1. VISVESVARAYA TECHNOLOGICAL UNIVERSITY
A PROJECT PRESENTATION ON
Submitted in fulfillment of the requirement for the award of the degree Bachelor of Engineering
In
BIOTECHNOLOGY
Project carried at
Sir M Visvesvaraya Institute Of technology
Bangalore – 562 157
Submitted by
AMSHUMALA. S (1MV11BT003)
TEJASHREE. B (1MV11BT048)
Guided by
Dr. C Rajendra Singh
Department of Biotechnology, Sir MVIT 1
2. CONTENTS
SERIAL NO. CONTENT
1 Introduction
2 About the Plant
3 Literature survey
4 Overview
5 Materials & methods, Results
6 Observation
8 Acknowledgment
9 Bibliography
2
3. • Herbalism is a traditional medicinal or folk medicine practice based on the use of plants and
plant extracts.
• Written records about medicinal plants dated back at least 5,000 years to the Sumerians, who
described well-established medicinal uses for such plants as laurel, caraway, and
thyme, archeological studies have shown that the practice of herbal medicine dates as far back
as 60,000 years ago in Iraq and 8,000 years ago in China.
http://www.hindawi.com/journals/ecam/2014/525340/
• The Old Testament also mentions herb use and cultivation in regards to Kashrut(Jewish dietary
laws)
• Many herbs and minerals used in Ayurveda were described by ancient Indian herbalists such
as Charaka and Sushruta.
• Greek compilers of existing and current herbal knowledge include Pythagoras
, Hippocrates, Aristotle, Theophrastus etc.
• Arabic indigenous medicine developed from the conflict between the magic-based
medicine and the Arabic translations of the Hellenic and Ayurvedic medical traditions.
INTRODUCTION
4. 4
CASSIA GRANDIS
TAXONOMY:
Common Names:
Pink Shower
Carao
Stinking Toe
Classification:
Cassia grandis L. f.
Kingdom: Plantae-Plants
Subkingdom: Tracheobionta-Vascular plants
Superdivision: Spermatophyta-Seed plants
Division: Magnoliophyta-Flowering plants
Class: Magnoliopsida-Dicotyledons
Subclass: Rosidae
Order: Fabales
Family: Fabaceae - Pea family
Genus: Cassia L. - Cassia
Species: Cassia grandis L. f. - Pink Shower
(National Plant Database. 2005.)
5. 5
DESCRIPTION:
Appearance : Dense, umbrella-shaped canopy and smooth
pale gray bark.
Leaf : Large, each composed of about 16 pairs of
leaflets. Has two-toned coloration with green
above and maroon below.
Between March and April, the tree produces abundant
flowers in long axillary racemes. Displays pastel shades
of pink and orange, hence the name Pink Shower. Each
flower has five large, lavender sepals and five
rounded, peach colored petals, three large stamens and
a long, curved pistil. The petals are not uniform in shape
and the uppermost petal has a yellow patch in the end.
Flower
:
Fruits are produced from the long pistils as they begin to
expand. First, they are visible as green strings then they
turn brown and start drying, becoming woody. Each fruit
is about 40cm long and cylindrical in shape.
Fruit :
6. 6
GEOGRAPHICAL DISTRIBUTION: C. grandis can be found in lowland and riparian, semi
deciduous forests, mixed tree systems, and roadsides,
occurring naturally from Mexico to South America (ICRAF,
2014; JSTOR Global Plants, 2014). It has also been widely
grown in urban and disturbed areas and cultivated land as an
ornamental and agro forestry plant, and as a wood source in
tropical regions around the world (ICRAF, 2014).
USES :
Animal feed, fodder, forage
Environmental :
Agro forestry, Boundary, barrier or support ,Ornamental,
Revegetation
Fuels :
Charcoal, Fuel wood
Human food and beverage
Beverage base, Fruits
Materials :
Carved material, Gums, Wood/timber
Medicinal, pharmaceutical :
Source of medicine/pharmaceutical
INDEGENOUS PRACTICES :
The seeds are used for crafting
beautiful leis in Hawaii.
7. LITERATURE SURVEY
7
•Volatile compounds were isolated from Cassia grandis fruit by simultaneous distillation-solvent
extraction. The volatile concentrate was examined by GC and GC/MS. A total of 108 compounds
were identified from which linalool (31.5% of the total volatiles) was the major compound. (J. A.
Pino et. Al,2010)
•Cassia grandis Linn. f. seed galactomannan: structural and crystallographical studies and the seed
was found to possess 50% endosperm gum and possess the characteristics of becoming potential
source of seed gum. (Harsha Joshi, Virendra P Kapoor 2003).
•In vitro anti oxidant properties of cassia grandis was studied and substantial scavenging properties
was reported. (M. K. MEENA et.al, 2009).
•The aqueous and ethanolic extracts of C. grandis (Family: Leguminosae) were evaluated for
antidiabetic activity by aqueous and ethanolic extracts,These extract showed that significantly
lowered the blood glucose levels compared to normal. (Sandesh R.,2010)
• Cassia grandis was also screened for treatment for dermatophytic infections and was found to be
highly active. (A. Cáceres et. Al., 1993) .
•A new anthraquinone had been isolated from the pods of C. grandis and was identifies as 1,3,4
trihydroxy- 6,7,8-2 methyl anthraquinone Structure was elucidated using chemical and
spectroscopic methods. (R.P. Varma et. Al, 2006)
•Chemical examination of the stems of C. grandis : 3 compounds palmitic acid, beta sito sterol and
emodin – 9- anthrone have been isolated.(Meena Rani et Al, 2015).
•A new trans 3- methoxy- 4,5- methylene- dioxy cinnamaldehyde has been isolated from arial parts
of C.grandis. (Gonzalezag et. Al, 1996)
8. OVERVIEW OF THE PROJECT
LITERATURE
SURVEY &
LOCATION
OF PLANT
COLLECTING LEAVES,
EXTRACTION, YIELD
CALCULATION
PHYTOCHEMICAL
ANALYSIS
ANTI MICROBIAL
STUDY
TLC
COLUMN
CHROMATO-
GRAPHY
SEED
GERMINATION
STUDIES
CONCLUSION
8
10. 10
COLLECTION & PROCESSING OF PLANT SAMPLE
The leaves from the plant Cassia grandis was
collected from the plant. The leaves were
transported in polythene bags to the lab, where
the leaves were subjected to drying.
• About 1kg of fresh leaves was collected and
spread on different trays.
• The leaves were then kept for air drying in the
lab at room temperature of 30°C for 3 days.
• The dried leaf samples were taken and ground
using an electric blender to obtain a fine
powder.
• The powdered samples were stored in a clean
glassware container until needed for analysis.
11. EXTRACTION & YIELD CALCULATION
The method used for extraction from the leaves of Cassia grandis is solvent extraction.
Solvents used for extraction are :
1. Water
2. Acetone
3. Ethanol
4. Methanol
5. Ethyl acetate
6. Petroleum ether
7. Chloroform
11
12. 12
ORGANOLEPTIC STUDY OF THE EXTRACT
EXTRACT COLOR UNDER
VISIBLE LIGHT
COLOR UNDER
UV LIGHT
TEXTURE
POWDER+WATER Mustard yellow Light gray Dry, shiny
appearance
POWDER+ACETONE Bottle green Dark pink Completely dry
POWDER+ETHANOL Peacock green Dark pink Completely dry
POWDER+METHANOL Parrot green Violet Completely dry
POWDER+ETHYL ACETATE Peacock green Dark pink Sticky
POWDER+PETROLEUM ETHER Light green Baby pink Sticky
POWDER+CHLOROFORM Brown Dark pink Sticky
SMELL OF THE POWDER : Pungent odour
15. PHYTOCHEMICAL SCREENING
TEST FOR NAME OF THE
TEST
PROCEDURE POSITIVE RESULT
ALKALOIDS WAGNER’S
TEST
Extract + 3-5 drops Wagner’s reagent
(1.27g of Iodine+2g KI in 100ml
water)
Formation of reddish
brown precipitate
immediately
CARBOHYDRATE MOLISCH’S
TEST
Extract+ α- napthol, add conc.
Sulphuric acid carefully on the sides
of the test tube
Formation of violet ring at
the junction of the 2
layers
CARDIAC
GLYCOSIDE
KELLER-
KELLIANI’S TEST
Extract + 2ml of glacial acetic acid
+drop of ferric chloride solution.
underlayed with 1ml of conc.
sulphuric acid.
Brown ring at the
interphase indicates
presence of deoxy sugar
characteristics
FLAVONOIDS ALKALINE
REAGENT TEST
Extracts + 20% Sodium hydroxide
solution. Formation of intense yellow
color, which becomes colorless on
addition of dil. HCl
Intense yellow color,
diappearing on addition
of dil. HCl
PHENOLS FERRIC
CHLORIDE TEST
Extract was treated with aqueous 5%
ferric chloride
Formation of Deep blue/
black colr
PHLOBATANNINS - Extract of the plant sample was
boiled with 1% aqueous hydrochloric
acid
Formation of blue- purple
color
15
16. TEST FOR NAME OF THE
TEST
PROCEDURE POSITIVE RESULT
SAPONINS FOAM TEST 2ml of extracts was added 6ml of
water in a test tube. The mixture was
shaken vigorously
Formation of persistent
foam
STEROLS &
TRITERPENOIDS
LEIBERMANN
BURCHARD TEST
Extract + few drops of chloroform,
acetic anhydride & conc. Sulphuric acid
Formation of reddish –
pink color immediately
TANNINS BRAYMER’S TEST 2ml of extracts was treated with 10%
alcoholic ferric chloride solution
Blue or greenish
solution observed
QUINONES - Extract + conc. HCl Formation of yellow
precipitate
OXALATES - Extract + glacial acetic acid Greenish – black color
formed
TERPENOIDS SANKOWSKI’S
TEST
Extract + 1ml chloroform+ conc.
Sulphuric acid
Formation of reddish
brown precipitate
immediately16
19. THIN LAYER CHROMATOGRAPHY
Thin layer chromatography (TLC) is a
chromatographic technique used to separate the
components of a mixture using a thin stationary
phase supported by an inert backing.
TLC is an analytical tool widely used because of its
simplicity, relative low cost, high sensitivity, and
speed of separation.
TLC functions on the same principle as all
chromatography: a compound will have different
affinities for the mobile and stationary phases and
this affects the speed at which it migrates.
Goal of TLC - Well defined, well separated spots.
In our experiment we used,
Samples of plant extract in 7 different solvents was chosen.
Stationary Phase-TLC paper(Silica).
Mobile phase
Chloroform : methanol in the ratio 9:1 was taken on an trial and error
basis.
20. 20
TLC sheet 5*5cm was taken
Crude samples of plant extract dissolved
in solvents were loaded onto the strip.
Keep it for drying for 10 mins.
About half an inch of TLC sheet was immersed
in the solvent system consisting of 9:1
chloroform: methanol
Wait till the solvent moves 3/4th of the sheet
Remove the strip from the solvent system and let it air dry
for 15 minutes
TLC sheet was then observed under normal
light and UV light for visualizing the
separation.
21. Results for TLC:
Under Visible light Under UV light
Proper separation of molecules was observed in acetone and ethyl acetate
extract.
*NOTE: 1.WATER, 2.ACETONE, 3.ETHANOL, 4.METHANOL, 5.ETHYL ACETATE, 6.PETROLEUM
ETHER, 7.CHLOROFORM
1 2 3 4 5 6 7 1 2 3 4 5 6 7
22. COLUMN CHROMATOGRAPHY
Column chromatography is a method used to purify individual chemical compounds from
mixtures of compounds.
Silica powder(Silica gel matrix) was added to
the column(50g)
Solvent (Chloroform:methanol-1:9) was added to
the column through separating funnel(500 ml)
20 mins
Wait till silica gel forms
Ethyl acetate extract(5ml) was added
Wait till noticeable difference between bands are
formed
Collect the solvent according to the distance
calculation made from TLC experiment.
27. 27
ANTI MICROBIAL TEST
Anti microbial effect of the plant extract was studied using agar well diffusion method.
Anti microbial effect was studied on :
1. Staphylococcus aureus 2. Enterococcus faecalis
3. Escherichia coli 4. Streptococcus mutans
28. 28
Bacteria were grown in the respective
broth and incubated at 37°C overnight.
Culture was adjusted to Mc Farland (turbidity standard) 0.5.
Required media and other glassware and
micro tips were sterilized in an autoclave
at 15lbs/sq. inch for 15 minutes.
Media was cooled to 45°C, 25µl of bacterial
culture was taken in a loop and added to it.
This was poured into the plates and allowed
to solidify. Sterile inverted tips were used as
template to make wells in the plates.
50µl of sterile plant extract was added into the wells
along with a positive (CHX) and a negative control (d/w).
The plates were then incubated at 37°C overnight
and observed for zone of inhibition
29. 29
ANTI MICROBIAL TEST RESULTS
There was no anti microbial property observed.
Water extract Acetone extract Ethanol extract
Methanol extract Ethyl acetate extract
30. 30
STUDY OF ANTI MITOTIC PROPERTY THROUGH SEED GERMINATION STUDY
Green gram seeds weighing 50±1g were collected, stored in a glass jar.
Micro titre plates were cleaned thoroughly and different
concentrations of the 4 water soluble extracts were
added to the wells as a part of the range study.
The optimum range of inhibition of growth was observed after an
incubation for 24hrs at room temperature.
All the 4 solvent extracts were brought to 5ml and stock
concentration of 200mg/ml was prepared.
100%,80%, 60%, 40%, 20%,10% 5% of the 200mg/ml extract was
dispensed into the micro titre wells in sets of three. The last 3
wells were added with water.
50±1g of healthy green gram seeds were added into each of the wells.
Lid of the micro titre plate was closed and incubated for 16 hours
after which the seeds were weighed. The seeds were put back into
their respective wells for further incubation to get the growth profile.
Graph of inhibition v/s concentration was plotted
and compared with the growth profile.
31. 31
SEED GERMINATION STUDIES
WATER EXTRACT
Water extract
IC50 value-8µl
Potency of the drug-40units/ml
0
20
40
60
80
100
120
0 20 40 60 80 100 120
34. 34
Methanol extract
IC50 value-27µl
Potency of the drug-135units/ml
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Murthy, G. Satyanarayana, et al. "An assay for screening anti-mitotic activity of herbal extracts." Current Science (00113891) 100.9
(2011).
35. 35
OBSERVATIONS
The organoleptic study of extract was made and the characterstics of the extract was
studied.
The extract samples were observed under visible light and UV light where the
significant change in the colour was observed which indicates presence of different
types of molecules.
The yield calculations was made and the percentage of yield was significantly high in
Water>Methanol>Acetone>Ethanol>Ethyl acetate>Chloroform>Petroleum Ether.
Phytochemical analysis was conducted for twelve phytochemicals and their presence
was almost nil in Petroleum Ether and Chloroform.
Carbohydrates,cardiac glycosides,phenols,sterols,tannins and terpinoids were found
in high quantities.
Oxalates are completely absent.
Flavanoids,alkaloids,saponins,quinones were present in few of the extracts.
The TLC strip was observed both under visible and UV light,higher degree of
separation was observed in acetone and ethyl acetate extract.
16 different elutions were obtained after conducting column chromatography and
the elutions were observed under UV and visible light.The different colourations were
observed indicating the presence of different molecules.
Antimicrobial activity was nil in all the extract plates.
Antimitotic activity was observed in all the water soluble extracts though the
potency and IC50 values were different.
36. 36
SKILLS GAINED
Technical skills
• Precision in pipetting, measurements
• Microbiology techniques
• Solvent extraction
• Phytochemical screening, Reagent
preparation, observation
• TLC
• Lab safety measures
Interpersonal skills
• Team work
• Leadership & volunteering
• Diplomacy
•Keen observation & patience
•Importance of precision &
presence of mind
37. 37
ACKNOWLEDGEMENT
It gives us immense pleasure to submit this project presentation on
“Extraction, phytochemical analysis and bio activity study of Cassia
grandis”, as a part of the curriculum for the award of Bachelor of
Engineering Degree in the field of Biotechnology.
We are thankful to our project guide Dr. C Rajendra Singh for his
constant guidance and support. We would like to thank the valuable
support extended by Dr. Arul Selvan, HOD, Microbiology department,
KCDS and Dr. Chandrashekar Naik for his kind co-operation.
We are also thankful to Dr H G Nagendra, Professor and Head of the
Department, Biotechnology, Sir MVIT.
We take this opportunity to express my deep sense of gratitude towards
the faculty of the Department of Biotechnology, Sir MVIT, for their
constant guidance.
38. 38
BIBLIOGRAPHY
• Murthy, G. Satyanarayana, et al. "An assay for screening anti-mitotic activity of
herbal extracts." Current Science (00113891) 100.9 (2011).
• Harborne, J. B. (1984). Methods of plant analysis (pp. 1-36). Springer Netherlands.
• Hammer, Katherine A., C. F. Carson, and T. V. Riley. "Antimicrobial activity of
essential oils and other plant extracts." Journal of applied microbiology 86.6 (1999):
985-990.
• Systematic Screening for phytochemicals of various solvent extracts of” Thevetia
peruviana”
