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:100714517008M. Sc GENETICS

UNDER GUIDANCE OF PROF. A.ROJA RANI

HEAD, DEPT.OF GENETICS OSMANIA UNIVERSITY

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

INTRODUCTION MEDICINAL 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.

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

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.

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

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.

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.

METHODSExtraction 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

MethodsPHYTOCHEMICAL 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.

MethodsPHYTOCHEMICAL

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.

ROTOR VAPORCollection 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

ANTI-OXIDANTANTI-OXIDANT ACTIVITYACTIVITY

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.

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 100OC 7. O.D. values were taken at 660nm wavelength.

CYTOTOXICITY ACTIVITYCell 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.

TRYPSINIZATION

MCF-7 Cells from mother cultures, which are initially stored at -80 oC, 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 37oC 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

CYTOTOXICITY ACTIVITYMTT 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 CO2 Incubator at 37oC.

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

•

CYTOTOXICITY ACTIVITYMTT 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 37oC.

After 21 hours of incubation in CO2 Incubator at 37oC, 20ul of MTT reagent was added to all the wells was incubated in CO2 Incubator at 37oC 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

CYTOTOXICITY ACTIVITYMTT ASSY

Fig :CO2 incubator Fig:ELISA Reader

COLUMN COLUMN CHROMATOGRAPHYCHROMATOGRAPHY

COLUMN CHROMATOGRAPHYMethod: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

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.

RESULTS &

DISCUSSION

PHYTOCHEMICAL ANALYSISTest Phytochemicals Methanolic extract Ethyl acetate

extractCHLOROFORM 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

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

PHYTOCHEMICAL ANALYSIS

•

Fig .2.4 Ninhydrin test: Purple Color 

Fig.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

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

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

ANTI-OXIDANT ACTIVITY

Type of sample Concentration (μg/ml) % DPPH scavenging activityHexane 1 54.28Hexane 10 48.57Hexane 100 34.28Hexane 200 28.57Hexane 500 14.28Methanol 1 68.57Methanol 10 62.58Methanol 100 57.14Methanol 200 34.28Methanol 500 28.57Chloroform 1 68.57Chloroform 10 62.58Chloroform 100 54.28Chloroform 200 34.28Chloroform 500 28.57control   - -

TABLE 1. Anti-oxidant activity with plant extract obtained from respective solvent.

ANTI-OXIDANT ACTIVITY

ABSORBANCE

ABSORBANCE

ABSORBANCE

ANTI-OXIDANT ACTIVITY

ABSORBANCE

CONCENTRATION

H:CH:ME

ANTI-INFLAMMATORY ACTIVITYType of sample Concentration(μg/ml) %Inhibition

Hexane 1 55.55Hexane 100 33.33Hexane 200 22.22Hexane 500 11.11Methanol 1 44.44Methanol 100 33.33Methanol 200 33.33Methanol 500 22.22Chloroform 1 66.66Chloroform 100 55.55Chloroform 200 44.44Chloroform 500 33.33controle   -   -Ibuprofen 100 71.89

Anti-inflammatory activity with plant extract obtained from respective solvent.TABLE:2

ANTI-INFLAMMATORY ACTIVITY

ABSORBANCE

ABSORBANCE

ABSORBANCE

ANTI-INFLAMMATORY ACTIVITYHEXANE:METHANOL:CHLOROFORM

ABSORBANCE

CONCENTRATION

CYTOTOXICITY ACTIVITYMTT 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 100Table 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

CYTOTOXICITY ACTIVITY

MTT ASSAY

Graph 2. Graph 1.

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 

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.

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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[2]. Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur: Phytochemical screening and Extraction: A Review Internationale Pharmaceutica Sciencia Vol 1 Issue 1 Jan-Mar 2011 Pg 98-10.

[3]. Rahman MA, Bachar SC, Rahmatullah M (2010). Analgesic and antiinflammatory activity of methanolic extract of Acalypha indica Linn. Pak. J. Pharm. Sci., 23(3): 256-258.

[4]. Balakrishnan N, Panda A B, Raj N R, Shrivastava A and Prathani R : The Evaluation of Nitric Oxide Scavenging Activity of Acalypha indica Linn : Root Asian J. Research Chem. 2(2): April.-June, 2009 ISSN 0974-4169 Pg.148-150.

[5]. Nandhakumar M, Tamil Iniyan G, Senthilkumar M, Dinesh Kumar B, Mitra A.In vitro Assay of alpha amylase inhibitory activity of indian medicinal herb acalypha Indica.J ournal of Clinical and Diagnostic Research 2009 April; 3:1475-1478.  [6]. Govindarajan M, Jebanesan A, Reetha D, Anisath R, Pushpanathan T, Samidurai K. Antibacterial activity of Acalypha indica L. Eur Rev Med Pharmacol Sci. 2008;12(5):299-2.