extraction of bioactive compounds from plant sources using maceration process.Maceration is a technique used in wine making and has been adopted in medicinal plant research.

1. 15BT320L- INDUSTRIAL TRAINING 1
Guide name: Dr.venkateswari 1. RANJANI SANKAR
Lab : Medical biochemistry RA1511009010084
Duration :15 days 2. NIVAAS.V
RA1511009010078

3. INTRODUCTION
 Medical Biochemistry is the branch of medicine concerned
with biochemistry and metabolism of human health and
disease. The medical biochemist is trained in the operation
and management of clinical biochemistry laboratories, and
acts as a consultant in all aspects of their use.
 Plants are a source of large amount of drugs . The
researchers today are emphasizing on evaluation and
characterization of various plants and plant constituents
against a number of diseases.

4. TECHNIQUES LEARNT
EXTRACTION OF BIOACTIVE COMPOUNDS FROM PLANT SOURCES
 1. AIM: Extraction of bioactive compounds from plant sources
using maceration process.
 1.1 MACERATION
 1.1.1PRINCIPLE:
Maceration is a technique used in wine making and has been
adopted and widely used in medicinal plants research.
Maceration involves soaking of plant materials (coarse or
powdered) in a stoppered container with a solvent and allowed to
stand at room temperature for a period of minimum 3 days with
frequent agitation . The process softens and break the plant’s cell
wall to release the soluble phytochemicals. After 3 days, the
mixture is pressed or strained by filtration. The choice of solvents
will determine the type of compound extracted from the samples.

5. METHODOLOGY
 The powdered sample was taken in a beaker.
 Appropriate amount of solvent is added to the sample.
For example for 10g of solvent in 100 ml of solvent was
used.
 The sample and the solvent were mixed well with a
glass rod.
 The beaker was then wrapped air tight.
 The beaker with the solvent was left disturbed for few
days.
 The sample and solvent was stirred periodically.
 After few days the solvent was filtered using a filter
paper.
 The filtered solvent is allowed for heating and crude
extract is obtained.
 In this above process catharanthus roseus plant sample
was used.
 The solvent used was ethanol.
1.1.3 RESULT:
Bioactive plant compounds of c.roseus plant were extracted by
using maceration process

6. SOXHLET APPARATUS
 1.2.1 AIM:To separate the bioactive plant by using soxhlet
apparatus
 1.2.2PRINCIPLE:
 In this method, finely ground sample is placed in a porous bag or
“thimble” made from a strong filter paper or cellulose, which is
place, is in thimble chamber of the Soxhlet apparatus . Extraction
solvents is heated in the bottom flask, vaporizes into the sample
thimble, condenses in the condenser and drip back. When the
liquid content reaches the siphon arm the liquid contents emptied
into the bottom flask again and the process is continued.

7. 1.2.3 METHODOLOGY
 A solid material containing some of the desired compound is placed
inside a thimble made from thick filter paper, which is loaded into the
main chamber of the Soxhlet extractor.
 The Soxhlet extractor is placed onto a flask containing the extraction
solvent. The Soxhlet is then equipped with a condenser.
 The solvent is heated to reflux. The solvent vapour travels up a
distillation arm and floods into the chamber housing the thimble of solid.
 The condenser ensures that any solvent vapour cools, and drips back
down into the chamber housing the solid material.
 The chamber containing the solid material slowly fills with warm solvent.
Some of the desired compound will then dissolve in the warm solvent.
 When the Soxhlet chamber is almost full, the chamber is automatically
emptied by a siphon side arm, with the solvent running back down to the
distillation flask cycle is repeated.
 After many cycles the desired compound is concentrated in the
distillation flask.
 The advantage of this system is that instead of many portions of warm
solvent being passed through the sample, just one batch of solvent is
recycled.
 After extraction the solvent is removed, typically by means of a rotary
evaporator, yielding the extracted compound.
 The non-soluble portion of the extracted solid remains in the thimble,
and is usually discarded.
 In this process c.roseus plant sample was used.
1.2.4 Result:
Bioactive compounds of c.roseus plant were extracted using Soxhlet apparatus
and used for further processes.

8. 2. SEPARATION TECHNIQUE
 2.1 AIM:
 To separate volatile liquid from the extraction solution using steam distillation.
 STEAM DISTILLATION:
 Distillation is the process of separating components of a mixture by evaporating
and then condensing the vapor into liquid, taking advantage of the fact that
different elements or compounds have different boiling points. It has wide uses,
from water purification and air distillation to extracting oils from organic matter
and refining crude oil. Several distillation techniques have been developed over
the years, including steam distillation.
 2.1.1 PRINCIPLE:
 Traditional distillation techniques require direct heating of the mixture to
evaporate its contents while this works better for most inorganic solvents and
less in organic solvents. There are many organic compounds that decompose at
high temperature, including many natural essential oils and aromatic
compounds.
 Matter surface have high energy molecules that are in contact with the
atmosphere which exert a certain pressure against the atmosphere due to the
internal energies known as the vapour pressure. Since heating increase the
internal energy of those molecules it also increases vapour pressure.
 Most organic compounds are immiscible in nature. The steam distillation
process works on the principle when two mixture of immiscible liquid is heated
while ensuring that the surfaces of both the liquids are in contact with the
atmosphere, the vapour pressure exerted by the system is increased.

9. 2.1.2 METHODOLOGY
 1. set the distillation apparatus which include
round bottom flask , condenser , heating
mantle, water with motor to regulate water
flow in and out of the condenser.
 2. The solution containing the solvent extract
is taken in the round bottom flask and heated
based on the solvents boiling point.
 3. When the solution reaches its boiling point
the solvent evaporates and moves into the
condenser and is converted to its original
form. This is then collected in a separate
beaker while the extract stays in the flask
 4.After the required amount of solvent is
evaporated the solution is further heated in a
separate heating mantle to obtain the crude
extract.
 5. The extract is collected from the flask and
put in separate containers. The weight varies
from 2-6 grams per 600ml solvent.
2.1.3 Result
The required extract has been separated from the solvent chosen and is further
used for testing of useful phytochemical components.

10. 3.PHYTOCHEMICAL TECHNIQUES
 3.1AIM:
 To do qualitative analysis of the given plant extract C.roseus
 3.2PRINCIPLE:
 Plant-derived substances have recently become of great interest owing to their
versatile applications. Medicinal plants are the richest bio-resource of drugs of
traditional systems of medicine, modern medicines, nutraceuticals, food
supplements, folk medicines, pharmaceutical intermediates and chemical entities for
synthetic drugs . Extraction (as the term is pharmaceutically used) is the separation
of medicinally active portions of plant (and animal) tissues using selective solvents
through standard procedures. The products so obtained from plants are relatively
complex mixtures of metabolites, in liquid or semisolid state or (after removing the
solvent) in dry powder form, and are intended for oral or external use. These include
classes of preparations known as decoctions, infusions, fluid extracts, tinctures,
pilular (semisolid) extracts or powdered decoction, hot continuous extraction
(Soxhlet), aqueous-alcoholic extraction by fermentation, counter- current extraction,
microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid
extraction, and phytonic extraction (with hydrofluorocarbon solvents). For aromatic
plants, hydrodistillation techniques (water distillation, steam distillation, water and
steam distillation), hydrolytic maceration followed by distillation, expression and cold
fat extraction may be employed. Some of the latest extraction methods for aromatic
plants include headspace trapping, solid phase micro- extraction, protoplast
extraction, microdistillation, thermomicrodistillation and molecular distillation.

11. EXPERIMENT OBSERVATION INFERENCE
TEST FOR TANNINS:
1ml of sample was taken in a test tube
and 1ml of 0.008M potassium
ferricynide was added. 1ml of 0.02M
ferric chloride containing 0.1N HCl was
added and observed.
Appearance of blue black
colouration
Presence of tannins
TEST FOR SAPONINS:
Crude extract was mixed with 5ml of
distilled water in a test tube and it was
shaked vigorously. Some drops of olive
oil were added.
Absence of foam formation Absence of saponins
TEST FOR FLAVONOIDS:
5ml of dilute ammonia solution were
added to a portion of crude extract
followed by addition of conc. H2SO4.
Appearance of yellow
colouration.
Disappearance of yellow
colour when left undisturbed.
Presence of flavonoids

12. TEST FOR QUINONES:
Dilute sodium hydroxide was added to
1ml of crude extract.
Blue green or red colouration was
observed
Presence of quinones
TEST FOR TERPENOIDS:
(SALKOWSKI TEST)
5ml of extract was mixed with 2ml of
chloroform and 3ml of concentrated
sulphuric acid a
Was carefully added to from a layer.
Reddish brown colouration was formed
at the interface.
Presence of terpenoids
TEST FOR CARBOHYDRATES:
(FEHLINGS TEST)
The filtrate was treated with 1ml of
Fehling's A and B and heated in a
boiling water bath for 5-10 minutes.
Appearance of orange precipitate Presence of carbohydrates.
TEST FOR PROTEINS:
(BIURET TEST)
Equal volume of 5% solution of sodium
hydroxide and 1% copper sulphate was
added
Appearance of pink or purple colour. Presence of proteins.

13. RESULT:
PHYTOCHEMICAL SCREENING OF VARIOUS COMPOUNDS WAS DONE AND OBSERVED
FOR POSITIVE ARE NEGATIVE RESULTS
 .

14. CONCLUSION
 Various techniques related to extraction, separation were learnt.
Preliminary qualitative test for finding different phytochemical particles
were done . By analyzing various phytochemicals, isolation of
phytochemicals of required interest can be done and used for various
studies .some of them include cancer, diabetes etc.
 REFERENCE:
 1. www.researchgate.net
 2. www.phytojournal.com