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molecules Article

Promising Inhibitory Effects of Anthraquinones, Naphthopyrone, and Naphthalene Glycosides, from Cassia obtusifolia on α-Glucosidase and Human Protein Tyrosine Phosphatases 1B Hyun Ah Jung 1 , Md Yousof Ali 2 and Jae Sue Choi 2, * 1 2

*

Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Korea; [email protected] Department of Food and Life Science, Pukyong National University, Busan 608-737, Korea; [email protected] Correspondence: [email protected]; Tel.: +82-51-629-5845; Fax: +82-51-629-5842

Academic Editor: Derek J. McPhee Received: 6 December 2016; Accepted: 23 December 2016; Published: 27 December 2016

Abstract: The present work aims to evaluate the anti-diabetic potentials of 16 anthraquinones, two naphthopyrone glycosides, and one naphthalene glycoside from Cassia obtusifolia via inhibition against the protein tyrosine phosphatases 1B (PTP1B) and α-glucosidase. Among them, anthraquinones emodin and alaternin exhibited the highest inhibitory activities on PTP1B and α-glucosidase, respectively. Moreover, we examined the effects of alaternin and emodin on stimulation of glucose uptake by insulin-resistant human HepG2 cells. The results showed that alaternin and emodin significantly increased the insulin-provoked glucose uptake. In addition, our kinetic study revealed that alaternin competitively inhibited PTP1B, and showed mixed-type inhibition against α-glucosidase. In order to confirm enzyme inhibition, we predicted the 3D structure of PTP1B using Autodock 4.2 to simulate the binding of alaternin. The docking simulation results demonstrated that four residues of PTP1B (Gly183, Arg221, Ile219, Gly220) interact with three hydroxyl groups of alaternin and that the binding energy was negative (−6.30 kcal/mol), indicating that the four hydrogen bonds stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, resulting in more effective PTP1B inhibition. The results of the present study clearly demonstrate that C. obtusifolia and its constituents have potential anti-diabetic activity and can be used as a functional food for the treatment of diabetes and associated complications. Keywords: Cassia obtusifolia; PTP1B; α-glucosidase; anthraquinones; insulin resistance; alaternin

1. Introduction Type 2 diabetes (DM2) is characterized by resistance of insulin-sensitive tissues, such as muscles, liver and fat, to insulin action. Although the mechanism of the insulin resistance is unknown, it is tightly associated with obesity [1]. Protein tyrosine phosphatases 1B (PTP1B), a member of the PTP family, is thought to function as a negative regulator of insulin signal transduction. PTP1B directly interacts with an activated insulin receptor or insulin receptor substrate-1 (IRS-1) to dephosphorylate phosphotyrosine residues, resulting in down-regulation of insulin action [2]. PTP1B knockdown mice show enhanced insulin sensitivity in glucose and insulin tolerance tests, indicating that PTP1B is a major player in the modulation of insulin sensitivity [3,4]. PTP1B overexpression in rat primary adipose tissues and 3T3/L1 adipocytes has been shown to decrease insulin-sensitive GLUT4 translocation [5], and insulin receptor and IRS-1 phosphorylation [6], respectively. Therefore, PTP1B inhibitors are potential therapeutic candidates to restore insulin sensitivity and treat DM2 and obesity. One of the Molecules 2017, 22, 28; doi:10.3390/molecules22010028

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therapeutic approaches to restoration of insulin sensitivity is to decrease postprandial hyperglycemia by retarding the absorption of glucose by inhibition of carbohydrate-hydrolyzing enzyme, such as α-glucosidase [7]. To this end, many efforts have been made to search for more effective and safe Molecules 2017, 22, 28 2 of 15 inhibitors of α-glucosidase from natural materials in order to develop a physiological functional food to treat diabetes [8]. Thus, a therapeutic strategy focusing on suppression of postprandial hyperglycemia therapeutic approaches to restoration of insulin sensitivity is to decrease postprandial hyperglycemia and improvement of insulin signaling could a valuable treatment strategy for not only the treatment by retarding the absorption of glucose by be inhibition of carbohydrate-hydrolyzing enzyme, such as of diabetic patients, individuals withhave impaired glucose tolerance. Many researchers α-glucosidase [7].but To also this end, many efforts been made to search for more effective and safehave inhibitors of α-glucosidase fromPTP1B, natural amaterials inregulator order to develop a physiological functional food to previously attempted to develop negative of insulin, and α-glucosidase, an enzyme treat diabetes [8]. Thus, a therapeutic strategy focusing on suppression of postprandial hyperglycemia that catalyzes the cleavage of glycosidic bonds in carbohydrates, inhibitors for the treatment of diabetes, improvement insulin signaling could a valuable treatment for not only the treatment eitherand synthetically orofvia the exploitation ofbe foods and herbs usedstrategy in traditional Chinese medicine. of diabetic patients, but also individuals with impaired glucose tolerance. Many researchers have Cassia is a large tropical genus composed of about 600 species of herbs, shrubs, and trees. Species previously attempted to develop PTP1B, a negative regulator of insulin, and α-glucosidase, an enzyme of Cassia are a rich source of naturally-occurring bioactive compounds anthraquinones. These plants that catalyzes the cleavage of glycosidic bonds in carbohydrates, inhibitors for the treatment of diabetes, are reported to have anti-diabetic, laxative, purgative, antimalarial, ulcer healing, hepatoprotective, either synthetically or via the exploitation of foods and herbs used in traditional Chinese medicine. nephroprotective, activities, and of they are600 alsospecies used in the treatment of skin Cassia is aand largeantitumor tropical genus composed about of herbs, shrubs, and trees.infection Species and periodic fever throughout tropical and subtropical region [9–12]. Cassia obtusifolia L. is a leguminous of Cassia are a rich source of naturally-occurring bioactive compounds anthraquinones. These plants annual that grows tropical countries Asia, andantimalarial, its main active are anthraquinone areherb reported to haveinanti-diabetic, laxative,inpurgative, ulceringredients healing, hepatoprotective, nephroprotective, activities, and are alsoas used in theof treatment of skin infection with and the compounds [13–16]. and Its antitumor herbal ingredients arethey popular a kind functional beverage periodic fever throughout tropical region [9–12]. Cassiaanti-fungal, obtusifolia L.and is a neuroprotection leguminous effects of reducing serum levels of fatand andsubtropical cholesterol, anti-oxidation, annual herb that in tropical countries in Asia, its mainin active anthraquinone activities [17,18]. C.grows obtusifolia can also protect liverand function ratsingredients with liverare injury and alleviate compounds [13–16]. Its herbal ingredients are popular as a kind of functional beverage with the effects of obesity, insulin resistance, and non-alcoholic steatohepatitis by up-regulating the AMP-dependent reducing serum levels of fat and cholesterol, anti-oxidation, anti-fungal, and neuroprotection activities protein kinase [19,20]. It was previously reported that the seeds of Cassia species have anti-diabetic [17,18]. C. obtusifolia can also protect liver function in rats with liver injury and alleviate obesity, insulin effects on postprandial glucose control and insulin secretion from the pancreas of normal resistance, and non-alcoholic steatohepatitis by up-regulating the AMP-dependent protein kinase [19,20]. and streptozotocin-induced diabetic rats [21,22], as well as on the in vitro formation of advanced glycation It was previously reported that the seeds of Cassia species have anti-diabetic effects on postprandial end products formation [23]. secretion from the pancreas of normal and streptozotocin-induced diabetic glucose control and insulin Since the seeds of as C. on obtusifolia are formation widely used in traditional Chinese medicine, the present rats [21,22], as well the in vitro of advanced glycation end products formation [23]. work Since the of C. obtusifolia are widely used in traditional Chinese medicine, the present work was performed to seeds characterize the anti-diabetic potential of C. obtusifolia and its constituents on PTP1B was performed to characterize the anti-diabetic potential of C. obtusifolia and its constituents on PTP1B and α-glucosidase in vitro. Enzyme kinetic analyses of the most active constituent, alaternin, were and α-glucosidase vitro. Enzyme kinetic analyses ofplots the most activeto constituent, also also performed usinginDixon and Lineweaver-Burk in order confirm alaternin, the type were of enzymatic performed using Dixon and Lineweaver-Burk plots in order to confirm the type of enzymatic inhibition inhibition and to define guidelines for use of alaternin as an anti-diabetic agent. Since there is no and to define guidelines for use of alaternin as an anti-diabetic agent. Since there is no detailed information detailed information on PTP1B-alaternin molecular interactions, this study also proposes an approach on PTP1B-alaternin molecular interactions, this study also proposes an approach to develop alaternin to develop alaternin as a potent anti-diabetic drug candidate scrutinizing molecular docking as a potent anti-diabetic drug candidate by scrutinizing molecularby docking predictions and enzyme predictions kinetics. We alsoand found that alaternin emodin stimulated kinetics.and Weenzyme also found that alaternin emodin stimulatedand glucose uptake in HepG2glucose cells inuptake a in HepG2 cells in a dose-dependent dose-dependent manner (Figure 1).manner (Figure 1).

OH

O

OH

OH

O

OH OH

HO

CH 3 O Emodin

HO

CH 3 O Alaternin

Figure 1. Chemical structureofofemodin emodin and alaternin from Cassia obtusifolia. Figure 1. Chemical structure alaterninisolated isolated from Cassia obtusifolia.

2. Results 2. Results 2.1. α-Glucosidase PTP1B InhibitoryActivity Activityof of the the MeOH obtusifolia andand Its 2.1. α-Glucosidase andand PTP1B Inhibitory MeOHExtract ExtractofofC.C. obtusifolia Solvent-SolubleFractions Fractions Its Solvent-Soluble In order to evaluate the anti-diabetic potential of C. obtusifolia, the MeOH extract was tested via in In order to evaluate the anti-diabetic potential of C. obtusifolia, the MeOH extract was tested vitro α-glucosidase and PTP1B inhibitory assays. The results of the α-glucosidase and PTP1B inhibitory via in vitro α-glucosidase and PTP1B inhibitory assays. The results of the α-glucosidase and PTP1B activities of the MeOH extract are shown in the Figure 2A,B and Table 1. As shown in Figure 2A,B, inhibitory activities of the MeOH extract are shown in the Figure 2A,B and Table 1. As shown the MeOH extract showed dose-dependent manner α-glucosidase and PTP1B inhibitory activities

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in Figure 2A,B, the MeOH extract showed dose-dependent manner α-glucosidase and PTP1B inhibitory activities with IC50 (concentration required to decrease by 50%) values of 200.07 ± 7.90 and Molecules 2017, 22, 28 3 of 15 14.79 ± 0.31 µg/mL, respectively, compared to the positive control acarbose (123.54 ± 0.29 µg/mL) and ursolic acid (3.37 ± 0.18 µg/mL). Since the MeOH extract of of C.200.07 obtusifolia both α-glucosidase with IC50 (concentration required to decrease by 50%) values ± 7.90showed and 14.79 ± 0.31 μg/mL, and PTP1B inhibitory activity, it was further fractionated for detailed investigation. The MeOH respectively, compared to the positive control acarbose (123.54 ± 0.29 μg/mL) and ursolic acid (3.37extract ± of C. 0.18 obtusifolia was dissolved in extract H2 O and partitioned with CH2 Cl and n-BuOH μg/mL). Since the MeOH of C.successively obtusifolia showed both α-glucosidase and PTP1B inhibitory 2 , EtOAc, activity, it was further fractionated for detailed Theand MeOH extract of C. obtusifolia wasof the to obtain different solvent-soluble fractions. Theinvestigation. α-glucosidase PTP1B inhibitory activity dissolved in H 2 O and successively partitioned with CH 2 Cl 2 , EtOAc, and n-BuOH to obtain different individual fractions of C. obtusifolia was then evaluated. As shown in Table 1, the EtOAc fraction solvent-soluble The α-glucosidase PTP1B inhibitory activity of the individual fractions showed the highestfractions. α-glucosidase and PTP1Band inhibitory activity with IC50 values of 74.50 ± 4.93 and of C. obtusifolia was then evaluated. As shown in Table 1, the EtOAc fraction showed the highest 57.90 ± 0.92 µg/mL, respectively, followed by the CH2 Cl2 fraction with IC50 values of 359.36 ± 10.81 α-glucosidase and PTP1B inhibitory activity with IC50 values of 74.50 ± 4.93 and 57.90 ± 0.92 μg/mL, and 85.31 ± 3.43 µg/mL, compared to the positive controls acarbose and ursolic acid, with IC50 respectively, followed by the CH2Cl2 fraction with IC50 values of 359.36 ± 10.81 and 85.31 ± 3.43 μg/mL, values of 114.75 andcontrols 3.02 ± acarbose 0.20 µg/mL. In particular, fraction stronger compared to ± the2.95 positive and ursolic acid, withthe IC50EtOAc values of 114.75 ±showed 2.95 and 3.02 inhibitory potential compared to acarbose, a well-known α-glucosidase inhibitor used clinically. On the ± 0.20 μg/mL. In particular, the EtOAc fraction showed stronger inhibitory potential compared to otheracarbose, hand, the n-BuOH and H2 O fractions showed α-glucosidase and inhibitory a well-known α-glucosidase inhibitor used moderate clinically. On the other hand, thePTP1B n-BuOH and potential with IC50 valuesmoderate of 372.12 ± 11.88 and 12.61 µg/mL, ± 4.87 H2O fractions showed α-glucosidase and434.02 PTP1B ± inhibitory potentialand with172.82 IC50 values of and 372.12 ± 11.88 and 434.02 ± 12.61 μg/mL, and 172.82 ± 4.87 and 214.52 ± 3.42 μg/mL, respectively. 214.52 ± 3.42 µg/mL, respectively.

Figure 2. Concentration-dependent protein protein tyrosine 1B 1B (A)(A) andand α-glucosidase (B) (B) Figure 2. Concentration-dependent tyrosinephosphatase phosphatase α-glucosidase inhibitory activity of the MeOH extractofofC. C.obtusifolia. obtusifolia. Ursolic and acarbose are are positive controls. inhibitory activity of the MeOH extract Ursolicacid acid and acarbose positive controls.

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Table 1. Protein tyrosine phosphatase 1B and α-glucosidase inhibitory activities of the MeOH extract of Cassia obtusifolia and its solvent soluble fractions.

Test Samples MeOH extract CH2 Cl2 fraction EtOAc fraction n-BuOH fraction H2 O fraction Ursolic acid c Acarbose d

PTP1B a

α-Glucosidase b

IC50 (Mean ± SEM)

IC50 (Mean ± SEM)

14.79 ± 0.31 85.31 ± 3.43 57.90 ± 0.92 172.82 ± 4.87 214.52 ± 3.42 3.37 ± 0.18

200.07 ± 7.90 359.36 ± 10.81 74.50 ± 4.93 372.12 ± 11.88 434.02 ± 12.61 123.54 ± 0.29

a,b

Final concentration of test samples and positive controls were 100 µg/mL (for PTP1B) and 400 µg/mL (for α-glucosidase), dissolved in 10% DMSO: 50% inhibition concentrations (IC50 , µg/mL) are expressed as the mean ± SEM of triplicate experiments. c,d Ursolic acid and acarbose were used as positive controls for the PTP1B and α-glucosidase assays, respectively.

2.2. Inhibitory Activity of Anthraquinones, Naphthopyrone Glycosides, and a Naphthalene Glycoside from C. obtusifolia on PTP1B and α-Glucosidase In order to evaluate the anti-diabetic activity of the 16 anthraquinones, two naphthopyrone glycosides, and a naphthalene glycoside from C. obtusifolia, the inhibitory potential of the PTP1B and α-glucosidase was evaluated using pNPP and pNPG as substrate, and results are expressed as IC50 values and presented in Table 2. All of the tested compounds clearly showed strong PTP1B and α-glucosidase inhibitory activity. Notably, alaternin and emodin exhibited the most potent PTP1B and α-glucosidase inhibitory potential with IC50 values of 1.22 ± 0.03 and 3.51 ± 0.15, and 0.99 ± 0.02 and 1.02 ± 0.01 µg/mL compared to the positive controls ursolic acid and acarbose with IC50 values of 3.37 ± 0.18 and 123.54 ± 0.29 µg/mL. Chrysophanol, physcion, obtusin, questin, 2-hydroxyemodin-1 methylether, and chryso-obtusin, displayed significant PTP1B inhibitory activity with IC50 values of 5.86 ± 0.99, 7.28 ± 0.49, 6.44 ± 0.22, 5.69 ± 0.47, 5.22 ± 0.29, and 14.88 ± 0.77 µg/mL, respectively. Whereas cassiaside, obtusifolin, gluco-obtusifolin, aurantio-obtusin, gluco-aurantio obtusin, chryso-obtusin 2-glucoside, aloe-emodin, chrysophanol triglucoside, and toralactone gentiobioside showed moderate PTP1B inhibitory activity with IC50 values of 48.55 ± 1.27, 35.27 ± 0.98, 53.35 ± 0.44, 27.19 ± 0.31, 31.30 ± 0.97, 39.34 ± 1.07, 56.01 ± 0.76, 80.17 ± 1.77, and 81.15 ± 0.15 µg/mL, respectively. In addition, chrysophanol, chryso-obtusin, aurantio-obtusin, obtusin, gluco-obtusifolin, and toralactone gentiobioside exhibited moderate inhibitory activity against α-glucosidase with IC50 values of 46.81 ± 0.12, 36.01 ± 0.89, 41.20 ± 0.17, 20.92 ± 0.41, 23.77 ± 0.72, and 37.60 ± 0.79 µg/mL, respectively. 2.3. Kinetic Parameters of Alaternin In an attempt to explain the mode of enzymatic inhibition of the most active component, alaternin, kinetic analyses were performed at different concentrations of the corresponding substrate (pNPP for PTP1B and pNPG for α-glucosidase) and various inhibitor concentrations. Dixon plots are the graphical method (a plot of 1/enzyme velocity (1/V) against inhibitor concentration (I)) for determining the type of enzyme inhibition and the dissociation or inhibition constant (Ki ) of an enzyme-inhibitor complex, and they are easily determined. Table 3 and Figure 3A,D demonstrate the enzymatic kinetic analysis of alaternin. Alaternin showed competitive PTP1B inhibition with a respective Ki value of 1.70 µM, while it showed different inhibition modes with α-glucosidase and; mixed type inhibition with a respective Ki value of 0.66 µM. Since the Ki value represents the concentration needed to form an enzyme-inhibitor complex, this value plays an important role in the development of preventive and therapeutic agents.

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Table 2. Protein tyrosine phosphatase 1B and α-glucosidase inhibitory activities of anthraquinones, naphthopyrone glycosides and a naphthalene glycoside from Cassia obtusifolia.

Test Compounds

PTP1B a

α-Glucosidase b

IC50 (Mean ± SEM)

IC50 (Mean ± SEM)

Anthraquinones Physcion Chrysophanol Emodin Alaternin Obtusifolin Obtusin Questin Chryso-obtusin Aurantio-obtusin 2-Hydroxyemodin-1 methylether Gluco-obtusifolin Gluco-aurantio obtusin Chryso-obtusin-2-glucoside Chrysophanol triglucoside Chrysophanol tetraglucoside

7.28 ± 0.49 5.86 ± 0.99 3.51 ± 0.15 1.22 ± 0.03 35.27 ± 0.98 6.44 ± 0.22 5.69 ± 0.47 14.88 ± 0.77 27.19 ± 0.31 5.22 ± 0.29 53.35 ± 0.44 31.30 ± 0.97 39.34 ± 1.07 80.17 ± 1.77 103.89 ± 1.22

2.38 ± 0.77 46.81 ± 0.12 1.02 ± 0.01 0.99 ± 0.02 142.12 ± 0.77 20.92 ± 0.41 136.19 ± 0.01 36.01 ± 0.89 41.20 ± 0.17 5.65 ± 0.20 23.77 ± 0.72 142.19 ± 1.22 178.85 ± 0.55 197.06 ± 1.09 228.79 ± 0.91

Naphthopyrone glycosides 48.55 ± 1.27 81.15 ± 0.15

Cassiaside Toralactone gentiobioside

129.23 ± 0.98 37.60 ± 0.79

Naphthalene glycoside 103.89 ± 1.22 56.01 ± 0.76 3.37 ± 0.18

Cassitoroside Aloe-emodin Ursolic acid c Acarbose d

172.59 ± 0.74 1.40 ± 0.27 123.54 ± 0.29

a,b

Final concentration of test samples and positive controls were 100 µg/mL (for PTP1B) and 400 µg/mL (for α-glucosidase), dissolved in 10% DMSO: 50% inhibition concentrations (IC50 , µg/mL) are expressed as the mean ± SEM of triplicate experiments. c,d Ursolic acid and acarbose were used as positive controls for the PTP1B and α-glucosidase assays, respectively.

Table 3. Enzyme kinetics analysis of alaternin with PTP1B and α-glucosidase. Test Sample a

IC50 (µM) Ki b Inhibition type c a

PTP1B

α-Glucosidase

1.61 1.70 Competitive

1.31 0.66 Mixed

50% inhibition concentrations are expressed as the mean ± SEM of duplicate samples; b The inhibition constants (Ki ) were determined by interpreting the Dixon plot; c Inhibition type were determined by interpreting the Dixon plot and Lineweaver-Burk plot.

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1000

B

A

1 mM 0.5 mM 0.25 mM

800

800

4.0 μΜ 0.8 μΜ 0 μΜ

600

1/V

1/V

600

400

400

200

200

0 -1.0

-0.5

0.0

0.5

1.0

0

1.5

-4

-2

0

1/[pNPP] (mM−1)

2

4

6

Concentration (μΜ)

500

600

C

3.90 μΜ 1.95 μΜ 0.97 μΜ

400

D

2.5 mM 1.25 mM 0.625 mM

500

400

1/V

1/V

300 300

200 200 100

100

0 -4

-3

-2

-1

0 0

1/[pNPG] (mM−1)

1

2

3

-2

-1

0

1

2

3

4

5

Concentration (μΜ)

Figure3.3.(A) (A)Lineweaver-Burk Lineweaver-Burkplot plotof ofPTP1B PTP1B inhibition inhibition of of alaternin alaternin was Figure was analyzed analyzed in in the thepresence presenceof different concentration of sample as follows: 0 μM (▼), 0.8 μM (○) and 4.0 μM (●) of alaternin. (B) of different concentration of sample as follows: 0 µM (H), 0.8 µM (#) and 4.0 µM ( ) of alaternin. Dixon plots of PTP1B inhibition by alaternin: 1 mM (●); 0.5 mM (○) and 0.25 mM (▼) of pNPP. (C) (B) Dixon plots of PTP1B inhibition by alaternin: 1 mM ( ); 0.5 mM (#) and 0.25 mM (H) of pNPP. Lineweaver-Burk plotplot of α-glucosidase inhibition of alaternin was analyzed in the presence of different (C) Lineweaver-Burk of α-glucosidase inhibition of alaternin was analyzed in the presence of concentration of sample follows: 0.97 μM 0.97 (▼),µM 1.95(H μM (○) and μM (●) alaternin. (D) Dixon different concentration of as sample as follows: ), 1.95 µM 3.90 (#) and 3.90for µM ( ) for alaternin. plots of α-glucosidase inhibitioninhibition by alaternin: 2.5 mM (●); 0.625 mM 0.625 (▼) ofmM pNPG. (D) Dixon plots of α-glucosidase by alaternin: 2.51.25 mMmM ( ); (○) 1.25and mM (#) and (H) of pNPG.

2.4. Molecular Docking Study of the Inhibitory Activity of Alaternin against PTP1B 2.4. Molecular Docking Study of the models InhibitoryofActivity of Alaternin against PTP1B The molecular docking alaternin (magenta color) and 3-({5-[(N-acetyl-3-{4[(carboxycarbonyl)(2-carboxyphenyl)amino]-1-naphthyl}L -alanyl)amino]pentyl}oxy)-2-naphthoic acid The molecular docking models of alaternin (magenta color) and 3-({5-[(N-acetyl-3-{4(compound 23) (cyan color) are illustrated in Figure 4A. The ligand–enzyme complexes acid with [(carboxycarbonyl)(2-carboxyphenyl)amino]-1-naphthyl}L-alanyl)amino]pentyl}oxy)-2-naphthoic alaternin/or compound 23 were stably positioned in the same pocket of the PTP1B by Autodock 4.2 (compound 23) (cyan color) are illustrated in Figure 4A. The ligand–enzyme complexes with (http://autodock.scripps.edu/downloads). As illustrated in pocket Figure of 4B, corresponding ligand alaternin/or compound 23 were stably positioned in the same thethe PTP1B by Autodock 4.2 interactions of alaternin at the active site of PTP1B are the three hydrogen-bonding interactions between (http://autodock.scripps.edu/downloads). As illustrated in Figure 4B, the corresponding ligand the Gly183,of Arg221, Ile219, andactive Gly220 residues of the andhydrogen-bonding the three hydroxylinteractions groups at Cinteractions alaternin at the site of PTP1B areenzyme the three 1, 2, and of alaternin, while theand twoGly220 residues Val184, andenzyme Thr263and of the participated between the6Gly183, Arg221, Ile219, residues of the the enzyme three hydroxyl groupsin hydrophobic interactions with the methyl group of alaternin. On the other hand, the five residues Tyr20, at C-1, 2, and 6 of alaternin, while the two residues Val184, and Thr263 of the enzyme participated Lys116, Arg24, Arg254, and Gln262 of the enzyme participated in hydrogen-bonding interactions with in hydrophobic interactions with the methyl group of alaternin. On the other hand, the five residues the carboxylate anions of compound binding energies of both compounds were Tyr20, Lys116, Arg24, Arg254, and Gln26223. of Moreover, the enzymethe participated in hydrogen-bonding interactions negative (−6.30 kcal/mol for alaternin and −10.18 kcal/mol for compound 23), indicating that additional with the carboxylate anions of compound 23. Moreover, the binding energies of both compounds hydrogen bonding stabilize the openand form of thekcal/mol enzyme and potentiate tighter binding to the were negative (−6.30would kcal/mol for alaternin −10.18 for compound 23), indicating that active site of PTP1B, resulting in more effective PTP1B inhibition. additional hydrogen bonding would stabilize the open form of the enzyme and potentiate tighter binding to the active site of PTP1B, resulting in more effective PTP1B inhibition.

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(A)(A)

(B)(B)

Figure 4.4.(A) Molecular docking models ofofthe PTP1B inhibition ofofalaternin (magenta color) and Figure (A) Molecular docking models thethe PTP1B inhibition alaternin (magenta color) and Figure 4. (A) Molecular docking models of PTP1B inhibition of alaternin (magenta color) and compound 23 (cyan color); (B) Ligand interaction diagram of alaternin in the active site of the PTP1B compound 23 (cyan color); (B) Ligand interaction diagram of alaternin in the active site of the compound 23 (cyan color); (B) Ligand interaction diagram of alaternin in the active site of the PTP1B enzyme. PTP1B enzyme. enzyme.

2.5. Effects ofofAlaternin and Emodin on Uptake 2.5. Effects of Alaternin and Emodin on Glucose Uptake 2.5. Effects Alaternin and Emodin onGlucose Glucose Uptake Before determining the insulin resistance ofofalaternin and emodin, the cytotoxicity ofofalaternin Before determining insulin resistance of alaternin and emodin, cytotoxicity of alaternin Before determining thethe insulin resistance alaternin and emodin, thethe cytotoxicity alaternin and emodin on HepG2 cells was first measured by the MTT assay. HepG2 cells were pretreated with and emodin HepG2 cells was first measured the MTT assay. HepG2 cells were pretreated with and emodin onon HepG2 cells was first measured byby the MTT assay. HepG2 cells were pretreated with alaternin at a concentration up to 50 μM and emodin at a concentration up to 12.5 μM, following alaterninatat a concentrationupuptoto5050 μM and emodin a concentration 12.5 μM, following alaternin a concentration µM and emodin at at a concentration upup to to 12.5 µM, following incubation for 2424h. and emodin did not possess any cytotoxicity up toto50 and 12.5 μM, incubation 24 h. Alaternin and emodin did not possess any cytotoxicity to50μM 50µM μM and 12.5 μM, incubation forfor h.Alaternin Alaternin and emodin did not possess any cytotoxicity upup and 12.5 µM, respectively. These concentrations were, thus, used in subsequent glucose uptake assays. To investigate respectively. These concentrations were, thus, used subsequent glucose uptake assays. investigate respectively. These concentrations were, thus, used in in subsequent glucose uptake assays. ToTo investigate the ability of andand emodin increase glucose uptake, a 2-[N-(7-nitrobenz-2-oxa-1,3-diazolability of alaternin emodin to increase glucose uptake, a 2-[N-(7-nitrobenz-2-oxa-1,3-diazolthethe ability of alaternin alaternin and emodin totoincrease glucose uptake, a 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) 4-yl) amino]-2-deoxyD-glucose (2-NBDG) uptake assay was performed insulin-resistant HepG2 4-yl) amino]-2-deoxyD-glucose (2-NBDG) uptake assay was performed with insulin-resistant HepG2 amino]-2-deoxyD -glucose (2-NBDG) uptake assay was performed withwith insulin-resistant HepG2 cells. cells. The positive control metformin at a concentration of 10 μM significantly increased insulincells. The positive control metformin at a concentration of 10 μM significantly increased insulinThe positive control metformin at a concentration of 10 µM significantly increased insulin-stimulated stimulated glucose uptake in insulin-resistant cells. Concentrations of 12.5, 25,alaternin, andand 50 μM stimulated glucose uptake in insulin-resistant HepG2 cells. Concentrations of 25, 50 μM glucose uptake in insulin-resistant HepG2 cells.HepG2 Concentrations of 12.5, 25, and 50 12.5, µM and alaternin, and 3.125, 6.25, and 12.5 μM emodin, significantly enhanced the insulin-stimulated uptake alaternin, and12.5 3.125, and 12.5 μM emodin, significantly enhanced the insulin-stimulated uptake 3.125, 6.25, and µM6.25, emodin, significantly enhanced the insulin-stimulated uptake of 2-NBDG in ofinsulin-resistant 2-NBDG in insulin-resistant HepG2 cells compared the control (Figure 5A,B). of 2-NBDG in insulin-resistant HepG2 compared to the control (Figure 5A,B). HepG2 cells compared tocells the control to (Figure 5A,B). A A

150 125 100 75 50 25

B B

150

*** ***

125

** **

*

100

*** ***

150

125

100

*

75

75

50

50

25

25

0 0 Insulin (10-6mol/L) Insulin (10-6mol/L) 2-NBDG (40 µM) + 2-NBDG (40 µM) Metformin (10 µM) ‒ Metformin (10 µM) ‒ Alaternin (µM) Alaternin (µM)

+ ‒ ‒

+ + ‒ ‒

+ + ‒ ‒

+ + + ‒

+ + + + + + + + ‒ ‒ + 12.5 ‒ 25 ‒ 12.5

+ + ‒ 25

+ + ‒ 50

+ + ‒ 50

150

*** ***

125

** ** *** ***

100

* 75

*

50

25

0 0 Insulin (10-6 mol/L) -6 Insulin 2-NBDG (40(10 µM)mol/L) + 2-NBDG (40 µM) Metformin (10 µM) ‒ Metformin (10 µM) Emodin (µM) ‒ Emodin (µM)

+ ‒ ‒

+ + ‒ ‒

+ + ‒ ‒

+ + + ‒

+ + + + + + + + + + + + + + ‒ ‒ ‒ + ‒ ‒ ‒ 3.125 6.25 12.5 ‒ 3.125 6.25 12.5

Figure 5. Effect of alaternin (A) (A) andand emodin (B) on insulin-stimulated glucose uptake in insulin-resistant Figure Effect alaternin emodin insulin-stimulated glucose uptake insulin-resistant Figure 5. 5. Effect ofof alaternin (A) and emodin (B)(B) onon insulin-stimulated glucose uptake in in insulin-resistant HepG2 cells. A glucose uptake assay was performed using the fluorescent D-glucose analogue 2-NBDG, HepG2 cells. glucose uptake assay was performed using the fluorescent D-glucose analogue 2-NBDG, HepG2 cells. AA glucose uptake assay was performed using the fluorescent D-glucose analogue 2-NBDG, −6 mol/L concentration of insulin was used for insulin resistance. Insulin-resistant HepG2 cells and a a1010 −6−6mol/L mol/L concentration Insulin-resistant HepG2 cells and a 10 and concentration of ofinsulin insulinwas wasused usedfor forinsulin insulinresistance. resistance. Insulin-resistant HepG2 were treated withwith different concentrations of alaternin emodin or emodin metformin 24 the24 insulinwere treated different concentrations of alaternin and emodin or metformin forh,24and h, for and the cells were treated with different concentrations of and alaternin and or for metformin h,insulinand stimulated 2-NBDG uptake waswas measured. Values are are the the mean ± SD the experiments. values 2-NBDG uptake measured. Values mean ± of SD ofmean the experiments. values thestimulated insulin-stimulated 2-NBDG uptake was measured. Values are the ± SD ofStatistical theStatistical experiments. * Statistical p