the plant acalypha indica showing the all activities againist cell lines(breast cancer)

1. Evaluation of
phytochEmical, anti-oxidant,
anti-inflammatory and cytotoxicity
activity
of
Acalypha indica (L.)
plant Extract on brEast cancEr(mcf-7)
cEll linEs
SUBMITTED BY
P.HANUMANTHA RAO
Roll no:100714517008
M. Sc GENETICS
UNDER GUIDANCE OF
PROF. A.ROJA RANI
HEAD, DEPT.OF GENETICS
OSMANIA UNIVERSITY

2. INTRODUCTION
 Acalypha indica is an erect, simple or branched, slightly
hairy annual herb.
 It is a species of plant having catkin type of
inflorescence.
 Family: Euphorbiaceae
 Distribution- It occurs throughout tropical Africa and
South Africa, in India and Sri Lanka, as well as in
Yemen and Pakistan.
 It is a common herb growing up to 40-75 cm tall with
ovate leaves.
 Vernacular names:
 Sanskrit: Harithamanjari, Muktavarchas, Rudra, and
Aritamanjari
 Telugu: Moorkonda, kuppi chettu, Pappantichettu, and
Mulakan-dachettu

3. INTRODUCTIONMEDICINAL VALUE:
Acalypha indica constitute an effective source of both traditional and modern
medicines.
About 80% of rural population depends upon the herbal medicine for their primary
health care.
Useful in treating pneumonia, asthma, rheumatism and several other ailments.
Dried leaves of Acalypha indica was made into a poultice to treat bedsores and
wounds.
Acalypha indica root is prescribed as a tonic, astringent, febrifuge and strong
purgative.
Leaves posses anti periodic and laxative properties, the leaves are used in jaundice,
piles, ulcers and also externally skin eruptions, ring worms, eczema.
The roots are used in chest pain, joint pain, and migraine and blood dysentery and
the extract of the root lowered the blood sugar level up to 30%.
Other properties of the herb include anti-inflammatory, anti-helmintic, anti-
bacterial, anti-fungal, anti-oxidant, neuro-protective, anti-venom and antiulcer
activity.

4. Taxonomic Classification
Kingdom: Plantae
Subkingdom: Tracheobionta
Super division: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Rosidae
Order: Euphorbiales
Family: Euphorbiaceae
Subfamily: Acalyphoideae
Genus: Acalypha
Species: Acalypha
indica

5. OBJECTIVES
 To obtain plant leaf powder and leaf extracts using soxhlet
apparatus with different organic solvents.
 To perform phytochemical analysis for different solvent extracts
and list out the types of phytochemicals have actively dissolved
in different organic solvents.
 To perform rotor vapor for different solvent extracts to obtain
crude extract.
 To perform anti-oxidant activity of different solvent extracts
 To perform the anti-inflammatory activity of different solvent
extracts.
 To perform column chromatography of different solvent
extracts.
 To perform Cell Culture and Cytotoxicity Assay (MTT Assay) by
crude extracts.

6. WORK flow
Collection of plant materials
Fine powder
Soxhlet extraction
Crude collection
Phytochemical screening Rotor evaporation
Anti-oxidant
activity
Anti-inflammatory
activity
Cytotoxicity
activity
Column chromatography
Fractions
Sub fractions
MCF-7 cells

7. METHoDOLOGY
 Collection of Plant material from CIMAP (Central Institute of Medicinal and
Aromatic Plants) and authenticated in BOTANY Department.
 Extraction from the plant powder with different organic solvents( polar, mid-polar,
non-polar) like methanol, Hexane, petrolium ether, ethyl acetate and chloroform by
soxhlet extraction method.
 Phytochemical screening for identification of secondary metabolites in different
organic crude extracts.
 These different organic crude extracts converted into crude compounds by Rotary
evaporator method
 Anti-Oxidant Activity of different organic crude compounds by DPPH method
 Anti-inflammatory Activity of different organic crude compounds by BSA method.
 Column chromatography for separation of secondary metabolites in different
organic crude extracts.
 Cell cytotoxicity activity on breast cancer cell lines MCF-7 with different organic
crude compounds by MTT assay method.

9. METHODS
Collection of Plant Sample:
FIG 1:Acalypha indica plants FIG 2:shade dried leaves FIG 3:fine powder
Different plant materials of Acalypha indica were collected and leaves were
separated.
The separated leaves were washed in a tray and shade dried for 3-5 days.
 This shade dried leaves after 3 days were milled to obtain a fine powder.
Always shade drying is preferred as shade drying prevents denaturation of
important phytochemicals when compared to sun drying.
 The weight of this fine powder was calculated.

10. METHODS
Extraction from the plant powder
 Soxhlet extraction is used
when the desired compound
has a limited solubility in a
solvent, and the impurity is
insoluble in that solvent.
 About 250 ml of solvent
was loaded in the round
bottom flask.
 About 30 grams of dried
fine plant powder was placed
in between cotton in
extraction chamber.
 continuous extraction for 48
hours.
 Three different kinds of
solvents were used
individually for extraction
namely methanol,
chloroform and hexane.
Water out
Water in
Fig 1: soxhlet apparatus
thimble
Boiling flask

11. Methods
PHYTOCHEMICAL SCREENING
 Phytochemical examinations were carried out for all the extracts as per the standard methods.
1.Detection of alkaloids:-
Extracts were dissolved individually in dilute Hydrochloric acid and filtered. The following tests
were carried out.
– a) Mayer’s Test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide).
Formation of a yellow coloured precipitate indicates the presence of alkaloids.
– b) Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide).
Formation of brown/reddish precipitate indicates the presence of alkaloids.
2.Detection of carbohydrates: -
Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were
used to test for the presence of carbohydrates.
- a) Benedict’s test: Filtrates were treated with Benedict’s reagent and heated gently. Orange
red precipitate indicates the presence of reducing sugars.
- b) Fehling’s Test: Filtrates were hydrolyzed with dil. HCl, neutralized with alkali and heated
with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of
reducing sugars.

12. Methods
PHYTOCHEMICAL
SCREENING3. Detection of phytosterols:-
a) Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were
treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of
golden yellow colour indicates the presence of triterpenes.
4. Detection of phenols :-
a) Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution.
Formation of bluish black colour indicates the presence of phenols.
5. Detection of flavonoids:-
a) Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide
solution. Formation of intense yellow colour, which becomes colourless on addition of dilute
acid, indicates the presence of flavonoids.
b) Lead acetate Test: Extracts were treated with few drops of lead acetate solution.
Formation of yellow colour precipitate indicates the presence of flavonoids .
6. Detection of proteins and aminoacids:-
a) Ninhydrin Test: To the extract, 0.25% w/v ninhydrin reagent was added and
boiled for few minutes. Formation of blue colour indicates the presence of amino acids.

14. ROTOR VAPOR
Collection of crude plant extract:-
 Liquid plant extract was obtained in the
bottom flask. This plant extract obtained
from three different solvents were collected
in three different glass bottles.
 The content in each bottle was transferred to
individual round bottom flasks and subjected
to rotor vapor individually to obtain
respective solvent crude extract [ex:
methanolic crude extract] etc.
 Each plant crude extract from different flasks
were collected in different tubes by scraping
the flask and respective solvent labeled for
further analysis of the plant crude extract.
Fig: Rotor vapor

15. ANTI-OXIDANTANTI-OXIDANT
ACTIVITYACTIVITY

16. ANTI-OXIDANT ACTIVITY
1. 5 test tubes (trilplicate) were taken and to each 3 ml
DPPH was added.
2. 1 ml of water was added to all test tubes.
3. 5 eppendorf tubes were taken and volumes of 1μl, 10μl,
100μl, 200μl & 500μl of plant extract was added to tubes
individually and volume was made upto 1ml by adding
respective solvent. This makes a total 1ml of plant extract.
4. Then each 1ml plant extract in 5 different eppendorf tubes
were added to respective test tubes and reaction mixture
was incubated for 20 mins at dark condition.
5. UV absorbance was recorded at 517 nm.

18. ANTI-INFLAMMATORY
ACTIVITY
1. 5 test tubes(triplicate) were taken and 5ml of 0.2 % BSA
was added to each.
2. To all the test tubes, 2.5ml of 0.2M phosphate buffer (pH
6.6) was added
3. 10000 μg/ml of all extracts were prepared by using ethanol
as a solvent.
4. 5 eppendorf tubes were taken and volumes of 1μl/ml,100
μl/ml, 200 μl/ml and 500 μl/ml of plant extract was added
to tubes individually and volume was made upto 1ml by
adding respective solvent. This makes a total 1ml of plant
extract.
5. This 1ml of plant extract 50 μl was added to all the test
tubes.
6. Then the tubes were heated in water bath for 10-15
minutes at 100O
C
7. O.D. values were taken at 660nm wavelength.

20. CYTOTOXICITY ACTIVITY
Cell culture and trypsinization:
Preparation of complete media for cells
500 ml of DMEM media was taken and to this 5ml
of antibiotic solution was added.
This makes the whole volume to 505ml and from
this 45 ml of volume is taken and to this 45ml, 5ml
of FBS was added making a total of 50ml of
complete media.
Plain media is direct usage of DMEM only.

21. TRYPSINIZATION
 MCF-7 Cells from mother cultures, which are initially
stored at -80o
C, are sub cultured into T-25 flask with
complete media.
 After 6 hours, the media used by the cells (spent media)
is discarded and cells are washed with 3 ml of PBS.
 Then PBS is discarded and 2 ml of trypsin is added and
the cells in the flask are incubated at 37o
C in CO2
Incubator for 3 minutes.
 The cells in the flasks are detached and digestion of
extracellular matrix (which holds cells together) is done
by trypsinization.
 Then the cells are visualized under inverted microscope.
2 ml of fresh complete media is added and contents in
the T-25 flask are transferred into centrifuge tubes and
centrifuged at 2000 rpm for 2 minutes.
 The supernatant (which contains media and trypsin) is
discarded.
 The pellet is washed with 3ml PBS.
Fig 1:T-25 flask;
trypsinization of MCF-7
cell lines
Fig 2. MCF -7 cells in under
fluorescence microscope

22. CYTOTOXICITY ACTIVITY
MTT ASSY
 To the 96 wellplate, 100 ul of
media with pellet cells is added to
12 wells (4 wells are working
wells, with three for compound
addition and one control and the
work is done for triplet hence a
total of 12 wells).
 This was subjected to 6 hours of
incubation in CO2Incubator at 37o
C.
 Three concentrations of plant
extracts 10mg/ml, 25mg/ml and
50mg/ml were prepared by
dissolving the crude leaf extract,
obtained after rotary evaporator, in
PBS.
Fig; 96 – well plate for MTT assay
•

23. CYTOTOXICITY ACTIVITY
MTT ASSY
 To the 1st
triplet (1st
three vertical wells), 100ul of
10mg/ml plant extract was added.
 Similarly to the 2nd
and 3rd
triplets 100ul of
25mg/ml and 100ul of 50mg/ml of leaf extract
was added.
 Only PBS was added to the control wells without
leaf extracts.
 Now this 96 wellplate is subjected to 21 hours of
incubation in CO2 Incubator at 37o
C.
 After 21 hours of incubation in CO2 Incubator at
37o
C, 20ul of MTT reagent was added to all the
wells was incubated in CO2 Incubator at 37o
C for 3
hours.
 The concentration of MTT used is 0.5mg/ml.
 After 3 hours of incubation, 100ul of DMSO was
added to all the wells.
 O.D values were noted using ELISA reader at
570nm.
Fig; 96 – well plate for MTT assay

24. CYTOTOXICITY ACTIVITY
MTT ASSY
Fig :CO2 incubator Fig:ELISA Reader

25. COLUMNCOLUMN
CHROMATOGRAPHYCHROMATOGRAPHY

26. COLUMN CHROMATOGRAPHY
Method:
1.Column was taken and filled to about one third with solvent (Ethyle acetate 200 ml)
2.In a beaker, measure the required amount of silica gel (125 g).
3.In a separate flask or beaker, measure solvent approximately one and a half times the
volume of silica.
4.Add the silica to the solvent, a little at a time, while swirling. Use a Pasteur pipette or
glass rod to mix the slurry.
5.Pour or pipette some of the slurry into the column. Allow the solvent to drain to prevent
overflowing.
6.Tap the column gently to encourage bubbles to rise and the silica to settle.
7.Continue to transfer the slurry to the column until all the silica is added.
8.Rinse the inside of the column by pipetting solvent down the inside edge.
9.Drain the solvent until the solvent level is just even with the surface of the stationary
phase.
10.The desired compound is loaded and isolated

27. COLUMN CHROMATOGRAPHY
Fig 1,2,3,4:Column chromatography fraction collection
Fig 7.POLAR fractionFig 6.MID POLAR fractionFig 5. NON-POLAR fraction
Fig 1. Fig 2. Fig 3. Fig 4.

28. RESULTS
&
DISCUSSION

29. PHYTOCHEMICAL ANALYSIS
Test Phytochemicals Methanolic extract Ethyl acetate
extract
CHLOROFORM
EXTRACT
Hexane extract
Mayer’s test Alkaloids ++ ++ + +++
Wager’s test Alkaloids ++ ++ ++ ++
Benedict’s test Carbohydrates - - - -
Fehling’s test Carbohydrates - - - -
Salkowski’s test Phytosterols - - - -
Ferric chloride
test
Phenols ++ - - -
Foam test Saponins + ++ - -
Alkaline Reagent
Test
Flavanoids ++ - - +++
Lead acetate test Flavanoids - + + -
Ninhydrin test Amino acids + +++ - +
Phytochemical analysis was performed using standard protocols. The results are in table 1.
Table 1: - = Absence + = Presence ++ = Moderate Presence +++ = More Presence

30. PHYTOCHEMICAL ANALYSIS
Fig.1 Mayer’s test: Yellow coloured ppt Fig.1.2 Wagner’s test: Darkbrown precipitate
Fig.1.3 Alkaline test: Yellow Color
Fig .1.4 Ninhydrin test: Purple Color
Hexane extract

31. PHYTOCHEMICAL ANALYSIS
•
Fig .2.4 Ninhydrin test: Purple ColorFig.2.3 Foam test: Foam Presence
Fig.2.1 Mayer’s test: Yellow precipitate Fig.2.2 Wagner’s test: Darkbrown precipitate
Ethyl acetate extract

32. PHYTOCHEMICAL ANALYSIS
Fig.3.1 Mayer’s test: Yellow Cream precipitate Fig.3.2 Wagner’s test: Darkbrown precipitate
Fig.3.3 Alkaline test: Yellow Color Fig.3.4 Ninhydrin test: Purple Color
Methanolic extract

33. PHYTOCHEMICAL ANALYSIS
Fig .4.1 Mayer’s test: Yellow precipitate Fig.4.2 Wagner’s test: Darkbrown precipitate
Fig.4.3 Alkaline test: Yellow Color
Chloroform extract

34. ANTI-OXIDANT ACTIVITY
Type of sample Concentration (μg/ml) % DPPH scavenging activity
Hexane 1 54.28
Hexane 10 48.57
Hexane 100 34.28
Hexane 200 28.57
Hexane 500 14.28
Methanol 1 68.57
Methanol 10 62.58
Methanol 100 57.14
Methanol 200 34.28
Methanol 500 28.57
Chloroform 1 68.57
Chloroform 10 62.58
Chloroform 100 54.28
Chloroform 200 34.28
Chloroform 500 28.57
control - -
TABLE 1. Anti-oxidant activity with plant extract obtained from respective solvent.

35. ANTI-OXIDANT ACTIVITY
A
B
S
O
R
B
A
N
C
E
A
B
S
O
R
B
A
N
C
E
A
B
S
O
R
B
A
N
C
E

36. ANTI-OXIDANT ACTIVITY
A
B
S
O
R
B
A
N
C
E
CONCENTRATION
H:CH:ME

37. ANTI-INFLAMMATORY ACTIVITY
Type of sample Concentration(μg/ml) %Inhibition
Hexane 1 55.55
Hexane 100 33.33
Hexane 200 22.22
Hexane 500 11.11
Methanol 1 44.44
Methanol 100 33.33
Methanol 200 33.33
Methanol 500 22.22
Chloroform 1 66.66
Chloroform 100 55.55
Chloroform 200 44.44
Chloroform 500 33.33
controle - -
Ibuprofen 100 71.89
Anti-inflammatory activity with plant extract obtained from respective solvent.TABLE:2

38. ANTI-INFLAMMATORY ACTIVITY
A
B
S
O
R
B
A
N
C
E
A
B
S
O
R
B
A
N
C
E
A
B
S
O
R
B
A
N
C
E

39. ANTI-INFLAMMATORY ACTIVITY
HEXANE:METHANOL:CHLOROFORM
A
B
S
O
R
B
A
N
C
E
CONCENTRATION

40. CYTOTOXICITY ACTIVITY
MTT ASSAY
The following are the O.D values obtained for hexane solvent extract at 570nm:
Table 1. Different O.D values of hexane solvent extract at 570nm
Sample 10mg/ml 25mg/ml 50mg/ml Control Control 2 Control 3
O.D Value 0.358 0.304 0.193 0.457 0.468 0.472
O.D Value 0.347 0.319 0.182 0.462 0.459 0.452
O.D Value 0.338 0.299 0.257 0.445 0.443 0.461
O.D AVG 0.347 0.307 0.210 0.454 0.456 0.455
Sample 10mg/ml 25mg/ml 50mg/ml Control
% of Cell Viability 76.78 67.62 46.25 100
Table 2. % Cell Viability
Sample Control 1 Control 2 Control 3 10mg/ml 25mg/ml 50mg/ml
% Compound
Toxicity 0 1.08 1.30 23.22 32.38 53.75
Table 3. % Cell toxicity

41. CYTOTOXICITY ACTIVITYMTT ASSAY
Graph 2.Graph 1.

42. CYTOTOXICITY ACTIVITY
Fig1. Normal MCF 7- cells Fig 2. Trypsinization of MCF -7 cells
Fig3.Drug treatment to MCF-7 cells Fig4. Crystal formation of MCF -7 cells

43. CONCLUSION
 Medicinal plants are plants containing inherent active ingredients and produce
bioactive compounds which act on different system of animals and man and
interfere in the metabolisms of microbes infecting them. The medicinal
properties of plants could be antioxidant, antimicrobial, anti-inflammatory
based on the phytochemicals in them.
 In the present work, phytochemical work shows that most of the important
plant phytochemicals were effectively dissolved in hexane and methanol
followed by ethylacetate and chloroform.
 Antioxidant work shows that the maximum anti-oxidation capacity is with
hexane extracts followed by methanol, and chloroform.
 MTT assay was performed using extracts of hexane extract and the percentage
toxicity values were increasing.
 This states that hexane extract has potent capacity in killing the breast cancer
cells which can be explored for isolation of active compounds for breast cancer
and drug designing.

44. ACKNOWLEDGEMENTS
• I would like to express my sincere thanks to the DEPARTMENT OF
GENETICS,OSMANIA UNIVERSITY.
• I sincerely express my deep sense of gratitude and indebtedness towards my
guide PROF. A. ROJA RANI, HEAD DEPARTMENTOF GENETICS.
• I feel pleasure in acknowledging the cooperation rendered by
Mr.CHEKURI SUDHAKAR, Mr. K.VENKATESH, Mr. SHIVA whose help
led to the completion of this project.
• Grateful thanks to our faculty, for their help, cooperation, and encouragement.

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