ASSIGNMENT OF PLANT

PHYSIOLOGY

Aqsa Khalil

Topic:Extraction of Phenolics from Plant

Material:

What are Phenolics?

Phenolic are aromatic benzene ring compounds with one or more hydroxyl groups produced by plants mainly for protection against stress.

Plant Phenolics:• Broadly distributed in plant kingdom.• Most abundant secondary metabolites of

plants.• Comprise 40% soluble protein of plants.• More than 8000 structures of phenolics

are known.• Lignin(polyphenol) is second most

abundant compound in plant.

Sources of Phenolics:• Fruits• Vegetables• Cereals• Olive• Legumes• Chocolate• Tea• Coffee• Wine

Classification of Phenolics:

Types of Phenols:• Simple Phenols: Coumarins Phenolic acids Hydroxybenzoic acids Hydroxycinnamic acids.• Polyphnols Tannins Lignins Flavenoids

Different Methods of Extraction:

There are different methods of extraction of phenolics from plant materials,•Conventional method•Soxhlet Extraction•Ultrasound Assisted Extraction(UAE)•Microwave Assisted Extraction(MAE)•Ultrasound/Microwave Assisted Extraction (UMAE)

1-Conventional Method of Extraction:

• Extract 5g of homogenized material with 50ml of appropriate solvent.

• Continue magnetic stirring(magnetic size 4.0-5.0cm) at 700rpm for 1hr at room temperature.

• The extract was then filtered(paper no.89)• Do the extraction process in triplicate.

2-Soxhlet Extraction:• Finely ground crude drug is placed in a

porous bag or “thimble” made of strong filter paper, of the Soxhlet apparatus.

• Heat the extracting solvent and condense it in a condenser.

• The condensed extractant drips into the thimble containing the crude drug, and extracts it by contact.

• When the level of liquid in chamber rises to the top of siphon tube , the liquid contents of chamber siphon is put into flask.

• This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated.

Soxhlet Apparatus

3-Sonication:• The procedure involves the use of

ultrasound with frequencies ranging from 20 kHz to 2000 kHz.

• An appropriate solvent is mixed with a sample and sonicated under controlled temperature for a specified time.

• This increases the permeability of cell walls and produces cavitation hereby releasing cell contents.

Probe and Bath Sonicators:• Probe sonicators are constantly in contact

with the sample and make reproducibility and repeatability difficult. In addition, the risk of sample contamination and foam production is higher.

• Bath sonicators can act on a range of samples simultaneously and allow for higher reproducibility

Example:• Extraction of isoflevones from Soy Beans

by sonication has done with 40%-60% Ethanol for 20 min.

• The yield was 1.353(mg GAE b/g) .

Probe and Bath Soncators:

Bath Sonicators: Probe Sonicators:

4-Microwave Assisted Extraction:

• Microwaves are non-ionizing radiation• (frequencies between 300 MHz and 300

GHz).• They induce molecular motion in materials or

solvents with dipoles resulting in sample heating .

• The heating causes plant cells to lose moisture through evaporation; swells and eventually ruptures the cells, releasing their active components.

Procedure:• Make 80%v/v solution of ethanol.• Take 10-40ml of solvent per g palnt

material.• Irradiate the solution for 5-30min at 70–130

°C and 200–1000 W microwave power.• The results revealed that MAE has the

power to give the highest yield compared to other methods

Example:• The extraction of polyphenols from Tea

was done using 12ml of 60% Ethanol per g of tea material for 10 min at 80°C using 600W microwave radiation.

Microwave Apparatus:

5-Ultrasound/Microwave Assisted Extraction (UMAE)

• The coupling of two powerful radiation techniques (ultrasonic and microwave).

• It consume lower volumes of solvents and result in higher extraction yields than conventional extraction.

• Lou et al. applied microwaves with ultrasonic extraction (UAME) and maceration to extract phenolics from Burdock leaves. The final optimized UMAE method gave a phenolic yield of 9 mg/g while less than 0.5 mg/g was achieved using maceration.

UMAE Apparatus:

6-Supercritical Fluid Extraction (SFE)

• Supercritical Fluid Extraction (SFE) is the process of separating one component (the extractant) from another (the matrix) using supercritical fluids as the extracting solvent. Extraction is usually from a solid matrix, but can also be from liquids.

• A SCF is a type of solvent that forms when the temperature and pressure of the fluid increase above its critical point .

• The SCF generated has the penetration power of the gas form and density of the liquid form.

• The usual SCF applied in SFE are methane, carbon dioxide, ethane, propane, ammonia, ethanol, benzene and water.

• Cylindrical extraction vessels are used.• The collection of the extracted analyte from

SFE is another important method. Significant analyte can lost during this step.

• CO2 is used as extracting solvent.• Organic solvents are frequently added to

CO2 extracting fluid to alleviate the polarity limitations.

• The component recovery rate generally increased with increase in temperature and pressure,

• For example the extraction of phenolic contents from guava seed through SFE is done at 50°C with at pressure of 200-300bar for120min using Ethyl acetate and Ethanol as modifier solvent with CO2.

SFE Apparatus:

7-Subcritical Water Extraction (SCWE)

• Water becomes subcritical when the temperature is 100–347 °C applied under sufficient pressure (normally 10–60 bar) to preserve its liquid form .

• The dielectric constant of water reduces under subcritical conditions due to the breakdown of intermolecular hydrogen bonds.

• At these subcritical conditions water shows polarity and dielectric constant equal to organic solvents like ethanol and methanol.

• For extraction of anthraquinones from Morinda citrifolia, the effectiveness of SCWE compared to that of other extraction methods, such as ethanol extraction in a stirred vessel.

Example:• Extraction of phenolic compound from Rice

bran through SCWE is done at 120-200°C for 5min at a pressure of 20bar using 2.5ml of solvent per gram material.

• SCWE extracts gave almost the same antioxidant activity as Soxhlet extracts, but SCWE extracts contained higher antioxidant activity than ethanol extracts and ultrasound-assisted extracts

SCWE Apparatus:• SCWE could be a

good alternative industrial method to use for extraction of large amounts of phenolic compounds without toxic organic solvent residues.

8-High Hydrostatic Pressure Extraction (HHPE)

• This method utilizes non-thermal super-high hydraulic pressure (1,000–8,000 bar).

• It works on the basis of mass transport phenomena.

• The pressure applied increases plant cell permeability.

• The cell components diffuse under applied pressure.

• HHPE creates pressure difference b/w the cell membrane interior and exterior.

• It allows solvent to penetrate into the cell causing leakage of cell components.

Example:• A higher yield of soluble polyphenols

in the juice of cashew apples has been obtained

using HHPE compared to other methods

HHPE Apparatus:

Importance:• Involved in defense against insect, herbivores and fungi.• Some have allelopathic function (Caffeic acid & ferulic acid).• Some are pigments and give color to plant parts

(anthocyanins).• Act as antiflamatory and antioxidant(blue anthocyanins).• Provide protection against UV(flavones & flavonles).• Antimicrobial properties and also involved in signaling

(isoflavonoids).• In humans phenolic compounds do not allow constriction of

blood vessels (tannins in tea& caffeine).

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