to the conformational distortion of PTP1B and 3W37 structures overall, thus leading to the deterioration of their enzymatic activity observed in assay-based experiments. This study encourages further investigations either to develop appropriate alternatives for diabetes treatment or to verify the role of amino acids Arg254 and Arg676. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: Tetradium ruticarpum; tyrosine phosphatase 1B (PTP1B); α-glucosidase (3W37); molecular This article is an open access article docking simulation distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Molecules 2021, 26, 3691. https://doi.org/10.3390/molecules26123691 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 3691 2 of 18

1. Introduction Diabetes mellitus (DM) is on the rise in the public. It has steadily become prevalent worldwide, most markedly in middle-income countries. The World Health Organization (WHO) estimated that diabetes was directly responsible for about 1.6 million deaths in 2015 and predicted that the increasing death toll would lead to the disorder becoming the seventh leading cause of death by 2030 [1]. Furthermore, evidence has suggested a link between diabetes and the cause of cardiovascular disease, blindness, kidney failure, stroke, and limb amputation [2]. In particular, type 2 DM, known as a non-insulin-dependent disorder, results from ineffective use of insulin in the body, accounting for 90–95% of total DM sufferers [3]. Therapeutic treatments for type 2 diabetes mainly relate to inhibition of insulin- and -based enzymes. The former attempts to compensate for defects in insulin secretion and insulin action by approaches for insulin signaling regulation; meanwhile, the latter is used for controlling postprandial hyperglycemia by mitigating the activity of , thus reducing gut glucose absorption [4]. First, tyrosine phosphatase (PTP1B) is a major glucose-homeostasis and energy- regulator, thus regarded as an attractive drug target for therapeutic intervention in type 2 diabetes and obesity [5]. In detail, it blocks insulin receptor substrate-1 and dephosphorylates phosphotyrosine residues, thus causing insulin insensitivity or even a cut-off of intracellular insulin signaling; meanwhile, regarding the leptin signaling pathway, PTP1B binds and dephosphorylates leptin receptor Janus kinase 2 (JAK2), thereby causing the malfunctioning of energy balance to emerge [6]. PTP1B structure was already well determined, and information on it is publicly available at UniProtKB, archived under entry ID: UniProtKB- A0A0U1XP67. Second, α-glucosidase, an exoenzyme found in animal, , bacterial, and fungual organisms, breaks down starch and disaccharides to glucose [7]. A study suggested that the can only yield the monosaccharides by catalyzing the hydrolysis of α-(1→4) and α-(1→6) bonds [8], confirming the sources of α-glucosidase from sugar beet seeds [9]. Protein structural data of the enzyme can be referenced at Worldwide under entry PDB-3W37. Third, oligo-1,6-glucosidase, often called isomaltase, is a debranching endoenzyme, hydrolyzing only the α-1,6 linkage in starch and glycogen to produce sugars with an α-configuration [10]. They cannot break the α-1,4 linkage. Although some bacterial species, such as Bacillus cereus, can synthesize oligo-1,6- glucosidase, this enzyme is present mainly in the animal kingdom [11]. In humans, it is located on the small intestine brush border [12]. Information on isomaltase crystal structure can be downloaded directly from Worldwide Protein Data Bank database under entry PDB-3AJ7. Therefore, PTP1B, 3W37, and 3AJ7 (Figure1) are considered highly promising drug targets for the effective treatment of type 2 diabetes. In principle, multiple inhibitions of PTP1B and glycoside proteins are a promising strategy to simultaneously suppress hyperglycemia and improve insulin sensitization. Tetradium ruticarpum (A. Juss.) T.G. Hartley (Ngô thù,Xà lạp in Vietnamese) is a flowering plant in the family. This plant, previously called ruticarpa (Wu Zhu Ru in Chinese and Goshuyu in Japanese), is a species of , fruit-bearing in Tetradium. The other synonym names were Ampacus ruticarpa (A. Juss.) Kuntze, and Evodia ruticarpa (A. Juss.) Hook. F. and Thomson, they are found widely in North India and China. Both the former genus name and the species name are often misspelled, and the plant usually appears in sources dealing with traditional Chinese medicine as “Evodia€ rutaecarpa”, and thus, Evodia rutaecarpa is presently the most used name in the herbal list in several countries. This species (A. Juss.) was first described by T.G. Hartley in the literature in 1981 [13]. In Vietnam, the tree grows widely in Pho Bang, Ha Giang province in the North of Vietnam, and is also grown in several medicinal gardens [14]. Its fruit (Fructus Tetradii Rutaecarpi) has been clinically used to relieve several irregular symptoms such as headache, vomit, diarrhea, abdominal pain, dysmenorrhea, and pelvic inflammation for thousands of years in Traditional Chinese Medicine and also was used as an herbal medicine for centuries in East Asia, including Japan, Korea, Thailand, and Vietnam. Several secondary metabolites were isolated from this plant such as alkaloids, terpenoids, flavonoids, steroids, and Molecules 2021, 26, 3691 3 of 18

phenylpropanoids, which demonstrated the effects on cancer, inflammation, cardiovascular diseases, and bacterial infection [15]. T. ruticarpum and its ingredients also exhibited pharmacological effects on obesity and diabetes. The hot water extract from E. rutaecarpa collection in Japan and its active compound, rhetsinine, inhibited aldose reductase in a dose-dependent manner, and aldose reductase inhibitors have been considered as having potential for the treatment of diabetic complications. In particular, rhetsinine inhibits sorbitol accumulation by 79.3% at 100 µM. Discussion of the results showed that rhetsinine might be potentially useful in the treatment of diabetic complications [16]. Moreover, a major quinolone alkaloid compound, , extracted from E. rutaecarpa reduced obesity and insulin resistance in obese/diabetic KK-Ay mice via signal [17], suggesting the improvement of glucose tolerance and prevention of the progress of insulin resistance associated with obese/diabetic through inhibition of mTOR-S6K signaling and IRS1 serine phosphorylation in adipocytes for this compound. Furthermore, evodiamine was also found to activate peroxisome proliferator-activated receptors (PPARs), ligand-activated Molecules 2021, 26, x 3 of 21 transcription factors that are involved in regulating glucose and lipid homeostasis, which were considered as a potential therapeutic target in Type 2 diabetes [18].

FigureFigure 1. (a) 1.α-glucosidase(a) α-glucosidase protein protein 3W37; 3W37; (b ()b Oligo-1,6-glucosidase) Oligo-1,6-glucosidase proteinprotein 3AJ7; 3AJ7; (c )(c Protein) Protein tyrosine tyrosine phosphatase phosphatase 1B. 1B.

TetradiumIn silico techniquesruticarpum based (A. Juss.) on computational T.G. Hartley simulation (Ngô thù, and Xà computing lạp in Vietnamese) calculation is a floweringare currently plant seeing in the a Rutaceae gain in popularity family. inThis medical plant, science previously as prescreening called Euodia research. ruticarpa They (Wu Zhureduce Ru in theChinese cost and and time Goshuyu of wet in laboratory Japanese), experiments is a species by of predicting deciduous, the fruit-bearing compounds tree with undesirable properties and the most promising candidates. The former substances in genus Tetradium. The other synonym names were Ampacus ruticarpa (A. Juss.) Kuntze, are deemed to be eliminated from the next analysis or further-developed research, while andthe Evodia latter ruticarpa justify the (A. selection. Juss.) RegardingHook. F. and ligand–protein Thomson, interaction,they are found molecular widely docking in North Indiasimulation and China. is an Both effective the former method genus to investigate name and the the potency species of aname ligand are as often an inhibitor misspelled, andtowards the plant its targetedusually protein. appears The in methodsources can dealing estimate with ligand–target traditional binding Chinese energy medicine and as “Evodia€intermolecular rutaecarpa interaction,”, and thus, thus Evodia predicting rutaecarpa the static is presently stability of the the most inhibitory used systems.name in the herbalEffectively list in several inhibited countries. by external This ligands, species the enzyme(A. Juss.) is likelywas first to undergo described conformational by T.G. Hartley in thechanges literature and thus in the1981 inevitably [13]. In ensuingVietnam, loss the of enzymatictree grows functionality. widely in ThisPho might Bang, help Ha toGiang provincemitigate in the the amount North ofof glucose Vietnam, catalytically and is also synthesized grown in and several excreted medicinal into the bloodstream.gardens [14]. Its Regarding ligand–protein inhibition simulated by molecular docking simulation, an associ- fruit (Fructus Tetradii Rutaecarpi) has been clinically used to relieve several irregular ated docking score (DS) under −3.2 kcal·mol−1 indicates good binding capacity [19–22]. In symptomsprinciple, such thefigure as headache, is calculated vomit, by adiarrhea sum of all, abdominal intermolecular-interaction pain, dysmenorrhea, free energy. and In pelvic inflammationparticular, the for affinity thousands stems fromof years hydrophilic in Traditional bonding, Chinese i.e., various Medicine hydrogen-bond and also types, was used as anand herbal hydrophobic medicine binding, for centuries i.e., van derin East Waals Asia, forces. including Furthermore, Japan, a root-mean-squareKorea, Thailand, and Vietnam.deviation Several (RMSD) secondary measures metabolites the average distance were isol betweenated from internal this atoms plant of such an inhibitory as alkaloids, terpenoids, flavonoids, steroids, and phenylpropanoids, which demonstrated the effects on cancer, inflammation, cardiovascular diseases, and bacterial infection [15]. T. ruti- carpum and its ingredients also exhibited pharmacological effects on obesity and diabetes. The hot water extract from E. rutaecarpa collection in Japan and its active compound, rhetsinine, inhibited aldose reductase in a dose-dependent manner, and aldose reductase inhibitors have been considered as having potential for the treatment of diabetic com- plications. In particular, rhetsinine inhibits sorbitol accumulation by 79.3% at 100 μM. Discussion of the results showed that rhetsinine might be potentially useful in the treatment of diabetic complications [16]. Moreover, a major quinolone alkaloid com- pound, evodiamine, extracted from E. rutaecarpa reduced obesity and insulin resistance in obese/diabetic KK-Ay mice via signal [17], suggesting the improvement of glucose tol- erance and prevention of the progress of insulin resistance associated with obese/diabetic through inhibition of mTOR-S6K signaling and IRS1 serine phosphorylation in adipo- cytes for this compound. Furthermore, evodiamine was also found to activate peroxi- some proliferator-activated receptors (PPARs), ligand-activated transcription factors that are involved in regulating glucose and lipid homeostasis, which were considered as a potential therapeutic target in Type 2 diabetes [18]. In silico techniques based on computational simulation and computing calculation are currently seeing a gain in popularity in medical science as prescreening research. They reduce the cost and time of wet laboratory experiments by predicting the com- pounds with undesirable properties and the most promising candidates. The former

Molecules 2021, 26, x 4 of 21

Molecules 2021, 26substances, x are deemed to be eliminated from the next analysis or further-developed re- 4 of 21

search, while the latter justify the selection. Regarding ligand–protein interaction, mo- lecular docking simulation is an effective method to investigate the potency of a ligand as an inhibitor towardssubstances its targeted are deemed protein. to Th methodeliminated can fromestimate the ligand–targetnext analysis orbinding further-developed re- energy and intermolecularsearch, while interaction, the latter thus justify predicting the selection. the static Regarding stability of ligand–protein the inhibitory interaction, mo- systems. Effectivelylecular inhibited docking by simulation external ligands, is an effective the enzyme method is likelyto investigate to undergo the potencycon- of a ligand as formational changesan inhibitor and thus towards the inevitably its targeted ensuing protein. loss of Th enzymatice method functionality.can estimate ligand–target This binding might help to mitigateenergy the and amount intermolecular of glucos interaction,e catalytically thus synthesized predicting theand static excreted stability into of the inhibitory the bloodstream.systems. Regarding Effectively ligand–protein inhibited inhibition by external simulated ligands, by the molecular enzyme isdocking likely to undergo con- simulation, an associatedformational docking changes score and (DS) thus under the inevitably −3.2 kcal·mol ensuing−1 indicates loss of enzymatic good bind- functionality. This ing capacity [19–22].might In help principle, to mitigate the figure the amount is calculated of glucos bye acatalytically sum of all synthesizedintermolecu- and excreted into lar-interaction freethe energy. bloodstream. In particular, Regarding the affinity ligand–protein stems from inhibition hydrophilic simulated bonding, by i.e., molecular docking various hydrogen-bondsimulation, types, an associatedand hydropho dockingbic binding,score (DS) i.e., under van −der3.2 kcal·molWaals forces.−1 indicates good bind- Furthermore, a root-mean-squareing capacity [19–22]. deviation In principle, (RMSD) the measures figure is the calculated average bydistance a sum be- of all intermolecu- tween internal atomslar-interaction of an inhibitory free energy. system. In Inhibition particular, failure the affinity is proposed stems fromif this hydrophilic value is bonding, i.e., Moleculesover2021 ,326 ,Å; 3691 meanwhile,various docking hydrogen-bond success is justified types, andby its hydropho associatedbic RMSD binding, value i.e., ≤2 vanÅ.[23] der 4 ofWaals 18 forces. In addition, inhibitoryFurthermore, morphology a root-mean-square and in-pose interaction deviation are(RMSD) visually measures illustrated. the averageDe- distance be- scriptive symbolstween regulated internal by MOE2015.10atoms of an inhibitory are given system.in Figure Inhibition 2. failure is proposed if this value is system. Inhibition failure is proposed if this value is over 3 Å; meanwhile, docking success over 3 Å; meanwhile, docking success is justified by its associated RMSD value ≤2 Å.[23] is justified by its associated RMSD value ≤ 2 Å [23]. In addition, inhibitory morphology and In addition,in-pose interaction inhibitory are visually morphology illustrated. and Descriptive in-pose symbols interaction regulated are byvisually MOE2015.10 illustrated. De- scriptiveare given symbols in Figure regulated2. by MOE2015.10 are given in Figure 2.

Figure 2. Descriptive denotation for in-pose interactions projected by MOE2015.10 molecular docking simulation.

In this study, a search for the anti-diabetic agents from Vietnamese medicinal Figurewas 2.Figure Descriptive carried 2. Descriptive out. denotation Two denotation natural for for in-pose in-pose products interactions were projected pr isolatedojected by MOE2015.10 by from MOE2015.10 T. molecularruticarpum molecu docking, larstructurally simulation. docking simulation. elucidated, and in vitro tested for their inhibition effects (on tyrosine phosphatase and α-glucosidase) by assay-basedIn this study, experiments. a search for theDensity anti-diabetic functional agents from theory Vietnamese and molecular medicinal plants wasIn carriedthis study, out. Twoa search natural for products the anti-diabetic were isolated agents from fromT. ruticarpum Vietnamese, structurally medicinal plants docking techniquewas wereelucidated, carried utilized out. and intoTwo vitropredict naturaltested the for stabilityproducts their inhibition of were the effects ligandsisolated (on tyrosineandfrom simulate T. phosphatase ruticarpum the and, structurally interaction betweenelucidated, αthe-glucosidase) studied and inhibitory byin assay-basedvitro tested agen experiments. tsfor and their the inhitargeted Densitybition functional proteins effects theory (on(3W37, tyrosine and 3AJ7, molecular phosphatase and and PTP1B). α-glucosidase)docking technique by assay-based were utilized toexperiments. predict the stability Density of the functional ligands and theory simulate and the molecular dockinginteraction technique between were the studied utilized inhibitory to predict agents the and stability the targeted of proteinsthe ligands (3W37, and 3AJ7, simulate the and PTP1B). 2. Results and Discussioninteraction between the studied inhibitory agents and the targeted proteins (3W37, 3AJ7, 2.1. Experimental andResults2. PTP1B). Results and Discussion 2.1. Experimental Results The fruit of T. ruticarpum was extracted with MeOH and then partitioned with ethyl The fruit of T. ruticarpum was extracted with MeOH and then partitioned with ethyl acetate to obtain2. an Results EtOAc and fraction. Discussion By us ing liquid–liquid partition and column chro- acetate to obtain an EtOAc fraction. By using liquid–liquid partition and column chromato- matographic separation,2.1.graphic Experimental compounds separation, Results compounds 1 and 2 were1 and purified2 were purified from this from EtOAc this EtOAc fraction. fraction. All All the the elucidated structuralelucidatedThe formulae fruit structural of T. are ruticarpum formulae presented are was presentedin Figureextracted in 3. Figure with3. MeOH and then partitioned with ethyl acetate to obtain an EtOAc fraction. By using liquid–liquid partition and column chro- O matographic separation, compounds 1 and 2 were purified7 from this EtOAc fraction. All 5 4 2 9 10 the elucidated structural formulae are5' presented in Figure1 3. 6 3 3 8 OH 2' 4' 6' 1' 7 O HO 6 9 N 2 3' O 4 7 8 1' 5 3' 4 5' 7' 4' 2' 5 2 9 10 5' 1 6 3 3 8 OH 122' 4' 6' 1' 7 HO 6 9 N 2 3' O 4 Figure 3. ChemicalFigure structure 3.8Chemical of isolated structure1' compounds of isolated3' compounds5' 1 and 27' 1fromand 2 T.from ruticarpumT. ruticarpum4' . 2'. 5 Compound121 was obtained as a white crystal and showing a positive reaction with Dragendorff’s reagent test. The molecular formula of 1 was determined to be C17H23NO + Figurebased 3. onChemical the ion atstructurem/z 258.1 of [M isolated + H] incompounds the fast atom 1 and bombardment 2 from T. ruticarpum mass spectrometry. (FAB-MS). The presence of an N-methyl group [δH 3.72 (3H, s)/δC 34.3], an olefinic group (δH 6.21 (1H, s, H-3)/δC 154.9 (C-2) and 111.3 (C-3)), a benzene ring (δH 8.43 (2H, dd, J = 8.0, 1.6 Hz, H-5), 7.36 (1H, brt, J = 8.0 Hz, H-6), 7.64 (1H, dt, J = 1.6, 8.0 Hz, H-7), and 7.49 (1H, brd, J = 8.0 Hz, H-8)/δC 126.8 (C-5), 123.4 (C-6), 132.2 (C-7), 115.5 (C-8), 142.1 (C-9), 1 13 and 126.7 (C-10)), and a ketone group at δC 178.0 (C-4) in the H and C NMR spectra indicated 1 to be N-methyl-4(1H)-quinolone type alkaloids (Figure3 and Supplementary data) [24,25]. The 1H NMR spectrum of 1 showed a signal of a heptyl moiety (2.69 (2H, t, J = 7.6 Hz, H-10), 1.67 (2H, q, J = 7.6 Hz, H-20), 1.27-1.42 (8H, m, H-30/H-40/H-50/H-60), 0.88 (3H, t, J = 6.8 Hz, H-70)), indicating the C7 side chain of 1 (Figure1). The 13C NMR and heteronuclear multiple quantum correlation (HMQC) spectra of 1 showed 17 carbon Molecules 2021, 26, 3691 5 of 18

signals, including one N-methyl carbon at δC 34.3 (N-CH3) and seven carbon signals of a 0 0 0 0 0 0 heptyl group (δC 34.9 (C-1 ), 28.7 (C-2 ), 29.4 (C-3 ), 29.2 (C-4 ), 31.8 (C-5 ), 22.8 (C-6 ), and 0 14.2 (C-7 ) (Figure1 and Supplementary data)). The HMBC correlations of N-CH3 to C-2 and C-9; H-3 to C-4 and C-10; H-5 to C-4, C-7, and C-9, as well as H-8 to C-6 and C-10, were observed, indicating the quinoline moiety of 1. Furthermore, the location of the heptyl group was located at C-2 based on the HMBC correlations from H-3 to C-10 and H-20 to C-2 (Figure4 and Supplementary data). Based on the above evidence and in the comparison with the published data, compound 1 was identified as schinifoline [25]. This compound 1 belongs to the alkylquinolin-4(1H)-one type alkaloid derived in the Rutaceae species and Molecules 2021, 26, x 6 of 21 was first identified from Zanthoxylum schinifolium [25]. This study has presented for the first time the purification of natural schinifoline from T. ruticarpum.

FigureFigure 4.4.HMBC HMBC andand COSYCOSY correlationscorrelations ofof compoundscompounds1 1and and2 2from fromT. T. ruticarpum ruticarpum..

CompoundGenetic and2 biochemicalwas obtained studies as colorless have shown needles. that ThePTP1B1H is NMR a key and negative COSY regulator spectra ofof 2leptinshowed and signals insulin of signaling a 1,4-disubstituted pathways which benzene are ringresponsible (δH 77.32 for (2H, glucose d, J =homeostasis, 8.4 Hz, H- 2/H-6)control andbody 6.87 weight, (2H, d,andJ =energy 8.4 Hz, expenditure H-3/H-5)), [26]. assigned This reveals to an A the2B2 -spinpotential system use andof nat- a transural-3-hydroxy-1-propenyl products with PTP1B group activity [(δ Hinhibition6.55 (1H, brd,or geneJ = 16.0 expression Hz, H-7), reduction 6.21 (1H, dt, effectsJ = 16.0 for, 6.0treating Hz, H-8), type and2 diabetes 4.30 (2H, and dd, obesityJ = 6.0, [3]. 1.2Our Hz, in H-9))vitro study (Figure showed1). The that signals compounds at δH 4.59 1–2 0 0 0 0 (2H,potentially brd, J =inhibited 6.4 Hz, H-1the PTP1B), 5.77 (1H,enzyme m, H-2activity), 4.09 with (2H, IC50 s, values H-4 ), of and 24.3 1.77 ± 0.8 (3H, and s, H-547.7 )± 1 in1.1 the μM,H NMRrespectively spectrum (Table were 1). arranged Ursolic toacid, be thea characteristic signal for 4-methylhydroxy-2-butenyl ursane-type triterpenoid 0 0 groupmainly (see found Supplementary in persimmon, data). was used The cross-peakas a positive between control H-1showingand the H-2 ICin50 value the COSY of 3.5 spectrum± 0.3 μM. further Furthermore, confirmed schinifoline the existence (1 of) 4-methylhydroxy-2-butenylexhibited a potential inhibitory group (effectFigure on3 13 andα-glucosidase Supplementary enzyme data). activity The withC NMR,an IC50 distortionless value of 92.1enhancement ± 0.8 μM, two by times polarization stronger transferthan acarbose, (DEPT) the and positive heteronuclear control single used quantum for this assay correlation having (HSQC) an IC50 spectra value of of 152.42 showed ± 0.6 theμM. presence Integrifoliodiol of 14 signals, (2) showed including similar six carbons activity for with the benzene acarbose, ring whose (δC 129.5 IC50 (C-1), value 127.6 was (C-2/C-6),167.4 ± 0.4 114.9μM. (C-3/C-5), and 158.4 (C-4)), three carbon signals for a trans-3-hydroxy- 1-propenyl group (δC 130.9 (C-7), 126.2 (C-8), and 63.9 (C-9)), and five carbon signals for 0 0 0 0 aTable 4-methylhydroxy-2-butenyl 1. PTP1B and α-glucosidase group inhibitory (δC 64.3activities (C-1 of), compounds 119.7 (C-2 ),(1‒ 140.12) isolated (C-3 from), 67.8 T. (C-4ruti- ), 0 0 0 0 andcarpum. 14.0 (C-5 )). The correlations of H-7 to C2 and C-6; H-8 to C-1; H-5 to C-2 , C-3 , and C-40, as well as H-2/H-6 and H-10 to C-4, in the HMBC spectrum confirmed the structure PTP1B α-glucosidase of compoundCompounds2 (Figure 4 and Supplementary data). The FAB-MS of compound 2 showed the pseudo-molecular ion at m/z 257.03 [M +IC Na]50, +µM, indicating a the molecularIC50, formulaµM a of C14H18O3. Based1 on this observation, together24.3 with ± 0.8 the comparison with 92.1 the ± published 0.8 data [25], compound2 2 was identified as intergrifoliodiol.47.7 ± 1.1 To the best of our167.4 knowledge, ± 1.4 this compoundUrsolic has acid been b isolated from T. ruticarpum 3.5 ± for0.3 the first time in the literature, - given our reachableAcarbose referencing. b - c 152.4 ± 0.6 a ResultsGenetic are expressed and biochemical as IC50 values studies (μM), have determined shown thatby regression PTP1B is analysis a key negativeand expressed regulator as the ofmeans leptin ± SD and of insulinthree replicates. signaling b Positive pathways control. which c Data are not responsible determined. for glucose homeostasis, control body weight, and energy expenditure [26]. This reveals the potential use of natural productsThe withexperimental PTP1B activity assays inhibitionreveal that or 1 and gene 2 expressionpossess good reduction inhibition effects activity for treatingtowards typeα-glucosidase 2 diabetes and obesityprotein [PTP1B,3]. Our ingiven vitro bystudy their showed significant that IC compounds50 value in1 comparison–2 potentially to inhibitedthose of the the control PTP1B drugs. enzyme In detail, activity the with corresponding IC50 values values of 24.3 accord± 0.8 andwith47.7 the order± 1.1 µ1 M,> 2 respectivelyregarding either (Table of1 ).the Ursolic targeted acid, proteins. a characteristic ursane-type triterpenoid mainly found in persimmon,Schinifoline was (SF) used was as known a positive as a 4-quinolinone control showing alkaloid the IC and50 value possessed of 3.5 some± 0.3 activi-µM. Furthermore,ties. Lu et al. reported schinifoline that ( 1schinifo) exhibitedline inhibited a potential the inhibitory negative effecteffects on of αCandida-glucosidase albicans en- in vivo by regulating lysosomal pathway-related genes that accelerate the metabolism and degradation of abnormal proteins [27]. In addition, schinifoline (1) and intergrifoliodiol (2) did not show in vitro inhibitory effect on nitric oxide production [28]. Wang et al. in- vestigated the radio-sensitizing effect of schinifoline on A549 cells. The cell viability re- sults indicated cytotoxicity of SF on A549 cells with IC50 values of 33.7 ± 2.4, 21.9 ± 1.9, and 16.8 ± 2.2 μg/mL after 6, 12, and 24 h treatment, respectively, with different concentra- tions. The results of cell proliferative inhibition and clonogenic assay showed that SF enhanced the radio-sensitivity of A549 cells when applied before 60Co γ-irradiation, and this effect was mainly time- and concentration-dependent [29]. Other studies showed that schinifoline have no antibacterial activity on Staphyloccocus aureus (ATCC25923), Staphylococcus epidermidis (ATCC12228), and Bacillus subtilis (ATCC6633) and no cyto- toxicity against four human cancer cell lines (HepG2, Hela, BEL7402, and BEL7403) [30],

Molecules 2021, 26, 3691 6 of 18

zyme activity with an IC50 value of 92.1 ± 0.8 µM, two times stronger than acarbose, the positive control used for this assay having an IC50 value of 152.4 ± 0.6 µM. Integrifoliodiol (2) showed similar activity with acarbose, whose IC50 value was 167.4 ± 0.4 µM. Table 1. PTP1B and α-glucosidase inhibitory activities of compounds (1-2) isolated from T. ruticarpum.

PTP1B α-Glucosidase Compounds a a IC50, µM IC50, µM 1 24.3 ± 0.8 92.1 ± 0.8 2 47.7 ± 1.1 167.4 ± 1.4 Ursolic acid b 3.5 ± 0.3 - Acarbose b - c 152.4 ± 0.6 a Results are expressed as IC50 values (µM), determined by regression analysis and expressed as the means ± SD of three replicates. b Positive control. c Data not determined.

The experimental assays reveal that 1 and 2 possess good inhibition activity towards α-glucosidase and protein PTP1B, given by their significant IC50 value in comparison to those of the control drugs. In detail, the corresponding values accord with the order 1 > 2 regarding either of the targeted proteins. Schinifoline (SF) was known as a 4-quinolinone alkaloid and possessed some activities. Lu et al. reported that schinifoline inhibited the negative effects of Candida albicans in vivo by regulating lysosomal pathway-related genes that accelerate the metabolism and degrada- tion of abnormal proteins [27]. In addition, schinifoline (1) and intergrifoliodiol (2) did not show in vitro inhibitory effect on nitric oxide production [28]. Wang et al. investigated the radio-sensitizing effect of schinifoline on A549 cells. The cell viability results indicated cyto- toxicity of SF on A549 cells with IC50 values of 33.7 ± 2.4, 21.9 ± 1.9, and 16.8 ± 2.2 µg/mL after 6, 12, and 24 h treatment, respectively, with different concentrations. The results of cell proliferative inhibition and clonogenic assay showed that SF enhanced the radio-sensitivity of A549 cells when applied before 60Co γ-irradiation, and this effect was mainly time- and concentration-dependent [29]. Other studies showed that schinifoline have no antibacterial activity on Staphyloccocus aureus (ATCC25923), Staphylococcus epidermidis (ATCC12228), and Bacillus subtilis (ATCC6633) and no cytotoxicity against four human cancer cell lines (HepG2, Hela, BEL7402, and BEL7403) [30], nor Jurkat T cells clone E6.1 [31], using the MTT method. Furthermore, SF can be used to treat experimental liver cancer cells in rats by influencing cytoskeleton [32] and can treat experimental hepatocarcinogenesis by inhibiting hepatoma cell’s DNA synthesis and preventing the cytodiaeresis [33]. Although schinifoline (1) and integrifoliodiol (2) possessed some biological activities, their PTP1B inhibitory activity has not been investigated to date. Our study showed that compounds 1 and 2 potentially inhibited the PTP1B enzyme activity with IC50 values of 24.3 ± 0.8 and 47.7 ± 1.1 µM, respectively. This is the first time that compound 2 has been identified from T. ruticarpum and the PTP1B inhibitory activity of these compounds has been investigated, given our reachable referencing.

2.2. Computational Results Geometrically optimized structures of 1–2 are shown in Figure5, their frontier molec- ular orbitals (HOMO and LUMO) are presented in Figure6, and the related quantum parameters are summarized in Table2. Firstly, it can be noticed that 1 contains carbonyl and N-heterocyclic groups, while 2 is functionalized with groups -OH and C-O-C. These were reported to be highly conducive to polarity and solubility of the host compound [34,35], thus implying promising inhibitability towards protein molecules based on polar interac- tions with highly polarized amino acids. Although C-O-C might contribute insignificantly to the compound polarity [36], their well-demonstrated antitumoural activity still incen- tivizes the use for cytological applications [37,38]. Furthermore, bonding analysis on frontier molecular orbitals suggests that the compounds are suitable for intermolecular inhibition. In fact, electrons of their HOMO and LUMO are densely distributed and largely space-occupying in a certain region of the molecules, mainly localizing at those of cyclic Molecules 2021, 26, x 7 of 21

nor Jurkat T cells clone E6.1 [31], using the MTT method. Furthermore, SF can be used to treat experimental liver cancer cells in rats by influencing cytoskeleton [32] and can treat experimental hepatocarcinogenesis by inhibiting hepatoma cell’s DNA synthesis and preventing the cytodiaeresis [33]. Although schinifoline (1) and integrifoliodiol (2) pos- sessed some biological activities, their PTP1B inhibitory activity has not been investi- gated to date. Our study showed that compounds 1 and 2 potentially inhibited the PTP1B enzyme activity with IC50 values of 24.3 ± 0.8 and 47.7 ± 1.1 μM, respectively. This is the first time that compound 2 has been identified from T. ruticarpum and the PTP1B inhibi- tory activity of these compounds has been investigated, given our reachable referencing.

2.2. Computational Results Geometrically optimized structures of 1–2 are shown in Figure 5, their frontier mo- lecular orbitals (HOMO and LUMO) are presented in Figure 6, and the related quantum parameters are summarized in Table 2. Firstly, it can be noticed that 1 contains carbonyl and N-heterocyclic groups, while 2 is functionalized with groups -OH and C-O-C. These were reported to be highly conducive to polarity and solubility of the host compound [34,35], thus implying promising inhibitability towards protein molecules based on polar interactions with highly polarized amino acids. Although C-O-C might contribute insig- nificantly to the compound polarity [36], their well-demonstrated antitumoural activity

Molecules 2021, 26, 3691 still incentivizes the use for cytological applications [37,38]. Furthermore, bonding7 ofanal- 18 ysis on frontier molecular orbitals suggests that the compounds are suitable for inter- molecular inhibition. In fact, electrons of their HOMO and LUMO are densely distributed and largely space-occupying in a certain region of the molecules, mainly localizing at groups.those of Thiscyclic indicates groups. This that indicates the molecules that the are molecules able to initiate are able intermolecular to initiate intermolecular inhibition frominhibition certain from approaching certain approaching manners that manners uphold that electron-transferring uphold electron-transferring interactability. interac- In addition,tability. In there addition, are no there significant are no differentials significant differentials between the between parameter the values parameter calculated values forcalculated each compound. for each compound. In particular, In particular, the energy the gaps energy (∆E gapsGAP) ( ofΔE 1GAP and) of 2 1 are and− 26.884 are −6.884 and −and7.102 −7.102 eV, respectively. eV, respectively. These These are considered are considered relatively relatively low values low values and thus and likelythus likely con- duciveconducive to chemical to chemical reactivity reactivity and and inhibitory inhibitory stability stability [39,40 [39,40].]. The The reason reason is thought is thought to be to thatbe that electrons electrons of inhibitor of inhibitor molecules molecules are easilyare easily activated activated and transferredand transferred to their to their surface, sur- readyface, ready for intermolecular for intermolecular activities. activiti Furthermore,es. Furthermore, electronegativity electronegativity (χ), or(χ), the or chemicalthe chem- potentialical potential (µ) in ( aμ) negative in a negative value, value, could could be considered be considered as a reliable as a reliable inhibition inhibition indicator indicator since itsince presents it presents an electron-attracting an electron-attracting tendency. tenden In principle,cy. In principle, a higher a electronegativity higher electronegativity implies aimplies stronger a attractionstronger ofattraction electrons of towards electrons the towards host molecule. the host Therefore, molecule. the Therefore, compounds the seemcompounds promising seem for promising docking investigation. for docking investigation.

Molecules 2021, 26, x 8 of 21

Figure 5. Optimized structures of the isolated compounds 1 and 2 calculated by DFT using basis Figure 5. Optimized structures of the isolated compounds 1 and 2 calculated by DFT using basis M052X/6-311++G(d,p). M052X/6-311++G(d,p).

FigureFigure 6. 6.HOMO HOMO and and LUMO LUMO of of isolated isolated compounds compounds1 and 1 and2 in 2T. in ruticarpum T. ruticarpumcalculated calculated by DFT by DFT at the at levelthe level of theory of theory M052X/def2-TZVP. M052X/def2-TZVP. The different The different colours colours (orange (orange and green) and green) refer to refer the deformation to the defor- ofmation electron of densities,electron densities, i.e. from greeni.e. from to orange.green to Nevertheless, orange. Nevertheless an in-depth, an disscusionin-depth disscusion is unnecessary is un- necessary for quantum-unrelated analysis. for quantum-unrelated analysis.

Table 2. Quantum chemical parameters of the isolated compounds 1–2 from T. ruticarpum calculated by NBO analysis at level BP86/def2-TZVPP including HOMO energy (EHOMO), LUMO energy (ELUMO), energy gap (ΔEGAP); ionization potential (I); electron affinity (A); electronegativity (χ); chemical potential (μ).

ΔEGAP = ELUMO − μ = −χ = Compound EHOMO (eV) ELUMO (eV) I = −EHOMO A = −ELUMO χ = (I + A)/2 EHOMO −(∂E/∂N)ν(r) 1 −7.238 −0.354 −6.884 7.238 0.354 3.796 −3.796 2 −7.374 −0.272 −7.102 7.374 0.272 3.823 −3.823

The quaternary structure of the targeted proteins 3W37 (also known as α-glucosidase), PTP1B (also known as tyrosine phosphatase 1B), 3AJ7 (oli- go-1,6-glucosidase), and their approachable sites by investigated compounds 1 and 2 are virtually represented in Figure 7. The corresponding in-pose amino acid residues are listed in Table 3. The results of prescreening on the inhibitability of compounds 1 and 2 (and a commercialized drug voglibose) towards these potential sites are summarized in Table 4. There are four sites found for the investigated compounds to entry, assigned as site 1 (yellow), site 2 (cyan), site 3 (grey), and site 4 (blue). All sites comprise large num- bers of different amino acids, meaning that they can be considered highly conducive to peripheral interactions. Significantly, site 2 of protein 3W37 comprises 21 different amino acids, a predominant number in comparison to others, implying its most effective inhib- itability. In fact, prescreening results also indicate that it is the most for the investigated compounds given by the lowest DS values (varying from −13.2 to −14.1 kcal·mol−1) and the number of interactions created (at least 3). Regarding protein PTP1B, there are no noticeable differences between the corresponding numbers of its detected sites. The lowest DS value and the corresponding number of interactions are found at site 1. In terms of 3AJ7, sites 1 and 3 are expected, providing better inhibitability than the others due to their dominant numbers of in-pose amino acids, i.e., 36 and 23, respectively. They are also the most effective inhibitory regions for 2 and 1, respectively. In addition, the comparable equivalency of the figures in comparison to those of the commercialized drug voglibose (D) justifies their inhibitory potentiality towards these diabetes-related proteins. Therefore, these sites opt for more in-depth analysis in the attempt to retrieve a structure–activity relationship between experiment-based and computer-based research studies.

Molecules 2021, 26, 3691 8 of 18

Table 2. Quantum chemical parameters of the isolated compounds 1–2 from T. ruticarpum calculated by NBO analysis at level BP86/def2-TZVPP including HOMO energy (EHOMO), LUMO energy (ELUMO), energy gap (∆EGAP); ionization potential (I); electron affinity (A); electronegativity (χ); chemical potential (µ).

EHOMO ELUMO µ = −χ = Compound ∆EGAP = ELUMO − EHOMO I = −EHOMO A = −ELUMO χ = (I + A)/2 (eV) (eV) −(∂E/∂N)ν(r) 1 −7.238 −0.354 −6.884 7.238 0.354 3.796 −3.796 2 −7.374 −0.272 −7.102 7.374 0.272 3.823 −3.823

The quaternary structure of the targeted proteins 3W37 (also known as α-glucosidase), PTP1B (also known as tyrosine phosphatase 1B), 3AJ7 (oligo-1,6-glucosidase), and their approachable sites by investigated compounds 1 and 2 are virtually represented in Figure7 . The corresponding in-pose amino acid residues are listed in Table3. The results of pre- screening on the inhibitability of compounds 1 and 2 (and a commercialized drug voglibose) towards these potential sites are summarized in Table4. There are four sites found for the investigated compounds to entry, assigned as site 1 (yellow), site 2 (cyan), site 3 (grey), and site 4 (blue). All sites comprise large numbers of different amino acids, meaning that they can be considered highly conducive to peripheral interactions. Significantly, site 2 of protein 3W37 comprises 21 different amino acids, a predominant number in comparison to others, implying its most effective inhibitability. In fact, prescreening results also indicate that it is the most active site for the investigated compounds given by the lowest DS values (varying from −13.2 to −14.1 kcal·mol−1) and the number of interactions created (at least 3). Regarding protein PTP1B, there are no noticeable differences between the corresponding numbers of its detected sites. The lowest DS value and the corresponding number of interactions are found at site 1. In terms of 3AJ7, sites 1 and 3 are expected, providing better inhibitability than the others due to their dominant numbers of in-pose amino acids, i.e., 36 and 23, respectively. They are also the most effective inhibitory regions for 2 and 1, respectively. In addition, the comparable equivalency of the figures in comparison to those of the commercialized drug voglibose (D) justifies their inhibitory potentiality towards these diabetes-related proteins. Therefore, these sites opt for more in-depth analysis in the attempt to retrieve a structure–activity relationship between experiment-based and computer-based research studies. The detailed docking simulation results for the inhibitory duos are summarized in Table5. Overall, both compounds exhibit better inhibitability towards PTP1B than towards the carbohydrate- (3W37 and 3AJ7). In fact, DS values obtained from calculations for 1-PTP1B and 2-PTP1B inhibitory complexes are −14.9 and −14.7 kcal·mol−1, markedly lower than those of 1-3W37 and 2-3W37, i.e., −13.2 and −13.8 kcal·mol−1, respectively. The former also contains more either hydrophilic or hydrophobic interactions between the ligands and the in-pose amino acids than the latter. In particular, the PTP1B-based inhibitions are formed by five hydrogen bond interactions and eight or nine van de Waals interactions; meanwhile, the corresponding figures for 3W37-based inhibitory systems are three and nine, respectively. All the inhibitory systems are considered biologically rigid since their RMSD value are all under 2 Å. Furthermore, the computed data regarding oligo-1,6-glucosidase (1-3AJ7 and 2-3AJ7) are included for further reference as the enzyme is well-known as an important target in diabetes-related research, despite the fact that it is not included in the experimental section in this study. These altogether can explain the experimental results that the studied compounds, i.e., 1 and 2, can inhibit enzyme tyrosine phosphatase 1B at significantly lower IC50 values than the carbohydrate-hydrolase counterpart. Although the primary parameters extracted from computational output (i.e., DS value and number of interactions) are highly consistent with the overall inhibition activity towards the enzymes (i.e., tyrosine phosphatase 1B and α-glucosidase) obtained from experimental assays, the specific orders regarding each of these proteins are still not clearly represented. Molecules 2021, 26, 3691 9 of 18 Molecules 2021, 26, x 9 of 21

Figure 7.7. QuaternaryQuaternary structuresstructures of of proteins proteins (a ()a 3W37,) 3W37, (b )(b 3AJ7,) 3AJ7, and and (c) ( PTP1Bc) PTP1B with with their their approachable approachable sites sites by investigated by investi- Moleculescompoundsgated compounds 20211, 26and, x 2 1: and site 2 1: (yellow),site 1 (yellow), site 2 (cyan),site 2 (cyan), site 3 (grey),site 3 (grey), and site and 4 (blue). site 4 (blue). 10 of 21 Molecules 2021, 26, x 10 of 21 Molecules 2021, 26, x 10 of 21 Molecules 2021, 26, x 10 of 21 Table 3. In-site amino acid residues of proteins 3W37, 3AJ7, and PTP1B. Table 3. In-site amino acid residues of proteins 3W37, 3AJ7, and PTP1B. Site Colour Residues of ProteinTable 3W37 3. In-siteIn-site Residuesaminoamino acidacid of residuesresidues Protein of 3AJ7of proteins Residues 3W37, 3AJ7, of Protein and PTP1B. PTP1B Site Colour Residues of ProteinTable 3W37 3. In-siteAsp69 amino Tyr72 acid Residues His112residues Lys156 of of Proteinproteins 3AJ73W37, 3AJ7, and PTP1B. Residues of Protein PTP1B Site Colour Residues of Protein 3W37 Residues of Protein 3AJ7 Residues of Protein PTP1B Site Colour Residues of Protein 3W37 Ser157 Tyr158 Residues Phe159 of Leu177 Protein 3AJ7 Residues of Protein PTP1B Site Colour Residues of Protein 3W37 ResiduesAsp69 Tyr72 of Protein His112 Lys1563AJ7 Ser157 Tyr158 Residues of Protein PTP1B Phe178 Gln182Asp69 Arg213 Tyr72 His112 Lys156 Ser157 Tyr158 Asp215 Val216Phe159 Ser240 Leu177 Ser241 Phe178Arg24 Gln182 Ala27 Arg213 Ser28 Asp29 Asp215 Asp69Phe159 Tyr72 Leu177 His112 Phe178 Lys156 Gln182 Ser157 Arg213 Tyr158 Asp215 Tyr360 Phe367 Pro395 Ile396 Asp242 Glu277Phe159 Gln279 Leu177 Phe178Phe30 Gln182 Pro31 Arg213 Cys32 Lys36 Asp215 Arg24 Ala27 Ser28 Asp29 Phe30 Pro31 Tyr360 Phe367 Pro395 Ile396 Leu397 Phe159Val216 Ser240Leu177 Ser241 Phe178 Asp242 Gln182 Glu277 Arg213 Gln279 Asp215 Arg24 Ala27 Ser28 Asp29 Phe30 Pro31 1 1 Tyr360Leu397 Phe367 Ile454 Phe457Pro395 Arg458 Ile396 Leu397His280 Phe303Val216 Thr306 Ser240 Ser241Asp48 Asp242 Val49 Glu277 Phe52 Ile219Gln279 Arg24Cys32 Lys36Ala27 Asp48Ser28 Asp29 Val49 Phe30Phe52 Pro31Ile219 1 Ile454Ile463 Phe457 Ile466 Arg458 Ile463 Ile466Asp307 Thr310His280 Ser311 Phe303 Thr306Arg254 Asp307 Arg257 Thr310 Met258 Ser311 Cys32 Lys36 Asp48 Val49 Phe52 Ile219 Tyr360Ile454 Phe457 Phe367 Arg458 Pro395 Ile463Ile396 Ile466Leu397 Val216His280 Ser240Phe303 Ser241 Thr306 Asp242 Asp307 Glu277 Thr310 Gln279 Ser311 Arg254 Arg257 Met258 Gly259 Gln262 1 Ile454 Phe457 Arg458 Ile463 Ile466Pro312 Leu313His280 Phe314 Phe303 Thr306Gly259 Asp307 Gln262 Thr310 Ser311 Cys32 Lys36 Asp48 Val49 Phe52 Ile219 Ile454 Phe457 Arg458 Ile463 Ile466 His280Pro312 Phe303Leu313 Thr306Phe314 Asp307Arg315 Thr310Tyr316 Ser311His351 Arg254 Arg257 Met258 Gly259 Gln262 Arg315 Tyr316Pro312 His351 Leu313 Phe314 Arg315 Tyr316 His351 Arg254 Arg257 Met258 Gly259 Gln262 Asp352 Gln353Asp352 Glu411 Gln353 Ile440 Glu411 Ile440 Arg442 Arg446 Pro312Asp352 Leu313 Gln353 Phe314Glu411 Arg315Ile440 Arg442 Tyr316 Arg446His351 Arg442 Arg446Asp352 Gln353 Glu411 Ile440 Arg442 Arg446 Glu301 Tyr659 Thr662 Leu663 Asp666 Asp352 Gln353 Glu411 Ile440 Arg442 Arg446 Glu301Glu301 Tyr659 Tyr659 Thr662 Thr662 Leu663Leu663 Asp666Val369 Ile370 Lys373 Pro488 Glu301Arg670 Tyr659Ile672 Arg676 Thr662 Ile697Leu663 Gly698 Asp666 Val369 Ile370 Lys373 Pro488 Asn489 Ser490 Ala35 Lys36 Leu37 Pro38 Asn40 Lys41 Arg670Asp666 Ile672 Arg670 Arg676 Ile672 Ile697 Arg676 Gly698Asn489 Ser490Val369 Asn493 Ile370 Lys373Ala35 Pro488 Lys36 Asn489 Leu37 Pro38Ser490 Ala35 Lys36 Leu37 Pro38 Asn40 Lys41 2 Arg699Ile697 Gly700 Gly698 Arg699Ile701 Gly700Ile754 Asn758Phe494 Glu497Asn493 Leu561 Phe494 Glu497Asn40 Leu561 Lys41 Asn44Glu562 Arg45 Phe563 Asn44 Arg45 Tyr46 Arg47 Asp48 2 Arg670 Ile672 Arg676 Ile697 Gly698 Val369 Ile370 Lys373 Pro488 Asn489 Ser490 Ala35 Lys36 Leu37 Pro38 Asn40 Lys41 2 Arg699Ile701 Gly700 Ile754 Asn758 Ile701 Ile759 Ile754 Asn758Glu562 Phe563Asn493 Gly564 Phe494 Glu497Tyr46 Leu561 Arg47 Asp48Glu562 Phe563 Asn44 Arg45 Tyr46 Arg47 Asp48 Ile759 Val760 Ala761 Thr790 Gly791 Gly564 Asn565 Tyr566 Pro567 Lys568 Val571 Val49 Ser50 2 Arg699Ile759Val760 Val760 Gly700 Ala761 Ala761 Thr790Ile701 Thr790Ile754 Asn758Gly791Asn565 Tyr566Asn493Gly564 Pro567 Asn565Phe494 Tyr566Glu497Val49 Pro567Leu561 Ser50 Lys568 Glu562 Val571 Phe563 Asn44Val49 Ser50Arg45 Tyr46 Arg47 Asp48 Ile759 Val760 Ala761 Thr790 Gly791 Gly564 Asn565 Tyr566 Pro567 Lys568 Val571 Val49 Ser50 Ile759Glu792Gly791 Val760 Glu792 Ala761 Thr790 Gly791Lys568 Val571Gly564 Asn565 Tyr566 Pro567 Lys568 Val571 Val49 Ser50 Glu792 Glu792 Lys156 Ser157Lys156 Tyr158 Ser157 Phe159 Tyr158 Phe159 Gly160 Gly161 Leu71 Lys73 Met74 Glu75 Ala77 Gln78 Tyr319 Pro658 Tyr661 Gln763 Arg773Gly160 Gly161Lys156 Asp233 Ser157 Tyr158Leu71 Phe159 Lys73 Gly160 Met74 Glu75Gly161 Leu71 Lys73 Met74 Glu75 Ala77 Gln78 Tyr319 Pro658 Tyr661 Gln763 Arg773 Lys156Asp233 Ser157Asn235 Tyr158 Ser236 Phe159 Thr237 Gly160 Trp238 Gly161 Ser311 Leu71Arg79 Lys73Ser80 Ser203Met74 Leu204Glu75 Ala77 Ser205 Gln78 3 Phe777Tyr319 Leu793 Pro658 Tyr661Phe794 Gln763 Leu795 Asp796Asn235 Ser236Asp233 Thr237 Asn235 Trp238 Ser236Ala77 Thr237 Gln78 Trp238 Arg79 Ser80 Ser311 Arg79 Ser80 Ser203 Leu204 Ser205 3 Tyr319Phe777 Pro658Leu793 Tyr661 Phe794 Gln763 Leu795 Arg773 Asp796 Leu313 Phe314 Asn317 Asn415 Ala418 Ile419 Pro206 His208 Gly209 Pro210 Val211 3 3 Phe777Arg773 Leu793 Phe777 Phe794 Leu793 Phe794Leu795 Asp796Ser311 Leu313Asp233 Phe314 Asn235 Ser236Ser203 Thr237 Leu204 Trp238 Ser205 Ser311 Pro206 Arg79 Ser80 Ser203 Leu204 Ser205 Trp841 Leu313 Phe314 Asn317 Asn415 Ala418 Ile419 Pro206 His208 Gly209 Pro210 Val211 3 Phe777Trp841Leu795 Leu793 Asp796 Phe794 Trp841 Leu795 Asp796Asn317 Asn415Glu422 Ala418 His423 Ile419 Glu428His208 Glu429 Gly209 Lys432 Pro210 Val211 Leu233 Lys237 Trp841 Glu422 His423Leu313 Glu428 Phe314 Asn317Leu233 Asn415 Lys237 Ala418 Ile419 Pro206 His208 Gly209 Pro210 Val211 Trp841 Glu422 His423 Glu428 Glu429 Lys432 Leu233 Lys237 Glu429 Lys432Glu422Lys13 Trp15 His423 Asn259 Glu428 Ile262 Glu429 Glu271 Lys432 Ile272 Met273 Leu233 Lys237 Tys331 Arg332 Asp333 Ile358 Asp359 Lys13 Trp15 Asn259 Ile262 Glu271 Ile272 Met273 Lys73 Met74 Glu75 Glu76 Ala77 Tys331 Arg332 Asp333 Ile358 Asp359Lys13 Trp15Lys13Thr274 Asn259 Trp15 Tyr289 Ile262 Asn259 Thr290 Ile262 Ser291 Glu271 Ala292 Ile272 Arg294 Met273 Lys73 Met74 Glu75 Glu76 Ala77 4 Tyr360Tys331 Met361 Arg332 Asp362 Asp333 Ile358Ala363 Phe364Glu271 Ile272Thr274 Met273 Tyr289 Thr274 Thr290 Ser291 Ala292 Arg294 Thr230 Leu234 Lys248 Val249 Glu252 4 Tys331Tyr360 Met361Arg332 Asp333Asp362 Ile358Ala363 Asp359 Phe364 His295 Glu296 Leu297Lys73 Ser298 Met74 Asp341 Glu75 Glu76 Cys342 Lys73Thr230 Met74 Leu234 Glu75 Lys248 Glu76 Val249 Ala77 Glu252 4 Tyr360Asp359 Met361 Tyr360 Asp362 Met361 Asp362Ala363 Phe364Tyr289 Thr290Thr274 Ser291 Tyr289 Ala292 Thr290 Ser291 Ala292 Arg294 Thr230 Leu234 Lys248 Val249 Glu252 4 4 Asp370 His373 Phe374 Arg629 His295 Glu296 Leu297Ala77 Ser298 Thr230 Asp341 Leu234 Cys342 Lys248 Lys255 Phe256 Tyr360Asp370Ala363 Met361 His373 Phe364 Asp362Phe374 Asp370 His373Arg629Ala363 Phe364Arg294 His295Trp343 Glu296 Thr230Lys255 Leu234Phe256 Lys248 Val249 Glu252 Asp370 His373 Phe374 Arg629 His295 Glu296 Leu297Val249 Ser298 Glu252 Asp341 Lys255 Cys342 Phe256 Lys255 Phe256 Phe374 Arg629 Leu297 Ser298Trp343 Asp341 Asp370 His373 Phe374 Arg629 Lys255 Phe256 Cys342 Trp343Trp343

Molecules 2021, 26, 3691 10 of 18

Table 4. Prescreening results on inhibitability of investigated compounds 1, 2 and the commercialized drug voglibose (D) towards the potential sites on proteins 3W37, 3AJ7, and PTP1B.

Protein 3W37 Protein 3AJ7 Protein PTP1B Compound Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 ENENENENENENENENENENENEN 1 −11.4 2 −13.2 3 −12.1 2 −10.9 3 −10.6 3 −11.4 2 −13.1 3 −11.7 3 −14.9 4 −12.4 3 −13.1 2 −11.8 3 2 −12.3 2 −13.8 3 −11.4 3 −11.7 2 −14.3 5 −12.8 3 −11.9 4 −11.3 3 −14.7 5 −13.4 4 −12.6 3 −11.4 4 D −11.7 3 −14.1 4 −12.5 3 −10.6 3 −13.6 4 −10.4 3 −11.5 3 −12.4 4 −12.4 3 −14.5 4 −11.8 3 −10.7 3 E: DS value (kcal ∗ mol−1); N: Number of interactions. Molecules 2021, 26, 3691 11 of 18

Table 5. Molecular docking simulation results for inhibitory complexes between the ligands (compounds 1 and 2) and the targeted proteins (3W37, 3AJ7, and PTP1B): 1-3W37, 1-3AJ7, 1-PTP1B, 2-3W37, 2-3AJ7, and 2-PTP1B.

Ligand-Protein Complex Hydrogen Bond Van der Waals Interaction Name DS RMSD L P T D E O N Arg676 H-acceptor 3.46 −1.0 Arg814, Gly698, Leu663, Arg699, Tyr659, Asp666, Gly791, 1-3W37 −13.2 1.84 O N Arg676 H-acceptor 3.02 −4.0 Ile672, Glu792 O N Arg676 H-acceptor 3.39 −0.9 C O Asp233 H-donor 2.91 −0.7 Asn235, Asn415, Ser236, Gly160, Lys156, Ile419, Phe314, 1-3AJ7 −13.1 1.33 O O Ser311 H-acceptor 3.09 −1.3 Leu313, Ala418, His423, Glu422 O N Asn317 H-acceptor 2.94 −1.1 C S Met258 H-donor 3.83 −0.8

O N Arg24 H-acceptor 3.09 −0.7 Ile219, Asp48, Gly259, Ser28, Gln262, Asp29, 1-PTP1B −14.9 1.25 O N Arg254 H-acceptor 2.92 −4.4 Gln21, His25 6-ring C Met258 π-H 3.44 −1.6 O O Asp666 H-donor 2.89 −2.1 Glu792, Gly698, Tyr659, Arg699, Leu663, Glu301, Met302, 2-3W37 −13.8 O O Thr299 H-acceptor 2.89 −1.3 1.07 Arg676, Ile672 O N Arg676 H-acceptor 3.18 −2.5 O O Asp307 H-donor 2.98 −1.2 C O Glu277 H-donor 3.48 −0.6 Tyr72, Asp215, Asp362, Val216, Gln353, Gln279, Thr306, 2-3AJ7 −14.3 O O Asp69 H-donor 2.73 −3.6 0.83 Phe303, Tyr158, Arg315, Phe159, His112,Gln182 O N Arg442 H-acceptor 2.82 −4.3 C 6-ring Phe178 H-π 3.69 −0.6 O N Arg24 H-acceptor 2.86 −2.6 O N Arg24 H-acceptor 3.07 −1.0 Arg47, Asp48, Arg254, Tyr20, Ile261, Gly259 Ser50, 2-PTP1B −14.7 O N Gln262 H-acceptor 2.85 −0.8 1.62 Asp29, Val49 O N Lys36 H-acceptor 3.27 −2.1 6-ring C Met258 π-H 4.20 −0.9 DS: Docking score energy (kcal·mol−1); RMSD: Root-mean-square deviation (Å); L: Ligand; P: Protein; T: Type; D: Distance (Å); E: Energy (kcal·mol−1). Molecules 2021, 26, 3691 12 of 18

Nevertheless, a brief structure relationship for the inhibition strength of the isolated compounds 1 and 2 from T. ruticarpum on each enzyme can be reached based on certain Molecules 2021, 26, x 15 of 21 hydrogen bonding interactions. First, the docking simulation technique expects that there exists a strong hydrogen bond formed between an oxygen atom of the ligand 1 and a nitrogen atom of amino acid Arg254 within the distance 2.92 Å in 1-PTPB1B. The bonding registers an exceptionallyThe inhibitory high value structures of Gibbs and free their energy in-pose−4.4 kcalintermolecular·mol−1. While interactions there are are visually five predictedshown hydrogen in Figure bonds 8. that Although contribute the tosites the are formation observably of theuncapacious inhibitory for system either macromol- 2-PTPB1, the aminoecule to acid enter is not or involved.for simultaneous This highly inhibitions, correlates the withinvestigated the results compounds obtained are still pre- dicted to geometrically fit in the structural topography of their inhibiting sites given their from the enzyme assays on tyrosine phosphatase 1B inhibition; i.e., 1 (IC50 24.3 ± 0.8 µM) > continuous proximity contours. This indicates a high degree of complementarity. In ad- 2 (IC50 47.7 ± 1.1 µM). Similarly, amino acid Arg676 is likely to play an important role in the formation of 1-3W37dition, inhibitorythe simulation complex for the as itcommercialized interacts with thedrug ligand voglibose via three (D) was hydrogen- also screened, and bonding interactions,the corresponding whose Gibbs results free are energy included in total in suppl is −5.9ementary kcal·mol data−1 .(Table Meanwhile, S1 and Figure S16) 2 only createsfor one optional of the hydrophilic reference. bonding with its targeted protein to form 2-3W37 inhibitory structure. The value of −2.5 kcal·mol−1 corresponds to the free Gibbs energy of this interaction. This can also be related to the differences observed between the enzyme assays on α-glucosidase inhibition. Therefore, the sufficient binding carried out on these amino acids, i.e., Arg254 and Arg676, seems to induce serious conformational changes on the enzymes PTP1B and 3W37, respectively, thus leading to the loss of their enzymatic functionality that is experimentally demonstrated in enzyme assays by the increase in IC50 values. In terms of oligo-1,6-glucosidase-based inhibitory systems (1-3AJ7 and 2-3AJ7), the concern is thought to relate to amino acid Arg442. The intuitive speculation still requires further relevant experimental work specifically targeting these amino acids to verify the proposed inhibition models. The inhibitory structures and their in-pose intermolecular interactions are visually shown in Figure8. Although the sites are observably uncapacious for either macromolecule to enter or for simultaneous inhibitions, the investigated compounds are still predicted to geometrically fit in the structural topography of their inhibiting sites given their continuous proximity contours. This indicates a high degree of complementarity. In addition, the simu- lation for the commercialized drug voglibose (D) was also screened, and the corresponding results are included in supplementary data (Table S1 and Figure S16) for optional reference.

Figure 8. VisualFigure presentation 8. Cont. and in-pose interaction map of ligand-3W37, ligand-3AJ7, and ligand-PTP1B inhibitory isolated compounds 1 and 2: (a) 1-3W37, (b) 1-3AJ7, (c) 1-PTP1B, (d) 2-3W37, (e) 2-3AJ7, (f) 2-PTP1B. In molecular ren- dering , black, gray (white), blue, and red are standard colours for carbon, hydrogen, nitrogen, and oxygen atoms, re- spectively.

3. Materials and Methods 3.1. Experimental Methods 3.1.1. General Experimental Procedures

Molecules 2021, 26, x 15 of 21

The inhibitory structures and their in-pose intermolecular interactions are visually shown in Figure 8. Although the sites are observably uncapacious for either macromol- ecule to enter or for simultaneous inhibitions, the investigated compounds are still pre- dicted to geometrically fit in the structural topography of their inhibiting sites given their continuous proximity contours. This indicates a high degree of complementarity. In ad- dition, the simulation for the commercialized drug voglibose (D) was also screened, and the corresponding results are included in supplementary data (Table S1 and Figure S16) for optional reference.

Molecules 2021, 26, 3691 13 of 18

Figure 8. VisualFigure presentation 8. Visual presentation and in-pose and interaction in-pose interaction map of ligand-3W37, map of ligand-3W37, ligand-3AJ7, ligand-3AJ7, and ligand-PTP1B and ligand- inhibitory isolated compoundsPTP1B inhibitory 1 and 2: ( isolateda) 1-3W37 compounds, (b) 1-3AJ71, (cand) 1-PTP1B2:(a) ,1-3W37 (d) 2-3W37,(b), 1-3AJ7(e) 2-3AJ7,(c), 1-PTP1B(f) 2-PTP1B,(d. )In2-3W37 molecular, ren- dering , black,(e) 2-3AJ7 gray (white),,(f) 2-PTP1B blue, and. In molecularred are standard rendering, colours black, for carbon, gray (white), hydrogen, blue, nitrogen, and red and are oxygen standard atoms, re- spectively. colours for carbon, hydrogen, nitrogen, and oxygen atoms, respectively.

3. Materials and3. Materials Methods and Methods 3.1. Experimental3.1. Methods Experimental Methods 3.1.1. General Experimental3.1.1. General ProceduresExperimental Procedures Proton and carbon nuclear magnetic resonance (NMR) were measured on a Varian Unity Inova 400 and/or 500 MHz spectrometers. Mass spectrometry (MS) data were obtained from a Varian FT-MS spectrometer and (Bruker Daltonics, Ettlingen Germany). Normal-phase and reverse-phase silica gels (F254, 40–63 mesh) were purchased from Merck, St. Louis, MO, USA. NP and RP thin-layer chromatography (TLC) plates were from Merck (St. Louis, MO, USA). High-performance liquid chromatography (HPLC, Santa Clara, CA 95051, USA) was carried out using a 1260 Agilent HPLC System: G1311C pump, G2260A auto-sampler, G1316A Thermo, and G1315D detector. Optima_Pak C18 column (10 × 250 mm, 10 and/or 5 µm particle sizes), RS Tech, Korea, and/or Zorbax eclipse XDB-C18 (9 × 250 mm, 5 µm particle size) were used for purification.

3.1.2. Plant Material Tetradium ruticarpum material was obtained in 2019, and the plant sample was identi- fied by Dr. Quoc-Binh Nguyen (Vietnam National Museum of Nature, Vietnam Academy of Science and Technology—VAST, Ha Noi, Vietnam). The specimen voucher of this plant was stored at INPC, VAST, Vietnam.

3.1.3. Isolation and Purification The buds (1.2 kg) were extracted with methanol (5.0 L × 3 times) at 45 ◦C using sonication for 5 h. After filtration, the extracted solution was then evaporated by a rotary system to remove the solvent. The total methanol crude extract obtained was further suspended in distilled water (1.0 L) and partitioned with EtOAc (1.0 L × 4 times). The resulting fractions were concentrated under reduced pressure in a rotary evaporator to give the EtOAc and an H2O residue, respectively. An activity-guided fractionation study resulted in the EtOAc extract being chosen for further study (Table S3 in Supplementary data). The EtOAc fraction was thus directly chromatographed on an open silica gel column (5.0 × 60 cm; 63–200 µm particle size) using a stepwise gradient of hexane/acetone (from 20:1 to 0:1) to give ten fractions (TR-1 to TR-10). By the α-glucosidase activity-guided Molecules 2021, 26, 3691 14 of 18

isolation (Table S3), fraction 2 (TR-2) was further selected for purification by diluting to methanol until saturation and then kept still for precipitation. The supernatant was then separated from the precipitate (TR-2P) and evaporated to give a crude fraction (TR-2S). These two sub-fractions further tested the alkaloid-containing compound by reaction with Dragendorff reagent on a TLC plate. Fraction TR-2S was prior purified by an open silica gel column (2.0 × 60 cm, 40–63 µm particle size) due to its alkaloid-containing fraction, eluted with a gradient solvent system of hexane/EtOAc (from 15:1 to 10:1), and afforded compounds 1 (23.9 mg) and 2 (19.5 mg). + 1 Schinifoline (1): White crystal; FAB-MS m/z: 258.1 [M + H] (C17H23NO); H-NMR (400 MHz, CDCl3) δH (ppm): 6.21 (1H, s, H-3), 8.43 (2H, dd, J = 8.0, 1.6 Hz, H-5), 7.36 (1H, brt, J = 8.0 Hz, H-6), 7.64 (1H, dt, J = 1.6, 8.0 Hz, H-7), 7.49 (1H, brd, J = 8.0 Hz, H-8), 2.69 (2H, t, J = 7.6 Hz, H-10), 1.67 (2H, q, J = 7.6 Hz, H-20), 1.27-1.42 (8H, m, H-30/H-40/H-50/H-60), 0 13 0.88 (3H, t, J = 6.8 Hz, H-7 ), 3.72 (3H, s, N-CH3); C-NMR (100 MHz, CDCl3) δC (ppm): 154.9 (C-2), 111.3 (C-3), 178.0 (C-4), 126.8 (C-5), 123.4 (C-6), 132.2 (C-7), 115.5 (C-8), 142.1 (C-9), 126.7 (C-10), 34.9 (C-10), 28.7 (C-20), 29.4 (C-30), 29.2 (C-40), 31.8 (C-50), 22.8 (C-60), 14.2 0 (C-7 ), 34.3 (N-CH3). + Integrifoliodiol (2): Colourless needles; FAB-MS m/z: 257.03 [M + Na] (C14H18O3); 1 H-NMR (400 MHz, CDCl3) δH (ppm): δH: 7.32 (2H, d, J = 8.4 Hz, H-2/H-6), 6.87 (2H, d, J = 8.4 Hz, H-3/H-5), 6.55 (1H, brd, J = 16.0 Hz, H-7), 6.21 (1H, dt, J = 16.0, 6.0 Hz, H-8), 4.30 (2H, dd, J = 6.0, 1.2 Hz, H-9), 4.59 (2H, brd, J = 6.4 Hz, H-10), 5.77 (1H, m, H-20), 4.09 0 0 13 (2H, s, H-4 ), 1.77 (3H, s, H-5 ); C-NMR (100 MHz, CDCl3) δC (ppm): 129.5 (C-1), 127.6 (C-2/C-6), 114.9 (C-3/C-5), 158.4 (C-4), 130.9 (C-7), 126.2 (C-8), 63.9 (C-9), 64.3 (C-10), 119.7 (C-20), 140.1 (C-30), 67.8 (C-40), 14.0 (C-50).

3.1.4. Protein Tyrosine Phosphatase 1B Inhibition Assay Protein tyrosine phosphatase 1B (human recombinant), i.e., PTP1B, was purchased from Biomol International LP, Plymouth Meeting, Pennsylvania, PA, USA, and the in- hibitory activities of the tested samples were evaluated using the method as described. [26] In a typical procedure, 0.05–0.1 µg of PTP1B (BIOMOL International L.P., Plymouth Meet- ing, Pennsylvania, PA, USA) and 4 mM p-NPP in a buffer containing 1 mM dithiothreitol, 0.1 M NaCl, 1 mM EDTA, and 50 mM citrate (pH 6.0), with or without test compounds, were added, with 100 µL final volume to each of the 96 wells. After the reaction mixture was incubated at 37 ◦C for 30 min, 10 M NaOH was added to quench the reaction. PTP1B enzyme activity was determined by the amount of produced p-nitrophenol at 405 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control, which contained no PTP1B enzyme. Ursolic acid (UA) was used as a positive control.

3.1.5. α-Glucosidase Inhibition Assay The inhibitory activity of α-glucosidase was determined according to the modified method with a readily available enzyme. In a typical procedure, α-glucosidase (0.1 U/mL, Wako) was dissolved in 100 mM sodium phosphate buffer (pH 6.8) and used as an enzyme solution. A total of 1 mM p-Nitrophenyl-α-D-glucopyranoside (pNPG) in the same buffer (pH 6.8) was used as a substrate solution. Then, 95 µL of enzyme solution, 10 µL of extracts, and 10 µL of ethanol and PBS solution (1:1) were mixed in the same 96-well plate for each concentration (250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL) and measured absorbance at 405 nm after incubation for 5 min. After substrate solution (95 µL) was added, the sample solution was incubated for another 10 min at 37 ◦C incubator. Enzymatic activity was quantified by measuring absorbance at 405 nm using an enzyme- linked immunosorbent assay (ELISA) microplate reader (D.I Biotech Ltd., Seoul, Korea). The IC50 value was defined as the concentration of α-glucosidase inhibitor that inhibited 50% of α-glucosidase activity. Acarbose was used as a positive control, and all assays were conducted in triplicate. Molecules 2021, 26, 3691 15 of 18

3.1.6. Statistical Analysis The collected data were analyzed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Average values and percentages were calculated. Differences in mean values were compared using analysis of variance (ANOVA), for normally distributed data, and the Mann–Whitney U test, for non-normally distributed data. Differences were considered significant at p < 0.05.

3.2. Computational Methods 3.2.1. Quantum Chemical Calculation Density functional theory (DFT) was utilized to investigate quantum properties of the isolated compounds 1 and 2 from T. ruticarpum. Their molecular geometry was optimized using Gaussian 09 without symmetry constraints [41] at the level of theory M052X/6- 311++G(d,p) [42]. Calculation of vibrational frequencies on the molecules was used to confirm that their structures were in global minimum on the potential energy surface (PES). Single-point energies at the M052X/6-311++G(d,p)-level-optimized geometries were calculated with the frozen-core approximation for non-valence-shell electrons by a larger basis set def2-TZVPP [43]. Each run of the optimization was based upon resolution-of- identity (RI) approximation. Frontier orbital analysis was implemented at the level of theory BP86/def2-TZVPP, providing localized molecular orbitals and orbital energy. NBO 5.1, available in Gaussian 09, was responsible for the analysis [44]. Bonding analysis revealed information of molecular electron density distribution. The highest occupied molecular orbital (HOMO) energy, EHOMO, represents intermolecular electron donation tendency; meanwhile, the electron-accepting ability of a molecule can be inferred from its value ELUMO (for lowest unoccupied molecular orbital—LUMO). Energy gap ∆E = ELUMO − EHOMO is considered as an indicator for intermolecular reactivity since it exhibits the formation of excited-state electrons towards the molecular surface. Ionization potential (I) and electron affinity (A) were calculated using Koopmans’ theorem [45], which negatively correlated with HOMO and LUMO energy as I = −EHOMO and A = −ELUMO. They then were used to yield the electronegativity (χ) of a molecule via the equation χ = (I + A)/2. Regarding an N-electron system with total electronic energy (E) and external potential ν(r), electronegativity (χ) is defined as the negative value of chemical potential (µ)[46,47]. This can be expressed by the following equation: χ = −µ = −(∂E/∂N)ν(r).

3.2.2. Molecular Docking Simulation MOE 2015.10 software was responsible for molecular docking simulation. Its requi- site input includes structural information of docking participants, i.e., two ligands 1-2, PTP1B, and glycoside-hydrolase proteins (3W37 and 3AJ7). The simulated ligand-protein inhibitory structures were evaluated based on docking score (DS) energy as the main indicator. Root-mean-square deviation (RMSD) and intermolecular interactions were also analyzed and discussed. The typical procedure of a molecular docking simulation followed three steps [19–22]: (a) Pre-docking preparation: Structural information of the targeted proteins can be referenced at UniProtKB and Worldwide Protein Data Bank: tyrosine phosphatase pro- tein PTP1B (entry: UniProtKB—A0A0U1XP67), alpha-glucosidase protein 3W37 (DOI: 10.2210/pdb3W37/pdb), and oligo-1,6-glucosidase protein 3AJ7 (DOI: 10.2210/pdb3AJ7/pdb). The protein structure and their 3D protonation were prepared using Quickprep tool. Their active sites were determined based on a ligand–amino acid radius of 4.5 Å. The protein structures obtained were saved in format *.pdb. The investigated adducts were optimized based on the configuration: Conj Grad for minima energy; termination for energy change = 0.0001 kcal·mol−1; max interactions = 1000; modify charge: Gasteiger–Huckel. (b) Docking investigation: After input preparation, intermolecular interaction simu- lation was performed on MOE 2015.10 system and simulated ligand–protein inhibitory structures were saved in format *.sdf. The docking simulation parameters were config- Molecules 2021, 26, 3691 16 of 18

ured: poses retained for intermolecular interaction probing = 10; maximum solutions per iteration = 1000; maximum solutions per fragmentation = 200. (c) Post-docking analysis: Docking score (DS) energy represents binding affinity of ligands and their targeted proteins in the site–site distance of a certain duo-system, thus considered as the primary indicator of inhibitability. Intermolecular interactions formed between the ligands and in-pose amino acids of the proteins included hydrophilic binding, e.g., electron-transferring (H-acceptor/donor), cation-arene (H-π), arene-arene (π-π), and ionic and hydrophobic interaction, also known as van der Waals forces. The simulation results in bonding amino acids, bonding lengths, and their Gibbs free energy regarding these interactions. Furthermore, static conformation of an inhibitory complex was predicted by its root-mean-square deviation (RMSD) value. This is based on the fact that RMSD represents the average between neighboring atoms; therefore, a smaller value means a more tightly bound conformation is formed. In addition, ligand conformation and orientation in its inhibited-protein active site were visualized on 2D and 3D planes.

4. Conclusions Two isolated natural compounds were successfully identified by structural elucidation as schinifoline (1) and integrifoliodiol (2). Enzyme assays demonstrated that the alkaloid compound (1) is the stronger inhibitor towards the both proteins tyrosine phosphatase 1B (PTP1B) and α-glucosidase (3W37). The corresponding orders are 1 (IC50 24.3 ± 0.8 µM) > 2 (IC50 47.7 ± 1.1 µM) regarding the former and 1 (IC50 92.1 ± 0.8 µM) > 2 (IC50 167.4 ± 0.4 µM) regarding the latter. The primary parameters obtained from molecular docking simulation, also known as DS value and number of interactions, confirm these experimental yields. In particular, in-depth analyses on structure–activity relationships imply that amino acids Arg254 and Arg676 might play an important role in the overall conformational changes of PTP1B and 3W37, respectively. This speculation still needs more specifically experimental work to verify, thus encouraging further continuous research from the readership.

Supplementary Materials: The following are available online. Figure S1: 1H NMR spectrum of compound 1, Figures S2 and S3: 1H NMR spectrum of compound 1 (expanded), Figure S4: 13C NMR spectrum of compound 1, Figure S5: 13C NMR spectrum of compound 1 (expanded), Figure S6: HMQC spectrum of compound 1, Figure S7: HMBC spectrum of compound 1, Figure S8: Mass spectrum of compound 1, Figure S9: 1H NMR spectrum of compound 2, Figure S10: 13C NMR spectrum of compound 2, Figure S11: DEPT spectrum of compound 2, Figure S12: COSY spectrum of compound 2, Figure S13: HSQC spectrum of compound 2, Figure S14: HMBC spectrum of compound 2, Figure S15: Mass spectrum of compound 2, Figure S16: Visual presentation and in-pose interaction map of voglibose-protein inhibitory complexes: (a) D-3W37, (b) D-3AJ7, (c) D-PTP1B, Table S1: Molecular docking simulation results for inhibitory complexes between the controlled drug voglibose (D) and the proteins 3W37, 3AJ7 and PTP1B, Table S2: Cartesian coordinates of the optimized 1 and 2 by DFT using basis M052X/6-311++G(d,p), Table S3. PTP1B and α-glucosidase inhibitory activities of total methanol extracts and its fractions from T. ruticarpum. Author Contributions: Investigation, T.-T.D. and D.-C.T.; methodology, N.T.A.N. and P.-T.Q.; soft- ware, P.-P.H. and T.-N.N.; data curation: Q.-T.T. and N.T.A.N.; validation, T.Q.B.; formal analysis, T.-T.D. and D.-C.T.; writing—original draft preparation, M.-H.T. and N.T.A.N.; writing—review and editing, P.-H.N. and D.-C.T.; supervision, P.-H.N. and T.-D.N.; funding acquisition, P.-H.N.; resources: T.-H.N. and H.-T.N. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a grant from the Vietnam Academy of Science and Technology (VAST), (Project code number: ÐLTE00.04/19-20). T.D.N is grateful for the support of the research program of the Thai Nguyen University of Agriculture and Forestry (TUAF) supported by the Ministry of Education and Training (MOET) through the project CT2020.03.DTN-07. Data Availability Statement: The data presented in this study are available in this article. Acknowledgments: Nguyen Thi Ai Nhung acknowledges the partial support of Hue University under the Core Research Program, Grant No. NCM.DHH.2020.04. Molecules 2021, 26, 3691 17 of 18

Conflicts of Interest: The authors declare no conflict of interest. Code Availability: (1) Gaussian09: EM64L—G09RevC.01—Named keywords: Density Functional Theory, BP86/def2-SVP, BP86/def2-TZVPP//BP86/def2-SVP; (2) MOE 2015.10 License Information— Feature: MOE; Version limit: 2016.02—Unlimited; Quickprep tool following configuration: Tether— Receptor with the strength of 5000; Refine of 0.0001 kcal·mol−1·Å−1, radius = 4.5 Å. Configuration: Conj Grad for minima energy; termination for energy change = 0.0001 kcal·mol−1; max interactions = 1000; modify charge: Gasteiger-Huckel. Docking score energy (DS), root-mean-square deviation (RMSD), binding interactions: hydrogen bonds, cation–π, π–π bonds, ionic interactions, van der Waals interactions. Sample Availability: Samples of the compounds are available from the authors.

References 1. World Health Organization. Global Report on Diabetes; WHO Press: Geneva, Switzeland, 2016; ISBN 978-92-4-156525-7. 2. Washburn, W.N. Development of the Renal Glucose Reabsorption Inhibitors: A New Mechanism for the Pharmacotherapy of Diabetes Mellitus Type 2. J. Med. Chem. 2009, 52, 1785–1794. [CrossRef] 3. Moller, D.E. New Drug Targets for Type 2 Diabetes and the Metabolic Syndrome. Nature 2001, 414, 821–827. [CrossRef][PubMed] 4. Ha, M.T.; Seong, S.H.; Nguyen, T.D.; Cho, W.K.; Ah, K.J.; Ma, J.Y.; Woo, M.H.; Choi, J.S.; Min, B.S. Chalcone Derivatives from the Root Bark of Morus alba L. Act as Inhibitors of PTP1B and α-Glucosidase. Phytochemistry 2018, 155, 114–125. [CrossRef] 5. Krishnan, N.; Bonham, C.A.; Rus, I.A.; Shrestha, O.K.; Gauss, C.M.; Haque, A.; Tocilj, A.; Joshua-Tor, L.; Tonks, N.K. Harnessing Insulin-and Leptin-Induced Oxidation of PTP1B for Therapeutic Development. Nat. Commun. 2018, 9.[CrossRef] 6. Johnson, T.O.; Ermolieff, J.; Jirousek, M.R. Protein Tyrosine Phosphatase 1B Inhibitors for Diabetes. Nat. Rev. Drug Discov. 2002, 1, 696–709. [CrossRef][PubMed] 7. Tomasik, P.; Horton, D. Enzymatic Conversions of Starch, 1st ed.; Elsevier Inc.: Amsterdam, The Netherlands, 2012; Volume 68, ISBN 9780123965233. 8. Sun, Z.; Henson, C.A. Degradation of Native Starch Granules by Barley α-Glucosidases. Plant Physiol. 1990, 94, 320–327. [CrossRef][PubMed] 9. Matsui, H.; Chiba, S.; Shimomura, T. Substrate Specificity of an α-Glucosidase in Sugar Beet Seed. Agric. Biol. Chem. 1978, 42, 1855–1860. [CrossRef] 10. Schaechter, M. Encyclopedia of Microbiology; Academic Press: Cambridge, MA, USA, 2009; ISBN 0123739446. 11. Watanabe, K.; Hata, Y.; Kizaki, H.; Katsube, Y.; Suzuki, Y. The Refined Crystal Structure of Bacillus Cereus Oligo-1,6-Glucosidase at 2.0 Å Resolution: Structural Characterization of Proline-Substitution Sites for Protein Thermostabilization. J. Mol. Biol. 1997, 269, 142–153. [CrossRef] 12. Hauri, H.P.; Quaroni, A.; Isselbacher, K.J. Biogenesis of Intestinal Plasma Membrane: Posttranslational Route and Cleavage of -Isomaltase. Proc. Natl. Acad. Sci. USA 1979, 76, 5183–5186. [CrossRef][PubMed] 13. Hartley, T.G. Tetradium Ruticarpumo (A.Juss.); Gardens’ Bulletin: Singapore, 1981. 14. Nguyễn, T.B.; Nguyễn, Q.B.; Vũ, V.C.; Lê, M.C.; Nguyễn, N.C.; Vũ, V.D.; Nguyễn, V.D.; Trần, Ð.D.; Nguyễn, K.D.; Nguyễn, T.D.; et al. Tên Cây Rừng Việt Nam; Nhà xuất bản Nông nghiệp: Hanoi, Vietnam, 2000. 15. Li, M.; Wang, C. Traditional Uses, Phytochemistry, Pharmacology, Pharmacokinetics and Toxicology of the Fruit of Tetradium Ruticarpum: A Review; Elsevier B.V.: Amsterdam, The Netherlands, 2020; Volume 263, ISBN 0860215132251. 16. Kato, A.; Yasuko, H.; Goto, H.; Hollinshead, J.; Nash, R.J.; Adachi, I. Inhibitory Effect of Rhetsinine Isolated from Evodia rutaecarpa on Aldose Reductase Activity. Phytomedicine 2009, 16, 258–261. [CrossRef][PubMed] 17. Wang, T.; Kusudo, T.; Takeuchi, T.; Yamashita, Y.; Kontani, Y.; Okamatsu, Y.; Saito, M.; Mori, N.; Yamashita, H. Evodiamine Inhibits Insulin-Stimulated MTOR-S6K Activation and IRS1 Serine Phosphorylation in Adipocytes and Improves Glucose Tolerance in Obese/Diabetic Mice. PLoS ONE 2013, 8, e83264. [CrossRef] 18. Tachibana, K.; Ishimoto, K.; Takahashi, R.; Kadono, H.; Awaji, T.; Yuzuriha, T.; Tanaka, T.; Hamakubo, T.; Sakai, J.; Kodama, T.; et al. Development of a Ligand Screening Tool Using Full-Length Human Peroxisome Proliferator-Activated Receptor-Expressing Cell Lines to Ameliorate Metabolic Syndrome. Chem. Pharm. Bull. 2019, 67, 199–202. [CrossRef][PubMed] 19. Ngo, T.D.; Tran, T.D.; Le, M.T.; Thai, K.M. Computational Predictive Models for P- Inhibition of in-House Chalcone Derivatives and Drug-Bank Compounds. Mol. Divers. 2016, 20, 945–961. [CrossRef][PubMed] 20. Chandra Babu, T.M.; Rajesh, S.S.; Bhaskar, B.V.; Devi, S.; Rammohan, A.; Sivaraman, T.; Rajendra, W. Molecular Docking, Molecular Dynamics Simulation, Biological Evaluation and 2D QSAR Analysis of Flavonoids from Syzygium Alternifolium as Potent Anti-Helicobacter Pylori Agents. RSC Adv. 2017, 7, 18277–18292. [CrossRef] 21. Thai, K.M.; Le, D.P.; Tran, N.V.K.; Nguyen, T.T.H.; Tran, T.D.; Le, M.T. Computational Assay of Binding Affinity with Original and Mutant Influenza 9 Using Molecular Docking. J. Theor. Biol. 2015, 385, 31–39. [CrossRef][PubMed] 22. Tarasova, O.; Poroikov, V.; Veselovsky, A. Molecular Docking Studies of HIV-1 Resistance to Reverse Transcriptase Inhibitors: Mini-Review. Molecules 2018, 23, 1233. [CrossRef][PubMed] 23. Ding, Y.; Fang, Y.; Moreno, J.; Ramanujam, J.; Jarrell, M.; Brylinski, M. Assessing the Similarity of Ligand Binding Conformations with the Contact Mode Score. Comput. Biol. Chem. 2016, 64, 403–413. [CrossRef] Molecules 2021, 26, 3691 18 of 18

24. Zhao, N.; Li, Z.L.; Li, D.H.; Sun, Y.T.; Shan, D.T.; Bai, J.; Pei, Y.H.; Jing, Y.K.; Hua, H.M. Quinolone and Indole Alkaloids from the Fruits of Euodia rutaecarpa and Their Cytotoxicity against Two Human Cancer Cell Lines. Phytochemistry 2015, 109, 133–139. [CrossRef][PubMed] 25. Liu, Z.L.; Chu, S.S.; Jiang, G.H. Feeding Deterrents from Zanthoxylum schinifolium against Two Stored-Product Insects. J. Agric. Food Chem. 2009, 57, 10130–10133. [CrossRef] 26. Nguyen, P.H.; Yang, J.L.; Uddin, M.N.; Park, S.L.; Lim, S.I.; Jung, D.W.; Williams, D.R.; Oh, W.K. Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors from Morinda citrifolia (Noni) and Their Insulin Mimetic Activity. J. Nat. Prod. 2013, 76, 2080–2087. [CrossRef] 27. Lu, L.; Li, Z.H.; Shan, C.Y.; Ma, S.H.; Nie, W.; Wang, H.B.; Chen, G.Q.; Li, S.H.; Shu, C.J. Whole transcriptome analysis of schinifoline treatment in Caenorhabditis elegans infected with Candida albicans. Mol. Immunol. 2021, 135, 312–319. [CrossRef] 28. Nguyen, P.H.; Zhao, B.T.; Kim, K.W.; Lee, J.H.; Choi, J.S.; Min, B.S.; Woo, M.H. Anti-inflammatory terpenylated coumarins from the of Zanthoxylum schinifolium with a-glucosidase inhibitory activity. J. Nat. Med. 2016, 70, 276–281. [CrossRef][PubMed] 29. Wang, C.F.; Fan, L.; Tian, M.; Qi, X.S.; Liu, J.X.; Feng, J.B.; Du, S.S.; Su, X.; Wang, Y.Y. Radiosensitizing Effect of Schinifoline from Zanthoxylum schinifolium Sieb et Zucc on Human Non-Small Cell Lung Cancer A549 Cells: A Preliminary in Vitro Investigation. Molecules 2014, 19, 20128–20138. [CrossRef][PubMed] 30. Wang, X.X.; Zan, K.; Shi, S.P.; Zeng, K.W.; Jiang, Y.; Guan, Y.; Xiao, C.L.; Gao, H.Y.; Wu, L.J.; Tu, P.F. Quinolone alkaloids with antibacterial and cytotoxic activities from the fruits of Evodia rutaecarpa. Fitoterapia 2013, 89, 1–7. [CrossRef][PubMed] 31. Fang, Z.; Jun, D.Y.; Kim, Y.H.; Min, B.S.; Kim, A.K.; Woo, M.H. Cytotoxic Constituents from the Leaves of Zanthoxylum schinifolium. Bull. Korean Chem. Soc. 2010, 31, 1081–1084. [CrossRef] 32. Bai, J.W.; Zhang, Y.; Yang, M.J. Effects of schinifoline on cytoskeleton of experimental hepatoma in rats observed by whole mount cell transmission electron microscopy. Beijing Zhongyiyao Daxue Xuebao 2000, 23, 27–29. 33. Bai, J.W.; Zhang, Y.; Wu, J. Flow cytometric analysis of the schinifoline inhibition on rat hepatoma cell induced by DEN. Beijing Zhongyiyao Daxue Xuebao 1999, 22, 34–35. 34. Thomas, G. Medicinal Chemistry: An Introduction; John Wiley & Sons: Hoboken, NJ, USA, 2011; ISBN 111996542X. 35. Sessler, J.L.; Gale, P.A.; Cho, W.-S. Anion Receptor Chemistry; Royal Society of Chemistry: London, UK, 2006; Volume 8, ISBN 0854049746. 36. Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933–956. [CrossRef] 37. Carvalho, A.A.; Andrade, L.N.; Batista, É.; Sousa, V.; De Sousa, D.P. Antitumor Phenylpropanoids Found in Essential Oils. BioMed Res. Int. 2015, 2015, 392674. [CrossRef] 38. Bezerra, D.P.; De Morais, M.C. The Dual Antioxidant/Prooxidant Effect of and Its Action in Cancer Development and Treatment. Nutrients 2017, 9, 1367. [CrossRef] 39. Kharkyanen, V.N.; Petrov, E.G.; Ukrainskii, I.I. Donor-Acceptor Model of Electron Transfer through Proteins. J. Theor. Biol. 1978, 73, 29–50. [CrossRef] 40. Bui, T.Q.; Phuong Loan, H.T.; Ai My, T.T.; Quang, D.T.; Phuong Thuy, B.T.; Nhan, V.D.; Quy, P.T.; Van Tat, P.; Dao, D.Q.; Trung, N.T.; et al. A Density Functional Theory Study on Silver and Bis-Silver Complexes with Lighter Tetrylene: Are Silver and Bis-Silver Carbenes Candidates for SARS-CoV-2 Inhibition Insight from Molecular Docking Simulation. RSC Adv. 2020, 10, 30961–30974. [CrossRef] 41. Frisch, H.B.M.J.; Schlegel, G.E.; Scuseria, M.A.; Robb, J.R.; Cheeseman, G.; Scalmani, V.; Barone, B.; Mennucci, G.A.; Petersson, H.; Nakatsuji, M.; et al. Gaussian 09; Gaussian Inc.: Wallingford, CT, USA, 2009. 42. Markovi, Z.S.; Dimitri, J.M. Mechanistic Study of the Structure—Activity Relationship for the Free Radical Scavenging Activity of Baicalein. J. Mol. Model. 2011, 17, 2575–2584. [CrossRef][PubMed] 43. Weigend, F.; Ahlrichs, R. Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys. Chem. Chem. Phys. 2005, 7, 3297–3305. [CrossRef] 44. Reed, A.E.; Weinstock, R.B.; Weinhold, F. Natural Population Analysis. J. Chem. Phys. 1985, 83, 735–746. [CrossRef] 45. Koopmans, T. Über Die Zuordnung von Wellenfunktionen Und Eigenwerten Zu Den Einzelnen Elektronen Eines Atoms. Physica 1934, 1, 104–113. [CrossRef] 46. De Chimie, L.; Theorique, P. Chemical Reactivity Indexes in Density. J. Comput. Chem. 1999, 20, 129–154. [CrossRef] 47. Obot, I.B.; Macdonald, D.D.; Gasem, Z.M. Density Functional Theory (DFT) as a Powerful Tool for Designing New Organic Corrosion Inhibitors. Corros. Sci. 2015, 99, 1–30. [CrossRef]