Dr. R. K. Duary Assistant Professor, Tezpur University Gallic acid functionalized starch: in silico studies against alpha amylase, slow digestibility, molecular structure and antioxidant activity Dr. R. K. Duary Assistant Professor, Tezpur University

INTRODUCTION Gallic acid GA, 3, 4, 5 -trihydroxy benzoic acid Starch composed of the two gluco-homopolysaccharides. It consist of two form: Amorphous and crystalline forms. -Amylose and Amylopectin It has retrogradation, gelatinization, swelling ,solubility properties. Due to consumption of more starchy products the level of glycemic index increases. One strategy to reduce the glycemic index of food products could be the use of phenolic compounds to reduce starch digestibility. Concept of slow digestibility. Gallic acid GA, 3, 4, 5 -trihydroxy benzoic acid Antioxidant properties, antiviral, anticancer, diabetic patients. Able to generate pancreatic β-cells (Latha et al., 2011)

Validation experiment Original Montbretin A Validated and original Montbretin A Original gallic acid Redocked Montbretin A Original Montbretin A Both original & redocked Redocked Montbretin A

In silico studies Active site cavity - CLC drug discovery & PyMol Human pancreatic alpha-amylase Complex with gallic acid Complex with MontbretinA

Molecular Simulation GROMACS 4ns 0ns 2ns 10ns 6ns 8ns Protein RMSD Ligand RMSD

Protein RMSF Protein gyration

Formation of starch and gallic acid Synthesis and grafting process : Catalizing agent : ascorbic acid and oxidizing agent : hydrogen peroxide Cirillo et al., 2010 Starch gallic acid conjugate

Methods for antioxidant properties Total phenolic content: Soluble starch (S-S) Intermediate starch (I-S) Starch gallic acid conjugate (S-G-C) Malik & Singh, 1980   Sl.No. SampleName Total phenolic content(µg GAE/g) 1,2 Gelatinized Non-gelatinized 1. SS 82.31± 0.097a 32.15±0.061a 2. IS 86.3± 0.063b 38.17±0.02b 3. SGC 149.89± 0.072c 70.95±0.043c 1Values reported as Mean ± Standard deviation of three replications. 2Means followed by same small letter superscripts within a column are non significantly different (p>0.05) In both gelatinized and non- gelatinized, it shows that conjugate has more TPC content but better results has been found in non-gelatinized form.

Samples (Gelatinized) Scavenging effect on the DPPH radical: Less IC50 value indicates more is the inhibitory activity. Conjugate contains less IC50 which shows that it has more inhibition activity than soluble starch without antioxidant as comparison done with the ascorbic acid. (Blois, 1958; Brand et al., 1995) Cirillo et al., 2012 Samples (Gelatinized) IC50 (mg/ml) S-S 282.069 I-S 342.713 S-G-C 76.647 Mean and Standard deviation values of the samples and also expressed in gallic acid (GAE) equivalent weight.

DPPH activity (mgGAE/g) 1,2 Sl. No. Samples DPPH activity (mgGAE/g) 1,2   S-S I-S S-G-C 1. 12.5 3.66±1.923a 6.55±0.193a 24.35±0.370a 2. 25 5.97±0.193b 7.13±0.385ab 34.46±0.147b 3. 37.5 7.26±0.294b 8.03±0.294b 39.62±0.294c 4. 50 10.08±0.294c 9.37±0.294c 43.59±0.147d 5. 62.5 11.17±0.193cd 10.78±0.51d 45.51±0.147e 6. 75 12.90±0.385d 13.55±0.29e 47.42±0.294f 7. 87.5  17.92±0.193e 17.15±0.38f 52.74±0.294g 1Values reported as Mean ± Standard deviation of three replications. 2Means followed by same small letter superscripts within a column are not significantly different (p>0.05)

Total antioxidant capacity (TAC) Sl. No. Samples % inhibition 1,2 1. S-S 18.97±0.69a   2. I-S 24.04±0.74b 3. S-G-C 47.96±0.47c 1Values reported as Mean ± Standard deviation of three replications. 2Means followed by different small letter superscripts within a column are significantly different (p ≤ 0.05) The total antioxidant activity expressed in mgGAE/g gives increase amount in conjugate sample than in soluble starch.

Morphological characteristics 1. SEM images SEM images shows perforation in the Soluble starch granules and the gaps are filled in the Starch gallic acid conjugate granules.

FT-IR Analysis Wavenumber (cm-1) Raw data of the FTIR results showing from range 4000 cm-1 to 500 cm-1

Deconvoluted FT-IR: Soluble starch Range of 900 – 1200 cm-1. Black curve: original spectra; dark blue curve: peak at 947 cm-1; 1022 cm-1; light blue curve peak at 995 cm-1; and green curve peak at 1047 cm-1.

Intermediate starch Range of 900 – 1200 cm-1. Black curve: original spectra; dark blue curve: peak at 947 cm-1 ; 1022 cm-1; light blue curve peak at 995 cm-1; 1022 cm-1 and green curve peak at 1047 cm-1.

Starch gallic acid conjugate Range of 900 – 1200 cm-1. Black curve: original spectra; dark blue curve: peak at 947 cm-1; 1022 cm-1; light blue curve peak at 995 cm-1; and green curve peak at 1047 cm-1.

Different Area fits, centre max and ratio of deconvoluted FT-IR Analysis Low ratio of 1047 and 1022 cm-1 and high ratio of 1022 and 995 cm-1 represents lower molecular order which means that decrease in crystallinity and molecular order. From 947 cm-1 V-complexes has been reduced in the conjugate.

DSC graphs: The DSC graphs of the above samples shows a sharp endotherm in the soluble starch which is not visible in the conjugate starch and intermediate starch. Cirillo et al., 2012

TGA profiles TGA results depicts that the conjugate shows more thermal stability than the soluble starch.

Study on effect of starch-gallic acid conjugate on starch digestibility 1. α-Amylase activity: 1Values reported as Mean ± Standard deviation of three replications. 2Means followed by different small letter superscripts within a column are significantly different (p≤0.05) Sl. No. Sample Name1,2 Mean±SD % inhibition 1 Intermediate starch (I-S) 1.427±0.025a 6.140±0.050c 2 Starch gallic acid conjugate (S-G-C) 1.343±0.040b 13.151±0.040b 3 Soluble starch 1.520±0.020c  - When compared with the soluble starch, the starch gallic conjugate shows more α-Amylase inhibitory activity. So it can show good activity in reduction of glycaemic index.

Intermediate starch (I-S) Starch gallic acid conjugate 2. Simulated in vitro starch digestion: Sample SDS % (Mean±SD) Soluble starch (S-S) 17.45±0.89a Intermediate starch (I-S) 24.55±0.95b Starch gallic acid conjugate 29.72±1.06c A :S-S (20mins) A’:S-S (120mins) B :I-S (20mins) B’:I-S (120mins) C :S-G-C (20 mins) C’ :S-G-C (120 mins) A B C A’ B’ C’

Starch hydrolysis % with digestion time and stages in minutes This depicts that the in soluble starch the starch hydrolysis increases and in conjugate it decreases which fulfilled our objective. With increase in SDS% the starch hydrolysis decreases.

Functional properties of soluble starch and its characteristics: Solubility and swelling power Sl. No. Starch sample Solubility1,2   Swelling power1,2 1. 2. 3. S-S I-S S-G-C 3.39± 0.03a 2.03±0.05b 2.22±0.06c 1.59±0.88d 0.62±0.01e 0.40±0.01f 1Values reported as Mean± SD of three replications. 2Means followed by different small letter superscripts within a column are significantly different (p ≤ 0.05). The samples shows more hydrophobicity in intermediate and conjugate as compared to soluble starch after 1 day of hydrolysis.

Conclusions: This study provides that gallic acid can be a potent molecule in incorporation for functionalized starch products. The conjugate provide good antioxidants properties and inhibition activity. The starch hydrolysis can be reduced by starch- antioxidant molecule when targeted with the enzymes which provides less glycemic index. New food product like thickening agent of starch based with antioxidant molecule incorporated in it can be formed. Functional foods like cereal based products can also be formed.