Extraction of Curcumin from Trumeric, HPLC

1. Estimation of Bioactive & Secondary Metabolites
from Plants Extract through HPLC Analysis
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Practical Session conducted in
Winter School
on
Innovative Tools and Techniques for Biofortifications
Dr. H. B. Patel
Associate Professor
Dept. of Pharmacology & Toxicology
College of Veterinary Pharmacology & Toxicology
Sardarkrushinagar Dantiwada Agricultural University

2. Secondary metabolites
• Products of primary metabolism
• Low-molecular weight,
• Structurally complex chemical compounds.
• Synthesized in plants as a means of protection
and adaptation to the environment.
• Produced under specific abiotic stresses and
pathogenic attacks;
• Impart survival tactics to plants.
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Secondary metabolites
• Do not directly participate in growth,
development and reproduction of plants hence
named “secondary metabolites”.
• Deployed as taxonomic markers because of having
limited distribution in taxonomic groups.
• Among secondary metabolites some of these
substances have effect on biological systems
which are considered as bioactive.
• Thus, bioactive compounds = ‘secondary plant
metabolites eliciting pharmacological or
toxicological effects in human and animals’.

4. • Drugs, oils, waxes, perfumes, flavouring
agents, dyes and many other commercially
important materials.
• Fascinating library of bioactive compounds
with many pharmacological activities like
antioxidant, antimicrobial, anti-diabetic, anti-
parasitic, anti-tumour
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Classification
Four major categories as classified by British Nutrition
Foundation.
Terpenoids:
such as carotenoids, sterols, cardiac glycosides and plant volatiles
Phenolics:
such as lignans, phenolic acid, tannins, coumarins, lignins,
stilbenes and flavonoids)
Nitrogen containing compounds:
Such as non-protein amino acids, cyanogenic glucosides and
alkaloids
Sulphur containing compounds:
such as phytoalexins, thionins, defensins and lectins)
The four important large molecule families are terpenoids,
steroids, flavonoids and alkaloids.

6. This is the separation of medicinally active
portions of plant from the inactive components
by using selective solvents in standard
extraction procedures.
Extract:
This is a preparation of crude drug which
contains all the constituents which are soluble
in the solvent used in making the extract.
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Extraction

7. Journey from Plant to Prescription
From Plant to Drug
O
O
OH
OH
OH
OH
HO
O
OH
OH
OH
Plant Crude
Extract
Pure
Compound
Molecular
Structure
Biological Activity
=
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8. 8
Plant constituents are usually contained inside
the cells.
Therefore, The solvent used for extraction must
diffuse into the cell to dissolve the desired
compounds.
BASIC PRINCIPLE IN EXTRACTION

9. CHOICE AND QUALITY OF EXTRCTION SOLVENT
The ideal solvent for a certain pharmacologically active
constituent should:
1. Dissolve the substance to be extracted, yet it must be
only sparingly soluble in the solvent from which the
desired substance is to be extracted.
2. Extract only the desired substance or as small an amount
as possible of any other substance present.
3. Not react chemically with the solute in an undesirable
way,
4. Be easily separated from the desired solute after
extraction.
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10. QUALITY AND CHOICE OF EXTRCTION SOLVENT
Ethyl alcohol is the solvent of choice for obtaining
classic extracts such as tinctures and fluid, soft
and dry extracts.
The ethyl alcohol is usually mixed with water to
induce swelling of the plant particles and to
increase the porosity of the cell walls which
facilitates the diffusion of extracted substances
from inside the cells to the surrounding solvent.
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11. COMMON METHODS OF PLANT EXTRACTION
 Infusion
 Decoction
 Maceration
 Percolation
 Digestion
 Continuous hot extraction
 Aqueous alcoholic extraction by fermentation
 Counter-current extraction
 Ultrasound extraction (sonication)
 Supercritical fluid extraction
 Phytonics process
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12. METHODS OF PLANT EXTRACTION
 Infusion
 The plant material
(herbal tea) is placed in
a pot and wetted with
cold water.
 boiling water is poured
over it, then left to
stand.
 It is then covered with a
lid, for about fifteen
minutes after which the
tea is poured off. 12

13. COMMON METHODS OF PLANT EXTRACTION
Decoction:
In this process, the crude
drug is boiled in a specified
volume of water for a
defined time.
It is then cooled and
filtered. This procedure is
suitable for extracting
water-soluble, heat-stable
plant constituents.
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14. COMMON METHODS OF PLANT EXTRACTION
• Maceration:
 Whole or coarsely powdered
plant material is placed in closed
container with the extracting
solvent and allowed to stand at
room temperature for a period of
time with frequent agitation
until the soluble matter is
dissolved.
 The mixture then is filtered, the
marc (the damp solid material)
is pressed.
 The combined liquids are
clarified by filtration or
decantation .
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15. COMMON METHODS OF PLANT EXTRACTION
Percolation:
In this method, the
plant material is
subjected to a slow flow
of fresh solvent at
intervals until sufficient
active ingredient is
extracted.
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16. COMMON METHODS OF PLANT EXTRACTION
Digestion:
Digestion is also
considered as
maceration but, at a
relatively elevated
temperature.
This method is suitable
for hard barks or woods
which are difficult for
water to penetrate.
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17. COMMON METHODS OF PLANT EXTRACTION
 Continuous hot extraction method:
 This is the most common
method used for the extraction
of organic constituents from
dried plant tissue.
 It can be used both on
laboratory and industrial
scales.
 In the lab, the powdered
material is continuously
extracted in a Soxhlet
apparatus with a range of
solvents of increasing polarity.
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18. COMMON METHODS OF PLANT EXTRACTION
• Aqueous alcoholic extraction by fermentation
This extraction procedure involves soaking the
crude drug,in from of either a powder or a
decoction,for a specific period of time,during
which it undergoes fermentation and
generates alcohol in situ; this facilitates the
extraction of the active constituents contained
in the plant material.
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19. • In Ayurveda, this method is not yet
standardized but, with the extraordinarily high
degree of advancement in fermentation
technology, it should not be difficult to
standardize this technique of extraction for
the production of herbal drug extracts.
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20. COUNTER – CURRENT EXTRACTION
• In this process, the material to be extracted is
moved in one direction (generally in the form of
a fine slurry) within a cylindrical extractor where
it comes in contact with extraction solvent.
• The process is highly efficient, requiring little
time and posing no risk from high temperature.
• Finally, sufficiently concentrated extract comes
out at one end of the extractor while the marc
(practically free of visible solvent) falls out from
the other end.
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21. ADVANTAGES OF CCE
a) A unit quantity of the plant material can be
extracted with much smaller volume of solvent
as compared to other methods like maceration,
decoction, percolation.
b) CCE is commonly done at room temperature,
which spares the thermolabile constituents
from exposure to heat which is employed in
most other techniques.
c) The extraction procedure has been rated to be
more efficient and effective than continuous
hot extraction.
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22. Ultrasound Extraction (Sonication)
• The procedure involves the use of ultrasound with
frequencies ranging from 20 kHz to 2000 kHz; this
increases the permeability of cell walls and produces
cavitation.
• Although the process is useful in some cases, like
extraction of rauwolfia root, its large-scale application is
limited due to the higher costs.
• One disadvantage of the procedure is the occasional but
known deleterious effect of ultrasound energy (more
than 20 kHz) on the active constituents of medicinal
plants through formation of free radicals and
consequently undesirable changes in the drug molecules.
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23. Supercritical Fluid Extraction
• Supercritical fluid extraction (SFE) is an
alternative sample preparation method with
general goals of reduced use of organic solvents
and increased sample throughput.
• The factors to consider include temperature,
pressure, sample volume, analyte collection,
modifier (cosolvent) addition, flow and pressure
control, and restrictors. Generally, cylindrical
extraction vessels are used for SFE and their
performance is good beyond any doubt.
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25. ADVANTAGES OF SFE
a) The extraction of constituents at low
temperature, which strictly avoids damage
from heat and some organic solvents.
b) No solvent residues.
c) Environmentally friendly extraction
procedure.
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26. Phytonics Process
• A new solvent based on hydrofluorocarbon-
134a and a new technology to optimize its
remarkable properties in the extraction of
plant materials offer significant
environmental advantages and health and
safety benefits over traditional processes for
the production of high quality natural
fragrant oils, flavors and biological extracts.
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27. • Advanced Phytonics Limited (Manchester,
UK) has developed this patented technology
termed “phytonics process”.
• The products mostly extracted by this
process are fragrant components of
essential oils and biological or
phytopharmacological extracts which can be
used directly without further physical or
chemical tretment.
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28. ADVANTAGES OF THE PROCESS
• Unlike other processes that employ high
temperatures, the phytonics process is cool and
gentle and its products are never damaged by
exposure to temperatures in excess of ambient.
• It is less threatening to the environment.
• The solvents used in the technique are not
flammable, toxic or ozone depleting.
• The solvents are completely recycled within the
system.
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29. FILTRATION OF MACERATES
• The extract so obtained is
separated out from the marc
(exhausted plant material)
by allowing it to drip/drop
into a beaker or a reservoir.
• The marc is retained at the
false bottom, and the extract
is received in the holding
tank.
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30. CONCENTRATION OF FILTRATE TO DRYNESS
The filtered plant part (liquid portion) must be
reduced to paste-like, cakey, and finally powdered
form through standard techniques.
The essence of concentrating filtrate to dryness is to;
 Calculate the active ingredient present after the
extraction by calculation of percentage yield.
 To obtain dose of plant extract in milligram or gram
that can be further constituted in a known volume of
vehicle for experimental animals dosing.
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32. EXTRACTION

33. THIN LAYER CHROMATOGRAPHY

34. HPLC

37. HPLC

38. SPECTROPHOTOMETRY

39. CONCLUSION
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40. REFERENCES
• Agrawal, S.S. Paridhavi, M.H. Herbal drug technology pg
no:321-350, 3.
• EMEA, 2006, Guideline on Quality of Herbal Medicinal
Products/Traditional Herbal.
• Harborne, J.B. Phytochemical methods- a guide to modern
techniques of plant analysis pg no:5-15.
• Medicinal Products, EMEA/CVMP/814/00 Rev 1, European
Medicines Agency, London, U.K., p. 1-11.
• Renu, S. and Badri P. N. (2012). New methods for extracting
phytoconstituents from plants. International Journal of
Biomedical and Advance Research. 03(10) . 770-774.
• Stefani D. H. and Susanne V. From plant to drugs. Center for
Toxicology. The University of Arizona.
• WHO, 2003, WHO Monographs on Selected Medicinal Plants,
Vol. III, World Health. Organization, Geneva.By Erhirhie Earnest O. 40