Antioxidant properties

1. COMPREHENSIVE REPORT
On
RESEARCH PROJECT REPORT
Studies on Antioxidant Properties of Methanolic and Aqueous extract of different
medicinal plants.
Submitted by
Khare Sachin Nandu
Reg. No. 2015BTGD030
Course No. : HOT 481
Course Title : Hands on Training
Project Guide
Mr.Akhilesh lokhande,
Assistant Professor
Department of Bioinformatics,
College of Agricultural Biotechnology, Gandheli, Aurangabad.
Submitted to
MGM COLLEGE OFAGRICULTURAL BIOTECHNOLOGY,
GANDHELI, AURANGABAD
AFFILIATED TO
VASANTRAO NAIK MARATHWADA KRISHI VIDYAPEETH, PARBHANI
(ISO 9001:2008 and ISO 14001: 20015 Certified

2. CONTENTS
•Introduction
• Materials and Methods
• Results
List of Plates
• Outcome of the programme
• Future Line of Work
• References
• Industrial Tour Report

3. INTRODUCTION
• Plant have play a significant role in maintaining human health and improving the
quality of human life for thousands of years and have served humans well as
valuable components of medicines.
• Antioxidants are chemicals that can prevent or slow cell damage. An antioxidant is
an actually not a substance; it’s a behavior. Any compound that can donate electrons
counteract free has antioxidant properties.
• Natural antioxidant are mainly found in fruits and vegetables. There are thousands
of antioxidant compounds out there, but the most common dietary once are vitamins
A, C and E. Beta carotene and lycopene antioxidant can also be produced
artificially and consumed in supplement form. ( Katalinic et al. 2006).

4. • Antioxidants are molecules that inhibit the oxidation of other molecules and thus,
prevent free radicals, which cause damage to cell.
• A lot of type surrounds a group of compounds found in food called antioxidants.
• They are thought as everything from disease fighters to memory protectors to the
antidote.
• Different fruit are taken to evaluate their antioxidants properties with the help of
aqueous and methanolic extract. Fruit peel are taken for checking their antioxidant
properties are. curcuma longa(Haldi), ,Zingiber officinal(Adrak), Coriandrum
sativum(Dhaniya), Cuminum cyminum(Zeera), Allium sativum,(Lahsun), Elettaria
cardomomum(Elaichi), Syzygium aromaticum(Lavang), Murraya
koenigii(Kadipatta), Illicum verum(Badishop), Brassica nigra (Mohri).

5. By considering above points in the view to determine antioxidant activity of different
medicinal plant. an experiment entitled “Effect of antioxidant properties of aqueous and
methanolic extract of different medicinal plants ” will be conducting at department of
Biochemistry and molecular biology, MGM College of Agricultural Biotechnology, Gandheli,
Aurangabad with following objectives :
1. To formulate aqueous and methanolic extract of different medicinal plants.
2. To study antioxidant activity, superoxide anion radicial scavenging activity and
reducing power of methanolic and aqueous extract of different medicinal plants.
OBJECTIVES

6. MATERIAL AND METHODOLOGY
The details of various materials and methods were adopted during the course of present
investigation will narrating in this chapter under appropriate heads:
Experimental site:
The experiment will be conducting in Department Biochemistry and Molecular
Biology laboratory MGM College of Agricultural Biotechnology, Gandheli,
Aurangabad during 2019-2020 summer session.
Statistical design : Factorial Randomized Block Design (FRBD)
No. of treatments : 20 (Combination of 10 different medicinal plant & 2 extracts)
No. of replication : 03

7. Sr.No. Symbol Treatment Details
1 T1
Zingibar officinale (Adrak)
2 T2
Curcuma longa (Haldi)
3 T3
Coriandrum sativum (Dhania)
4 T4
Cuminum cyminum (Zeera)
5 T5
Allium sativum (Lahsun)
6 T6
Elettaria cardamomum (Elaichi)
7 T7
Murraya koenigii (Kadi patta)
8 T8
Syzygium aromaticum (Lavang)
9 T9
Illicium verum (Badishop)
10 T10
Brassica nigra (Mohri)
Treatment Details: Factor 1:- medicinal plants used for formulation of extracts

8. Sr.No. Symbol Treatment Details
1 E1 Methanolic Extract
2 E2 Aqueous Extract
Factor 2:- Types of extracts
Collection of fruits:
.
The fresh fruit of curcuma longa(Haldi), ,Zingiber officinal(Adrak), Coriandrum sativum(Dhaniya),
Cuminum cyminum(Zeera), Allium sativum,(Lahsun), Elettaria cardomomum(Elaichi), Syzygium
aromaticum(Lavang), Murraya koenigii(Kadipatta), Illicum verum(Badishop), Brassica nigra (Mohri),,
were collected from the local area of aurangabad maharastra.

9. Preparation of methanolic extracts:
medicinal plant adrak,dhaniya. Zeera, lahsun,haldi,elichi,lavang,kadipatta,
badishop,mohri,were collected in aurangabda area.
.
The fruits were washing in tap water and air dried at room temperature.
Dried fruits 5 gram by quantity will grinding to produce fine homogenous
mixture.
The fine fruit mixture will soaking in 40 ml of 95% Methanol at room
temperature for 72 hours in dark .
The solution will then filtered through Whattmann filter paper and evaporated
to dryness using rotary evaporator at temperature below 400 C and final filtrate
was used for further procedure.( Kanatt et al.,2007 )

10. Preparation of aqueous extract:
medicinal plant adrak,dhaniya. Zeera, lahsun,haldi,elichi,lavang,kadipatta,
badishop,mohri,were collected in aurangabda area.
The fruits were washing in tap water and air dry at room temperature.
.
The aqueous extract of the medicinal plants were obtain by adding 10 ml of
boiling water to 5 gm of powder plant material in glass flask and incubated at
room temperature for 8 hours on a rotating shaker (200 rpm).
The aqueous extract will filter using whatmann no. 1.( Ao et al., 2008 )

11. Antioxidant activity (DPPH Free radical scavenging activity) of
extract:
DPPH assay will performe according to the method of
Yamaguchi, Takamura, Matoba, and Terao (1998).
The dilute extract (200 µl) will mix with 800 µl of Tris–HCl buffer (100
mM, pH 7.4).
To this will add 1 ml of 500 µM DPPH in ethanol (final
concentration of 250 µM) and the whole vortex vigorously.
tubes were then incubate at room temperature for 20 min under dark
conditions and the absorbance will measure at 517 nm.x
(Yamaguchi et al., 1998 ).
Percent DPPH-scavenging activity will calculate as:
[(Control absorbance - Extract absorbance) ∕ (Control absorbance)] × 100.

12. Superoxide anion radical-scavenging activity of extract :
Superoxide anion-scavenging activity determined according to the method of
Liu, Ooi, and Chang (1997) with some modifications.
The reaction mixture consist of 1 ml of NBT (156 µM in 0.1 M potassium
phosphate buffer pH 7.4), 1.0 ml of NADH (468 µM in 0.1 M potassium phosphate
buffer pH 7.4) and 0.5 ml of an appropriately dilute sample.
The reaction will initiate by addition of 100 µl of PMS (60 µM in 0.1 M
potassium phosphate buffer pH 7.4) to the mixture.
The tubes were incubate at ambient temperature for 5 min and the absorbance
willl measure at 560 nm.
Decrease absorbance of the reaction mixture indicate increase superoxide anion-
scavenging activity ( Liu et al.,1997 ).
The percentage inhibition of superoxide anion generation will calculate using
the following formula:
% Inhibition = [(A0 – AS ) / A0 ] × 100 ,
where A0 is absorbance of the control and AS is absorbance of the sample.

13. Reducing power of extract :
The reducing power of the extracts will determine according to the
method of Oyaizu (1986).
An aliquot (2.5 ml) will mix with 2.5 ml of 200 mM sodium phosphate
buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide and the mixture will
incubate at 500C for 20 min.
Ten percent TCA (2.5 ml) will add and the mixture will centrifuge at 650g
for 10 min.
The upper layer (5 ml) will mix with 5 ml of distil water and 1 ml of 0.1%
ferric chloride and the absorbance will measure at 700 nm ( Oyaizu, 1986 ).

14. Medicinal plants (Factor
A)
Extract ( Factor B )
Methanolic Extract E1 Aqueous Extract E2
T1
14.86 10.66
T2
13.66 9.80
T3
14.51 13.66
T4
11.19 9.80
T5
11.93 8.80
T6
11.56 8.70
T7
12.22 8.07
T8
11.06 9.03
T9
12.00 10.15
T10
11.50 9.15
RESULT OF THE PROGRAMME
The results obtained in the presents investigation on “Studies on Antioxidant Properties of
Aqueous and Methanolic Extract of different Medicinal Plants ” are presented under
following heads .
3.1 Antioxidant activity
Data on mean antioxidant activity recorded after DPPH radical scavenging assay
presented in Table 3.1 and depicted in Figure 1.
Table 3.1 Percentage of antioxidant activity shown by different medicinal plants.

15. 0
2
4
6
8
10
12
14
16
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
DPPH
reduction
medicinal
plants
Methanoli Extracts
Aqueous extracts
:
levels of methanolic and aqueous extactcs
Fig 1:- Percentage of DPPH reduction shown by different medicinal
plants.
SE± CD at 1%
FACTOR A 0.03 0.09
FACTOR B 0.01 0.04
A×B 0.003 0.003

16. 3.2 Superoxide anion radical-scavenging activity of extract :
Data on mean Superoxide anion radical-scavenging assay presented in Table 3.2 and
depicted in Figure 2
Table 3.2 Percentage of Superoxide anion radical-scavenging activity shown
by different medicinal plants:
Medicinal plants
(Factor A )
Extract ( Factor B )
Methanolic Extract
E1
Aqueous Extract
E2
T1 9.19 12.98
T2 10.98 6.55
T3 9.38 9.06
T4 11.48 7.49
T5 13.18 11.78
T6 10.58 9.45
T7 10.34 5.95
T8 8.88 11.65
T9 12.54 7.33
T10 6.88 7.68

17. 0
2
4
6
8
10
12
14
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10
Superoxide
radical
scavenging
anion
reduction
of
medicinal
plants
Methanolic
Aqueous
Levels of Methanolic and Aqueous extract of medicinal plants
Fig. 2 : Percentage of superoxide anion scavenging reduction shown by
different medicinal plants:
SE± CD at 1%
FACTOR A 0.03 0.096
FACTOR B 0.017 0.043
A×B 0.003 0.004

18. 3.3Reducing power of extract :
Data on mean Reducing power of extract presented in Table 3.3 and depicted in Figure 3
Table 3.3 Reducing power of extract shown by different medicinal plants:
Medicinal plants
(Factor A )
Extract ( Factor B )
Methanolic Extract
E1
Aqueous Extrac
E2
T1 3.43 3.10
T2 2.10 1.03
T3 1.30 1.10
T4 2.76 1.40
T5 1.86 0.60
T6 3.00 1.26
T7 1.00 0.53
T8 3.00 0.60
T9 0.50 0.86
T10 1.53 1.43
SE± CD at 1%
FACTOR A 0.023 0.068
FACTOR B 0.010 0.030
A×B 0.020 0.095

20. PLATES
Plate 1 : Sample of different medicinal plants
Plate 2 : Different powder of medicinal plants

21. Plate 3: Aqueous extract of different medicinal plants sample

22. Plate4: Methanolic extract of different medicinal plants sample

23. OUTCOME OF THE PROGRAMME
After completion of project the final outcome of investigation is as following:
The Methanolic extract of zingibar officinale has highest DPPH reduction 14.86%.
The methanolic extract has significantly superior antioxidant reduction compare to
aqueous extract of medicinal plants.
The Methanolic extract of Allium sativum has highest superoxide anion radical scavenging
reduction 13.18% .
The Methanolic extract has significantly superior superoxide anion radical scavenging
reduction compare to aqueous extract of medicinal plants.
The Methanolic extract of Zingibar officinale has highest reducing power activity 3.43.
The methanolic extract has significantly superior reducing power activity compare to
aqueous extract of medicinal plants.
Zingibar officinale has highest DPPH antioxidant potential.

24. SUMMARY OF THE PROGRAMME
Present investigation entitled “Studies on antioxidant properties of aqueous and
methanolic extract of different medicinal plants” was carried out during December 2018 -
April 2019 MGM College of Agricultural Biotechnology, Aurangabad.
Experiment was laid out in Factorial Randomized Block Design (FRBD) with ten
treatments and three replications of different medicinal plants. Curcuma longa(Haldi),
,Zingiber officinal(Adrak), Coriandrum sativum(Dhaniya), Cuminum cyminum(Zeera),
Allium sativum,(Lahsun), Elettaria cardomomum(Elaichi), Syzygium aromaticum(Lavang),
Murraya koenigii(Kadipatta), Illicum verum(Badishop), Brassica nigra (Mohri).
The healthy disease free medicinal plants were selected. Fruits were cut and
separate the pulp. The medicinal plants were collected and were washed in tap water and air
dried at room temperatur

25. FUTURE LINE OF WORK:
Further studies are required to better evaluate the effect of these extracts. In- vivo
clinical testing is essential to confirm in-vitro results. As the evidence base of the medicinal
plants evolves, so will our understanding. More research into medicinal plants extracts and
antioxidant synergistic effect.
The various serious diseases such as neurodegenerative disorders, cancer, liver
cirrhosis, cardiovascular diseases, atherosclerosis, cataracts diabetes and inflammation can
be controlled using medicinal plants.
Superoxide anion radical-scavenging activity and reducing power is also studied in this
experiment.
The methanolic extract of Zingibar officinale showed highest DPPH reduction and Zingibar
officinale highest reducing power.
The aqueous extract of Allium sativum showed highest Superoxide anion radical-scavinging
reduction.

26. REFERENCES
Ao, C., Li. A., Elazaawely A.A., Xuan. D. T, Tawata. S 2008. Evalvation of antioxidant
and antibacterial activities of ficus Microcarpa L.fil. extract. 5 (19): 940-
948.
Chan,E.W.C., Lim Y.Y., Chong K.L., Tan J.B.L., Wong S.K. 2010. Antioxidant
properties of tropical and temperate herble teas. Journal of food
composition and analysis. 2 (3): 185-189.
Chand, S. and Dave.R. 2009. Invitro models for antioxidant activity evaluation and
some medicinal plants possessing antioxidant properties: An overview.
African Journel of Microbiology Research. 3 (13): 981-996.S
Kanatt,R.S., Chander.R., Sharma.A. 2007. Antioxidantal potential mint (Mentha
spicataLl.) in radiation processed lamb meat. 10 (1): 451-458.
Katalinic,V., Milos.M., Kusilic.T., Jukic.M., 2006. Screening of 70 medicinal plant
extracts for antioxidant capacity and total phenols. 94 (1): 550-557
Kaur,C and Kapoor .C.H. 2002 .Antioxidant activity and total phenolic content of
some Asian vegitables. 3 (7): 153-161.

27. INDUSTRIAL TOUR REPORT
LIST OF VISITED INSTITUTE
1. ISHVED BIOTECH PVT. LTD.
2. BEJO SHEETAL BIO-SCIENCE FOUNDATION

28. RELEATED PHOTOGRAPHS
Visit to Ishved Biotech Pvt. Ltd.

29. Visited to Bejo Sheetal Pvt. Ltd

30. Thank You……………