PHAR301: Plant Cell wall materials and Ergastic cell contents

Asst. Prof. Dr. Emmanuel Mshelia Halilu

CYPRUS INTERNATIONAL UNIVERSITY

Faculty of Pharmacy
 THE CELL WALL
The cell-wall is a constant feature of all plant cells. Each cell is bounded by a non-living wall, thick or thin, according to
the nature of cell, forming an elastic or semi-rigid frame work around it. This frame work is called cell-wall. The cell-wall
maintains the form of the cell and provide requisite protection to protoplasm (i.e. the living content of the cell). Besides,
cell-wall form the skeleton of the plant body and are responsible for its strength, rigidity and flexibility. The cell-wall is a
laminated structure, i.e. it consists of layers laid down one against another by the protoplasm. The cell-wall is of two types.
They are, primary and secondary cell- walls.

A Simple diagram for a primary and secondary cell-wall

Chemical Nature of the Cell-Wall (cell wall materials)
The chemical substances of which the cell-wall is composed are mainly pectin, cellulose, lignin, cutin, suberin and mucilage.
Many mineral matters may also be introduced into the cell-wall.

Cellulose
Cellulose, is an insoluble carbohydrate, it is universally present in the cell-walls of all plant cells with the exception of fungi.
Infact, cellulose is the main constituent of the cell-wall.

Sometimes, cellulose is associated with hemicellulose and pectose at the stage of formation of primary wall, thereby making
the wall soft and elastic. Almost pure cellulose occur in the secondary wall formed later, making the wall stiff but flexible.
Later, cellulose may be associated with lignin, cutin, suberin, etc in a special cases.

Cellulose is a soft, elastic and transparent substance, and it is readily permeable to water but insoluble in it. Seed fibres like
cotton is made of pure cellulose.

Woody tissues are made predominantly of cellulose impregnated with lignin (lignocellulose). Chemically, cellulose is a
polysaccharide represented by the formula (C 6H10O5)n. Cellulose molecules are mostly long straight complex chains of D-
glucose units.
Chemical Tests for cellulose
Cellulose can be recognized by the reaction with the following reagents:

Chlor-zinc-iodine solution: True cellulose give a blue colour with chlor-zinc-iodine

Ammoniacal solution of copper oxide: True cellulose is dissolved by ammoniacal solution of copper oxide solution, and when
the alkaline liquid is poured into dilute H 2SO4, the cellulose is precipitated.

Uses of Cellulose
Cellulose is a very important substance in many respects. Many microorganisms utilize cellulose as food. They secrete an
enzyme cellulase to hydrolyse it. It forms major food to herbivorous animals.

Cellulose can not be digested by human beings. Articles like paper, cellophane, rayon (artificial silk) etc., are made from
cellulose for commercial use all over the world.
Hemicellulose
It is a mixture of different organic compounds (and not chemically allied to cellulose). It occurs in cell-walls which may
sometime be heavily thickened. It is hydrophilic (i.e., it adsorbs water). There is a thick deposit of hemicellulose in the cell-
wall endosperm of date palm seed and certain other palm seeds.

It is stored there as a reserved food for the use of the embryo, and it is often called reserve cellulose. When seed germinates, it
becomes converted into glucose and other compounds by the action of the enzyme, cytase.

Chemical Tests for Hemicellulose.

Hemiellulose can be recognized by the reaction with the following reagents:
Iodine solution: When hemicellulose is treated with iodine solution, it gives blue colour.
Ruthenium Red: Hemicellulose stains red with ruthenium red.

Pectic Compounds
Pectic compounds occur in plants in three forms: insoluble pectose (protopectin), soluble pectin and insoluble pectic acid. In
water, pectin swells into mucilage. It is responsible for the setting of jellies made from certain fruits.
Protopectin present in the primary cell-walls act as a binding or cementing material holding together the cells of the plant
body.

Chemical Tests for Pectic compounds

Pectic compounds can be recognized by the reaction with the following reagents:
Methyl blue: They give blue colour with methyl blue solution.
Chlor-zinc-iodine Solution: They give yellow colour with Chlor-zinc-iodine Solution.
Lignin
It is an organic compound of high carbon content, distinct from carbohydrates. It is a complex phenyl propanoid (C6-C3)
polymer with –O-CH3 and –OH groups.

Lignin is deposited in the meshes of the network formed by cellulose microfibrils in the cell-wall, and it is responsible for the
considerable thickening and strengthening of the secondary cell-wall, as in xylem elements.

It may however, be present in the middle lamella, primary wall and secondary wall, as in woody tissues, i.e. the whole wall
may be lignified. Lignin is a hard and chemically complex substance.

Lignified cells are usually thick-walled and always dead. Although hard, lignin is permeable to water. Sclerenchyma,
sclereids, bast fibres, tracheids, wood vessels and wood fibres are common lignified structures.

The Cell-wall can be lignified if it is incrusted with lignin. The process of conversion of cellulose into lignin is called
lignification .

Chemical Tests for lignin.

Lignin can be recognized by the reaction with the following reagents:
Aniline hydrochloride solution: Lignified cell walls stains bright yellow when treated with Aniline hydrochloride solution.
Safranin solution: Lignified cell-walls stains permanent red with Safarin solution.
Chlor-zinc-iodine Solution: Lignified cell-walls stains yellow or deep brown with Chlor-zinc-iodine Solution.
Phloroglucinol: Lignified cell-walls stains pink or reddish-violet when treated with phloroglucinol and followed by conc.
HCl.
Cutin
Cutin is a mixture of some pectic compounds. It forms a definite layer, sometimes of considerable thickness, called cuticle,
on the skin (outer surface of the epidermal layer) of the stem, leaf and fruit.

Cutin makes the cell-wall impermeable or very slightly permeable to water. Its function, therefore, is to prevent or check
evaporation of water from the exposed surfaces of the plant body and provide thermal insulation to the cell-wall. The
process of impregnation of the walls with cutin is known as cutinization, and the formation of cuticle as cuticularization.

Chemical Tests for cutin

Cutin can be recognized by the reaction with the following reagents:
3% Methanolic KOH Solution dissolves and saponifies cutin.
Sudan IV: Cutin gives red colour with sudan IV.
Tincture of alkanna: Cutin gives red colour with tincture of alkanna.
Chlor-zinc-iodine solution: Cutin gives brown or yellow colour with Chlor-zinc-iodine solution.
Suberin
The cell-walls of certain tissues may be charged with another waxy substance, called suberin. Like cutin, suberin is a mixture
of waxy substances and is therefore, allied to cutin.

The constituent fatty acid acids are however, different in two cases. Suberin occurs in the walls of cork cells and also in the
endodermis and exodermis of the roots of several plants. Being waxy in nature, it makes the cell-wall almost impervious to
water and therefore, like cutin, suberin prevents or check evaporation of water and provides thermal insulation to the cell-wall.
The phenomenon of impregnation of cell-wall with suberin is referred to as suberization.

Chemical Tests for Suberin

Suberin can be recognized by the reaction with the following reagents:
3% NaOH Solution dissolves and saponifies suberin.
Sudan IV: suberin gives red colour with sudan IV.
Tincture of alkanna: Suberin gives red colour with tincture of alkanna.
Chlor-zinc-iodine solution: Suberin gives brown or yellow colour with Chlor-zinc-iodine solution.
Mucilage (mucilagenous cell-wall)
It is a complex carbohydrate. It is a slimy substance widely distributed in various parts of plants. It absorbs water greedily,
retains it tenaciously and forms a viscous mass.

Chemical Tests for mucilage

Mucilage can be recognized by the reaction with the following reagents:
KOH solution: Mucilage cell-wall swell when treated with KOH solution.
Chlor-zinc-iodine solution: Mucilage cell-wall stains blue or purple with Chlor-zinc-iodine solution.
Ruthenium red: Mucilage cell-wall stains pink with Ruthenium red.
Corallin Soda: Mucilage cell-wall stains red with corallin soda.

Callose Cell-wall
Certain cell-walls are covered with a membranous adcrusting carbohydrate substance called callose. It is composed of a kind
of hemicellulose which is a polymer of β-glucose units linked 1:3 carbon atoms.

Chemical Tests for callose

Callose is soluble in 5% alkali solutions and stains red with corallin soda and stains blue with aniline blue.
 CELL INCLUSIONS (Ergastic Substances)
INTRODUCTION
Various chemical substances occur as non-living inclusions in the cytoplasm as products of metabolism or by-products.
Ergastic substances are non-protoplasm materials found in cells. Ergastic substances may appear in the protoplasm, in
vacuoles, or in the cell wall. These compounds include: Carbohydrates, Proteins, Fats and Oils, Alkaloids and purines,
Glycosides, Volatile oils, Gums and Mucilages, Resins, Tannins, Calcium oxalates, Calcium carbonate and Silica. These
substances, being non-living, they are referred to as ergastic substances.

In Pharmacognosy, the cell contents with which we are concerned are those which can be identified in vegetable drugs by
microscopical examination, chemical and physical tests.

Cell inclusions are very important in drug identification because, the cell inclusions of a given drug are of fairly constant shape
and size range, yet they differ from those of other drugs.
Principally, the cell inclusions are classified as follows:
a) Food products
b) Secretory products
c) Waste products.
Food Products
They are substances manufactured by the protoplasm of the cell from simple inorganic substances like carbon dioxide and
water and are stored in the cells as food materials. The food thus manufactured is partly used up to make new protoplasm and
partly its broken down to provide necessary energy, and the remainder of it is stored as reserve food material in the protoplasm
of cells. The reserve food materials may be classified as follows:

Carbohydrates
They are non-nitrogenous food products. They contain Carbon, Hydrogen and oxygen. Among these elements, hydrogen and
oxygen occur in the same proportion as they do in water. The general chemical formula of carbohydrates is C x(H2O)y. They are
derived more or less directly from carbon dioxide (CO 2) and water (H2O) during photosynthesis. Some carbohydrates are
insoluble while some are soluble in water. The most important insoluble product is starch and the soluble products are inulin,
sugar, etc.
 Starch grains: 1. Compound starch gain. 2.
Simple starch grain. 3. From cotyledon of
Pisum sativum seed. 4. From flesh of Musa
spp. 5. From tuberous root of Ipomoea batatas.
6. From endosperm of Oryza sativa grains. 7.
From endosperm of Zea mays grains. 8. From
cotyledon of Cicer seed. 9. From tuber of
Solanum tuberosum. 10. Same in situ.

Diagrams of the various forms of starch grains

Note: The position and form of the hilum, and the presence or absence of well-defined striations, are the most
important features of starch grains. For the microscopical examination of starch in drugs, the sample must be
mounted in glycerol or cresol as starch is soluble in chloral hydrate solution.
 Inulin
This a carbohydrate of the polysaccharide type. It is a soluble carbohydrate, usually found in the cell wall. Inulin has been
reported from the roots of many Compositae. It is commonly found in the tuberous root of Dahlia (a plant) and Helianthus
tuberosus. It can easily be precipitated by keeping the Dahlia roots for six to seven days, in ethanol, in the form of spherical,
Star-shaped or wheel-shaped crystals. These are observed when viewed under microscope. Inulin has the same chemical
composition as starch, viz. (C6H10O5)n.

NITROGENUOS FOOD PRODUCTS

The important nitrogenous food materials are proteins and amino acids.
Proteins
Proteins are very complex, nitrogenous, organic compounds, essentially containing carbon, hydrogen, Nitrogen and oxygen. All
plant proteins also contain sulphur and complex ones contain phosphorus too. Of all the organic compounds, with the exception
of protoplasm and nucleus, proteins have the most complex chemical composition, and various kinds are found in the plant
body. A common form of protein found in the endosperm of castor seed is known as aleurone grains.

Each aleurone grain is a solid, ovate or rounded body which usually encloses a crystal-like body in it, known as crystalloid and
a globule like body known as globoid. The crystalloid is proteinaceous in nature and occupies the major portion of aleurone
grain, whereas the globoid is double phosphate of calcium and magnesium and occupies the narrower part of the grain. The
aleurone grains vary in their shape and size in different plants. When they are in oily seed of castor they are larger in size,
while when found with starch they are very minute in size.
Diagrams of aleurone grains
Aleurone in Maize seed
Aleurone in Castor seeds under lıght Mıcroscope
Test for proteins
Iodine solution: Proteins turn yellowish brown with strong iodine solution.

Xanthoproteic reaction: A precipitate is formed with the addition of some strong nitric acid. It turns yellow on boiling. After
cooling, add a little strong ammonia and the yellow colour changes to orange.

Millon’s reaction: Add millon’s reagent (nitrate of mercury) and a white precipitate is formed; on boiling, it turns brick red.

Biuret reaction: Add an excess of caustic soda (NaOH) followed by a few drops of copper sulphate (CuSO 4) produces a violet
colour, which deepens on heating.

Test for protein from castor seed

Treat a thin section of castor endosperm with 90% ethanol for 3-4 minutes and then with strong iodine solution. Mount it in
thick glycerine and note under the microscope that the aleurone grains and cystalloids turn deep brown, while the globoids
remain colourless. Add 1% or 2% caustic soda solution to a fresh section and note that the aleurone grains get dissolved, while
the globoids remain unaffected. Treat another section with dilute acetic acid (ethanoic acid) and observe that only the globoids
get dissolved.
AMINO COMPOUNDS
They are simple nitrogenous food materials. They are found in the form of amino acids and amines which occur in the cell sap.
These are abundantly found in the growing apices of the plants, while less frequently occur in storage tissues. There are about
25 known amino acids. Amino acids are peculiar in that, they have both basic and acidic properties. They are constituted of
carbon, hydrogen, nitrogen and oxygen.

They have the formula R-CH(NH2)-COOH, where R is an alkyl group. An amino group is always attached to the carbon atom
next to the carboxyl group. In amino acids like cystine and methionine, sulphur is also present. Some of the other amino acids
are glycine, alanine, glutamic acid, aspartic, leucine, lysine, tyrosine, etc.

In plants, when translocation is necessary, proteins become converted into amines and amino acids. The amines and amino
acids travel to the growing regions, where the protoplasm is very active, and they are directly assimilated by it. They are also
the initial stages in the formation of proteins.
FATS AND FATTY ACIDS (Fixed oils)
The fats and fatty oils in plants are composed of glycerine and organic acids. They occur in the form of minute globules in the
protoplasm. Special types of fats and fatty oils are found in the seed and fruits of flowering plants. Fats and fatty oils form an
important reserve food with a considerable amount of energy stored in them. Their energy value is more than double of that of
carbohydrate. When fats are decomposed, the energy stored in them is liberated an utilized by protoplasm for its manifold
activities. Fats and oils are composed of carbon, hydrogen and oxygen. They are characterized by a small percentage of
oxygen as may be seen from the formula of such common fats as stearin- C 57H110 O6 and palmitin-C51H98O6, olein-C57H104O6,
and linolein-C57H98O6. Owing to the very small percentage of oxygen contained in fats, the oxidation of fats produces large
amounts of energy. Fats and fatty oils are insoluble in water but are soluble in ether, chloroform and petroleum.
Comparatively few of them are soluble in ethanol e.g castor oil. Fats that are liquid at ordinary temperature are known as oils.
In plants, fats are usually present in the form of oils. Oils are of two kinds, they are: Fixed (or non-volatile) oils and essential
(or volatile) oils. Fats may be solid or liquid (fatty oils) according to the temperature.
Tests for fats and oils
Osmic acid (1% aqueous solution) stains black.
Alcoholic solution of sudan (iii)and sudan (iv) stains them red.
Alcoholic solution alkanet (or alkanin) stains them red.
They leave permanent greasy mark on it when pressed against paper.
Secretory Products
Besides food materials, several other products are also secreted by protoplasm which are not useful as nutritional products but
they may help or accelerates the various reactions in the cell. The secretory products are as follows:
Enzymes
They are soluble nitrogenous substances secreted by the protoplasm. They are digestive in function and convert the insoluble
substances into soluble and complex compounds into simpler ones, e.g., diastase converts starch into sugar. Because of the
action of this enzyme, an insoluble substance is converted soluble one. The enzyme lipase breaks up fats into their components,
glycerine and fatty acids. Papain converts proteins into amino acids.
Colour in cell
The substances that give colour to cells are usually found in the plastids. Chlorophyll is a green colouring matter secreted by
chloroplasts and performs the phenomenon of photosynthesis. Chlorophyll is not a single but it is a mixture of two pigments
known as chlorophyll-a and chlorophyll-b. The yellow pigments, carotenoids are also found in cell sap and give colours to the
petals of the flowers. The anthocyanins are also secretory products of the protoplasm and are stored in the cell sap; they also
impart colour to the petals of the flowers.
Nectar
The nector is another useful secretion of protoplasm. It is secreted by special glands or organs of the flower, called nectaries.
Nectar is a sweet substance secreted by special glands, nectaries, in flowers and certain leaves.
Osmophors
The fragrance of flowers, is generally produced by volatile substances distributed throughout the epidermal region of the
perianth parts. However, In some plants, the fragrance originates in special glands known as osmophors.
Examples of such special glands are found in the family of plants such as Asclepiadaceae, Aristolochiaceae, Araceae,
Burmanniaceae and Orchidaceae.

Essential or volatile oils

The essential or volatile oils frequently occur in oil glands. These oils are volatile and are usually very odouriferous. Well
known examples are eucalyptus oil, oil from orange peel and and oil from lemon. They are also present in the petals of the
flowers of many plants e.g jasmines. The fragrant odour of such flowers is due to the presence of essential oils formed in
them. The essential oils differ from the fatty oils in their chemical composition, as well as in being volatile. They are
sufficiently soluble in water to impart their taste and odour. Being volatile, essential oils are obtained by mere application of
pressure. Like fatty oils, they are readily soluble in ether, petroleum, etc. They are soluble in alcohols but fixed oils are not.
Other examples of volatile oil include: lemon grass oil, clove oil, lavender oil, sandal wood oil and thyme oil.

Oleoresins (resins)
The term ‘resin’ is applied to more or less solid amorphous product of complex nature. On heating, they soften and finally
melt.
Resins are often associated with volatile oils and known as oleoresins (oleo means oil). When resins are associated with gums,
they are known as gum resins or when resin is associated with oil and gum it is known as oleo-gum-resin. Mastic and
ammoniacum, which are usually considered as a resin and gum resin respectively, both contain volatile oil. Resins, oleoresins
and gum resins are usually secreted into secretory cavities or ducts. They stain slowly with dilute tincture of alkanna.
Mucilage
Mucilage is a slimy substance widely distributed in various parts of plants. Chemically, it is a complex carbohydrate. Its
physical property is that it adsorbs water greedily, retains it tenaciously and forms a viscous mass. But when dry, it is very hard
and horny. Mucilage is abundantly found in the fleshy leaves of Indian aloe. It is also abundant in the flower of china rose, in
fruits of lady’s finger, in the branches and leaves of Indian spinach, and in the seeds of linseed (linum), flea seed (plantago).
Mucilage also occur in the fleshy leaves of desert plants.

Test for mucilage

The official solution of Ruthenium red stains the mucilage of senna and buchu leaves, althaea, linseed and mustard. A lead
acetate medium can be used to prevent undue swelling or solution of the substance being tested. Some forms of mucilage are
stained by the BP alkaline solution of corallin e.g that found in squill. Others are stained by chlor-zinc-iodine or methylene blue
dissolved in alcohol and glycerine.
WASTE PRODUCTS
They are excretion of the plant cells. Usually these products are stored in the dead cells. These products are formed as a result
of metabolic activities of the protoplasm, and therefore, they are known as metabolic wastes of plants. Usually there are two
types of metabolic wastes. They are:
Non-nitrogenous waste products
Nitrogenous waste products.

Non-nitrogenous waste products

The non-nitrogenous waste products include tannins, calcium carbonate, calcium oxalate crystals, latex, gums resins and
organic acids.

Tannins
They are non-nitrogenous complex compounds, commonly found dissolved in the cell sap. They are derivatives of phenol and
usually glycosides. They are found in the cell walls, in the dead cells, in the heart wood and in the bark. They are also found
in abundance in the leaves and unripe fruits. The tannins possess a bitter taste and their presence in the tea leaves make the tea
decoction bitter. They are used in tannin industry. As regards their function, they act as protectants of protoplasm against
injury, decay, termites and pests; as reserve substances related in some manner to the starch metabolism; as substances
associated with the formation and transport of sugars; as antioxidants; and as protective colloids maintaining the homogeneity
of the cytoplasm. Tannins turn blue-black with iron salt e.g., ferric chloride (FeCl 3).
Mineral crystals
The mineral crystals include: calcium oxalate crystals, calcium carbonate and silica. They occur in the cell cavity or in the cell
wall. The crystals of calcium oxalate are the most common and are very widely distributed among various plants.

Calcium oxalate
Oxalic acid (C2O4H2) rarely occurs in the free state in most plants but extremely common as its calcium salt in the form of
crystals. It is conspicuously seen on the leaves and other organs of the plants. The calcium oxalate occur in various forms as:

Raphides: They are needle-like, long slender crystals usually lying parallel to each other in a bundle, which is sometimes
found in a special sack-like cell. The raphides can also occur singly. The raphides are commonly found in water hyacinth,
balsam, water lettuce and aroids, such as Colocasia, Alocasia and Amorphophallus. They are frequently shut off by a cell wall
and prevented from coming in contact with the protoplasm.

Druses, Rosette Crystals or Cluster crystals (i.e., Conglomerate crystals or Sphaero-crystals): This is one of the commonest
compound crystal having the appearance of a rosette. This cluster of crystals radiate from a common centre. They are found in
water lettuce (pistia), taro (colocasia), eucalyptus, nerium, etc.

Prismatic Crystals: They are single calcium oxalate crystals found in various plants. They may be square, prismatic,
rhomboidal or pyramid-like in shape. They can be readily seen in dry scales of onions.

Idioblasts: They are stellate calcium oxalate crystals usually found in the aerenchyma of aquatic plants to give support to the
tissue. They are quite common in Limnanthemum, Nymphaea, Atropa, etc.
Calcium Carbonate
This may be found embedded in or incrusted in the cell walls. It occurs in the form of a crystalline mass, often pear-shaped in
appearance, in the leaf of ficus. This crystalline mass (CaCO 3) is called cystoliths. Cystoliths are found in many plants of
Acanthaceae, Moraceae, Urticeae, Ulmaceae, Cannabinaceae, etc. Also well formed cystoliths are seen in enlarged upper
epidermal cells and in the clothing hairs of the lower epidermis of the leaf of Cannabis sativa. When the mineral substance of
the cystoliths is dissolved out in dilute acid, there remains a small, often stratified, basis composed of cellulose.

Tests for Calcium carbonate

Calcium carbonate can be identified by the fact that it dissolves with effervescence in acetic acid (ethanoic acid), hydrochloric
acid or sulphuric acid. If 50% H2SO4 is used, a needle shaped crystals of calcium sulphate (CaSO 4) gradually separate out.

Silica
It is deposited mostly in the cell wall, but some times it forms bodies in the lumen (cavity) of the cell. The Gramineae is the
best known example of a plant group having silica in both the cell walls and the cell lumina. Silica is abundantly found in the
leaves and stem of horsetail (Equisetum), several grasses, e.g., lemon grass, rice straw, wheat straw, etc., and in several algae
with silicified structures, e.g., diatoms. Silica is insoluble in all acids except hydrofluoric acid (HF). It may be examined by
igniting the material and treating the ash with hydrochloric acid, the silica remaining unaltered.
Thank you