06 | Isolation of Chitin and Glucosamine

Objectives
• Describe the structure of chitin and glucosamine and correlate their structure with their
physicochemical properties
• To isolate chitin and glucosamine from biological samples

Chitin
• Major constituent in the shells of crustaceans (crabs, lobster, insects, tahong)
• A homopolysaccharide composed of repeating units of b1-4 linked N-acetylglucosamine.
o (NAG) linked by beta-glycosidic bond
• Strength depends on the hydrogen bonding between
adjacent molecules producing rigid sheets
• Chemical deactylation yields chitosan
o Chitosan is important in water purification
o Chelating properties and used in wound healing
preparations

Glucosamine
• Derived from acid-catalyzed deacetylation and hydrolysis
of chitin
o Used as an anti-arthritis drug in a variety of
supplements

Chitosan
• Most important derivative of chitin
• Soluble in acidic aqueous media
N-acetyl-glucosamine
• extensively used as a dietary supplement in the treatment for osteoarthritis, knee pain, and back
pain, and does not affect glucose metabolism

Perna viridis – Philippine tahong

• Calcinated and can be used as commercial CaCO3 for commercial use
o CaCO3 is used as calcium supplement, phosphate binder, antacid

Crustacean shells -> Size reduction (pulverize) -> Washing demineralization (HCl) this is
decalfication of CaCO3 -> Maceration (room temperature) -> Transfer and wash with H2O ->
No longer acidic -> Chitin
Demineralization
• is generally performed by acid treatment using dilute hydrochloric acid (preferred).
• involves the decomposition of calcium carbonate into the water-soluble calcium salts with the
release of carbon dioxide as shown in the following equation:

2 HCl + CaCO3 → CaCl2 + H2O + CO2 ↑

Chitin -> Acid hydrolysis (conc. HCl) -> Filtration of solution (pale yellow color) -> Add ethanol and
cool in ice bath -> Recrystallization -> Filtration, washing drying -> Glucosamine

Digestion of chitin is
• for acid hydrolysis of chitin
• The hydrolysis process involves two acid-catalyzed hydrolysis reactions: the glycosidic linkage
(depolymerisation) and the N-acetyl linkage (deacetylation).
 Chitin Glucosamine

Insoluble
Cold water Soluble
(hydrophobic property of acetyl)

Hot water Insoluble Soluble

Ethanol Insoluble Slightly soluble

Diethyl ether Insoluble Insoluble

 07 | Isolation of Pectin
Objectives
• Describe the structure of pectin and correlate it to its physicochemical properties
• Isolate pectin

Pomelo (Citrus grandis)

• Citrus fruit
• Consists of peels/rind skin (source of pectin) that can be separated from the pulp (edible portion)

Pectin
• Methylated ester of polygalacturonic acid that contains 1,4-linked a-D-galacturonic acid residues,
arabinan, galactans
• Commonly found in cell walls and lamellae of plants
o They function with cellulose and hemicellulose as intercellular cementing material
o Used as thickener, emulsifier, texturizer, and stabilizer
o Influences blood cholesterol (6g of pectin a day to reduce cholesterol)
o Acts as a natural prophylactic substance against poisoning with toxic cations
o Effective in removing lead and mercury from GIT and respiratory organs

Factors affecting percentage yield of pectin

1. Extraction condition
2. Temperature
3. Extraction time
4. pH
5. Type of extraction solvent
6. Drying method
§ Commercial pectins are extracted from the inner portion of the rind of citrus fruits
using an acid extraction method with a yield of 10%
Commercial pectin for the production of jellied food products is standardized to the convenient “150
jelly grade” by addition of dextrose or other sugars, and sometimes contains sodium citrate or other
buffer salts.

USP Identification test for distinguishing pectins from gums

• Heat 1g of pectin with 9ml of water on a steam bath until a solution is formed, replacing water
lost by evaporation; it forms a stiff gel on cooling
• To a solution (1 in 100) add an equal volume of alcohol; a translucent, gelatinous ppt is formed.
(distinction from most gums)
• To 10 mL of a solution add 1mL of thorium nitrate TS. Stir, allow to stand for 2 mins. A stable ppt
or gel forms (distinction from tragacanth)
• Acidify the gel from the preceeding test w 3N HCl and shake; a voluminous, colorless, gelatinous
precipitate forms w/c upon boiling becomes white flocculent (pectic acid)
 Peel and separate the mesocarp layer -> Cubes -> Digestion with sulfuric acid ->
Strain (cheesecloth), collect filtrate -> repeat digestion -> Add ethanol to precipitate

Pectin extracted from various materials can be different in molecular structure (i.e., molecular weight,
degree of esterification and acetyl content) and therefore, possesses different functional properties

Pectin designates those water soluble pectinic acid of varying methyl ester content and degree of
neutralization which is capable of forming gels with sugar and acids under suitable conditions.

Acid (1M H2SO4) was added for maintaining different pH medium as reagents. Several acids can be
utilized for the extraction of pectin. The percentage yield of pectin is based on the acid collection.
 08 | Mucilage Swelling Factor in Gums
Objectives
• Define mucilage swelling factor
• Differentiate gums from mucilage
• Importance of mucilage swelling factor in choosing viscosity enhancing agents for suspensions

Gums and Mucilage

• Natural plant hydrocolloids that may be classigied as anionic (nonionic) polysaccharides
• Salts of polyssacharides
• Upon hydrolysis, yields a mixture of sugars and uronic acid
• Heteropolysaccharides, they are sulfated
o The swell when they absorb water
o Used as viscosity enhancing agents (suspensing agents) in pharmaceutical applications

Gums
• Pathological products formed upon the injury of the plant
• Mainly secreted as exudates when the bark is damaged or as a result of a breakdown of cell
walls due to unfavorable conditions such as drought
o Exudates serve to seal the wound or the plant incision
o Prevents the dehydration of plant
• Examples: acacia, tragacanth

Mucilage
• Generally normal physiological products formed within the cell
o Used as food and water reservoir, protection for germinating seeds, lubrication for the
growing tip, adhesive in seed dispersal
• Found in epidermal cells, seed coats, roots and barks
§ Malvaceae, Sterculiaceae, Ulmaceae
Algaes
• Contain marine gums as components of their cell walls and membrane/ intercellular regions
o Serves as food storage material

Marine gums
• Agar
• Carrageenan
• Alginates

Microbial gums
• For fermentation
• Exopolysaccharides can be isolated from fermentation broth

Mucillage swelling (swelling index)

• Important physical characteristic of pharmaceutical gums
• Forms the basis in choosing the appropriate viscosity-enhancing agent in certain pharmaceutical
preparations

Official cellulose derivatives

• Cellulose ether derivatives and cellulose ester derivatives

v Gums are non-glycogenetic and may be used in the preparation of diabetic syrups.
• Mucilage relieves irritation of mucous membrane by forming a protective film.

Agar
• produced from red seaweeds which have been freeze dried then formed into bars called
kanten.
• The kanten bars are sometimes broken up into flakes or ground into a powder.
Carrageenans
• gel forming polysaccharides found naturally in the whole plant form of Irish moss.
o used commercially as a stabilizer in products ranging from dairy to toothpaste.
• Extraction of carrageenan for high volume commercial use involves the use of strong acids
which can be irritating to the GI tract and potentially carcinogenic.
o Types of carrageenan:
1. Lota Carrageenan - In the presence of calcium, forms a soft gel
2. Kappa Carrageenan - In the presence of calcium, forms a stiff and brittle gel. With potassium
salts, forms very firm and elastic gels.
3. Lambda Carrageenan - will not form a gel, but can be used as a thickener.
Acacia Acacia is the dried gummy exudation obtained from the stems and branches of Acacia
Senegal L.
Agar It is the dried gelatinous substance obtained by extraction with water from Gelidium amansii
or various species of red algae like Gracilaria and Pterocladia, belonging to family
Gelidiaceae.
Alginic acid It is a polyuronic acid composed of reduced mannuronic and glucoronic acid, which are
obtained from the algal growth of the species of family Phaeophyceae.

Sodium alginate Sodium Alginate is the sodium salt of alginic acid.

Carageenan It is the sulphated polysaccharide obtained from the seaweed called Irish moss, the red
algae Chrondrus crispus Linn.

Guar Gum Guar gum is a seed gum produced from the powdered endosperm of the seeds of
Cyamopsis tetragonolobus Linn. Belonging to the family of Leguminosae.

Karaya Gum Gum karaya is a dried, gummy exudates obtained from the tree Sterculia urens (Roxburgh),
Sterculia villosa, Sterculia tragacantha (Lindley) or other species of Sterculia belonging to
family Sterculia.
Pectin Pectin, in general, is a group of polysaccharides found in nature in the primary cell walls of
all seed bearing plants and are invariably located in the middle lamella.
Psyllium Psyllium is a soluble fiber used primarily as a gentle bulk-forming laxative in products such
as Metamucil. It comes from a shrub-like herb called Plantago ovata that grows worldwide
but is most common in India.
Starch Starch consists of polysaccharide granules obtained from the grains of maize, rice or wheat.

Tragacanth It is the air dried gummy exudates, flowing naturally or obtained by incision, from the stems
and branches of Astragalus gummifer Labill and certain other species of Astragalus,
belonging to family Leguminosae
Xanthan Xanthan is a microbial polysaccharide produced from Xanthomonas campestris
 03 | Microscopic Evaluation of Crude Drugs
Objectives
• Importance of microscopic evaluation of crude drugs
• Interpret histological description of vegetable drugs in monographs
• Compare actual samples with official descriptions to confirm identification, quality, and detect
adulteration and contamination
• Write a description of the histology of the crude plant drugs and the botanic characteristics of
powdered drugs

Microsopic Evaluation
• Analytical technique
• Preliminary identification of herbal drugs
• Plants possess characteristic tissue structure demonstrated through botanical sections/mounts
• Includes:
o Determination of size/shape; Position of cells and tissues
o Determination of chemical nature of cell and cell walls

Philippine pharmacopoeia official monograph (Histology)

• Describe microscopic properties
• Official requirement for vegetable drugs
• Includes:
o Particular tissue within an organ
o Arrangement/type of cells within tissue
o Presence/type of secretory canal, oil, or resin duct or laticifers within an organ
o Number of epithelial cells surrounding a secretory canal
o Presence of starch, inulin, fat globules, fluids and other materials in cytoplasm, organelles,
vacuoles, cavities or cell walls
Microscopic Evaluation of vegetable drugs
• Microscopic linear measurements
o Size of starch grains
o Length and width of fibers, trichomes
o Determination of leaf constants (Stomatal number, stomatal index, palisade ratio)
• Quantitaive microscopy
o Lycopodium Spore method

Cassava used as a matrix in preparing tissue sections

For fresh samples, use 1:1 glycerin-alcohol solution, or chloral hydrate TS
For powdered drugs, add 50% v/v glycerin solution
For dry plant samples, Dry or gently boil in water until soft, treat as fresh sample then

Stomatal studies
• prepare an epidermal peel on both adaxial (upper) and abaxial (below) sides of leaf
• count the number of stomata (two guard cells per square mm of epidermis)

Determination of Stomatal number

• Stomatal number is the average number of stomata per square mm of the epidermis of the leaf.
• Stomatal number is affected by various factors like age of the plant, size of the leaf,
environmental conditions etc.
• Stomatal index is not much affected by thesef actors. It is relatively constant. Hence it is more
significant in the evaluation of a leaf drug.
 • Histological studies may be conducted using tissue culture; the ability to visualize or
differentially identify microscopic structures is frequently enhanced through the use
of histological stains. Histology is an essential tool of biology and medicine.
• Histochemistry refers to the science of using chemical reactions between laboratory chemicals
and components within tissue.
• The chloral hydrate will be used to optically clear parts of the plant.
• Defatting helps in removing non polar as well as coloring matter like chlorophyll contents from
the plant.
• Light capturing is the specialization of the adaxial or upper surface and the abaxial or lower
surface is specialized for gas exchange.
o The adaxial stomata are exposed to more direct radiation, whereas the abaxial ones are
shaded by the leaf itself and receive the light transmitted through the mesophyll and
reflected from the surroundings.
• The top layer of cells in a leaf are called the palisade leaf cells. They are specially adapted to
make the most of the light conditions they receive. These are Rod shaped cells that contain
large numbers of chloroplasts for photosynthesis.
o These cells are located close to the leaf surface to maximise light absorption.

Lycopodium Spore Method

• This is an important technique employed in identification of crude drug when chemical and
physical methods are inapplicable. Using this, one can determine the proportions of the
substance present by means of the microscope.

Microchemistry
• is the study of the constituents by application of chemical methods to small quantities of drugs
in powdered form or to histological sections of the drug. The techniques like microscopic linear
measurements, determination of leaf constants and quantitative microscopy are also used in the
evaluation of plants.
 05 | Phytochemical screening of crude drugs
Objectives
• List down chemical identification tests for groups of phytochemicals
• Explain and interpret principles of phytochemical screening

Chemical evaluation of crude drugs

• Includes qualitative/quantitative chemical tests and instrumental analysis of fundamental
physicochemical properties or constants of the whole plant drug.
• Chemical methods of isolation, purification and identification
• Qualitative tests for constituents such as alkaloids, glycosides, tannins

Phytochemical analysis
• Important in quality control and drug discovery
• Phytochemical screening is the qualitative testing of the chemical constituents of plants (in
crude extracts)
• Helps in the choice of extraction method and solvent in isolation and purification of active
constituents

Preliminary treatment
o Dissolve the dried extract in an extracting solvent
§ Distilled water
§ Ethanol
o Transfer the filtrate and add diethyl ether until the pigment is removed from
aqueous/alcoholic layer
o Collect the clear aqueous/alcoholic layer for phytochemical testing.
 Test for Carbohydrates
Test For Reagent Positive result
GENERAL TEST MOLISCH ɑ-naphthol in 95% Reddish
- Monosaccharides gives a rapid (+) REAGENT ethanol violet/purple
colored ring
Molisch
- Disaccharides and polysaccharides Dehydrating agent at the junction of
test
react slower SULFURIC two liquids
ACID

Carbohydrates are dehydrated to produce an aldehyde (furfural/furfural derivatives.)

These compounds condense with ɑ-naphthol to form purple ring.
Oligosaccharides/polysaccharides are first hydrolysed then dehydrated.
REDUCING SUGARS FEHLING’S A FEHLING’S B Brick red
- Differentiate reducing sugars from non- CuSO4 5H2O .
Potassium sodium precipitate
reducing sugars, not specific for tartrate (Rochelle
of Cu2O (Cuprous
aldehydes Distilled water salt) oxide)
Fehling’s
test Deep blue solution
Sulfuric acid NaOH (Cu2+)

Distilled Water

TEST FOR REDUCING SUGARS BENEDICT’S CuSO4 . 5H2O Brick red

- Same principle as Fehling’s test but REAGENT precipitate
Benedict’s reagent is much stable than Sodium citrate
of Cu2O (Cuprous
Benedict’s Fehling’s reagent (Complexes with oxide)
test Cu2+) Deep blue solution
(Cu2+)
Sodium carbonate
(Alkali conditions)
Reducing sugars under alkaline conditions tautomerize to form enediols (reducing agents).
They reduce cupric ions to cuprous form and themselves converted to sugar acids.
 Test for Carbohydrates
Test For Reagent Positive result
DIFFERENTIATION TEST SELIWANOFF Resorcinol Cherry red color
REAGENT (Condensation
- Distinguishes glucose (aldose) agent)
and fructose (ketose).
Seliwanoff
- Positive for ketohexoses Distilled water
(fructose, sucrose…)
HCl (Dehydrating
agent)
Keto hexoses on treatment with HCl form 5-hydroxy methyl furfural that condenses with resorcinol.
SPECIFIC TEST BIAL’S Orcinol Bluish product
- This test is specific for pentoses. REAGENT
All other colors indicate
Ferric Chloride a negative result for
Bial’s test - Hexoses generally react to form pentoses
green, red or brown products HCl (Dehydrating
agent)

The test reagent dehydrates to form furfural. Furfural further reacts with orcinol and the iron ion present in the test
reagent to produce a bluish product.

For Molisch test

For Benedict test For Fehling test

For Seliwanoff
test

For Bial
 Test for Glycosides
Test For Reagent Positive result
Alkaline Reagent • Presence of flavanoid NaOH Intense yellow color
test (Flavonoids)
- Presence of flavanoid Magnesium ribbon Orange color (Flavones)

Crimson color
Shinoda test Conc. HCl (Flavonols)
(Flavonoids)
Magenta color
(Flavones)
- Anthraquinone glycosides are Ammonia Rose red layer
the ones whose aglycone
Ammonical layer indicates
component is a Diethyl ether
Modified antraquinone glycosides
polyhydroxyanthraquinone
borntrager test
derivative Ferric Chloride
- Stimulant cathartics
HCl
- Test to detect cholesterol Glacial acetic acid Bluish green color
Liebermann
(Steroidal glycosides)
burchard Presence of steroids
- Aka Acetic anhydride test Acetic anhydride

SPECIFIC TEST Sulfuric acid Lower reddish brown layer

- For cardiac glycosides
Lead acetate solution
Upper acetic acid layer that turns
Keller-Killiani test green
Chloroform
(2-Deoxy sugar)
Due to the presence of digitoxose
Glacial acetic acid

Ferric chloride
 Test for Saponins
Test For Reagent Positive result
• Presence of Saponin Saline solution Honeycomb-like foam or froth
Froth test (3cm, 30 mins)
Magnesium oxide
Test for Tannins and polyphenols
- For hydrolysable tannins Ferric chloride Blue black or green or blue
green solution or precipitate
3 kind of tannins: (hydrolysable tannins)
Ferric chloride test
1. Pseudo tannins
2. Hydrolysable tannins
3. Nonhydrolysable tannins
- or matchstick test Add HCl Pink or red color
- The matchstick contains lignin on the tootpick
- Catechin in the presence of
Test for catechins
acid produces phloroglucinol
with reacts with the lignin of
the wood

- Chlorogenic acid are esters of Add aqueous ammonia Green color

Test for polyphenolic caffeic acid and (upon exposure to air)
chlorogenic acid clyclitol

Test for plant acids

- For plant acids only
- Not for saponins because
Neutralization saponins possess soap-like
reaction qualities and produce a lather
when mixed with water
Boil in water bath Observe occurrence of
stable and dense froth
Do not perform if it
contains saponins Froth – small bubbles in liquid
caused by agitation,
Sodium carbonate fermentation
 Test for Proteins and Amino acids
Test For Reagent Positive result
• Test for amino acids, Triketohydrin Purple color
Ninhydrin test peptides in determining hydrate
amino acids
Biuret test • Tripeptides up to protein NaOH Pink to purple color
• Detect presence of Cupric sulfate
Alkaline peptide bonds Potassium tartrate
conditions, Cu2+ (Stabilize
forms violet complexes)
complex
Test for Alkaloids
Alkaloids possess N-containing hetero-cycle. They act as reservoir for protein synthesis and
detoxicating agents.
Reacts with potassium Potassium iodide Orange red or reddish
iodide in an acidic solution brown precipitate
Dragendorff’s
and forms potassium Glacial acetic acid
reagnet
bismuthiodide solution
Bismuth II nitrate
- Picric acid is a pale yellow Picric acid Yellow color precipitate
Hager’s reagent solution that is much
(Saturated stronger acid than phenol, Water
solution of picric it decomposes carbonates
acid) and may be titrated with
bases

Uses mercuric chloride and Mercuric Potassium Cream colored

Mayer’s Test
potassium iodide Iodide precipitate
 Gives yellow color
commonly amorphous
Valser’s test precipitates with alkaloids
except purine bases
Iodo-potassium iodide
Wagner’s
reagent Test for alkaloids even
purine bases
Potassium Iodide White precipitate
Mercuric iodide
Mercuric iodide teat
solution
Potassium Iodine in Reddish brown
Potassium iodide precipitate