EXTRACTION EVALUATION AND APPLICATION OF ...

EXTRACTION EVALUATION AND APPLICATION OF BIOACTIVE COMPOUNDS FROM Costus igneus LEAF

S. THIRUCHENDURAN B.Sc. (Applied Sciences) in Food Science and Technology (Special)

MASTER OF SCIENCE IN FOOD TECHNOLOGY

2016

EXTRACTION EVALUATION AND APPLICATION OF BIOACTIVE COMPOUNDS FROM Costus igneus LEAF BY THIRUCHENDURAN SOMASUNDARAM B.Sc. (Applied Sciences) in Food Science and Technology (Special)

THESIS SUBMITTED TO THE PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN FOOD TECHNOLOGY CHAIRPERSON: Dr. K. UMA MAHESWARI

INTER FACULTY P.G. PROGRAMME IN FOOD TECHNOLOGY DEPARTMENT OF FOODS AND NUTRITION PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY HYDERABAD – 500030 2016

DECLARATION I, THIRUCHENDURAN SOMASUNDARAM, hereby declare that the thesis entitled ―EXTRACTION

EVALUATION

AND

APPLICATION

OF

BIOACTIVE

COMPOUNDS FROM Costus igneus LEAF‖ submitted to Professor Jayashankar Telangana State Agricultural University for the award of Master of Science in Food Technology is the result of original research work done by me. I also certify that neither the thesis nor its part thereof has been previously submitted by me for a degree to any university.

Place: Hyderabad Date:

(THIRUCHENDURAN SOMASUNDARAM)

CERTIFICATE Mr. THIRUCHENDURAN SOMASUNDARAM has satisfactorily prosecuted the course of research and the thesis entitled ―EXTRACTION EVALUATION AND APPLICATION OF BIOACTIVE COMPOUNDS FROM Costus igneus LEAF‖ submitted is the result of original research work and is of sufficiently high standard to warrant its presentation to the examination. I also certify that neither the thesis nor its part thereof has been previously submitted by him for a degree of any university.

Place: Hyderabad Date:

(Dr. K. Uma Maheswari) Chairperson

CERTIFICATE This is to certify that the thesis entitled ―EXTRACTION EVALUATION AND APPLICATION OF BIOACTIVE COMPOUNDS FROM Costus igneus LEAF‖ submitted in partial fulfillment of the requirements for the degree of ―MASTER OF SCIENCE IN FOOD TECHNOLOGY‖ to the Professor Jayashankar Telangana State Agricultural University, Hyderabad is a record of the bonafide original research work carried out by Mr. THIRUCHENDURAN SOMASUNDARAM under our guidance and supervision. No part of the thesis has been submitted by the student for any other degree or diploma. The published part and all assistance received during the course of investigations have been duly acknowledged by the author of the thesis. Chairperson (Advisory Committee) Thesis approved by the Student Advisory Committee

Chairperson

:

.

Member

Member

:

:

Dr. K. Uma Maheswari Professor and University Head and Programme Director (Food Technology) Department of Foods and Nutrition Post Graduate and Research Center College of Home Science, Rajendranagar, Hyderabad-500 030

(Signature)

Dr. W. Jessie Suneetha Assistant Professor Department of Foods and Nutrition Post Graduate and Research Center College of Home Science, Rajendranagar, Hyderabad-500 030

(Signature)

Dr. B. Rajeswari Senior Scientist (Plant Pathology) Regional Agriculture Research Station, PJTSAU Jagital

(Signature)

Date of final viva-voce:

ACKNOWLEDGEMENTS The thesis titled “Extraction Evaluation and Application of Bioactive Compounds from Costus igneus leaf” is a result of collective efforts from a team for whom I always will be indebted. It is my duty to call upon their names here and thank for their invaluable service. First of all I express my sincere gratitude towards the authorities of PJTS Agricultural University and especially authorities of College of Home Science, for their untiring service extended to hold the entire university system together with effective and efficient functioning. If not for their invaluable service and successful management none of the work completed under this thesis was possible. It is a huge privilege to work under the guidance of Dr. K. Uma Maheswari, Professor and University Head and Programme Director (Food Technology) Department of Foods and Nutrition, PGRC, College of Home Science, Hyderabad. Without her intuitive guidance and timely encouragement, this work would have not been possible. The present work bears the impression of her concrete suggestions, careful and constructive criticism, constant encouragement and meticulous attention at all stages of the research work. I am always indebted for the care, secureness and positive vibrations extended by her. It was a great learning experience for me to conduct this thesis under her supervision. My profound gratitude to, Dr. W. Jessie Suneetha, Assistant Professor, Department of Foods and Nutrition, PGRC, College of Home Science, Hyderabad, for her interest and good will towards this work, encouragement, facilitating instruments on time, taking immense struggle to make sure their proper functioning and all other help extended during the course of this thesis. My heartfelt thanks are towards Dr. B. Rajeswari, Senior Scientist (Plant Pathology), RARS, Jagital for the advice and help given during my research work. I cannot miss thanking Dr. Anila Kumari, Assistant Professor, Department of Foods and Nutrition, PGRC, College of Home Science, Hyderabad for her help in procuring chemicals and glassware for the experiments. My sincere thanks to Dr. T.N.V.K.V. Prasad, Senior Scientist, Nanotechnology Laboratory, Institute of Frontier Technology, Tirupati for the short training he provided me in synthesis and characterization of Nanoparticle and Nanoencapsulation of bioactive compounds. His help at the initial part of this thesis was immeasurable. My sincere thanks to Dr. Susila, Senior Scientist, Herbal Garden, Hyderabad for her help in identification of the plant and preparation of nursery. My thanks to Ms. S. Logeswari for her help in data entry and statistical analysis of the data. I am grateful to her for her moral support and financial help too. I am thankful to my sensory panel members of first year MSc Food Technology students for lending me their time and taste buds for the thesis. I am thankful to BSc Food Science and Nutrition students for their help and participation in sensory analysis. I am thankful to everyone at the department of Foods and Nutrition and the University at large for their direct and indirect support in completing my work successfully. Finally but most importantly I am expressing my gratitude to those who financially supported me throughout the course work and research, especially my father Mr. A. Somasundaram, my mother Mrs. S. Kamalam and my sisters Mrs. S. Sathiyavani and Mrs. K. Dayalini. Without their monetary help and verbal support,

this work is impossible. My friend Er. S. Sudharshan for his financial support at most needy occasions. Indian Council for Agriculture Research, Indian Council for Cultural Relations, Ministry of External Affairs - India, Indian high commission to Sri Lanka and Ministry of Higher Education – Sri Lanka enabling me with the fellowship. They all helped me survive financially. Thank you, Almighty god who have always been with me in all my endeavors. Date: Place

(S. THIRUCHENDURAN)

LIST OF CONTENTS

CHAPTER

TITLE

I

INTRODUCTION

II

REVIEW OF LITERATURE

III

MATERIAL AND METHODS

IV

RESULTS AND DISCUSSION

V

SUMMARY AND CONCLUSIONS

VI

LITERATURE CITED

VII

APPENDICES

PAGE NO.

LIST OF TABLES

TABLE NO.

TITLE

Table 2.1

Major constituents in Costus igneus leaf oil

Table 3.1

Experimental design for preparation of tea bags

Table 4.1

Phytochemicals in C. igneus leaf extracts

Table 4.2

Screening of C. igneus leaf phytochemicals in ethanol extract using mobile phase pet ether: benzene: methanol (16:3:2) by TLC method

Table 4.3

Bioactive compounds identified in FDE of C. igneus leaf

Table 4.4

Experimental design for preparation of tea bags

Table 4.5

Mean CATA sensory scores and probability values of tea formulations

Table 4.6

Correlation coefficient between ‗Attributes‘ and ‗Overall Liking‘

Table 4.7

Mean hedonic score for tea formulations prepared with C. igneus leaf powder

Table 4.8

Sensory attribute from the most discriminating to the least discriminating power

Table 4.9

DPPH scavenging activity of solvent extracts of C. igneus leaf powder

Table 4.10

DPPH scavenging activity of tea prepared with C. igneus leaf powder

PAGE NO.

LIST OF ILLUSTRATIONS

FIGURE NO

TITLE

Figure 2.1

C. igneus plant a) Top view b) Side view

Figure 3.1

Extraction methods of C. igneus leaf

Figure 3.2

Freeze drying process of the C. igneus leaf extract

Figure 4.1

HMD and Dendrogram obtained from MFA for peak absorbance of solvent extracts at 200 nm to 700 nm (Correlation and classification of variables; solvents and wavelengths)

Figure 4.2

Phytol

Figure 4.3

Epoxyoctadecan-1-ol, Cis-9,10

Figure 4.4

Oleyl alcohol

Figure 4.5

(Z)-14-tricosenyl formate

Figure 4.6

Lauraldehyde or Dodecanal

Figure 4.7

Undecanal

Figure 4.8

Tridecanal

Figure 4.9

Hexadecanedi-1,16-ol

Figure 4.10

Undecanoic acid, 2-methyl

PAGE NO

Figure 4.11

Decanoic acid, 2-methyl

Figure 4.12

Octanoic acid, 4-methyl, methyl ester

Figure 4.13

Acetamide, 2-amino

Figure 4.14

Butylurea

Figure 4.15

Octanoic acid, 2-methyl

Figure 4.16

Butanoic acid, 2-methyl

Figure 4.17

Pentanoic acid, 2-methyl-,butyl ester

Figure 4.18

Gas chromatogram of C. igneus leaf extract

Figure 4.19

MS of compounds eluded at 17.18 min

Figure 4.20

Sensory profile of tea formulation prepared with C. igneus leaf powder

Figure 4.21

Two dimensional plot derived from MFA of CATA analysis data of six tea formulations

Figure 4.22

Two dimensional graph derived from Multiple Correspondence Analysis (PCoA) of CATA data for six tea formulations depicting the correlations of attributes and the liking scores of the tea formulations

Figure 4.23

Mean impact graph for sensory attributes of formulated tea

Figure 4.24

Percent discoloration of DPPH by C. igneus leaf extracts

Figure 4.25

Microgram

ascorbic

acid

equivalent

DPPH

radical

scavenging activity of prepared tea from tea formulations

LIST OF APPENDICES

APPENDIX NO A

TITLE

Proforma for modified CATA analysis of herbal tea Proforma for sensory analysis of prepared tea from tea

B formulations using nine-point hedonic scale C

Bioactive compounds identified from FDE of C. igneus leaf

PAGE NO

SYMBOLS AND ABBREVIATIONS DPPH

2,2-diphenyl-1-picrylhydrazyl

A549

Adenocarcinoma epithelial cell

AC

Acetone

50AC

Acetone:water (1:1, v/v)

80AC

Acetone:water (4:1, v/v)

C2H3OCl

Acetyl chloride

AAA

Acidified Acetone

HT 29

Adenocarcinoma cell

Α

Alpha

ANOVA

Analysis of variance

et al.

And others

Aq

Aqueous

Viz

As follows

B.C

Before Christ

Β

Beta

n-BuOH

Butanol

BHT

Butylated hydroxytoluene

CI

Costus igneus

COD

Calcium oxalate dehydrate

COM

Calcium oxalate monohydrate

CO2

Carbon dioxide

CVD

Cardio vascular diseases

CDC

Center for disease control

CDER

Center for Drug Evaluation and Research

Cm

Centimeter

CATA

Check all that apply

CHCl3

Chloroform

Conc.

Concentrated

R2

Correlation coefficient

Da

Dalton

DM

Diabetes mellitus

DAD

Diode array detector

EC

Elettaria cardamomum

EI

Electron Impact

eV

Electron volt

EAAE

Enzyme-assisted aqueous extraction

EACP

Enzyme-assisted cold pressing

ELISA

Enzyme-linked immunosorbent assay

ET

Ethanol

70ET

Ethanol:water (7:3, v/v)

EMBL-EBI

European Molecular Biology Laboratory European Bioinformatics Institute

F1

Factor one

F2

Factor two

FeCl3

Ferric chloride

HT 1080

Fibrosarcoma cells

FT-IR

Fourier transform infrared spectroscopy

FDE

Freeze dried extract

GC-MS

Gas chromatography and mass spectrometry

G

Gram

HPLC

High performance liquid chromatography

Hr

Hour

HIV

Human immuno deficiency virus

HCl

Hydrochloric acid

IEC

International Electrotechnical Commission

K

Kelvin

Kg

Kilogram

kHz

Kilo Hertz

KJ

Kilo Joules

LP

Leaf powder

LDL

Low density lippo protein

M

Mass

MS

Mass spectrometry

MHz

Mega Hertz

MFA

Multi factor analysis

MT

Methanol

70MT

Methanol:water (7:3, v/v)

Μg

Microgram

Μl

Microliter

MAE

Microwave assisted extraction

Mg

Milli gram

Ml

Milli liter

Mm

Millimeter

Min

Minutes

MAbs

Monoclonal antibodies

Nm

Nanometers

NCBI

National Center for Biotechnology Information

N.E.Br

Nicholas Edward Brown

MEP

Non-mevalonate pathway

Z

Number of electrostatic charge

%

Percentage

±

Plus or minus

pH

Potential hydrogen

PLE

Pressurized liquid extraction

PCA

Principal component analysis

PCoA

Principal coordinate analysis

PTP

Protein tyrosine phosphatase

PEF

Pulsed electric field

Rf

Retention factor

Rpm

Rotation per minute

S

Second

NaOH

Sodium hydroxide

S.K.L.T.S

Sri Konda Laxman Telangana State Horticultural University

H2SO4

Sulphuric acid

SFE

Supercritical fluid extraction

TGSC

The Good Scents Company

TLC

Thin layer chromatography

UAE

Ultrasound assisted extraction

UV-Vis

Ultraviolet and visible light

USA

United states of America

V

Voltz

v/v

volume by volume

WL

Wavelength

Wt

Weight

w/v

Weight by volume

WHO

World health organization

ZnCl2

Zinc chloride

ZO

Zingiber officinale

Name

: THIRUCHENDURAN SOMASUNDARAM

I.D No.

: FST/2014-018

Title of the Thesis

: EXTRACTION EVALUATION AND APPLICATION OF BIOACTIVE COMPOUNDS FROM Costus igneus LEAF

Degree to which it is submitted

: MASTER OF SCIENCE IN FOOD TECHNOLOGY

Major Field

: FOOD TECHNOLOGY

Faculty

: INTER-FACULTY P.G. PROGRAMME

Major Advisor

: DR. K. UMA MAHESWARI

University

: PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY

Year of Submission

: 2016

________________________________________________________________________

ABSTRACT Food as the basic connection for humans with nature can be utilized as one of the means to tackle the global non-communicable disease epidemics. High curative potentials lie within the biomolecules of nature‘s unexplored space. Designing foods or beverages with the incorporation of functional bioactive compounds from natural sources have been a successful method to tackle global non-communicable disease epidemics. The major issue with the functional foods is their sensory inferiority when compared to unhealthy foods. Costus igneus is one of the multifunctional herbal plants which have exhibited functional properties against many ailments. C. igneus leaves contain different bioactive compounds which have antidiabetic, anticancer, antihypertention and hypolipidemic properties. Development of functional food or beverage from C. igneus is one aspect which hasn‘t been explored yet. Extraction with different solvents and methods and consequent UV-Vis spectra analysis, phytochemical screening and thin layer chromatography (TLC) analysis confirmed the presence of flavones, flavonols, steroids, alkaloids, saponins, flavonoids, triterpenes, tannins, glycosides and reducing sugars.

Gas chromatography and mass spectrometry (GC-MS) analysis of the volatile organic compounds from the freeze dried extract (FDE) of C. igneus leaf powder (LP) results showed the presence of 15 volatile organic compounds which were known for their biological activities. Most of the bioactive compounds have been documented to have anti-inflammatory antidiabetic, anticancer, antihypertention and hypolipidemic properties. Among six different combinations of tea formulations, 0.1 g C. igneus, 0.2 g Z. officinale and 0.1 g E. cardamomum (‗308‘) was the most acceptable with a mean overall liking score of 7.4±0.5 in nine-point hedonic scale. The significant (p