Anti-diabetic properties of novel curcumin-polyhydroxy ...

Anti-diabetic properties of novel curcumin-polyhydroxy derivatives:

Synthesis, molecular docking, and their inhibitory effects on α-

glucosidase activity

Mohammad Ezati 1†, Hadi Adibi 2, Robabeh Nemati 1, Reza Khodarahmi 3*, Reza Hassan

Sajedi 1*

1 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran,

Iran2 Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical

Sciences, Kermanshah, Iran3 Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Kermanshah University of

Medical Sciences, Kermanshah, Iran

*Corresponding author’s email: [email protected], [email protected]

Paper Code: biotech12-06800588

Introduction

• Diabetes mellitus is a serious metabolic disorder in modern society due to long-term complications associated with chronic hyperglycemia

[Mehrabi et al., 2021]. Type 2 diabetes (T2DM), the most prevalent form of diabetes, is characterized by insulin resistance, relative lack of

insulin secretion, and elevated level of blood glucose (hyperglycemia). Therefore, a preventive approach to treating diabetes is to reduce

postprandial hyperglycemia by inhibiting the α-amylase and α-glucosidase enzymes that hydrolyze carbohydrates [Yousefi et al., 2015]. α-

Glucosidase located in the brush border of small intestine which breaks down starch and disaccharides into a single alpha-glucose

molecule. Glucosidase inhibitors are a common strategy that can effectively control α-glucosidase activity and reduce the incidence of

type 2 diabetes and its complications. Many chemically synthesized inhibitors (acarbose, voglibose, and miglitol) have good

hypoglycemic effects. However, these drugs also have a variety of side effects. Acarbose, for instance, is known to cause abdominal pain,

distension, and diarrhea [Tavaf et al., 2020].

• Natural molecules have become a more acceptable source of anti-diabetic agents compared with chemically synthetic drugs. Curcumin

(bis-α,β-unsaturated β-diketone), a hydrophobic polyphenol is a yellow color pigment in the food industry which derived from a turmeric

plant (Curcuma longa) and plays an important role in the prevention of various diseases, including cancer, neurodegenerative diseases,

arthritis, and diabetes [Pari et al., 2008]. This compound was also recommended by Chinese traditional medical prescriptions to be used

against diabetic complications [Hasaninezhad et al., 2020]. Accordingly, in the current study, the effects of new synthetic curcumin-

polyhydroxy derivatives on α-glucosidase inhibition were evaluated. Also, we investigated the stability and antioxidant properties of

curcumin derivatives.

1

Materials and Methods

2

• In this process, 0.5 mmol of curcumin and 0.5 mmol of different hexoses were heated in the presence of 1 g of borax as the catalyst and 25

mL of ethanol solvent for 48-72 h under reverse distillation. After confirming the completion of the reaction by TLC test, WORK UP steps

were performed to purify the final reaction product. The final products were synthesized in 76–88% yields and their FT-IR, and Mass

spectra were recorded to confirm the constitutions of the molecules.

• Purification of α-Glu from rat intestine was carried out using the method by Mehrabi et al. (2021). In order to measure the activity of rat α-

Glu, the reaction mixture containing rat α-Glu and different concentrations of compounds (0-200 µM) were pre-incubated for 10 min at

37°C. Then, the pNPG with final concentration of 10 mM, was added, and the reaction mixture was incubated 25 min at 37°C. Then,

protein content was precipitated using 5% trichloroacetic acid and centrifuged at 10000 rpm for 10 min.To determine the liberated glucose,

4 ml o-toluidine (6% (v/v) in glacial acetic acid) was added to the solution as the color reagent and further incubated in a boiling water

bath for 15 min. After cooling down to room temperature, the absorbance of samples was measured at 630 nm.

• To study the stability, curcumin-based compounds were dissolved in simulated gastric and intestinal fluids. The stability of compounds

were calculated by measuring the absorbance at λmax at different time intervals. Simulated gastric and intestinal fluid was prepared

without enzyme as followed by Tapal et al. (2012).

• In this study, the DPPH and FRAP methods have been used to measure the antioxidant activity of synthesized compounds as followed by

Mehrabi et al. (2021).

• Homology modeling and protein sequence for Baker’s yeast α-Glu (MAL12) were carried out using the method by Imran et al. (2015).

The molecular structure of compounds were constructed using ChemDraw and optimized by MM2. Auto Dock Vina was used to identify

the binding details of compounds to α-Glu. The docking results were visually analyzed using PyMol and Ligplot.

Results and Discussion

3

• The physical properties and the spectral data of the synthesized curcumin-based derivatives are as follows:

• L2: Molecular weight: 530.53 (g/mol); Molecular formula: C27H30O11; Yield: 88%; m.p.: 292 oC. FT-IR (KBr) υmax (cm-1): 3541-3414

(stretch OH, broad peak), 2924 (stretch C-H, aromatic), 2854 (stretch C-H, aliphatic), 1620 (stretch C=C, vinyl), 1550 (stretch C=C,

aromatic), 1396 (bending C-H, aliphatic ), 1288 (stretch C-O, phenol ), 1153 (stretch C-O, alkohol), 1026 (bending C-H, oop aromatic),

698 (bending O-H, oop phenol); MS (m/z, %): 530.6 (M, 1.72), 491.5 (M-39, 1.72), 422.4 (M-69, 1.72), 369.8 (M-52, 2.58), 335.3 (M-36,

3.44), 299.5 (M-36, 2.58), 239.6 (M-60, 7.75), 177.4 (M-62, 25.86), 137.4 (M-40, 34.48), 91.4 (M-46, 32.75), 77 (M-14, 36.2), 43.4 (M-

34, 100).





6.66), 277.6 (M-36, 2.85), 239.5 (M-38, 8.57), 191.2 (M-48, 9.52), 147.3 (M-44, 21.9), 91.3 (M-56, 43.8), 77 (M-14, 45.71), 43.4 (M-34,

100).





2.19), 313.7 (M-54, 12.28), 239.6 (M-74, 17.54), 171.4 (M-68, 4.38), 123.3 (M-48, 8.77), 91.3 (M-32, 7.89), 83.4 (M-8, 35.96), 77 (M-6,

5.26), 43.4 (M-34, 100).

Results and Discussion

4

• The poor clinical outcomes of current anti-diabetic medicine and the increasing prevalence of type-II diabetes mellitus are two important

reasons that led us to investigate the new therapeutic approaches focusing to control this metabolic disorder in its initial stages. In this

study, we investigated the α-glycosidase inhibitory potential of the curcumin derivatives and acarbose was used as a positive control. The

results revealed that all curcumin derivatives inhibited α-Glu activity in a dose-dependent pattern. The IC50 values of α-Glu inhibition for

acarbose, L1, L2, L3, and L4 were 4.7±0.6, 20.9±2.5, 14.2±1.2, 12.6±0.3, and 18.5±0.4 µM respectively.

• Previous studies have shown that approximately 90% of curcumin is rapidly degraded within 30 minutes in phosphate buffer systems. For

this purpose, the stability of curcumin and synthesized derivatives in the simulated intestinal medium with pH = 7.5 and the simulated

gastric medium with pH = 1.2 at 25 °C for 60 minutes was investigated. As such, the stability of synthesized derivatives was considered to

be 100% the moment of zero and measured after 60 min. Our result showed the stability of derivatives in the intestinal environment

follows the order of L1< L4 ≃ L2 < L3. Also, the stability study in gastric simulated environment follows the order of L1< L4 <L3 < L2.

• Recent studies have revealed that compounds with combined antioxidant and anti-glycation properties are more effective in the treatment

of diabetes mellitus. Therefore, we investigated the antioxidant activity of all the synthesized derivatives. The level of antioxidant activity

of compounds follows the order of L4 < L3 < L2 ≃ L1, and L4 ≃ L3 < L2 < L1 based on DDPH and FRAP methods respectively.

• Molecular docking analysis method was also used to determine the binding affinity and mode of interaction between the curcumin

derivatives and the predicted 3D model of α-Glu. Docking results showed that curcumin derivatives interact effectively with α-glucosidase

by different binding affinity energies. In this regard, Table 1 shows the docking energies and Hydrogen and Hydrophobic interactions

between curcumin derivatives and the enzyme.

Scheme 1. The chemical structures of curcumin-based compounds (L2–L4)

Figure or Table

5

Table 1. Docking results and hydrophobic and hydrogen interactions of synthesized compounds α-Glu

Hydrophobic InteractionsHydrogen bondDE (kcal/mol)Compound

F157,F158,F177,T215,N241,E276,A278,H279,F300,D408,R439R312-8.7L1

Y71,F157,F177,A278,H279,F300,P309,F310,F311,R312,H348,D408,R439R212,D214,H239,N241,H245,E276,D349,Q350 -9.4L2

Y71,F157,F177,H245,E276,A278,H279,F300,F311,R312,Y313,D408,R439D68,D214,H239,N241,H348,D349,Q350,R439-9.8L3

D68,Y71,F157,F177,T215,E276,H279,F300,P309,F311,R312,Y313D214,H239,N241,H348,D349,Q350,R439-9.2L4

Conclusion

6

• In summary, with the increasing prevalence of diabetes in the world’s population, the search for active compounds with α-Glu inhibitory

activity from medicinal plants has become an interesting field of science. In the current study, we have synthesized new curcumin-based

compounds and assessed their inhibitory action against α-Glu. These modifications in curcumin structure have improved its capacity in

inhibition of the α-Glu enzyme.

• All compounds were able to inhibit the α-Glu enzyme. Among the evaluated derivatives, compound L3 was identified as a potent inhibitor

of α-Glu enzyme than curcumin. On the other hand, considering the antioxidant characteristics of the synthesized compounds, all

derivatives displaying good antioxidant activity and improved anti-inflammatory properties are highly acceptable as antidiabetic

medicines to be used by diabetic patients. Further studies through the rational drug design on these structures, can be lead to the

exploration of a new class of powerful antidiabetic drugs in the future.

References

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