Pharmacognosy: Ayurvedic treatments

Pharmacognosy: Ayurvedic treatments: Acidity After the meals, take one piece of Clove (Laung) and suck on it slowly. This gives instant relief and helps i...

Ayurvedic treatments

Acidity	After the meals, take one piece of Clove (Laung) and suck on it slowly. This gives instant relief and helps in reducing other problems arising out of acidity.
Weight loss	Eat a tomato every day before your breakfast for a couple of months. This really helps. Next, drink a half glass of Nimbu Pani (lemon juice in water) every morning before tea.
Toothache	Bite a clove. Clove oil is an excellent remedy for toothaches.
Indigestion	Drink 1 tsp. of mint juice mixed with an equal amount of honey and lemon juice.
Cough & Cold	Dissolve a 1/2 tsp. of turmeric powder in half a cup of hot sweetened milk and drink for relief.
Chest Congestion	Boil tea leaves, mint & a bit of ginger. Mix with a dash of honey and drink it hot for relief.
Mouth Sores	Sprinkle a mixture of powdered cardamom and rock sugar (misri) over the tongue.
Bee Bite	Rub the area with a paste of soft mud and salt in order to avoid swelling.
Burns & scalds	Place a crushed onion on the affected part & cover with a bandage.
Stains in teeth	Add a drop of clove oil to the toothpaste before brushing your teeth OR Sprinkle a little table salt on your toothpaste before brushing your teeth.
Constipation	Have an apple or guava to cure constipation.
Nausea	Keep whole cardamom or clove in the mouth to subside the sensation of vomiting.
Acne & Pimples	Do not try to prick and peel the acne as this can leave permanent marks on the face. Rub the whole nutmeg with milk on a grinding stone and apply this paste. Leave until dry then wash.
Constipation	Take 10/12 pieces of Raisin (Munnakka), wash and remove the seeds and then boil them with a glass of milk. Drink this milk and eat the raisins. The next day you will have normal bowel movements.
Earache & infection	Take two cloves of Garlic. Remove the skin. Put them in two spoons of Mustard Oil in a small pan and warm it up on low heat. When the garlic starts turning black (burning) then turn off the heat. Sieve the contents. When the oil becomes lukewarm, then using a cotton stub, put 2 to 4 drops in the aching ear.
Throat Irritation	Take a teaspoon of table salt and mix with a cup of hot water. Gargling with this mix 3 - 4 times a day is helpful in giving relief to throat irritation.
Weak Memory	Take a few almonds and immerse them in a cup of water in the evening. The next morning, after removing the red skin, grind the almonds. Then mix the ground almonds with a glass of milk and boil. When it has boiled, then mix a spoonful of Ghee and one spoonful of sugar. When it is lukewarm, then drink it. Do this for 15 to 40 days. It helps in improving memory and also brings vitality to the body.
Cold	Drink a glass of warm water with 2-3 drops of lime juice. Do not eat anything for at least half an hour before & after taking it.
Wounds	Apply turmeric powder to the wound. It will stop the bleeding immediately and the wound will also heal faster.
Diarrhea	Take a teaspoon of ginger juice mixed with a pinch of salt.
Insect bites and stings	Wash the affected area with vinegar.
Cough	Take a spoonful of ginger juice mixed with equal parts of honey two or three times a day.
Itchy throat	Take honey. It will relieve the itchiness as it moisturizes the throat.

My self Dr Sandeep

I am Dr. Sandeep welcomes you all to visit my blogs, I will be concentrating mainly on Pharmacognosy, Phyto-Chemistry, and Biological activity as well I will focus about- 

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Quality control methods for medicinal plant materials

Quality control methods for medicinal

plant materials

My dear, I have taken this all material from  Quality control methods for medicinal plant materials, WHO Library Cataloguing in Publication Data, World Health Organization 1998

 Precision of measurement

Quantities and volumes

The quantities and volumes of the materials and reagents used in the tests must be measured with adequate precision, which is indicated in the following way:

A value of: 

20.0 means not less than 19.5 and not more than 20.5 

2.0 means not less than 1.95 and not more than 2.05

0.20 means not less than 0.195 and not more than 0.205.

Temperature

Temperature measurement is indicated in a manner similar to that given for quantities and volumes.

Storage conditions given in general terms refer to the following equivalent temperatures:

In a refrigerator                                     0-6 °C

Cold or cool                                           6-15 °C

Room temperature                                  15-25 °C,

or up to 30°C depending on climatic zones.

 

Solubility
Unless otherwise specified in the test procedure for the plant material concerned, the approximate solubility of medicinal plant materials should be determined at 20°C. Solubility is expressed in terms of "parts", representing the number of millilitres (ml) of the solvent, in which 1 g of the solid is soluble. Descriptive terms are sometimes used to indicate the solubility of a substance, with the following meanings:

 

very soluble                                  less than 1 part

freely soluble                               1-10 parts

soluble                                          10-30 parts

sparingly soluble                          30-100 parts

slightly soluble                             100-1000 parts

very slightly soluble                     1000-10 000 parts

practically insoluble                      more than 10000 parts

 

Storage

Medicinal plant materials must be stored under specified conditions in order to avoid contamination and deterioration.

Containers
The container and its closure must not interact physically or chemically with the material within in any way that would alter its quality. The following descriptive terms are used to indicate general requirements for the permeability of containers:

A well-closed container must protect the contents from extraneous matter or from loss of the material under normal conditions of handling, shipment, or storage.
A tightly closed container must protect the contents from extraneous matter, from loss of the material, and from efflorescence, deliquescence, or evaporation under normal conditions of handling, shipment, or storage. If the container is intended to be opened on several occasions, it must be designed to be airtight after reclosure.

Protection from light

Medicinal plant materials requiring protection from light should be kept in a light-resistant container that - either by reason of the inherent properties of the material from which it is made or because a special coating has been applied to it - shields the contents from the effects of light. Alternatively, the container may be placed inside a suitable light-resistant (opaque) covering and/or stored in a dark place.

Temperature
Materials that need to be stored at temperatures other than room temperature should be labelled accordingly.

Humidity

Low humidity may be maintained, if necessary, by the use of a desiccant in the container provided that direct contact with the product is avoided. Care must be taken when the container is opened in damp or humid conditions.

Size of cut

Medicinal plant materials are used either whole, or in cut or powdered form. Cut medicinal plant materials are prepared by cutting or crushing the plant into small pieces. The cut is graded according to the aperture size of the mesh of the sieve through which the material will pass, and is indicated as follows:

 

Aperture size                      (mm)

coarse cut                           4.00

medium cut                        2.80

fine cut                               2.00

 

Powder fineness and sieve size

Powders
The coarseness or fineness of a powder is classed according to the nominal aperture size expressed in hum of the mesh of the sieve through which the powder will pass, and is indicated as follows:

Descriptive term Particle size

Coarse (2000/355) All the particles will pass through a No. 2000 sieve, and not more than 40% through a No. 355 sieve

Moderately coarse (710/250) All the particles will pass through a No. 710 sieve, and not more than 40% through a No. 250 sieve

Moderately fine (355/180) All the particles will pass through a No. 355 sieve, and not more than 40% through a No. 180 sieve

Fine (180) All the particles will pass through a No. 180 sieve

Very fine (125) All the particles will pass through a No. 125 sieve

Sieves

The wire sieves used to sift powdered medicinal plant materials are classified by numbers that indicate their nominal aperture size expressed in μm. The sieves are made of wire of uniform circular cross-section, and have the following specifications:

Number of sieve Nominal size of aperture  Nominal diameter of wire.    

Approximate screening area

(μm)                      (mm)                                     (mm)                                       (%)

2000                      2.00                                        0.90                                         48

710                        0.710                                      0.450                                       37

500                        0.500                                      0.315                                       38

355                        0.355                                      0.224                                       38

250                        0.250                                      0.160                                       37

212                        0.212                                      0.140                                       36

180                        0.180                                      0.125                                       35

150                        0.150                                      0.100                                       36

125                        0.125                                      0.090                                       34

90                          0.090                                      0.063                                       35

75                          0.075                                      0.050                                       36

45                          0.045                                      0.032                                       34

The sieves recommended here have been selected from among those conforming to ISO standard 565, 1990.

Determination of foreign matter

Medicinal plant materials should be entirely free from visible signs of contamination by moulds or insects, and other animal contamination, including animal excreta. No abnormal odour, discoloration, slime or signs of deterioration should be detected. It is seldom possible to obtain marketed plant materials that are entirely free from some form of innocuous foreign matter.

However, no poisonous, dangerous or otherwise harmful foreign matter or residue should be allowed.
During storage, products should be kept in a clean and hygienic place, so that no contamination occurs. Special care should be taken to avoid formation of moulds, since they may produce aflatoxins.

Macroscopic examination can conveniently be employed for determining the presence of foreign matter in whole or cut plant materials. However, microscopy is indispensable for powdered materials.

Any soil, stones, sand, dust and other foreign inorganic matter must be removed before medicinal plant materials are cut or ground for testing.

Foreign matter is material consisting of any or all of the following:

- parts of the medicinal plant material or materials other than those named with the limits specified for the plant material concerned;

- any organism, part or product of an organism, other than that named in the specification and description of the plant material concerned;

- mineral admixtures not adhering to the medicinal plant materials, such as soil, stones, sand, and dust.

Sample size

It is difficult to prepare a pooled sample of foreign matter since most of it adheres to the medicinal plant materials which are intrinsically non-uniform.

Special procedures requiring considerable practice are therefore necessary. The problem is especially difficult when the samples of unbroken crude medicinal plant materials selected are small; they should be sufficiently large to be representative.

The following quantities constitute a sample, unless otherwise specified in the test procedure for the plant material concerned.

Plant material Sample size

roots, rhizomes, and bark                                                                                                500 g

leaves, flowers, seeds, and fruit                                                                                      250 g

cut medicinal plant materials (average weight of each fragment less than 0.5 g)         50g

Visual examination and odour

Wrinkled and contracted leaves, herbs or flowers should be softened and stretched flat ("Preliminary treatment"). Certain fruits and seeds may also require softening before dissection and observation of internal characteristics. No other preliminary treatment is required.

Recommended procedures

Size
A graduated ruler in millimetres is adequate for the measurement of the length, width and thickness of crude materials. Small seeds and fruits may be measured by aligning 10 of them on a sheet of calibrated paper, with 1 mm spacing between lines, and dividing the result by 10.

Colour

Examine the untreated sample under diffuse daylight. If necessary, an artificial light source with wavelengths similar to those of daylight may be used. The colour of the sample should be compared with that of a reference sample.

Surface characteristics, texture and fracture characteristics Examine the untreated sample. If necessary, a magnifying lens (6x to 10x) may be used. Wetting with water or reagents, as required, may be necessary to observe the characteristics of a cut surface. Touch the material to determine if it is soft or hard; bend and rupture it to obtain information on brittleness and the appearance of the fracture plane - whether it is fibrous, smooth, rough, granular, etc.

Odour

If the material is expected to be innocuous, place a small portion of the sample in the palm of the hand or a beaker of suitable size, and slowly and repeatedly inhale the air over the material. If no distinct odour is perceptible, crush the sample between the thumb and index finger or between the palms of the hands using gentle pressure. If the material is known to be dangerous, crush by mechanical means and then pour a small quantity of boiling water onto the crushed sample in a beaker. First, determine the strength of the odour (none, weak, distinct, strong) and then the odour sensation (aromatic, fruity, musty, mouldy, rancid, etc.). A direct comparison of the odour with a defined substance is advisable (e.g. peppermint should have an odour similar to menthol, cloves an odour similar to eugenol).

Taste
N.B. This test should be applied only if specifically required for a given plant material.
Inspection by microscopy

Once the material has been examined and classified according to external characteristics, inspection by microscopy can be carried out as the next step.

Equipment
The following are required: - a microscope equipped with lenses providing a wide range of
magnification and a substage condenser, a graduated mechanical stage, objectives with a magnification of 4x, 10x, and 40x, and colour filters of ground glass, blue-green; high eyepoint eyepieces are preferred for wearers of spectacles;

- a lamp, either separate or incorporated into the microscope;

- a set of polarizing filters;

- a stage micrometer and an ocular micrometer to be inserted into a 6x eyepiece and placed

on the diaphragm or, preferably, a micrometer eyepiece;

- a set of drawing attachments for the microscope;

- a microburner (Bunsen type);

- slides and cover-glasses of standard size;

- a set of botanical dissecting instruments.

Recommended procedures

Preliminary treatment

Select a representative sample of the material. Dried parts of a plant may require softening before preparation for microscopy, preferably by being placed in a moist atmosphere, or by soaking in water. For small quantities of material, place a wad of cotton-wool moistened with water into the bottom of a test-tube and cover with a piece of filter-paper. Place the material being examined on the paper, stopper the tube and allow to stand overnight or until the material is soft and suitable for cutting. Use a desiccator for larger quantities of material, placing water into the lower part instead of the drying agent. Bark, wood and other dense and hard materials usually need to be soaked in water or equal parts of water, ethanol and glycerol for a few hours or overnight until they are soft enough to be cut. Boiling in water for a few minutes may sometimes be necessary.

Any water-soluble contents can be removed from the cells by soaking in water. Starch grains can be gelatinized by heating in water. In certain cases, material can be moistened with water for a few minutes to soften the surfaces and allow sections to be cut.

Preparation of specimens

Powdered materials

Place 1 or 2 drops of water, glycerol/ethanol TS or chloral hydrate TS on a glass slide. Moisten the tip of a needle with water and dip into the powder. Transfer a small quantity of the material that adheres to the needle tip into the drop of fluid on the slide. Stir thoroughly, but carefully, and apply a cover-glass. Press lightly on the cover-glass with the handle of the needle, and remove excess fluid from the margin of the cover-glass with a strip of filter-paper. Other fluids may be used, if necessary, in the same manner.

If the specimen is to be freed from air bubbles, boil carefully over a small flame of a microburner until the air is completely removed. Care should be taken to replace the fluid that evaporates so that the space beneath the cover-glass is completely filled with fluid at the conclusion of the operation.

Surface tissues of leaves and flowers

To render pieces of thin leaves transparent, boil them directly on a slide. Cut a piece of leaf into two portions, turn one piece upper side down and add chloral hydrate TS. Boil the specimen carefully over a small flame of a microburner and, as soon as bubbles escape, remove the slide from the flame. When the bubbles have ceased to appear, boil again until the fragments are transparent.

For slightly thicker but still papery leaves, cut square pieces, about 6 mm from the edge of the leaf, if not otherwise specified. The pieces should be taken onethird to one-half of the way from the leaf-base and should include a midrib or large vein. In addition, cut 1 or 2 pieces from the edge including 1 or 2 indentations, where appropriate. For broken or cut leaves take suitable fragments as described above. Place the fragments in a test-tube containing chloral hydrate TS and boil for a few minutes until they become transparent. Transfer a fragment to a slide and cut it into two equal portions. Turn one piece upper side down and align the two pieces so that both upper and lower surfaces can be observed under the microscope. Add 1-2 drops of chloral hydrate TS and apply a cover-glass.
For thicker leaves, that do not become transparent enough when prepared by the method described above, clarify fragments by boiling with chloral hydrate TS in a test-tube. Transfer a fragment onto a slide, cut it into two equal portions and turn one portion upper side down. Scrape the surface of the two portions using a scalpel until only a single layer of epidermis remains. Wash the epidermis with drops of chloral hydrate TS or glycerol /ethanol TS to remove any residues. If possible, turn both parts of the epidermis upper side down, and add one of the above fluids.

For very thick or fleshy leaves, pull off the upper and lower parts of epidermis by winding the softened leaf around the index finger, pressing with the thumb and the middle finger against the index finger and carefully incising, catching the incised part with forceps, and bending the epidermis backwards carefully.

Petals and sepals of flowers may be treated in a similar manner.

Sections

Select representative pieces of the material being examined and cut into suitable lengths, one end of which is softened and smoothed. Prepare cross or transverse sections by cutting with a razor blade or microtome at a right angle to the longitudinal axis of the material. Prepare longitudinal sections by cutting in parallel with the longitudinal axis, either in a radial direction (radial section) or in a tangential direction (tangential section).

Thick materials, such as wood, woody stems, rhizomes and roots can be cut by holding the softened material between the thumb and index finger, supported by the middle finger or by holding it in the central hole of a hand microtome.

Thin materials such as leaves, petals and slender stems should be bound between two halves of a piece of elder-pith or other suitable support. If necessary, moisten the surface to be cut and the blade with ethanol (∼375 g/l) TS. Cut the sections as thinly and evenly as possible. Transfer the sections with a brush moistened with ethanol (∼150g/l) TS to a dish containing ethanol (∼150g/l) TS. Select satisfactory sections for the preparation of the slides. For certain materials a sliding microtome may be used. Seeds and fruits that are very flat, or that are small and spherical, and cannot be held in the manner described above may be inserted into a notch cut into a small rubber stopper or embedded in hard paraffin (paraffin wax) as follows. Prepare a hard paraffin block, rectangular in shape, measuring about 7 x 7 x 15 mm, and melt a small hole in the centre of one end using a heated needle or thin glass rod. Press the material, which should be dry or softened by exposure to moisture, into this hole. Then prepare sections with a microtome. For the examination of mucilage, aleurone grains or spherical aggregations of inulin, cut the material without using water.

Clarification of microscopic particles

The presence of certain cell contents, such as starch grains, aleurone grains, plastids, fats and oils, may render sections non-translucent and obscure certain characteristics. Reagents that dissolve some of these contents can be used in order to make the remaining parts stand out clearly or produce a penetrating effect. This renders the section more transparent and reveals details of the structures.

If the refractive index of the clarifying agent is close to that of the cell structure, the material being tested becomes almost invisible; if it differs appreciably, the material becomes markedly evident.

The most frequently used clarifying agents are described below (for the methods of preparation, see section 22, "Reagents and solutions").

Chloral hydrate TS

On gentle heating chloral hydrate TS dissolves starch grains, aleurone grains, plastids, and volatile oils, and expands collapsed and delicate tissue without causing any undue swelling of cell walls or distortion of the tissues. It has a refractive index ( n ) of 1.44 -1.48. It is the best reagent for rendering calcium oxalate clearly evident and is particularly useful for small crystals. However, when allowed to stand, it slowly dissolves calcium oxalate, owing to an increase in acidity.

 Measurement of specimens

Equipment
Use a microscope with an ocular micrometer to measure the size of small objects. The scales should be calibrated using a stage micrometer, consisting of a glass slide of usual size, upon which a scale is engraved or photographed, usually 1 or 2 mm long, in 0.1 and 0.01 mm divisions. The ocular micrometer consists of a small disc of glass, across the diameter of which a 100-line scale is engraved or photographed. The disc is placed into the eyepiece.

Calibration of the ocular micrometer

Place the ocular micrometer scale in the eyepiece of the microscope. Put the stage micrometer on the microscope stage and focus on the scale divisions. Both scales should be sharply defined in the field of view. Turn the eyepiece to place the scales in a parallel position and, if necessary, move the stage micrometer until the starting lines of both scales coincide (Fig. 1). Now find another point, as far along the scale as possible, where two other division lines are exactly superimposed. Count the number of divisions on the ocular micrometer and the corresponding length on the stage micrometer scale, in order to determine the length that is equivalent to one division on the ocular micrometer scale; for example, if 100 divisions on the ocular micrometer scale are equal to 30 divisions on the stage micrometer scale, since the divisions on the stage micrometer scale are 0.01 mm apart, 100 ocular micrometer divisions are equivalent to 0.30 mm, therefore each division on the ocular micro-meter scale represents 3.0 μm. Since the calibrations apply only for a particular lens combination, it is advisable to determine and record the ocular micrometer values for the most frequently used combinations.

Alignment of the stage micrometer and the ocular micrometer 

Method
Place the specimen on the microscope stage and focus on the object to be measured. Superimpose the ocular micrometer scale and read off the dimensions of the object. Multiply the number of scale divisions by the micrometer value to give the actual dimension in micrometres. By this method, using a 40x objective and a 6x eyepiece, measurements are correct to the nearest 2 μm, i.e. a dimension of 20 μm is liable to an error of 2 μm, or 10%, or ±5%; a dimension of 100 μm is liable to an error of 2% or ±1%.

For curved and elongated objects, the measurement of lengths may be made using a microscope equipped with a drawing apparatus or a camera lucida. The instrument should be set up so that the image of the object, the drawing paper and the pencil are in focus simultaneously. With the stage micrometer in place, trace the lines of the stage micrometer scale upon the paper fastened on a drawing board. Tilt the drawing board, if necessary, until the divisions drawn upon the paper are equally spaced. The magnification is determined by measuring the distance between selected lines on the drawing paper and dividing by the distance between the corresponding lines on the stage micrometer. Place the specimen on the microscope stage and trace the image of the object on the paper. Superimpose a coloured thread along the length of the object drawn on the paper and after straightening it, measure the length of the thread using a ruler graduated in millimeters; divide the measured length by the magnification to give the actual length of the object.

 Leaf stomata

Types of stoma

In the mature leaf, four significantly different types of stoma are distinguished by their form and the arrangement of the surrounding cells, especially the subsidiary cells: 

• The anomocytic or ranunculaceous (irregular-celled) type; the stoma is surrounded by a varying number of cells, generally not different from those of the epidermis.
• The anisocytic or cruciferous (unequal-celled) type; the stoma is usually surrounded by three or four subsidiary cells, one of which is markedly smaller than the others.
• The diacytic or caryophyllaceous (cross-celled) type; the stoma is accompanied by two subsidiary cells, the common wall of which is at right angles to the stoma.
• The paracytic or rubiaceous (parallel-celled) type; the stoma has two subsidiary cells, of which the long axes are parallel to the axis of the stoma.

In describing an epidermis where certain stomata differ from the predominant type, the term applying to the majority of stomata is used.

Determination of the stomatal index

Place fragments of leaves, about 5 x 5 mm in size, in a test tube containing about 5 ml of chloral hydrate TS and heat on a water bath for about 15 minutes or until the fragments are transparent. Transfer a fragment to a slide and prepare it as described earlier, the lower epidermis uppermost, in chloral hydrate TS; place a small drop of glycerol/ethanol TS at one side of the cover-glass to prevent the material from drying. Examine under a microscope with a 40x objective and a 6x eyepiece, equipped with a drawing apparatus. Mark on the drawing paper a cross (χ) for each epidermal cell and a circle (o) for each stoma. Calculate the stomatal index as follows:

stomatal index = S x100

                            -------------------

                                E+ S

where S = the number of stomata in a given area of leaf E= the number of epidermal cells (including trichomes) in the same area of leaf.

For each leaf sample, no fewer than ten determinations should be carried out and the average index calculated.

Qualitative chemical examination of extracts

Qualitative chemical examination of extracts

The extraction can be done by taking powdered drugs in

The general method followed for screening of the constituents present in the extract is as follows:-------------------------------------------------------------------------------------------------

Detection of carbohydrates:

Extracts were dissolved individually in 5 ml of distilled water and filtered. The filtrates were used to test the presence of carbohydrates.

    a)   Molisch’s test: (named after Austrian botanist Hans Molisch) Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube, and 2ml concentrated sulphuric acid was added carefully along the sides of the test tubes. The formation of a violet ring at the junction indicates the presence of carbohydrates.

    b) Benedict’s test (also called Benedict's solution or Benedict's test) is a chemical reagent named after an American chemist, Stanley Rossiter Benedict) (One liter of Benedict's reagent can be prepared from 100 g of anhydrous sodium carbonate, 173 g of sodium citrate, and 17.3 g of copper(II) sulfate pentahydrate. It is often used in place of Fehling's solution.)

Filtrates were treated with Benedict’s reagent and heated in a water bath. The formation of an orange-red precipitate indicated the presence of reducing sugars.

C)    Fehling’s test: The test was developed by German chemist Hermann von Fehling in 1849.^[2] Fehling's solution is always made just prior to the test. It is composed of equal parts of the following solutions:

• 69.28 grams copper(II) sulfate pentahydrate dissolved in 1 liter of distilled water (Fehling's solution A)
• 346 grams Rochelle salt (potassium sodium tartrate tetrahydrate) and 120 grams sodium hydroxide in 1 liter of distilled water (Fehling's Solution B)

Filtrates were hydrolyzed with dilute hydrochloric acids, neutralized with alkali, and heated with Fehlings A and B solutions. The formation of a red precipitate indicates the presence of reducing sugars.

d) Osazone formation: A sugar, on heating with Phenyl hydrazine hydrochloride, sodium acetate, and acetic acid, forms yellow crystals of osazone.

e)  Selivanoff’s test: (Resorcinol test for ketoses )  A crystal of resorcinol is added to the solution and heated with an equal volume of concentrated hydrochloric acid  Pink color is produced in the case of ketoses (eg. Fructose, honey, or hydrolyzed insulin).

f) Test for pentose: A solution of the material is heated with an equal volume of hydrochloric acid containing a little phloroglucinol. A red color is formed in the case of pentose.

g) Furfural test: The sample is heated in a test tube with a drop of syrupy phosphoric acid to convert it into furfural. A disk of filter paper moistened with a drop of 10% solution of aniline in 10% acetic acid is placed over the mouth of the test tube. The bottom of the test tube is heated for 30-60 seconds. A pink or red stain appears on the reagent paper.

h)  Barfoed's Test: The Barfoed's Test is a chemical test carbohydrates that detects the presence of monosaccharides. Placing one ml of a sample solution in a test tube, 3 ml of Barfoed's reagent, a solution of acetic acid, and cupric acetate solution are added. Heat the solution in a boiling water bath for three minutes, and if a formation of reddish precipitate appears, it tests positive for monosaccharides.

Detection of alkaloids: Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were tested carefully with alkaloid reagents.

a) Mayer’s test: Filtrates were treated with Mayers reagent (potassium mercuric iodide), formation of a yellow cream precipitate indicates the presence of alkaloids.

b) Wagner’s test: Filtrates were treated with Wagner’s reagent (iodine in potassium iodide) and observed. The formation of a brown/reddish brown precipitate indicates the presence of alkaloids.

c) Dragendroff’s test: Filtrates were treated with Dragendroff’s reagent (solution of potassium bismuth iodide). The formation of a red precipitate indicates the presence of alkaloids.

d) Hager’s test: Filtrates were treated with Hager’s reagent (saturated picric acid solution) Formation of a yellow-colored precipitate indicates the presence of alkaloids.

e)  Tannic acid test: A freshly prepared solution of tannic acid (55w/w) gives a precipitate with most of the alkaloids, which is soluble in dilute acid or ammonia solution.

f) Ammonia Reineckats test: A saturated aqueous solution of ammonia reineckate, slightly acidified with hydrochloric acid, gives a pink flocculent precipitate with most of the alkaloids.

Detection of phytosterols:

a) Salkowski’s test: The extracts were treated with chloroform and filtered separately. The filtrates were treated with a few drops of concentrated sulphuric acid, shaken, and allowed to stand. If the lower layer turns red, sterols are present. If the lower layer turns golden yellow triterpenes are present.

b) Liebermann Burchard test: The extracts were treated with chloroform solution and a few drops of conc. 1 ml of acetic anhydride solution followed by sulphuric acid. A blue-green colour shows the presence of phytosterols.

Detection of glycosides:

Extracts were hydrolyzed with dilute hydrochloric acid, and the hydrolysate was subjected to glycoside tests.

Test for cardiac glycosides:

   

 a. Legals test: To the hydrolysate, 1 ml of pyridine and a few drops of sodium nitroprusside solution are added, and then it is made alkaline with sodium hydroxide solution. Colour change shows the presence of glycosides.

b. Liebermann’s test: 3 ml. of extract with 3 ml. acetic anhydride was heated, cooled then a few drops of conc. sulphuric acid. The blue color appears in the presence of bufadenolide.

c. Test for deoxysugars (Keller killiani test): 2 ml. of extract, glacial acetic acid, one drop of 5% FeCl3, and conc. H2SO4 was added. A reddish-brown color appears at the junction of two liquid layers, and the upper layer appears bluish-green.

d. Xanthydrol test: The crude drug is heated with a 0.1 to 5% solution of Xanthydrol in glacial acetic acid containing 1% hydrochloric acid. A red color is produced due to the presence of 2-deoxysugar.

Test for anthraquinone glycosides:

a. Borntrager’s test: Hydrolysate is treated with chloroform, and the chloroform layer is separated. To this, an equal quantity of dilute ammonia solution is added. Color change in the ammoniacal layer shows the presence of O glycosides.

b. Modified Borntrager’s Test: The extracts were treated with ferric chloride solution and heated in a boiling water bath for about 5 min. The mixture was cooled and shaken with an equal volume of benzene. The benzene layer was separated and treated with half its volume of ammonia solution. The formation of pink or cherry red color in the ammonical layer indicates the presence of a C glycoside.

Detection of saponin:

a) Froth’s test: The extracts were diluted with distilled water to 20 ml, shaken in a graduated cylinder for 15 min. The formation of a 1 cm layer of foam indicates the presence of saponins.

b) Haemolytic test: Add drug extract or dry powder to one drop of blood placed on a glass slide. A hemolytic zone appears.

Test for Cyanogenic glycosides:

Ferriferrocyanide test: Macerate 1 g of the powdered drug with 5 ml of alcoholic KOH for 5 min. transfer it to an aqueous solution containing FeSO₄ and FeCl₃, and maintain at 60⁰-70⁰C for 10 minutes. Now transfer of the contents confirms the presence of HCN.

Detection of phenolic and tannins:

a. Ferric chloride test: The extract was treated with a few drops of neutral ferric chloride solution. The formation of a bluish-black color indicates the presence of a phenolic nucleus.

b.Gelatin test: To the extract, 1% gelatin solution containing sodium chloride was added. The formation of a white precipitate indicates the presence of tannins.

c. Goldbeater’s test: A small piece of goldbeater skin (membrane prepared from the intestine of an ox) is soaked in 20% Hydrochloric acid, rinsed with distilled water, and placed in a solution of tannins for 5 minutes. The skin pieces are washed with distilled water and kept in a solution of ferrous sulphate. A brown or black color is produced due to the presence of tannins.

d. Phenazone test: A mixture of aqueous extract of a drug and sodium acid phosphate is heated, cooled, and filtered. A solution of phenazone is added to the filtrate. A bulky-colored precipitate is formed.

e. Match stick test (Catechin test): a match stick is dipped in aqueous plant extract, dried near a burner, and moistened with concentrated hydrochloric acid. On warming near the flame, the matchstick wood turns pink or red due to the formation of phloroglucinol.

Test for flavonoids:

a. Lead acetate test: The extracts were treated with a few drops of lead acetate solution; the formation of a yellow precipitate indicates the presence of flavonoids.

b. Alkaline reagent test: The extracts were treated with a few drops of sodium hydroxide separately. The formation of an intense yellow color, which becomes colorless with the addition of a few drops of dilute acid, indicates the presence of flavonoids.

c. Shinoda test: The extracts were treated with a few fragments of magnesium metal separately, followed by a dropwise addition of concentrated hydrochloric acid. The formation of a magenta color indicates the presence of flavonoids.

Detection of proteins and amino acids:

a. Millon's test: (The reagent is made by dissolving metallic mercury in nitric acid and diluting it with water. The test was developed by the French chemist Auguste Millon (1812–67).

The extracts were treated with 2ml of Millon's reagent. The formation of a white precipitate, which turns to red upon heating, indicates the presence of proteins.

b. Ninhydrin test: (Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect fingerprints, as the terminal amines or lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin.) 

To the extracts, 0.25% ninhydrin reagent was added and boiled for a few minutes. The formation of blue color indicates the presence of amino acids.

c. biuret test: (The biuret reagent is made of potassium hydroxide (KOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, and blue to pink when combined with short-chain polypeptides.) 

An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often), followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5–160 mg/mL can be determined.

d.