Final Glory PPT.pptx

GC-MS Analysis and Inhibitory evaluation of
extracts of Kavuni rice on major enzymes
linked to Diabetes: An In-vitro & In-silico
Approach
Under the Guidance of
Prof. S. Umamaheswari
Presented by
E. Glory Arul Blessy
(Reg No. 20204012502105)

Diabetes mellitus
 Diabetes mellitus - group of metabolic
diseases characterized by chronic
hyperglycemia resulting from defects in insulin
secretion,/insulin action/both.
 Chronic hyperglycemia of diabetes is
associated with long-term damage, dysfunction,
and failure of different organs (eyes, kidneys,
nerves, heart & blood vessels).
 Diabetes mellitus is rising to an alarming
epidemic level.
 Major cause of Diabetes in recent years
Higher consumption of polished white rice
Ref: Akram and Hisham, 2015

White rice and Diabetes
• Undergoes higher degree of
milling process
• Devoid of essential
micronutrients
• Starchy endosperm-higher
carbohydrate levels
• High Glycemic index & High
Glycemic Load: increased risk
of type 2 diabetes
• Lower levels of bioactive
substances
• Lower levels of dietary fiber
and polyphenols
Ref: Q Sun et al., 2010

Why pigmented rice varieties is preferred?
• Valuable Nutrient Content
• Low Glycemic Index
• Rich in Antioxidants like
Anthocyanins
• Presence of Polyphenols-
Antioxidants
• Undergoes less degree of
milling process
• Reduce the risk of metabolic
disorders
• Richer in Dietary fiber,
minerals and vitamins
• Reduction of LDL Cholesterol
• Higher concentration of
bioactive substances
Ref: TS Rathna Priya et al., 2019

Red Kavuni Rice
 Pigmented and Traditional Rice Variety
Indigenous to Tamil Nadu
 Grown mainly in Tanjore, Tirunelveli,
Dharmapuri & Kanyakumari Districts
 Contains anthocyanin (antioxidant)
 Contains higher levels of amylose, dietary
fiber, protein, β- carotene, lutein and
polyphenols than white rice.
 Inhibitory potential against α-amylase and
β-glucosidase
Ref: Valarmathi et al., 2016

Objectives
Preparation of Rice Extract and
Phytochemical Screening
In vitro Antioxidant Assay and In vitro
Antidiabetic Assay
GCMS Analysis of the Rice Extract
Analysis of Drug-Likeness Property of
Phytocompounds using SWISSADME
Docking Studies of the Phytocompounds with
receptors related with Diabetes mellitus

Methodology
Rice Extract Preparation
Sample: Kavuni rice
Method: Soxhlet Extraction
Solvent: Methanol Ref: Choudhury et al., 2020
Phytochemical Screening
Tests the presence/absence of
Carbohydrates
Protein
Alkaloid
Phenol
Flavanoid
Tannin
Saponins
Anthocyanins Ref: Mannan et al., 2013

GC-MS Analysis
To profile the volatile bioactive compounds present in the methanol extract
of Kavuni rice.
(Ref: Ashok Kumar et al., 2020)
Analysis of Drug-Likeness Property of Bioactive compounds using
SWISSADME
To evaluate the pharmacokinetics, drug-likeness properties of the Bioactive
compounds present in the rice extract.
• Physicochemical properties
• Lipophilicity
• Water solubility
• Pharmacokinetics
• Drug-likeness
• Medicinal Chemistry
(Ref: Daina et al.,2017)

Molecular Docking Studies
To analyse the binding efficiency of the bioactive compounds of the rice
extract and to find out crucial residues involved in bond formation against
the enzymes (α-amylase & α-glucosidase)
Preparation of Receptor (α-amylase & α-glucosidase)
Preparation of Ligand
Docking studies using PatchDock
Interaction analysis using PyMol, LigPlot & Discovery Studio
(Ref: Ladokun et al., 2018)

Preparation of Rice Extracts
Two solvents Methanol and Ethanol were used for the
Extraction using Soxhlet.

Qualitative Phytochemical Screening
Phytochemical Tests Ethanol Methanol
Tannin + +
Saponins _ _
Flavonoid + +
Glycoside _ +
Phlobatannins _ _
Steroid _ +
Terpenoids _ +
Carbohydrate + +
Reducing sugar _ +
Alkaloids + +
“+”indicates the presence and “-” indicates the absence
of the Phytochemicals

Quantitative Analysis of Phytochemicals
Extract Anthocyanin (mg/g) Flavonoid (mg GAE/g) Phenol (mg GAE/g)
Methanol 0.0297±0.007 6.58±0.41 0.952±0.054
Ethanol 0.0212±0.004 5.16±0.22 0.652±0.24

Antioxidant Analysis
DPPH Scavenging Assay
Concentration
(µg/ml)
% Inhibition
Control Ethanol Extract of Kavuni Rice Methanol Extract of Kavuni rice
25 24.3±0.21 9.7±0.61 21.5±0.05
50 32.5±0.17 14.6±0.23 30.8±0.08
100 47.6±0.07 21.7±0.08 44.2±0.64
150 56.1±0.19 28.2±1.21 53.7±0.03
200 67.6±0.03 39.5±0.08 66.9±0.14
0
10
20
30
40
50
60
70
80
25 50 100 150 200
%
Inhibition
Concentration
Control
Ethanol Extract
Methanol Extract

Ferric Reduction Assay
Concentration
(µg/ml)
Absorbance @ 700nm
Control Ethanol Extract of Kavuni Rice Methanol Extract of Kavuni rice
25 0.36±0.01 0.20±0.01 0.44±0.07
50 0.54±0.13 0.37±0.05 0.49±0.13
100 0.79±0.03 0.56±0.03 0.78±0.02
150 1.21±0.02 0.94±0.01 1.19±0.14
200 1.53±0.22 1.11±0.03 1.49±0.16
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
25 50 100 150 200
Absorbance
@
700nm
Concentration
Control
Ethanol extract
Methanol extract

Antidiabetic Analysis
Alpha Amylase Inhibition
Concentration
(µg/ml)
% Inhibition by Ethanol
Extract
% Inhibition by Methanol
Extract
% Inhibition by Positive
Control
50 29.6±0.21 36.1±0.34 41.8±0.22
100 34.5±0.27 47.4±0.17 53.6±0.15
150 40.3±0.11 59.6±0.32 61.5±0.06
200 47.5±0.08 62.3±0.02 69.4±0.26
0
10
20
30
40
50
60
70
80
50 100 150 200
%
Inhibition
Concentration
Ethanol Extract
Methanol Extract
Positive Control

Alpha Glucosidase Inhibition
Concentration
(µg/ml)
% Inhibition by Ethanol Extract % Inhibition by Methanol
Extract
% Inhibition by Positive
Control
50 21.05±0.08 48.43±0.13 54.01±0.45
100 29.02±0.11 57.11±0.19 69.18±0.06
150 37.11±0.32 64.25±0.25 77.21±0.09
200 48.55±0.14 78.98±0.21 86.54±0.13
0
10
20
30
40
50
60
70
80
90
100
50 100 150 200
%
Inhibition
Concentration
Ethanol Extract
Methanol Extract
Positive Control

Glucose Uptake in Yeast Cells
Concentration of Standard
and Extract in µg/ml
% Glucose uptake in Yeast
cells (Standard)
cells (Ethanol Extract)
cells (Methanol extract)
100 37.02±0.07 17.51±0.21 32.21±0.31
200 41.21±0.32 24.22±1.07 36.28±0.74
300 49.21±0.11 30.17±0.27 42.65±0.22
400 57.05±0.32 36.52±0.14 53.47±0.26
500 68.07±0.24 41.11±0.19 61.09±0.54
0
10
20
30
40
50
60
70
80
100 200 300 400 500
%
of
Glucose
Uptake
Concentration
Standard
Ethanol Extract
Methanol Extract

GCMS Analysis
S.No Compound
Name
Structure Formula Molecular
weight (g/mol)
Compound
nature
Retention time
(Minutes)
1 Oleic acid C18H34O2 282.46 Monosaturated
Fatty acid
7.20
2 Butyl 8-
methylnonyl
ester
C22ssH34O4 362.50 Benzoic acid
esters
15.58
3 Ferulic acid C10H10O4 194.18 Phenolic
compound
20.57
4 1,2-
benzenedicarbo
xylic acid
C8H6O4 166.13 Unsaturated
polyester resin
23.08
5 Octadecanoic
acid
C18H36O2 284.48 Stearic acid 23.782
6 Disulfide,di-
tert-dodecyl
C24H5O52 402.78 Organic
disulphide
27.473
7 Trans-O-
Hydroxy
cinnamic acid
C9H8O3 164.16 Phenolic acid 28.50
8 Hexadecanoic
acid
C16H32O2 256.42 Saturated Fatty
acid
34.48

Swiss ADME-Radar image of the bioactive compounds

Docking studies with Alpha amylase
S.No Compounds Global
energy(kcal/mol)
Attractive
Vdw
(kcal/mol)
Repulsive
Value
(kcal/mol)
ACE
(kcal/mol)
1 Oleic acid -77.98 -20.53 8.04 -14.73
2 Butyl 8-methylnonyl ester -31.93 -9.82 6.80 -4.26
3 Ferulic acid -27.63 -18.53 3.49 -27.11
4 1,2-benzenedicarboxylic acid -29.95 -16.91 1.22 -8.25
5 Octadecanoic acid -30.54 -16.03 6.83 -8.53
6 Disulfide,di-tert-dodecyl -23.32 -13.32 9.79 -8.84
7 Trans-O-Hydroxy Cinnamic
acid
-65.07 -30.64 7.73 -25.13
8 Hexadecanoic acid -32.18 -15.66 5.20 -8.37

Docking Studies with Alpha Glucosidase
S.No Compounds Global
energy(kcal/mol)
Attractive
Vdw
(kcal/mol)
Repulsive
Value
(kcal/mol)
ACE
(kcal/mol)
1 Oleic acid -41.24 -23.50 4.63 -12.34
2 Butyl 8-methylnonyl ester -29.62 -14.54 3.28 -6.60
3 Ferulic acid -43.31 -20.80 6.22 -28.55
4 1,2-benzenedicarboxylic acid -39.47 -20.92 5.37 -8.67
5 Octadecanoic acid -39.52 -19.25 3.04 -9.13
6 Disulfide,di-tert-dodecyl -31.04 -18.61 10.53 -8.18
7 Trans-O-Hydroxy Cinnamic
acid
-30.22 -13.81 9.89 -28.34
8 Hexadecanoic acid -32.72 -19.47 6.66 -6.64

Binding of Ferulic acid with Alpha Amylase Interaction of Trans-O-Hydroxy Cinnamic acid
with Alpha Amylase
Interaction of Ferulic acid with Alpha
Glucosidase
Interaction of Ferulic acid with Alpha
Glucosidase
Binding Analysis of Ferulic acid and Trans-O-Hydroxy Cinnamic acid with
Alpha Amylase & Alpha Glucosidase

Interaction Analysis Using Discovery Studio
Interaction of Ferulic acid with Alpha Amylase Interaction of Trans-O-Hydroxy Cinnamic acid
with Alpha Amylase
Interaction of Ferulic acid with Alpha Glucosidase Interaction of Trans-O-Hydroxy Cinnamic acid with
Alpha Glucosidase

Conclusion
• Kavuni rice has medicinal values since it contained a number of secondary
metabolites with potent antioxidant and Antidiabetic properties
• The presence of bioactive compounds with potent biological and
pharmacological activity showed the therapeutic potential of the rice.
• Further, In-silico studies confirmed the potent inhibition of the alpha
amylase and alpha glucosidase with good binding energies.
• Hence it can be concluded that the methanol extract of Kavuni rice has
good medicinal properties and it will be employed in the treatment and
management of Diabetes Mellitus.

References
• Sun, Q., et al., White rice, brown rice, and risk of type 2 diabetes in US men and women. Arch Intern
Med, 2010. 170(11): p. 961-9.
• Rathna Priya, T., Eliazer Nelson, A.R.L., Ravichandran, K. et al. Nutritional and functional
properties of coloured rice varieties of South India: a review. J. Ethn. Food 6, 11 (2019).
• Choudhury, Paramita., et al., (2020). Assessment of nutritional value and quantitative analysis of
bioactive phytochemicals through targeted LC‐MS/MS method in selected scented and pigmented
rice varietals. Journal of Food Science. 85. 10.1111/1750-3841.15164.
• Mannan, Mohammad et al.,(2013). Screening of phytochemical compounds and antioxidant
properties in local and HYV of Bangladeshi rice (Oryza sativa L.) International Journal of
Biosciences (IJB). 3. 151-160. 10.12692/ijb/3.4.151-160.
• Ashokkumar K et al.,(2020) Comparative Profiling of Volatile Compounds in Popular South Indian
Traditional and Modern Rice Varieties by Gas Chromatography–Mass Spectrometry Analysis. Front.
Nutr. 7:599119. doi: 10.3389/fnut.2020.599119
• Daina, A., Michielin, O. & Zoete, V. SwissADME: a free web tool to evaluate pharmacokinetics,
drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep 7, 42717 (2017).
• Ladokun et al.,(2018). GC-MS and molecular docking studies of Hunteria umbellata methanolic
extract as a potent anti-diabetic. Informatics in Medicine Unlocked. 13. 10.1016/j.imu.2018.08.001.
• Mohammad Ajmal Ali., 2020.,Molecular docking and molecular dynamics simulation of anticancer
active ligand ‘3,5,7,3′,5′-pentahydroxy-flavanonol-3-O-α-L-rhamnopyranoside’ from Bauhinia
strychnifolia Craib to the cyclin-dependent protein kinase, Journal of King Saud University -
Science,Volume 32, Issue 1,Pages 891-895,ISSN 1018-3647,

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