Ultrasonication Assisted Extraction of Isolation Characterization of Berberine from Annamita Cooculs Stem & it’s biological activities

PROJECT TITLE
Ultrasonication Assisted Extraction of Isolation
Characterization of Berberine from Annamita
Cooculs Stem & it’s biological activities.
Submitted By
GUTTI . PAVAN
M.Sc -2 , Biochemistry
Under The Guidance of
Dr. UTKARSHA LEKHAK
Department of Biochemistry,
The Institute of Science Mumbai 400032

SN TOPIC SN TOPIC
1 Aims 10 Determination of berberine content
2 Phytochemicals introduction 11 Colum chromatography
3 Alkaloids 12 Spectra analysis of berberine
4 Berberine 13 Antioxidant studies
5 Anamirta cocculus 14 Anti inflammatory studies
6 Project overview 15 Antimicrobial studies
7 Preparation stem water extract 16 Conclusion
8 Phytochemical ( alokilds) qualitative analysis : 17 Acknowledgement
9 Determination of alkaloid by BCG method 18 Bibliography
INDEX

To detect the presence of alkaloids in extracts by qualitative
method.
Isolation and characterization of berberine.
To detect antioxidant & anti-inflammatory activity in plant
extracts.
Antimicrobial activity studies.
AIMS

PHYTOCHEMICALS INTRODUCTION
Phytochemical is a broad term meaning plant (photo) chemical
referring to a wide variety of biologically activity compounds that
occur naturally in plants. which produced health benefits for
humans as medicinal ingredients and nutrients .

Alkaloids
Alkaloids are natural
product that contains
heterocyclic nitrogen
atoms, are basic in
character.
Alkaloids are naturally
synthesis by a large
numbers of organisms,
including animals, plants,
bacteria and fungi

BERBERINE
Berberine is a quaternary ammonium salt from
the protoberber.ine group of benzylisoquinoline alkaloids.
Berberine is an alkaloid found in a wide variety of traditional
plants.
Berberine has a broad spectrum of pharmacological activities.
Berberine comes to us from India and China and, where it was
first used in traditional Chinese medicine and Ayurvedic medicine.
Berberine compound is supplemented for its anti-diabetic and
anti-inflammatory effects.

anamirta cocculus
Anamirta cocculus (Marathi: काकमारी) is a Southeast
Asian and Indian climbing plant. Its fruit is the source
of picrotoxin, a poisonous compound with stimulant
properties.
Chemical substances
• The stem and the roots contain quaternary alkaloids,
suchas berberine, palmatine, magnoflorine and columba
mine.
• The seeds deliver picrotoxin, a sesquiterpene, while the
seed shells contain the tertiary
alkaloids menispermine and paramenispermine.

anamitra cocculus
stem powder
Ultrasonication
assisted extraction
Column
chromatography
TLC
spectra analysis
pure compound
berberine
Antioxidant
studies
Antimicrobial
studies
PROJECT OVERVIEW

Preparation stem water
extract
Weight 2 grams of stem powder
with 50 ml distilled water in 250 ml
standard conical flask. mix well
properly. then keep the water flask in
sonication, at 37 c, for 30 mins.
 The crude aqueous extract was
acidified with 1N HCl and then
treated with 1N NaOH. The aqueous
layer was further extracted using
chloroform.
The extraction was repeated until
the last fraction did not give any
precipitate (orange color) with
Dragendorff’s reagent.

Phytochemical ( alokilds) Qualitative Analysis :
1. Dragondroff’s reagent test
• take 2 ml plant extract into the test tube and add 5 drops dragendorff reagent, The formation of an
orange-red colored precipitate indicated the presence of alkaloids.
2. Mayer's test
• take 2 ml plant extract into the test tube and add 5 drops Mayer's reagent reagent, The formation of an
cream precipitate indicate the presence of alkaloids.
3. Hager test
• take 2 ml plant extract into the test tube and add 5 drops hangers reagent, The formation of a yellow
precipitate indicate the presence of alkaloids.
4.wagner test
• take 2 ml plant extract into the test tube and add 5 drops Wagner reagent, The formation of a brown coal
precipitate indicates the presence of alkaloids.
5.scheibler test
• take 2 ml plant extract into the test tube and add 5 drops scheibler reagent, The formation of a light yellow
-white precipitate indicates the presence of alkaloids.
6.sonnensceins test
• take 2 ml plant extract into the test tube and add 5 drops sonnensceins reagent, The formation of a light
yellow precipitate indicates the presence of alkaloids.

A-DRAGENDORFF TEST : orange -red color ppt , B- HAGERS TEST: Yellow color PPT ,
C- MAYER TEST: Cream color ppt , D- WAGER TEST: Red- brown colored PPT ,
E-SCHEUBLER TEST: white with light yellow , F- SONNENSCEINS TEST: Light yellow color

Determination of Alkaloid content by BCG complex method

Determination of berberine content
• Take 1 ml plant extract
sample The absorbance for
test and standard solutions
were determined against
the blank ( only methanol )
at 420 nm .
• berberine sulphate Was
used as the standard
concentration of (40, 80,
120 ,160 and 200 μg/ml) .

colum chromatography
SAMPLE : load 1 ml crude sample ( 53
mg/ml).
SOLVENT SYSTEM : when finished the
loading sample into column, after runs with
column using different concentrations of
solvent system CHCl3: MeOH (in ratios of
98:2, 96:4, 95:5, 90:10, 80:20 and 70:30) .
SUCCESSIVE SOLVENT SYSTEM : The
CHCl3: MeOH (98:2) fraction yielded a
single peak in TLC, which was identified as
berberine by UV-Visible spectroscopy, FT-IR
and 1H NMR and LCMS.

UV data
The UV spectrum of
isolated berberine
sample and standard
berberine was found to
overlap, indicating the
purity of the spots.
The absorption
spectrum of berberine
in methanol solution is
424 nm.

IR DATA
FTIR spectra were recorded on
Shimadzu IR-Spectrometer (FTIR 8201)
central university of Hyderabad, at
room temperature within the wave
number range of 4000 to 400 cm−1
using KBr discs. The observed IR
absorption peak at ranges of
3500 cm−1 represents the N-H stretch
group, peak 2951.80cm−1 indicates
to C-H stretch, peak 1253cm−1 C-H
stretch aromatic, 2731 cm−1 shows C-
H stretch alkane, 1622 cm−1 peak
shows C-C stretch ring aromatic , peak
1328 cm−1 shows C-N stretch ,
mainly 1597 cm−1 shows benzene ,
1564 cm−1 indicates C=N, 972 cm−1
peak indicates O-C-O groups.

H1 NMR DATA
• 1H NMR spectra were recorded
in CDCl3. All assignments of the
proton atoms were found in
their expected region.
• The NMR spectra of berberine
was confirmed the absence of in
the berberine the signals
corresponding to two CH3
groups is observed at 1.68 and
1.64 ppm, (6H) C-H protons at
7.49 , 7.42, 6.83, 7.28, 7.24 and
6.85 ppm , (3H) CH2 protons at
1.64, 2.19 and 2.03 ppm.

Mass spectra data
Mass spectra provide an
essential clue for elucidating the
structure of compounds.
The esi mass spectra of the
berberin were recorded and
used to compare their
stoichiometry composition. The
berberin displays the prominent
molecular ion peak (m+) at 𝑚/𝑧
=336.1235

ANTI INFLAMMATORY ACTIVITY
• .
 Procedure The anti-inflammatory
activity was measured by the method
of (standard protocol, Plant Research
Scheme).
 Inflammation is a complex process,
which is frequently associated with
pain and involves occurrences
such as: the increase of vascular
permeability, increase of protein
denaturation and membrane
alteration.
 When cells in the body are
damaged by microbes, physical
agents or chemical agents, the
injury is in the form stress.
Inflammation of tissue is due to
response to stress

DPPH Radical Scavenging Activity
Procedure ferric reducing antioxidant
(FRAP)The activity was measured by the
method of (KalaisezhiyenPavithra, 2015.
Take different volume of plant(
5,10,20,40,80.100,200 and 300 ug/ml) extracts
were made up to 40 ul with DMSO and 2.96 ml
DPPH (0.1 mM) solution was added. The
reaction mixture was incubated in dark
condition at room temperature at 20 mins,
after 20 mins the absorbance of the mixture
was measured at 517 nm .
2ml of DPPH was taken as control. Prepare one
blank solution which contains the ml of 9 ml
reagent and 1ml 1 ml distilled water.
The following equation was used to determine
the percentage of the radical scavenging
activity of each extract.
Percentage of radical scavenging activity=[(OD
control- OD sample)/OD control] × 100

The antioxidant activity of samples was evaluated by
the green phosphomolybdenum complex formation
according to the method of Prieto(1999).
Ferric reducing antioxidant
power assay Phosphomolybdenum assay
Procedure ferric reducing antioxidant (FRAP)The
activity was measured by the method of (standard
protocol, Plant Research Scheme)

ABTS activity aassay:
• ABTS radical scavenging activity was
estimated by following re et.al(1999)
method.
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity of the
extracts was determined according to the
method reported by (Klein et al).

The chelating ability of the extract against iron was
determined using the method of Puntel et al.
Reducing power assay
• Reducing the power of MPE was
determined according to the
developed method
Hydrogen peroxide scavenging assay

Iron Chelation Assay
• The chelating ability of the extract against iron was determined using the method of
Puntel et al.

Steps
1.Collection of bacterial strains
• Pure cultures of all experimental bacteria were obtained from the
microbiology department of the Institute of science Mumbai,
Maharashtra. The pure bacterial cultures were maintained on nutrient
agar medium.
2.Bacterial Inoculums preparation
• Make The different bacterial suspensions( at lest 1ml) were adjusted
with sterile saline to a concentration of 1.0 X 108 CFU/ml. Inoculums
preparation Each bacterial strain was subcultured overnight at 35 C in
Mueller-Hilton agar slants. The bacterial growth was harvested using 5
ml of sterile saline water, its absorbance was adjusted at 580 um and
diluted to attain viable cell count of 108 CFU/ml using
spectrophotometer.

3.Media Preparation and Its Sterilization:
• Antimicrobial susceptibility was tested on solid Agar-agar media (gm/100
ml: peptone 1gm , NaCl 0.5gms, yeast extract 0.3 grams and agar 4.gms
maintain pH 7.1) was used for developing surface colony growth. take above
chemical into starization conical flask boiled to dissolve the contents of the
medium and after pack with sterilization cotton. It is sterilized by
autoclaving at 121ºC for 20 minutes at 15 Lbs. pressure.
4.Pour media into a petri dish, and sample loading
• take 20 ml media into which were already washed and sterilized petri dish.
Do under near flame and Petri dishes( contain media ) keep for turn to
semi-solid phase under sterilization conditions. and loaded one control disc
(chloramphenicol (30 µg/disc) , respectively. The plates were incubated for
24 h at 37 °C.
5.Measurement zone of inhibition
• The measured diameters of the zone of inhibition for berberine against
different bacteria were measured in millimetre by using scale .recorded and
considered an an indication for antibacterial activity.

1 = Almonella Typhi . Gram (-ve) 2= Almonella Typhi . Gram (-ve) 3= Escherichia coli. Gram (-ve
4= Enterocolitica. Gram (-ve) 5= S. aureus. Gram (+ve) 6= Shigella. Gram (-ve)
1A ,2A,3A,4A,5A,6A = Chloramphenicol( disk) zone of inhibition
1B ,2B ,3B,4B,5B ,6B = berberine ( disk) zone of inhibition

7 = Bacillus subtilis . Gram (+ve) 8 = Streptococcus pyogenes. 9 = Bacillus cereus . Gram (+ve)
10 = Pseudomonas aeruginosa. Gram (-ve) 11 = Vibrio cholerae . Gram (-ve)
7A ,8A ,9A ,10A ,11A = Chloramphenicol( disk) zone of inhibition , 7B ,8B ,9B,10B,11B = berberine( disk) zone of inhibition

CONCLUSION
 The berberine alkaloid was first time isolated at 370 C from stem Anamirta
cocculus plant using ultrasonication extraction method. The CHCl3: MeOH (98:2)
fraction yielded a single peak in TLC, which was identified as berberine by UV-
Visible spectroscopy. After the extraction of Berberine for the confirmation, TLC
was also performed to confirm the Rf value to be 0.52.
 The experimental values of IR, UV , MS and H1NMR are used to predict the
molecular structure, atomic stretching, possible molecular functional group, etc.,
for the confirmation of berberine alkaloid present in the Anamirta cocculus plant
and are confirmed. The theoretical values are good agreement with the
experimental values.
 The molecular structure is found by Gaussian software (V3) and also IUPAC name
by Chem Doodle software and is represented as : 5,6-dihydro-9,10-
dimethoxybenzo[g]-1,3-benzodioxolo[5,6a]quinolizinium). and the molecular
formula is C20H18NO.

 The Anamitra cooclus water stem extract has great potential as
antimicrobial compounds against microorganisms. Thus, they can
help to use in the treatment of infectious diseases in human and
fruit spoilage caused by resistant microbes.
 Food spoilage is often caused by the growth of many different
pathogenic bacterial strains. Prevention of food spoilage in the food
industry and foodstuff is mainly based on the application of
chemical preservatives.
 we concluded The anamirta cocculus plant water extracts which
proved to be potentially effective as (anamirta cocculus ) can be
used as natural alternative preventives to control food poisoning
diseases and preserve foodstuff avoiding healthy hazards of
chemically antimicrobial agent applications.

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ACKNOWLEDGEMENT
• I would like to express my special thanks of gratitude to my Guide
Hon’ble Dr.Utkarsha leahak, who gave me the golden opportunity
to do this wonderful PPT “Ultrasonication Assisted Extraction of
Isolation Characterization of Berberine from Annamita Cooculs
Stem & it’s biological activities.” for her constant support and
motivation that has encouraged me to come up with the
assignment. Secondly i would also like to thank my Parents and
Friends (M.Sc-2 Biochemistry IOS) and Non-teaching staff who
have rendered their whole hearted support to all times for the
successful completion of this assignment .
My Family
Ios Biochemistry non-teaching staff
My M.Sc Biochemistry friends
Hon’ble
Dr.Utkarsha leahak

Ultrasonication Assisted Extraction of Isolation Characterization of Berberine from Annamita Cooculs Stem & it’s biological activities

Ultrasonication Assisted Extraction of Isolation Characterization of Berberine from Annamita Cooculs Stem & it’s biological activities

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